A presentation on freeze-dried radiopharmaceutical kits for liver cancer therapy

1. Kit Based SIRT Agents: Preparation And
Quality Control
Madhava B Mallia
Radiopharmaceuticals Division, Radiochemistry and Isotope Group
Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085, INDIA

2. All options: Quick glance…
2
90Y-Glass microspheres
Therasphere®
90Y-resin microspheres
SIR-spheres®
166Ho-PLA microspheres
QuiremSpheres®
Images not to scale…
188ReN-DEDC/Lipiodol188Re-SSS/Lipiodol188Re-HDD/Lipiodol
131I-Lipiodol

3. SIRT AGENTS...
Microspheres
Therasphere®/SIR-spheres®/ QuiremSpheres®
O
O
I
I
Lipiodol
Lipiodol based agents
SIRT AGENTS KIT/Ready to use
131I-lipiodol Ready to use
188Re-lipiodol Kit formulation
3
Ready to use
Representative image

4. 90Y- Microspheres 188Re-Lipiodol 131I-Lipiodol
Half-life 64.1 h 16.9 h 8 days
Isotope energy 2.28 MeV (99.9 %) 2.12 MeV (71.4 %)
1.96 MeV (25.3 %)
0.60 MeV (71.4 %)
0.33 MeV (25.3 %)
Beta range (tissue)
(Max – Min)
11.9 - 2.5 mm 11.0 - 2.4 mm 0.3 – 2.0 mm
Imagable gamma Low abundance
positron
Yes Yes
Production Reactor Generator Reactor
Cost of therapy Very high Moderate to low Moderate to low
188Re  Poor man’s Yttrium
4

5. Cancer
patient
population
Cancer
patient
population
High
Income
Low
Income
90Y- SIR-Spheres® 188Re-Lipiodol
COST FACTOR
5

6. Options...
6
188Re-HDD/Lipiodol
• Lee YS, Jeong JM, et al. Nucl Med Commun.
2002; 23(3):237-42.
• Paeng JC, Jeong JM, et al. J Nucl Med. 2003
Dec;44(12):2033-8.
ReN kit DEDC
kit
188ReN-DEDC/Lipiodol
• Boschi A, Uccelli L et al. Nucl Med Commun.
2004;25(7):691-9.
188Re-SSS/Lipiodol
• Nicolas Lepareur et al., International Journal of
Molecular Imaging, doi:10.1155/2012/278306

7. 188Re-Radiopharmaceuticals for TARE:
General strategy
1.Prepare a lipophilic 188Re-complex
2.Extract into lipiodol
3.Assay and use it for TARE
7

8. 188Re-HDD/Lipiodol...
• HDD evolved from TDD
TDD
(2,2,9,9-Tetramethyl-4,7-diaza-1,10-decanedithiol)
HTDD
(4-Hexadecyl-2,2,9,9-Tetramethyl-4,7-diaza-1,10-
decanedithiol)
8
Jeong JM, Kim YJ, Lee YS, Ko JI, Son M, Lee DS, Chung JK, Park
JH, Lee MC. Lipiodol solution of a lipophilic agent, (188)Re-TDD, for
the treatment of liver cancer. Nucl Med Biol. 2001;28(2):197-204.
Paeng JC, Jeong JM, Yoon CJ, Lee YS, Suh YG, Chung JW, Park JH,
Chung JK, Son M, Lee MC. Lipiodol solution of 188Re-HDD as a new
therapeutic agent for transhepatic arterial embolization in liver cancer:
preclinical study in a rabbit liver cancer model. J Nucl Med. 2003 ;
44(12):2033-8.

9. 9
HTDD
(4-Hexadecyl-2,2,9,9-Tetramethyl-4,7-diaza-1,10-
decanedithiol)
AHTDD
(Acetylated 4-Hexadecyl-2,2,9,9-Tetramethyl-4,7-
diaza-1,10-decanedithiol)
Lee YS, Jeong JM, Kim YJ, Chang YS, Lee HJ, Son M, Lee
JW, Yoon HS, Kang WJ, Lee DS, Chung JK, Lee MC, Suh
YG. Development of acetylated HDD kit for preparation of
188Re-HDD/lipiodol. Appl Radiat Isot. 2007;65(1):64-9.
AHTDD kit

10. 10
AHDD
(Acetylated 4-Hexadecyl-4,7-diaza-1,10-
decanedithiol)
AHTDD
(Acetylated 4-Hexadecyl-2,2,9,9-Tetramethyl-4,7-
diaza-1,10-decanedithiol)
Ligand used at present
Banka VK, Moon SH, Jeong JM, Seelam SR, Lee YS, Kim YJ, Lee DS, Chung JK. Development of 4-hexadecyl-4,7-diaza-1,10-decanedithiol (HDD) kit for the preparation
of the liver cancer therapeutic agent Re-188-HDD/lipiodol. Nucl Med Biol. 2015 Mar;42(3):317-22.

11. Freeze-dried AHDD kit...
• AHDD – 1 mg
• SnCl2.2H2O – 6 mg
• Tartaric acid – 20 mg
• Mannitol – 10 mg
11
AHDD kit

12. 12
Preparation: Up to 3 mL of freshly
eluted sodium 188Re-perrhenate
added to AHDD kit vial and heated at
100C for 1 h.
100C, 60 min
AHDD kit
1. Vortex (10 min)
2. Centrifuge (10 min)
3. Separate lipiodol layer
3 mL lipiodol
Preparation of 188Re-HDD/Lipiodol...

13. Mixing of lipiodol phase: Minimizing the dose to
the operator...
13

14. Centrifuge and separate lipiodol phase...
14
Aqueous
phase
Lipiodol
phase
Automated
procedure for
separation of
lipiodol phase!!

15. Quality control:188Re-HDD/Lipiodol...
15
Banka VK, Moon SH, Jeong JM, Seelam SR, Lee YS, Kim YJ, Lee DS, Chung JK. Development of 4-hexadecyl-4,7-diaza-1,10-decanedithiol (HDD) kit for
the preparation of the liver cancer therapeutic agent Re-188-HDD/lipiodol. Nucl Med Biol. 2015 Mar;42(3):317-22.
Spot the lipiodol extract

16. Extraction efficiency...
16
AHTDD AHDD

17. 188Re-SSS/Lipiodol...
• Garin et al...2004
• 188Re-(S2CPh)(S3CPh)2 complex  188Re-SSS complex
• Higher yield
• Higher extraction into lipiodol compared to 188Re-HDD
• Lower intestinal and urinary excretion compared to 188Re-HDD
17
Garin E, Noiret N, Malbert C, Lepareur N, Roucoux A, Caulet-Maugendre S, Moisan A, Lecloirec J, Herry JY, Bourguet P. Development and biodistribution of
188Re-SSS lipiodol following injection into the hepatic artery of healthy pigs. Eur J Nucl Med Mol Imaging. 2004 Apr;31(4):542-6.

18. Freeze-dried SSS kit (Two-vial kit)...
Kit 1
• Sodium gluconate – 7.5 mg
• Ascorbic acid – 30 mg
• Potassium oxalate – 40 mg
• SnCl2.2H2O – 0.8 mg
Kit 2
• Sodium thiobenzoate – 20 mg
18
Kit 1 Kit 2
Sodium thiobenzoate

19. Step 1
• Reconstitute vial 1 with 1-2 mL (max.) of freshly
eluted Na188ReO4
• Incubate vial 1 for 15 min at ambient temperature
Step 2
• Reconstitute vial 2 with 0.5 mL of physiological
serum/saline and transfer to vial 1
• Heat vial 1 at 100C for 30 min
19
Preparation of 188Re-SSS/Lipiodol...
1. Vortex (10 min)
2. Centrifuge (10 min)
3. Separate lipiodol layer
3 mL lipiodol

20. Centrifuge and separate lipiodol phase...
20
Aqueous
phase
Lipiodol
phase

21. 21
Quality control:188Re-SSS/Lipiodol...
Point of spotting Solvent front
188Re-SSS complex
Rf = 0.6
(Representative TLC)
TLC plate - silica/aluminium 60 F254 gel plates (Merck, Darmstadt, Germany)
Sample - 188Re-SSS complex/lipiodol
Developing solvent – Petrolium ether/dichloromethane (6:4 v/v)

22. Extraction efficiency...
22
Labeling efficiency Final Yield
188Re-SSS/Lipiodol 97.3 ±2.1%. 87% ± 9.1
Garin E, Noiret N, Malbert C, Lepareur N, Roucoux A, Caulet-Maugendre S, Moisan A, Lecloirec J, Herry JY, Bourguet P. Development and biodistribution of
188Re-SSS lipiodol following injection into the hepatic artery of healthy pigs. Eur J Nucl Med Mol Imaging. 2004 Apr;31(4):542-6.

23. 188ReN-DEDC/Lipiodol...
Boschi A et al…2004
23
188ReN intermediate
Step 1
+
DEDC ligand
Step 2
188ReN-DEDC complex
• Higher yield
• Higher extraction into lipiodol compared to 188Re-HDD
• Lower intestinal and urinary excretion compared to 188Re-HDD
Features similar to 188Re-SSS complex
Boschi A, Uccelli L, Duatti A, Colamussi P, Cittanti C, Filice A, Rose AH, Martindale AA, Claringbold PG, Kearney D, Galeotti R, Turner JH, Giganti M. A kit formulation for
the preparation of 188Re-lipiodol: preclinical studies and preliminary therapeutic evaluation in patients with unresectable hepatocellular carcinoma. Nucl Med Commun.
2004 Jul;25(7):691-9. Erratum in: Nucl Med Commun. 2004 Sep;25(9):983.

24. Freeze-dried DEDC kit (Two-vial kit)...
24
Kit 1
• N-methyl-S-methyl dithiocarbazate – 2 mg
• Sodium oxalate – 28 mg
• SnCl2.2H2O – 0.8 mg
Kit 2
• Diethyl dithiocarbamate – 27 mg
• NaCO3/NaHCO3 buffer
Kit 1 Kit 2

25. 25
Step 2Step 1
Glacial acetic acid (150 L)
+
Na188ReO4 (up to 3 mL)
KIT 1
Incubate for 30 min
at room
temperature
2 mL saline
KIT 2
1.5 mL of
reconstituted solution
from kit vial 2
KIT 1
80C, 20 min
KIT 1
3 mL lipiodol
KIT 1
1. Vortex (10 min)
2. Centrifuge (10 min)
3. Separate lipiodol
layer
Step 3
Preparation of 188ReN-DEDC/Lipiodol…
Average time for preparation: ~ 60 min

26. Centrifuge and separate lipiodol phase...
26
Aqueous
phase
Lipiodol
phase

27. Quality control:188ReN-DEDC/Lipiodol…
27
Point of spotting Solvent front
188ReN-DEDC complex
Rf = 0.8
(Representative TLC)
TLC plate - silica/aluminium 60 F254 gel plates (Merck, Darmstadt, Germany)
Sample - 188ReN-DEDC/lipiodol
Developing solvent –dichloromethane

28. 28
Extraction efficiency...
Labeling efficiency Final Yield
188ReN-DEDC/Lipiodol 97% ± 2 96% ± 3
Quantitative Extraction!!!

29. Making the kit more user friendly...
29
Step 1
Glacial acetic acid (150
L)
+
Na188ReO4 (up to 3 mL)
Incubate for
30 min at
room
temperature
KIT 1
Original method
DTCz 2 mg
SnCl2.2H2O 0.8 mg
Sodium oxalate 28 mg
pH 3 to 4
DTCz 2 mg
SnCl2.2H2O 0.8 mg
Sodium oxalate 28 mg
Oxalic acid xx mg
pH 3 to 4
Step 1
Modified method
KIT 1
Na188ReO4 (up to 3 mL)
Incubate for
5 min at
room
temperature

30. 30
Kinetics of formation of [188ReN]2+...
188ReN core
188ReO4
-
188ReN core
HPLC of [188ReN]2+ intermediate prepared using oxalate buffer kit
HPLC of [188ReN]2+ intermediate prepared by conventional method
Mallia MB, Chirayil V, Dash A. Improved freeze-dried kit for the preparation of (188)ReN-DEDC/lipiodol for the therapy of unresectable hepatocellular carcinoma.
Appl Radiat Isot. 2018 Jul;137:147-153.

31. 31
Step 2
2 mL saline
KIT 2
1.5 mL of
reconstituted solution
from kit vial 2
KIT 1
80C, 20 min
KIT 1
3 mL lipiodol
KIT 1
1. Vortex (10 min)
2. Centrifuge (10 min)
3. Separate lipiodol
layer
Step 3
Average time for preparation: ~ 35 min
Save 25 mins!!
188ReN-DEDC complex: Prepared by
conventional kit using glacial acetic acid
188ReN-DEDC complex: Prepared by
oxalate buffer kit

32. The procedure...
32
188Re-lipiodol

33. 188Re-HDD Vs 188Re-SSS Vs 188ReN-DEDC…
33
188Re-HDD 188Re-SSS 188ReN-DEDC
Commercial availability Yes Yes No (will be available in
India soon)
Manufacturer Seoul National University,
South Korea
Institute for
Radioelements (IRE),
Belgium
Radiopharmaceuticals
division, BARC
Cost ~$294 (in India) ---- ~$ 75 (proposed)
Volume limit (Na188ReO4) 3 mL 2 mL 3 mL
(lab tested up to 4 mL)
Extraction efficiency in
lipiodol (reported)
90.2% ± 2.6 87% ± 9.1 96% ± 3
Extraction efficiency in
lipiodol (Clinical practice,
India)
50 – 55% 65 – 75% 70 – 75%

34. • Rhenium-188 is generator produced
• Availability of freeze-dried kits (multiple options): On demand
preparation possible in hospital radiopharmacy
• No need to keep the patient in isolation
• Moderate to low cost compared to 90Y/166Ho – microsphere
therapy
34
188Re-SIRT Agents: Advantages…

35. 188Re-SIRT Agents: Challenges…
• Cost of 188W/188Re generator
• Limited 188Re-Radiopharmaceuticals for clinical practice
• Competition from TACE and other modalities
• “Too many cooks spoil the broth”: Too many nuclear medicine
centers for 188Re-therapy affects economic viability at individual
centers…
35

36. THANK YOU...
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