Dr. Silvia Capuani, PhD CNR IPCF UOS Roma Nuclear Magnetic Resonance Laboratory Physics Department, “Sapienza” Universit

1. XCVI Congresso Nazionale, Bologna, 20 - 24 Settembre 2010 1H and 19F MRI and MRS to optimized the effectiveness of BNCT for malignant gliomas treatment Dr. Silvia Capuani, PhD CNR IPCF UOS Roma Nuclear Magnetic Resonance Laboratory Physics Department, “Sapienza” University of Rome Piazzale Aldo Moro 5, 00185 Rome, Italy E-mail: Silvia.Capuani@roma1.infn.it

2. Introduction Boron Neutron Capture Therapy (BNCT) is a promising radio-therapeutic modality, for the treatment of malignant gliomas and non-operable head/neck tumors. BNCT

3. Introduction Several clinical trials, using BNCT, have reported encouraging results in patients with malignant gliomas However, the clinical outcome of BNCT still remains largely unsatisfactory It is general opinion that one of the major limitations for BNCT effectiveness is the insufficient incorporation of 10B into the tumor cells Moreover, the relatively low specificity of 10B-carriers uptake in tumor cells as compared to the surrounding normal tissues limits the use of high dose BNCT protocols. A second limitation is due to a lack of an effective method to monitor, in vivo, the pharmacokinetic of boron compounds in order to obtain the optimal irradiation time

4. Aims Our goals were: 1) to develop an effective imaging method to monitor, in vivo, the bio-distribution of BPA 19F-MRI and 19F-MRS were used to obtain in vivo spatial distribution mapping and pharmacokinetic of BPA 2) to investigate the use of L-DOPA as enhancer for BPA uptake in C6-glioma cells The investigation was first performed in vitro using C6-glioma cells and then extended in vivo to the animal model using the C6-rat glioma model

5. 1) Materials and method 19F-BPA (98% enriched in 10B) Animal model: C6-glioma rat brain Administration of 19F-BPA-fr complex 14 days after tumour implantation rats were infused with a 19F-BPA-fr-complex solution (300mg/kg (bw)) within carotid artery Kabalka GW et al. 2000 Org. Prep. Proced.Int. 32 290-293; Reddy NK et al. 1985 J. Lab. Compd. Radiopharm. 33 599-600; Porcari P et al. 2006Phys. Med. Biol. 51 3141-3154

6. cerebellum frontal lobe bulbus olfactorius brainstem Rat atlas sagittal view Axial view NMR image Animal model FL CP 25 Male Wistar rats (300-350g) were anesthetized by intraperitoneal injection of ketamine (60mg/kg) and xylazina before being fixed in a stereotactic frame. A middle scalp incision was made and C6 cell suspension (106 cells in 10 µl) was slowly injected with a Hamilton syringe through a burr hole in the right hemisphere, 3 and 4 mm depth from the dura. Then, the syringe was slowly removed and the burr hole and the scalp sutured. Survival time of the rats was about 2-3 weeks after tumour implantation. All procedures related to animal care were strictly conformed in accordance with Decree 116/92 which represents the Italian enforcement of the European Directive 86/609/EEC.

7. Methods MRI measurements at 7T Tumor growth was monitored by Magnetic Resonance Imaging at 7T.

8. 2s 1s 3s 4s 1) In vivo Imaging results Axial 1H MR images of rat brain acquired two hours after 19F-BPA-fr complex infusion 19F axial image of rat brain acquired after 1H MR scan 2.5h after infusion P. Porcari, S. Capuani, E. D’Amore et al. 2008Phys. Med. Biol. 53:6979

9. 1) Results: 19F-BPA spatial bio-distribution mapping by means of 19F MRI Superimposition of 19F axial image (in colour level: low=blue, red=high) acquired 2.5 hours after infusion on the corresponding morphological 1H proton reference (in grey levels). P. Porcari, S. Capuani, E. D’Amore et al. 2008Phys. Med. Biol. 53:6979

10. 4h after infusion 2.5h after infusion rat brain rat brain • Results: pharmacokinetic of 19F-BPA • by means of 19F MRS blood samples extracted from the right femoral vein 4h 2.5h 1h P. Porcari, S. Capuani, E. D’Amore et al. 2008Phys. Med. Biol. 53:6979

11. 2) Materials and method BPA-fr to perform HPLC Measurements And 19F-BPA-fr to perform Imaging experiments a large clinical experience has been accumulated in the use of L-DOPA at different doses

12. 2) In vivo experiments: Methods All animals were sacrificed 2.5 hours after the end of BPA infusion. Assessment of BPA concentrations in: tumor tissue, normal brain, and blood samples was performed using High-Performance Liquid Chromatography (HPLC) A B N=15 N=10 Pre-treatment with L-DOPA: 50mg/Kg Intra-peretoneally A B Intra-carotid BPA-fr infusion: 300mg/Kg Intra-carotid BPA-fr infusion: 300mg/Kg

13. 2) In vivo L-DOPA results MRI 1H 19F L-DOPA preloading BPA uptake in C6-glioma model is dramatically increased by L-DOPA preloading HPLC Control BPA accumulation in tumor samples was significantly higher in treated group compared to control group (p<0.0001) 19F-BPA tumour signal was observed only in L-DOPA pre-treated rat but not in the other case confirming an increased 19F-BPA tumour uptake after L-DOPA administration S. Capuani, T. Gili, M.Bozzali et al. Int J Radiat Oncol Biol Phys 2008; 72:562 P. Porcari S. Capuani, E. D’Amore et al. Appl. Rad. Isot. 2009;67:S365.

14. Discussion and Conclusion 19F-MRI in combination with 1H-MRI selectively maps the spatial-distribution of 19F-BPA in C6 tumour-bearing rats The • 19F MRI is a useful method to investigate and evaluate the pharmacokinetics of the fluorinated-containing drugs • Correlation between 19F MRI and 19F MRS results highlights an improved understanding of 19F-BPA uptake in tumour and systemic circulation showing the optimal irradiation time L-DOPA pre-administration produced in the C6 glioma rat model an enhancement of tumor BPA accumulation which was 2.7 times higher than in the control condition Our study demonstrates that L-DOPA preloading induces a remarkable increase of BPA uptake in tumor but not in normal brain tissues Our results appear promising especially for their potential application to clinical therapeutic protocols Thank you for your attention

15. BNCT Study Group in Rome F.S. Pastore Department of Neuroscience, Institute of Neurosurgery, University “Tor Vergata” S. Capuani, P. Porcari CNR-IPCF Physics Department “Sapienza”, University of Rome, E. D’Amore Service for Biotechnology and Animal Welfare, Istituto Superiore di Sanità, Rome, Italy R. Marini Bettolo Department of Chemistry, Sapienza University of Rome M. Bozzali Neuroimaging Laboratory, IRCCS Santa Lucia Foundation; Research activities in BNCT field: MR Imaging and Spectroscopy applied to studies of 19F-BPA and BSH pharmacokinetics, with the aim to optimize the timing of neutronic irradiation according to the spatial distribution of Boron agents. In vivo and in vitro investigations on L-DOPA as an agent able to selectively increase the BPA uptake in glioma cells. Quantitative techniques employed are: NMR, HPLC, Dielelectric Spectroscopy. Recent publications: P. Porcari et al. Phys Med Biol 2006;51:3141. S. Capuani et al. Magn Reson Imaging 2008;26:987 S. Capuani et al. Int J Radiat Oncol Biol Phys 2008; 72:562. P. Porcari et al. Phys Med Biol 2008;53:6979 S. Capuani et al. Appl. Rad. Isot. 2009;67:S34. P. Porcari et al. Appl. Rad. Isot. 2009;67:S365.