1. R.F. Barth 1, W. Yang 1, R.J. Nakkula 1, W. Tjarks 2, K. Ishita 2, L.C. Wu 3, P.J. Binns 4 and K.J. Riley 5 1 Department of Pathology, 2 Division of Medicinal Chemistry, 3 Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210 4 Department of Radiology, Mt. Auburn Hospital, Cambridge, MA 02138 5 Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 02114 Evaluation of Carboranyl Thymidine Analogues as Potential Delivery Agents for Boron Neutron Capture Therapy

2. Introduction We have had a longstanding interest in carboranyl nucleosides as potential boron delivery agents for Neutron Capture Therapy (NCT). Among these are carboranyl thymidine analogues (CTAs), which are substrates for thymidine kinase-1 (TK1) and is only expressed in proliferating cells, including a wide variety of a malignant tumors. As has been previously reported by us, a panel of 3-carboranyl Thd analogues (3CTAs) have been designed and synthesized. One of these, designated N5-2OH, previously has been evaluated in vivo using two tumor model systems, the murine L929 tumor in nude mice to validate target specificity and the RG2 rat glioma in Fischer rats to evaluate its therapeutic efficacy. In the present report we describe additional studies with N5-2OH using the F98 and RG2 rat glioma model and data on two recently synthesized CTAs designated 18a and 18b.

3. Chemical Structures and Formulas of Carboranyl Thymidine Analogues

4. Western Blot Analysis of Thymidine Kinase 1 Expression in Glioma and L929 Cell Lines 25- 20- 37- 50- MW marker (kDa) 10 50 hTK1 (ng) F98 RG2 L929TK- L929TK+ TK1 beta-actin F98 RG2 L929TK- L929TK+

5. Target Validation of L929 TK1(+) and (-) Tumors Following i.t. Injection of N5-2OH and BNCT TK1(+) (  ); TK1(-) (  ); TK1(+) radiation control (  ); TK1(+) untreated control (  ). The vertical lines indicate the standard deviations (SD) of the mean tumor volumes. Animals bearing TK1(+) tumors, which had received N5-2OH by i.t. injection followed by BNCT, had a 15 fold inhibition in growth with a tumor volume of 247±151 mm 3 compared to 3603±1103 mm 3 for matched control animals that did not receive BNCT and 2225±1074 mm 3 for radiated controls that did not receive N5-2OH. Animals bearing L929 TK1(-) tumors showed modest reductions in tumor volumes (Proc. Natl. Acad. Sci. 105:17493-97, 2008).

6. F98 Rat Glioma Model

7. Biodistribution and Physical Radiation Doses Following Administration of N5-2OH Boron uptake (ID%/g) ‡ Physical dose (Gy) ¶ Group † TumorBrain * Blood Tumor/ Brain Conc. Ratio TumorBrain F98/CED N5-2OH17.3±4.3<0.5 34.6 5.71.9 F98/CED N5-2OH + i.v. BPA 28.0±4.54.0±1.35.5±1.57.0 8.22.7 F98/i.v. BPA10.7±1.7 3.8±1.15.2±1.3 2.8 4.22.6 F98/ Irradiation control (CED of DMSO) None -- 1.8 10 B enriched N5-2OH was administered i.c. by means of convection enhanced delivery over 24 h using ALZET ® pumps at a flow rate of 8.33 μL/h. BPA was administered intravenously 2.5 h prior to BNCT. † F98 glioma cells were implanted into rats intracerebrally. The rats were irradiated 14 days after implantation. ‡ Boron content was quantified by means of DCP-AES. Boron uptake was present percent injected dose per gram tissue ( ID%/g) * Boron concentrations for the tumor bearing cerebral hemisphere after excision of the tumor. ¶ Physical dose estimates include contributions from γ photons, 14 N (n,p) 14 C and 10 B (n,  ) 7 Li reactions

8. Survival Times of F98 Glioma-Bearing Rats Following CED of N5-2OH With or Without i.v. BPA Group † No Survival time (days) * % Increased life span Mean±SE Median RangeMeanMedian CED of N5-2OH + i.v. BPA 843.5±5.9 43.5 36-52 7167 CED of N5-2OH 937.9±6.8 38 29-514946 i.v. BPA 1036.7±3.2 37 32-424442 Irradiated Controls 831.3±3.9 32 27-3723 Untreated Controls 725.4±2.4 26 21-28-- * Mean and median survival times were determined for each group of 7-10 rats. ¶ Percent increased life span (%ILS) was defined relative to mean and median survival times of untreated controls. † N5-2OH and BPA were administered as described in footnotes in Tables 1 and 2.

9. Survival Plots of F98 Glioma Bearing Rats Survival times have been plotted for untreated animals (●), irradiated controls (○), and animals that received i.v. BPA (♦) or N5-2OH either alone (▲) or in combination with BPA (  ).

10. Survival Plots of RG2 Glioma Bearing Rats Kaplan-Meier survival plots for RG2 glioma bearing rats. Survival times have been plotted for for untreated animals (●), irradiated controls (○), and animals that received i.v. BPA (♦) or N5-2OH either alone (▲) or in combination with BPA (  ).

11. Chemical Structures and Formulas of Carboranyl Thymidine Analogues

12. Biodistribution of Carboranyl Thymidine Analogues N5-20H, 18a and 18b in RG2 Glioma Bearing Rats a Boron concentrations (µg/g) Ratios Test agent b % Boron c and µg Boron TumorR. BrainL. BrainBlood T/BrT/Bl N5-2OH20.45115.3 8.9 ± 2.92.1 ± 0.91.3 ± 0.51.0 ± 0.4 4.39.3 18a25.2971.7 32.5 ± 17.96.7 ± 8.41.5 ± 1.10.5 ± 0.0 4.962.4 18b23.2990.0 9.44 ± 3.80.9 ± 1.30.8 ± 0.70.5 ± 0.0 10.820.4 a RG2 gliomas (105 cells/10µl) were implanted stereotactically into the right caudate nucleus of syngeneic Fischer rats Biodistribution studies were initiated 14 d. later. b The test agents in a volume of 200 µl were administered i.c. by means of ALZET pumps (model #2001D) over 24hrs at a flow rate of 8.33µl/h. Rats were euthanized immediately thereafter, and their brains and blood were taken for boron determinations by ICP-OES. c % boron was based on the respective molecular weights of N5-2OH (528.65), 18a (427.51) and 18b (417.7).

13. Mass Spect Studies (neg mode) to Determine in vivo Phosphorylation of the RG2 Glioma [18a-MP + K] - Int.: 436

14. Conclusions Relating to CTAs as Boron Delivery Agents 1.Strong TK1 expression was demonstrated by Western blot analysis of the F98 and RG2 glioma and the L929 TK1 (+) cell lines. 2.Target validation previously was established using the L929 TK1 (+) and mutant TK1 (-) cell lines (PNAS 105:17493, 2008). 3. In vivo BNCT studies with the F98 glioma model have demonstrated that intracerebral CED of N5-2OH yielded survival data that were equivalent to those obtained with i.v. administration of BPA. 4.The combination of N5-2OH and BPA resulted in a modest increase in MST of F98 glioma bearing rats compared to a statistically significant increase (P= 0.0003) in RG2 glioma bearing rats 52.9 vs. 43.5d (PNAS 105, 2008).

15. 5. In vivo biodistribution studies of 18a following i.c. administration by Alzet pumps to RG2 glioma bearing rats showed enhanced uptake compared to N5-2OH. 6. Metabolic mass spectrometric studies indicated the possible formation of monophosphates of N5-2OH and 18a in RG2 glioma cells in vivo. 7. Enzyme kinetic studies have shown that 18a is a 2-3x better substrate of hTK1 than N5-2OH (J. Med. Chem. 48:1188, 2005). 8. Further in vivo studies are warranted to determine if 18a is superior to N5-2OH as a boron delivery agent for NCT.