1. Daisuke Kanoh, 1 Shoji Tachikawa, 1 Shinichi Sato, 2 Hiroyuki Nakamura*,2 1)Department of Chemistry, Faculty of Science, Gakushuin University, Japan 2) Synthetic Organic Division, Chemical Resources Laboratory Tokyo Institute of Technology, Japan Development of Albumin-bound closo- Dodecaborate and its Promising Boron Delivery Efficacy to Tumor

2. >25 ppm 10 B in Tumor, T/B and T/N >2.5 Large amount administration is required! ⇒ Significantly low toxic Different approach is needed in comparison with conventional antitumor drug design How can we deliver a large amount of 10 B to tumor? Use of Nanocarriers (Micelles, Liposomes…) In the clinical study: L-BPA (500 mg/kg) 1 hr before irradiation ⇒ Total dose: 30 g /person (60 kg body weight) Requirements for Efficient BNCT

3. Nano Carrier Drug Delivery System (DDS) Tumor tissue -Enhanced permeability and retention (EPR) effect → particle size of 100 nm - Escape from Reticulo-endothelial-system (RES) → PEG-conjugation Tumor blood vessel Tumorcells Abnormal architectures without tight junction Liposome

4. 10 B Ligand 10 B Liposomal Boron Agents for BNCT 1991- H. Yanagie, et al Boronated anti-CEA immunoliposome 2002- R. J. Lee, et al FA-PEG-Liposome 2003- E. B. Kullberg, et al EGF-PEG-Liposome 2004- H. Nakamura, et al Boron Lipid Liposome 1992- M. F. Hawthorne, et al Boronated DSPC Liposome 2004- K. Maruyama, et al TF-PEG-Liposome PEG Boron lipids 2013 H. Nakamura, et al High Boron Content Liposome 10 B DSBL Total lipid dose 147 mg/kg Total lipid dose 743 mg/kg 30 ppm (15 mgB/kg) 82 ppm (30 mgB/kg) 143 ppm (50 mgB/kg) Boron conc. in tumor H. Koganei, et. al., Bioconjugate Chem. 2013, 24, 124. 100 nm 2009- K, Tomizawa, H. Matsui et al Anti-EGFR-antibody-Liposome

5. 5 Antibody-conjugates Biopolymer-Based Boron Delivery BSD MoAb IB16-6 (B16-BL6 melanoma) 4.8 x 10 4 ~ 2.5 x 10 5 / cell R. F. Barth, et al, Bioconjugate Chem. 1994, 5, 58-66 Cetuximab BSD ~1100 boron atoms/BSD R. F. Barth, et al, Bioconjugate Chem. 2004, 15, 185-194 Growth factor-conjugates EGF (53 aminoacid residues) BSD BSD-EGF BNCT effect Rolf. F. Barth, et al, Cancer Res. 2002, 62, 3159. F98 glioma-bearing rats

6. 6 Biopolymer-Based Boron Delivery 12,00017,00029,000 68,000 (8 nm)150,000 (15 nm) EPR Effect was observed in the proteins with the MW over 68,000 Accumulation of Evans blue-stained albumin complex in tumor blood tumor skin

7. Albumin as a Biopolymer DDS Vehicle Abraxane® (an albumin-paclitaxel nanoparticle) -FDA approval in 2005 -Treatment of Breast cancer, non-small cell lung cancer, stomach cancer, and adenocarcinoma of the pancreas Structure of Abraxane A 130 nm albumin-bound particle form of paclitaxel Uptake of abraxane is presumably mediated by the EPR effect and the gp60 transcytosis. F. Kratz, B. Elsadek, J. Control. Release 161 (2012) 429–445 Albumin, a major plasma protein constituent, is composed approximately 55% of the human plasma protein, and has been extensively investigated as a versatile carrier for therapeutic and diagnostic agents.

8. 8 Maleimide Doxorubicin Conjugate in situ Binding to Albumin + Chemical structure of INNO-206, the (6-maleimidocaproyl)hydrazone derivative of doxorubicin Tumor targeting based on in situ binding to the circulating albumin after intravenous injection. C. Unger, B. Haring, M. Medinger, J. Drevs, S. Steinbild, F. Kratz, K. Mross, Clin. Cancer Res. 13 (16) (2007) 4858.

9. 9 Design of Maleimide Boron Cluster Conjugates Maleimide-closo-dodecaborate conjugate （ B 12 H 12, MID ） Easy to synthesize and low toxic BSA (bovine serum albumin) D.S. Wilbur, M. F. Hawthorne, Bioconjugate Chem. 2007, 18, 1226−1240 Maleimide-decaborate conjugate （ B 10 H 10 ） (COCl) 2 68% F(ab’) 2 antibody

10. 10 4 quant 87% 2 steps 321 9 V. I. Bregadze, Appl.Organometal. Chem., 2007, 21, 98-100 5 (MID-TBA) MID 77% 89% 2 steps Synthesis of Maleimide-closo-Dodecaborate Conjugate (MID)

11. 11 MID is low toxic! IC 50 (mM) >1 MTT assay (CT26 and B16 cell 、 72h) MID 4 BSH Cytotoxicity of closo-Dodecaborates

12. 12 The reaction proceeded quantitatively in an hour at rt. Reaction of MID with Cysteines + 6 (MID-TMA) 7a: R = H 7b: R = Ac 8a: R = H 8b: R = Ac MID-2TMA ESI-negative MS m/z 205.1 8a m/z 265.6 8b m/z 286.4 The reaction process was monitored by ESI-TOF MS.

13. 13 BSH was detected by the anti-BSH antibody in Western blot analysis. The intensity of BSH-conjugated BSA bands increased until the mixture of an 1:1 ratio and then became plateau. Conjugation of MID to BSA in vitro antibody* * anti-BSH antibody was kindly donated by Prof. Kirihata (Osaka Prefecture University)

14. 14 Cys34 Total number of Cys: 33 Structure of BSA

15. 15 +410 The peptide including Cys34 We could detect the conjugation of MID and the peptide fragment including Cys34 in BSA. MW 2,845 MW 2,835(+48) MID: MW 410 MID : BSA = 1 : 1 MID : BSA = 100 : 1 MID : BSA = 10 : 1 BSA only Analysis MID-BSA Conjugation by MALDI-TOF-MS BSA was digested with trypsin and the peptide fragments were analyzed by MALDI-TOF-MS.

16. CT26-bearing mice (Balb/c/ ♀ ; 5 weeks) i.v. 16 Tumor boron conc. reached 60 ppm in the mice treated with MID-BSA conjugates (10:1, 30 mgB/kg) 12 hours after injection from tail vein. Boron concentration in other organs was low compared to boron liposomes. Biodistribution of MID-BSA Conjugates in tumor-bearing mice Tumor 60 ppm 38 ppm

17. Biodistribution of BSH-Boron Lipid Liposomes B/P = 2.7 BSH-Liposome 10% DSBL 30 and 50 mg B/kg H. Koganei, M. Ueno, S. Tachikawa, L. Tasaki, H. S. Ban, M. Suzuki, K. Shiraishi, K. Kawano, M. Yokoyama, Y. Maitani, K. Ono, H. Nakamura, Bioconjugate Chem. 2013, 24, 124.

18. CT26-bearing mice (Balb/c/ ♀ ; 5 weeks) i.v. 18 Biodistribution of MID-BSA Conjugates in tumor-bearing mice Tumor 60 ppm 38 ppm Tumor boron conc. reached 60 ppm in the mice treated with MID-BSA conjugates (10:1, 30 mgB/kg) 12 hours after injection from tail vein. Boron concentration in other organs was low compared to boron liposomes. EPR effect was also observed even in the case of the injection of MID itself, suggesting that the conjugation of MID to serum albumin in circulating blood.

19. 19 MID was treated with the blood of mice for 1 h at 37 o C and the MID-conjugated protein was determined by Western blotting analysis using anti-BSH antibody. The higher accumulation of the MID-BSA (10 ： 1) conjugate is probably due to in situ binding of MID to circulating albumin after intravenous injection. Conjugation of MID to Serum Albumin in Blood Control (blood only) MID + blood Control (blood only) MID + blood ~60 kDa (serum albumin)

20. 20 Tumor growth of mice injected with MID-BSA conjugates at a dose range of 7.5-30 mg[B]/kg was suppressed after BNCT, and the tumor of some mice completely disappeared 14 days after neutron irradiation. BNCT Effect of Mice Injected with MID-BSA (10:1) Conjugates Neutron flux 1.5-2.2 x 10 12 n/cm 2 Kyoto University Reactor (KUR) thermal neutron Collaborated with Prof. M. Suzuki’s group at KUR 30 mg[B]/kg 15 mg[B]/kg 7.5 mg[B]/kg

21. 1.We succeeded in the synthesis of maleimide closo-dodecaborate (MID) for conjugation of boron sources to biopolymers. 2.MID is low toxic. 3.MID readily reacted with cysteines including Cys34 in BSA. 4. MID also reacted with serum albumin in blood of mice. 5. Tumor boron conc. reached 60 ppm in the mice treated with MID-BSA (30 mg[B]/kg) conjugates 12 hours after injection. 6. Tumor growth of mice injected with MID-BSA conjugates was suppressed after BNCT. 21 Conclusion Acknowledgment We thank Professor Mitsunori Kirihata (Osaka Prefecture University) for kindly donating anti-BSH antibody and Stella Pharma Co. Ltd. for the supply of 10 B 12 H 12. We also thank Professors Suzuki, Kondo, Sakurai, and Tanaka for their kind supports to proceed thermal neutron irradiation experiments at KUR.

22. 9F 7F Thank you very much for your kind attention! Thank you very much for your kind attention!