Boron-containing nucleosides as potential delivery agents for neutron capture therapy of brain tumors.

The purpose of the present study was to evaluate both in vitro and in vivo a series of boron-containing nucleosides that potentially could be used as delivery agents for neutron capture therapy. The rationale for their synthesis was based on the fact that proliferating neoplastic cells have increased requirements for nucleic acid precursors, and, therefore, they should preferentially localize in the tumor. A series of 3-carboranlyalkyl thymidine analogs has been synthesized and a subset, designated N4, N5, and N7, and the corresponding 3-dihydroxypropyl derivatives, designated N4-2OH, N5-2OH, and N7-2OH, have been selected for evaluation. Using these compounds as substrates for recombinant human thymidine kinase-1 and the mitochondrial isoenzyme thymidine kinase-2, the highest phosphorylation levels relative to thymidine were seen with N5 and the corresponding dihydroxypropyl analog N5-2OH. In contrast, N4, N4-OH, N7, and N7-OH had substantially lower phosphorylation levels. To compare compounds with high and low thymidine kinase-1 substrate activity, N5 and N7 and the corresponding dihydroxypropyl derivatives were selected for evaluation of their cellular toxicity, uptake and retention by the F98 rat glioma, human MRA melanoma, and murine L929 cell lines, all of which are thymidine kinase-1(+), and a mutant L929 cell line that is thymidine kinase-1(-). N5-2OH was the least toxic (IC50, 43-70 microm), and N7 and N7-2OH were the most toxic (IC50, 18-49 microm). The highest boron uptake was seen with N7-2OH by the MRA 27 melanoma and L929 wild-type (wt) cell lines. The highest retention was seen with L929 (wt) cells, and this ranged from 29% for N5-2OH to 46% for N7. Based on the in vitro toxicity and uptake data, N5-2OH was selected for in vivo biodistribution studies either in rats bearing intracerebral implants of the F98 glioma or in mice bearing either s.c. or intracerebral implants of L929 (wt) tumors. At 2.5 hours after convection-enhanced delivery, the boron values for the F98 glioma and normal brain were 16.2 +/- 2.3 and 2.2 microg/g, respectively, and the tumor to brain ratio was 8.5. Boron values at 4 hours after convection-enhanced delivery of N5-2OH to mice bearing intracerebral implants of L929 (wt) or L929 thymidine kinase-1(-) tumors were 39.8 +/- 10.8 and 12.4 +/- 1.6 microg/g, respectively, and the corresponding normal brain values were 4.4 and 1.6 microg/g, thereby indicating that there was selective retention by the thymidine kinase-1(+) tumors. Based on these favorable in vitro and in vivo data, neutron capture therapy studies will be initiated using N5-2OH in combination with two non-cell cycle dependent boron delivery agents, boronophenylalanine and sodium borocaptate.

Imaging of fluorine and boron from fluorinated boronophenylalanine in the same cell at organelle resolution by correlative ion microscopy and confocal laser scanning microscopy.

PURPOSE: There is a clear need for a technique that provides subcellular locations of fluorine and boron atoms from fluorinated neutron capture agents because positron emission tomography is being tested as a tool for providing tumor boron concentrations in boron neutron capture therapy. EXPERIMENTAL DESIGN: Ion microscopy was used in combination with confocal laser scanning microscopy to investigate the subcellular locations of fluorine and boron from fluorinated p-boronophenylalanine (F-BPA) in human glioblastoma T98G cells. The fluorinated compound was also compared with p-boronophenylalanine (BPA) for delivery of boron after a clinically relevant 6-h exposure. Mitochondria were identified by rhodamine 123 labeling. A strict cryogenic sample preparation was used, and measurements were made in fractured freeze-dried cells. RESULTS: The nucleus, a perinuclear mitochondria-rich cytoplasmic region, and the remaining cytoplasm were the three subcellular regions identified in individual T98G cells. In cells treated with F-BPA, the mitochondria-rich perinuclear cytoplasmic region exhibited significantly lower fluorine and boron signals than the remaining cytoplasm and the nuclei. Ion microscopy observations revealed a nearly 1:1 distribution of fluorine and boron in subcellular compartments. Quantitative subcellular observations indicated that there was no significant difference in boron delivery to subcellular compartments between the F-BPA and nonfluorinated BPA. CONCLUSIONS: These observations provide the first direct evidence that fluorine and boron from fluorinated BPA are cocompartmentalized in cells and that the fluorinated compound is as efficient for boron delivery as the nonfluorinated BPA at a clinically relevant time point. These observations provide strong support for the use of F-BPA in positron emission tomography biodistribution studies for boron neutron capture therapy.

Quantitative subcellular secondary ion mass spectrometry (SIMS) imaging of boron-10 and boron-11 isotopes in the same cell delivered by two combined BNCT drugs: in vitro studies on human glioblastoma T98G cells.

Ion microscopy was used for subcellular quantitative imaging of the isotopes 10B and 11B in the same cell to evaluate boron delivery using a mixture of two neutron capture therapy drugs, p-boronophenylalanine-fructose (BPA-F) and sodium borocaptate (BSH). The application of 10B-labeled BPA-F and 11B-labeled BSH allowed independent imaging of both 10B and 11B in the same cell using a CAMECA IMS-3f ion microscope. Mixed-drug treatments were compared to single-drug exposures given under identical conditions. 10BPA-F delivered 10B heterogeneously to T98G human glioblastoma cells, with a significantly reduced concentration in an organelle-rich perinuclear region. The intracellular distribution of 11B from 11BSH contrasted with that of the 10B from 10BPA-F, with 11B distributed nearly homogeneously throughout cells. The subcellular distributions of 10B and 11B were sustained in mixed-drug treatments and resembled their localizations after the single-drug treatments. In both single- and mixed-drug treatments, cellular levels of 10B from 10BPA-F nearly doubled between 1 h and 6 h, with a 3:1 intracellular to nutrient medium partitioning, while cellular levels of 11BSH remained essentially unchanged. The net effect of the combined treatment with 10BPA-F and 11BSH was an additive delivery of boron to cells. This study introduces a novel approach for checking potential synergistic, antagonistic or simple additive delivery of two mixed boronated compounds in cellular/subcellular compartments.