Biological Evaluation of Boron for Health Science for Research Purpose
Louis Pasteur Center for Medical Research
Experimental BNCT
Case Report 1: in-vitro surviving fraction & toxicity
carboranyl-thio-d-glucoses:TDG
The murine squamous cell carcinoma cell line (SCCVII)were incubated in subconfluent condition in standard medium of MEM (Nissui, Tokyo, Japan) supplemented with 10% fetal cow serum, in 36℃ 5% carbondioxicide atmosphere for overnight. Aliquots of Carboran sugar (open cage carboranyl-thio-d-glucoses:TDG) and 10BPA were added for 3 hrs into the petridishes respectively for uptaking into tumor cells in dose-dependent (8.8 and 17.6 ppm 10B of TDG, and 20 ppm 10B of BPA). Boron-10 concentrations in mediums were confirmed by prompt g spectroscopy. The tumor cells were washed in twice after boron loading. Aliquots containing 5×103cells/ml were pipetted into cylindrical Teflon tubes 1 cm in diameter and 3 cm high that did not generate any secondary radiation when subjected to thermal neutron. The inability of cells to adhere to Teflon allows precise quantitative manipulation of cells without trypsinization. The thermal neutron fluence was determined by averaging the activity of two gold films symmetrically attached to the Teflon tube surface along the thermal neutron axis. The thermal/epithermal neutron fluence ranged from 0 to 3.4×1012/ 6.1x1011n/cm2(absorbed equivalent dose <1.4 Gy). The g-ray dose was monitored by thermoluminescent dosimeters (TLDs) attached to the tubes and ranged from 0 to 0.6 Gy. Immediately after irradiation, 500 cells were seeded in 6-cm Petri dishes (Corning, NY) and incubated for 10 days in a humidified 36℃atmosphere of 5% carbon dioxide to allow colony formation. The colonies were fixed and stained with a 10% formaldehyde/1% toluidine blue solution and then counted microscopically. The IC50(moles/liter), i.e. the concentration that inhibited the growth of SCC VII cells by 50% after 3 day of continuous exposure, was determined via CellTiter 96®Aqueous One Solution Cell Proliferation Assay.
Statistics
Triplication of the in-vitroBNCT was carried out. Values were expressed as mean ±SE. Significant differences on the survival study were assessed by the student t-test. These statistical analysis was performed using Prism®, Graph Pad Soft Ware Inc., CA, USA.
Results
After boron loading with TDG, no any abnormal morphological findings on the tumor cells were observed via an ordinal microscope. IC50of TDG was very low 5.3x10-2M, that was almost half of that of BSH (2.75x10-2M) which has been clinically used for malignant brain tumors.
Conclusions
The toxicity and tumor cell killing effect was superior that those of BSH and BPA. TDG should be investigated for further bio-distribution study, and it might be a probable candidate for BNCT.
Figure 1. Survival fraction.
Boronated dipeptide borotrimethylglycyl—phenylalanine
For the treatment planning, boron concentration in tumor, normal brain and blood must be determined. Figure2 shows boron distribution after intra-venous administration of BGPA (highly hydrophilic form of borono-glycyl-phenylalanine) to brain tumor bearing rat by aautoradiography in which boron distribution is well visualized as high density area (figure upper) [Takagaki]. The boron distribution looks uniform macroscopically and tumor seems to be easily destroyed via thermal neutron bombardment. However, boron distribution is very heterogeneous microscopically especially in the marginal region of the tumor and also in the tumor for the presence of quiescent cells (resting cell on G0 mitotic stage) that has only weak essential metabolic activity. Furthermore thermal neutron distribution is also heterogeneous, since thermal neutron fluence strongly decrease in deep brain by scattering thermal neutrons in the brain parenchyma. Thus, after BNCT, the tumor was heterogeneously destroyed as mixture of lethal, sub-lethal, and intact tumor cells. “The tumor was overall near-killed.”
Figure 2 . Biodistribution of B-com visualized via alpha-autoradiographic technique.
Takagaki M, Powell W, Sood A, Spielvogel BF, Hosmane NS, Kirihata M, Ono K, Masunaga SI, Kinashi Y, Miyatake SI, Hashimoto N. Boronated dipeptide borotrimethylglycyl—phenylalanine as a potential boron carrier in boron neutron capture therapy for malignant brain tumors. Radiat Res. 2001 Jul;156(1):118-22.
Case Report 2: in-vivo surviving fraction & toxicity
GdNCT:GdDTPA
Subcutaneous melanoma were investigated in larger tumor models. Ten days after subcutaneous implantation 1 mm cubes of Green’s melanotic melanoma into the thighs of Syrian golden hamster model (N = 30), the melanomas had grown to the size of a thumb tip (approx. 12×12×12÷3 mm3= 576 mm3). For continuous infusion of GdDTPA, the femoral vein on the contralateral side was cannulated. During continuous 2 ml/kg/hr infusion of GdDTPA, thermal neutrons were irradiated on the thigh. During the protocols, the whole body of each animal was protected from neutron bombardment by a Li sheet (Figure 3). After the start of infusion, Gd concentration in blood was determined every 10 minutes by PGS. After GdNCT, tumor size was measured at intervals and tumor volume was estimated by dividing the result of multiplying the 3 measured dimensions by 3.
Figure 3. Hamster with cutaneous melanoma in thigh placed at the thermal neutron irradiation port of the Research Reactor Institute of Kyoto University.
As Figure 4 shows, thermal neutron irradiation without Gd had little effect on tumor growth, which recommenced after a single week of inhibition. By contrast, GdNCT was able to effectively destroy melanoma tissues. All tumor growth was strongly inhibited: tumor size dramatically decreased and was well restrained for more than 2 weeks after treatment. Complete destruction was also observed 2 of 10 hamsters. We concluded that remission can be achieved by GdNCT with continuous infusion of GdDTPA.
Figure 4. Effect of GdNCT on growth of subcutaneous melanoma in hamsters.
