1. Examination of the usefulness as the new boron compound of ACBC-BSH Gen Futamura 1 Shinji Kawabata 1 Shin-Ichi Miyatake 1 Toshihiko Kuroiwa 1 Yoshihide Hattori 2 Mitsunori Kirihata 2 Hiroki Tanaka 3 Yoshinori Sakurai 3 Shinichiro Masunaga 3 Koji Ono 3 1 Department of Neurosurgery, Osaka Medical College 2 Osaka Prefecture University 3 Kyoto university research reactor institute 16 th International Congress on Neutron Capture Therapy June 16, 2014 Helsinki, Finland

2. 1)1-amino-3-fluorocyclobutane-1-carboxylic acid (ACBC), unnatural amino acid was reported as the agent which showed intense uptake in glioblastoma 1)-4) What is ACBC ? 1)Washburn LC, et al, Cancer Res 1978;38:2271-2273 2)Washburn LC, et al, J Nucl Med 1979;20:1051-1055 3)Goodman MM, et al, J Labelled Compd Radiopharm 1994 4)Hubner K, et al, Mosby-Year Book, Inc 1992-10 Introduction *ACBC has high affinity to amino acid transporter. *ACBC is not metabolized in cells. *ACBC is not used for protein synthesis.

3. 2) In a clinical study, [ 18 F]-ACBC showed usefulness as a tracer of PET imaging 5) T/N ratio 5.0 MRI(Gd+ ） 18 F-ACBC PETFDG PET This PET imaging becomes useful as well as F-BPA PET imaging in clinical BNCT 5) Shoup, et al, J NucI Med 1999; 40: 331 Introduction

4. 2-2- ACBC-BSH Introduction ACBCBSH + We conjugate BSH to ACBC for further BNCT experiments.

5. *We have adopted Convection Enhanced Delivery(CED) for drug administration to solve this problem 6)-9) 2-2- ACBC-BSH BPA 6)Kawabata, et al, J Neurooncol 2011 June 1 7)Miyata et al, Neurosurgery 2011 68:1380-1387 9)Saito et al, Cancer Res 2004; 64(7):2572-2579 8)Hiramatsu et al, Lasers Surg Med. 2011 January *ACBC-BSH is high molecular weight and low electrical charge, so it is difficult for this agent to reach regions that tumor cells invade microscopically where the BBB seems to be intact. Introduction Molecular weighht:439.75Molecular weighht:208

6. *CED is one of the methods for local drug infusion into the brain. *Continuous slow infusion into a local site of the brain under a small constant positive pressure makes a ‘bulk flow’ in the interstitial space. *Bulk flow enables to distribute high molecular weight drugs to a large area in interstitial space, keeping high concentration without through the disrupted BBB. Convection Enhanced Delivery (CED) CED enables to distribute ACBC-BSH to tumor cells invade microscopically where the BBB seems to be intact. Introduction

7. Convection Enhanced Delivery (CED) Brain Brain F98 glioma Hamilton syringe

8. 1) in vivo biodistribution experiment To determine the biodistribution of ACBC-BSH following intracerebral (i.c.) administration by CED to F98 glioma bearing rats. Administration rate Administration rate: 0.33 µl/min, 30 min (CED30min) 0.13 µl/min, 24 h (CED24h) 2) in vivo BNCT therapy experiment To determine the efficacy of ACBC-BSH as boron delivery agents for BNCT in F98 glioma bearing rats. Objectives

9. *Animal: Fischer 344 male rat *Cell line: F98 rat glioma cells （ bearing 1×10 5 cells ） (provided by Rolf F. Barth, Department of Pathology, Ohio State University) *Way of administration: CED or iv *CED Administration rate: 0.33 µl/min, 30 min (total 10 µl) 0.13 µl/min, 24h (total 200 µl) *Evaluation of boron concentration: ICP-AES Materials and Methods 1) (Tumor, Blood, brain, skin, muscle, heart, lung, liver, spleen, kidney)

10. Boron compound *ACBC-BSH 1000 µg 10 B/ml *BPA 1000 µg 10 B/ml Injection methods *ACBC-BSH CED30min dose 10µl *ACBC-BSH CED24h dose 200µl *ACBC-BSH iv dose 2000µl *BPA iv dose 2000µl =250 mg 10 B/kg b.w. Materials and Methods 1)

11. i.c. tumor implantation CED over 24 h 1 h 6 h 24 h Timing of boron concentration measurement i.c. tumor implantation CED over 30 min 1 h 6 h24 h i.c. tumor implantation iv 1 h 6 h iv CED30min CED24h Materials and Methods 1)

12. Boron concentrations ± SD （ µg 10 B/g ） Ratios b Agent Time a (h)nBloodBrainTumorT/BloodT/Brain ACBC-BSH CED c 30min 140.8±0.30.1±0.010.7±3.013.3147.2 630.9±0.60.2±0.10.5± 2.8 2430.8±0.1 ±0.00.4±0.30.46.2 CED d 24h 1 33.3±2.21.5±1.321.1±3.36.414.2 6 31.3±0.00.4±0.317.8±10.113.742.4 2430.5±0.10.0± ±0.1 1.4 iv e 146.3±1.70.2±0.02.7±0.50.414.9 637.0±3.40.3±0.16.0±1.70.918.6 BPA iv f 138.5±0.33.0±0.619.7±1.42.36.7 634.1±1.33.1±1.314.4±3.43.64.7 Result Boron concentrations (Blood, Brain, Tumor) 1)

13. （ µg 10 B/g ） Boron concentration (Tumor) Tumor Boron concentrations Result 1)

14. Boron concentrations ± SD （ µg10B/g ） BPAACBC-BSH Organ i.v. CED 30min CED 24h Skin5.2±1.65.1±2.11.0±1.90.5±0.6 Muscle6.7±1.62.6±1.90.2±0.42.1±1.3 Heart7.8±0.51.4±0.50.1± 1.3±0.9 Liver9.6±1.420.7±5.40.3±0.11.9±1.8 Spleen14.6±5.36.1±3.13.0±1.94.4±2.0 Kidney45.7±12.3106.4±15.70.6±0.11.5±0.4 Lung8.5±2.111.7±8.20.3±0.11.2±0.8 Blood8.5±0.36.3±1.70.8±0.33.3±2.2 Brain3.0±0.60.2±0.00.1±0.01.5±1.3 Tumor 19.7±1.4 2.7±0.5 10.7±3.0 21.1±3.3 Boron biodistribution in normal tissue （ 1hour after intracerebral drug administration ） Result 1)

15. Immunostaining of F98 glioma cell （ in vitro ） ACBC-BSH BPA microscopeimmunostainingoverlay diffusely distuributed in the cytoplasm and cell nuclei aggregated on the fringe of the cell nuclei Result 1)

16. * The tumor boron concentrations of ACBC-BSH CED24h (21.1µg/g) was highest and was similar to BPA iv (19.7µg/g). * Immunostaining showed that ACBC-BSH was incorporated into the cytoplasm of the F98 cells and aggregated on the fringe of the cell nuclei compared with BPA. We expected that the BNCT using ACBC-BSH delivered by CED24h therapeutic effect would show greater therapeutic effect than BPA iv administration. Snmmary of biodistribution experiment 1)

17. *Animal: Fischer 344 male rat *Cell line: F98 rat glioma cells （ bearing 1×10 3 cells ） (provided by Rolf F. Barth, Department of Pathology, Ohio State University) *Boron delivery system: ACBC-BSH(1000 µg 10 B/ml) *Studies were initiated at KUR. *Way of administration for BNCT study a) untreated b) Irradiated （ neutron irradiation...1MW 1hour ） c) BPA iv + neutron irradiation d) ACBC CED24h + neutron irradiation e) ACBC CED24h + BPA iv + neutron irradiation Materials and Methods 2)

18. Boron concentrations±SD （ µg10B/g ） Physical radiation dose b (Gy ) Biological radiation dose c (Gy-eq) AgentRoute Time a (h) BrainTumorBrainTumorBrainTumor ACBC-BSH CDE24h11.5±1.321.1±3.31.1 3.7 2.1 d 12.3 d BPA iv13.0±0.619.7±1.41.3 3.5 2.3 11.6 Without compound s --0±00±00.9 -- Untreated Controls --0±00±0 00 00 Radiation doses Result 2)

19. Kaplan-Meier survival curves Survival rate Result 2)

20. 1) The CBE factor is specific for different tissues and for different boron compounds and depends on the microdistribution of 10 B in the cells. Therefore, therapeutic effect by the same boron concentration varies according to the localization. 10 )-12) Discussion B B B B B B B B B B B B B B B B cytoplasm nucleus As much as a boron compound accumulates it near the nucleus, the therapeutic effect becomes higher. Conceptual diagram of CBE = high CBE 12) Dagrosa MA, et al, International journal of radiation oncology. 2011;79:262-8. 11) Santa Cruz GA, et al, Radiation research. 2004;162:702-10. 10) Fox JC, et al, BJR supplement / BIR. 1992;24:48-52. alpha particle

21. 1)The therapeutic effect of ACBC-BSH CED24 and BPA iv was approximately same. The reason is that I think extracellular accumulations of ACBC-BSH was high. Discussion B B B B B B B B B B B B B B B B cytoplasm nucleus alpha particle ACBC-BSHBPA B B B B Conceptual diagram of CBE in this case

22. 2)We did not compete for the therapeutic effect by using ACBC- BSH together in BPA. Difference in drug distribution at the cell level was regarded as the cause. Discussion ACBC-BSH BPAmicroscopeimmunostainingoverlay

23. Conclusion *This study suggested the possibility that ACBC-BSH became the drug to add therapeutic effect to.

24. Thank you for your attention

26. Discussion 13) Yang W, et al, Journal of neuro-oncology. 2011;101:379-90 BNCT using ACBC-BSH CED24 and BPA iv expect higher therapeutic effect than existing treatment of BNCT. 3) The F98 brain tumor model that we used is the animal model that is resistant to standard treatment using temozolomide 13). Rolf F. BARTH et al 14) reported that the following survival data. ％ ILS *untreated.....................................................................Rat survival 24days (mean) - *irradiation (Physical dose 4.42Gy)…………….........Rat survival 29days (mean) 16 *BPA iv(250mg/kg) +BSH iv(30mg/kg)+BNCT..……Rat survival 41days (mean) 68 +17days duration of survival This time our study result *untreated……….........................................................Rat survival 26days (mean) - *irradiation (Physical dose 0.9Gy)...............................Rat survival 30days (mean) 13 *BPA iv+ BNCT..........................................................+10days duration of survival 40 *ACBC-BSH CED24h+BNCT....................................+11days duration of survival 43 *ACBC-BSH CED24h+BPA iv+ BNCT.....................+16days duration of survival 59 Other time our study result *untreated …………………………………................Rat survival 30days (mean) - *irradiation (Physical dose 1.0Gy)……………….......Rat survival 31days (mean) 3 *BPA iv(250mg/kg) +BSH iv(100mg/kg)+BNCT……+10days duration of survival 32 14) R.F.Barth, et al, Int.J.Radiation Oncology Biol.Phys.2000;209-218

27. *The therapeutic effect of both ACBC-BSH CED24h and BPA iv was approximately same. *The therapeutic effects by ACBC-BSH and BPA did not compete. in vivo BNCT efficacy study Summary of BNCT efficacy study

28. Agent/Route Survival Time %ILS b Groupnana Mean±SDMedianRangeMedian Untreated Controls6 27.2±2.426.525-31 - Irradiated Controls529.8±1.930.027-3213.2 BPA/i.v.537.4±2.637.034-4039.6 ACBC-BSH/CED24h737.0±5.238.031-4443.4 ACBC-BSH/CED24h + BPA/i.v. 644.3±8.042.038-6058.5 survival times of F98 glioma bearing rats * p<0.0005 * p<0.0015 * p<0.0013 p<0.0382 * Result in vivo BNCT efficacy study * log-rank test p<0.05 %ILS = MeST(untreated controls) MeST(each groups)-

29. ACBC BSH +

30. F98 in vitro Boron up take Figure4

31. Kyoto university research reactor institute

32. Discussion F98 glioma cell を用いた免疫染色 ACBC-BSH BPA顕微鏡写真 免疫 染色 重ね合わせ 細胞核を含む 細胞内全体に分布 細胞核周辺に凝集 B B B B B B B B B B B B B B B B 細胞質 核 核のより近くにホウ素が 集積した方が、殺腫瘍 細胞効果が高い α 粒子

33. F98 in vitro Boron up take 各化合物のモル濃度をそろえて 24 時間暴露