Masao Takagaki

Radiobiological evidence suggesting heterogeneous microdistribution of boron compounds in tumors: Its relation to quiescent cell population and tumor cure in neutron capture therapy

PURPOSE The heterogeneous microdistribution of boron compounds in tumors and its significance on tumor cure were examined by a radiobiological procedure. The role of quiescent (Q) cells in tumor was especially investigated. METHODS AND MATERIALS 10B-enriched paraboronophenylalanine (BPA) and mercaptoundecahydrododecaborate (BSH) were administered to SCCVII tumor bearing C3H/He mice by intragastric and i.v. injections, respectively. The continued effects of these boron compounds with thermal irradiations were studied by using colony formation and tumor control assays. Their effects on Q cells were also analyzed by the combined method of micronucleus frequency assay and an identification of proliferating (P) cells by BUdR and anti-BUdR monoclonal antibody. RESULTS 10B-concentration after BPA (1,500 mg/kg) and BSH (75 mg/kg) administration were 11 ppm at 3 h and 10.5 ppm at 30 min, respectively. Cell survival decreased exponentially with an increment of neutron fluence (phi). The exponential parts of the curves were: -InSF = -0.052+ 13.0x10(13)phi, -InSF = -0.032+7.68X10(-13)phi, and -InSF = -0.0005+2.68x10(-13)phi for BPA-BNCT, BSH-BNCT, and NCT alone, respectively. Fifty percent tumor control was obtained at the influence of 10.2 x 10(12) n/cm2 in BPA-BNCT. On the other hand, 11.4 x 10(12) n/cm2 of neutrons had to be delivered in BSH-BNCT. The normal nuclear division fraction defined as the cell fraction that did not express micronuclei at first mitosis after treatment was investigated. The surviving cell fraction and the normal nuclear division fraction were regarded as equal in NCT alone. However, the normal nuclear division factor following BPA-BNCT was greater than the surviving cell fraction, and the difference increased with an increase in neutron fluence. In Q cells, BSH-BNCT yielded higher micronucleus frequency than BPA-BNCT and NCT alone. The frequencies in Q cells following BPA-BNCT and NCT alone were almost same as that in total cell population after NCT alone. CONCLUSIONS Our data suggested that BPA distributed in tumors hetergeneously. Q cells especially might not accumulate BPA. To decrease the possible disadvantage of BPA-BNCT, the combination of BPA and BSH or other neutron capture element that emit particles with longer ranges, for example, gadolinium, would have to be investigated.

The combined effect of boronophenylalanine and borocaptate in boron neutron capture therapy for SCCVII tumors in mice.

PURPOSE To increase the effect of boron neutron capture therapy (BNCT) on tumors in vivo, the combined effects of para-boronophenylalanine (BPA) and borocaptate sodium (BSH) were investigated. METHODS AND MATERIALS 10B-enriched BPA and BSH were administered to C3H/He mice bearing SCCVII tumors by intragastric and intravenous injections, respectively. The colony formation and tumor control assays were employed for investigating antitumor effects of BNCT. The extent of homogeneity of tumor cell killing effect was examined by the distribution of frequencies of binuclear cells (BNC) producing a certain number of micronuclei (0,1,2,--,> or =5) to total number of BNC and by the comparison between surviving cell fraction (SF) in colony formation assay and the normal nuclear division fraction (NNDF) at first mitosis following BNCT. RESULTS The relationships between SF and radiation dose in Gy (D) at around 10 ppm of 10B in tumors were as follow: -InSF = -0.101 + 0.648 Gy(-1) x D, 0.0606+0.435 Gy(-1) x D, and -0.0155 + 0.342 Gy(-1) x D for BPA, BPA + BSH, and BSH, respectively. In tumor control assay, BPA was also more effective than BSH, but the difference of effectiveness significantly decreased: 1.9 times more effective in colony assay vs. 1.2 times in tumor control assay. The most effective treatment to achieve tumor cure was BNCT using BPA + BSH, and it was 1.9 times more effective than BSH-BNCT. In BSH-BNCT, NNDF decreased exponentially with radiation dose and was equal to SF. However, NNDF following BPA-BNCT showed a biphasic decrease with radiation dose, and SF was much lower than NNDF. In the combination of BPA and BSH, the discrepancy between NNDF and SF decreased in comparison with BPA-BNCT. The distribution of frequency of BNC with a certain number of micronuclei to total BNC was very close to Poisson distribution in BSH-BNCT tumors; however, it deviated from the Poisson in BPA-BNCT tumors. In combination with BPA and BSH, the distribution showed an intermediate pattern. These findings indicate that BSH distributes homogeneously with a heterogeneous distribution of BPA in tumors, and the heterogeneous effect of BPA-BNCT was improved by the combination of two boron compounds. CONCLUSION The heterogeneous cell killing effect of BPA-BNCT was improved by the combination of BSH, and increased tumor control rates. Therefore, this combination may improve clinical outcome of BNCT although the effects on normal tissues have to be examined before clinical application.

Boron and gadolinium neutron capture therapy for cancer treatment

Introduction Principles of Neutron Capture Therapy Major NCT Drug Prototypes Boron Compounds-Based BNCT Agents Neutron Sources for NCT NCT Dosimetry and Treatment Planning Selected in vitro and in vivo Studies Clinical Trials Summary The Future of NCT Appendix: Clinical State of BNCT by US Department of Energy (DOE) as of 1997 Acknowledgments References.

The effects of boron neutron capture therapy on liver tumors and normal hepatocytes in mice.

To explore the feasibility of employing boron neutron capture therapy (BNCT) to treat liver tumors, the effects of BNCT were investigated by using liver tumor models and normal hepatocytes in mice. Liver tumor models in C3H mice were developed by intrasplenic injection of SCCVII tumor cells. After borocaptate sodium (BSH) and boronophenylalanine (BPA) administration, 10B concentrations were measured in tumors and liver and the liver was irradiated with thermal neutrons. The effects of BNCT on the tumor and normal hepatocytes were studied by using colony formation assay and micronucleus assay, respectively. To compare the effects of BSH‐BNCT and BPA‐BNCT, the compound biological effectiveness (CBE) factor was determined. The CBE factors for BSH on the tumor were 4.22 and 2.29 using D10 and D0 as endpoints, respectively. Those for BPA were 9.94 and 5.64. In the case of hepatocytes, the CBE factors for BSH and BPA were 0.94 and 4.25, respectively. Tumor‐to‐liver ratios of boron concentration following BSH and BPA administration were 0.3 and 2.8, respectively. Considering the accumulation ratios of 10B, the therapeutic gain factors for BSH and BPA were 0.7‐1.3 and 3.8‐6.6, respectively. Therefore, it may be feasible to treat liver tumors with BPA‐BNCT.

Mutagenic effects at HPRT locus induced in Chinese hamster ovary cells by thermal neutrons with or without boron compound

CHO cells were exposed to thermal neutrons and their mutation frequency was determined. The Kyoto University Research Reactor (KUR), which has a very low level of contamination by gamma-rays and fast neutrons was used as a thermal neutron source. Cells were irradiated in the presence or absence of boric acid to determine mutation frequency and cell survival. Thermal neutron irradiation was 2.5 times as mutagenic as gamma-irradiation without boron. In the presence of boron, however, thermal neutron irradiation was from 4.2 to 4.5 times as mutagenic as gamma-irradiation. When the mutation frequency was plotted against the survival fraction, a higher degree of mutagenicity was observed in the presence than in the absence of boron. These results suggest that the enhancement of thermal neutron-induced mutation with boron is strongly associated with alpha-particles released by 10B(n, alpha)7 Li reaction.

Alteration in the hypoxic fraction of quiescent cell populations by hyperthermia at mild temperatures

We investigated oxygenation of quiescent (Q) tumour cells in vivo by mild heat treatment. C3H/He mice bearing SCC VII tumours received BrdU continuously for 5 days via implanted mini-osmotic pumps, to label all proliferating (P) cells. The tumours were then irradiated after treatment, and were excised, minced and trypsinized. The tumour cell suspension thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling was determined using immunofluorescence staining for BrdU. This MN frequency was then used to calculate the surviving fraction of unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P + Q) tumour cells. Thus, a cell survival curve could be determined for the cells not labelled with BrdU, which can be regarded as the Q cells in a tumour for all practical purposes. The MN frequency in total tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU. Assays performed immediately after irradiation of both normally aerated and hypoxic tumours showed that Q cells contained higher hypoxic fractions than the total tumour cell population. Mild heat treatment (40.0 degrees C, 60 min) before irradiation decreased the hypoxic fraction, even when is was combined with nicotinamide administration. In contrast, mild heating did not decrease the hypoxic fraction when the mice were placed in a circulating carbogen (95% O2/5% CO2) chamber. Therefore, mild heat treatment was thought to preferentially oxygenate the chronically hypoxic fraction.

Impact of the p53 Status of the Tumor Cells on the Effect of Reactor Neutron Beam Irradiation, with Emphasis on the Response of Intratumor Quiescent Cells

Human head and neck squamous cell carcinoma cells transfected with mutant p53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both the hind legs of Balb/cA nude mice. Tumor‐bearing mice received 5‐bromo‐2′‐deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. After administration of sodium borocaptate‐10B (BSH) or p‐boronophenylalanine‐10B (BPA), the tumors were irradiated with neutron beams. The tumors not treated with 10B‐compound were irradiated with neutron beams or γ‐rays. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (=quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN and apoptosis frequencies in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. Without 10B‐carriers, in both tumors, the relative biological effectiveness of neutrons was greater in Q cells than in total cells, and larger for low than high cadmium ratio neutrons. With 10B‐carriers, the sensitivity was increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of BSH‐treated Q cells. These sensitization patterns in combination with 10B‐carriers were clearer in SAS/neo than in SAS/mp53 tumors. The p53 status of the tumor cells had the potential to affect the response to reactor neutron beam irradiation following 10B‐carrier administration.

Boron neutron capture therapy : Preliminary study of BNCT with sodium borocaptate (Na2B12H11SH) on glioblastoma

To plan the optimal BNCT using BSH for glioblastoma patients, the10B concentration in tumor and blood was investigated in 11newly diagnosed glioblastoma patients. All patients received 20 mg BSH/kgbody weight 2.5–16 hrs prior to tumor removal. The quantitativedistribution of 10B was determined by prompt gamma rayspectrometry and/or α-track autoradiography. 10Bdistribution in tumors was heterogeneous, ± 25% of scatteringat the microscopic level, and the distribution was also heterogeneous at thetissue level. 10B concentration in blood decreased inbi-exponential decay as a function of the time after the end of theadministration. The T/B ratio showed non-exponential increase with largevariation. The maximum T/B ratio would be around 1. The tumor/normal brain(T/N) ratio of 10B concentration was 11.0 ± 3.2. The10B content in normal brain is originated in vascular10B in parenchyma, since the 10B content innormal brain to blood (N/B ratio) being compatible with the blood content inparenchyma. These values allow for BNCT, using thermal neutrons, on braintumors located less than approximately 3.3 cm in depth from the brainsurface of neutron incidence, providing that the dose on the normalendothelium is controlled to less than the tolerance limit. In ourpreliminary study of BNCT, a 31% 3-year survival was achieved overall for 16 glioblastoma patients and a 50% 2-year survival wasachieved on 8 glioblastoma patients in our recent dose escalation studybased on these data.

Boronated Dipeptide Borotrimethylglycylphenylalanine as a Potential Boron Carrier in Boron Neutron Capture Therapy for Malignant Brain Tumors

Abstract Takagaki, M., Ono, K., Masunaga, S-I., Kinashi, Y., Oda, Y., Miyatake, S-I., Hashimoto, N., Powell, W., Sood, A. and Spielvogel, B. F. Boronated Dipeptide Borotrimethylglycylphenylalanine as a Potential Boron Carrier in Boron Neutron Capture Therapy for Malignant Brain Tumors. Radiat. Res. 156, 118–122 (2001). A boronated dipeptide, borotrimethylglycylphenylalanine (BGPA), was synthesized as a possible boron carrier for boron neutron capture therapy (BNCT) for malignant brain tumors. In vitro, at equal concentrations of 10B in the extracellular medium, BGPA had the same effect in BNCT as p-boronophenylalanine (BPA). Boron analysis was carried out using prompt γ-ray spectrometry and track-etch autoradiography. The tumor:blood and tumor:normal brain 10B concentration ratios were 8.9 ± 2.1 and 3.0 ± 1.2, respectively, in rats bearing intracranial C6 gliosarcomas using α-particle track autoradiography. The IC50, i.e. the dose capable of inhibiting the growth of C6 gliosarcoma cells by 50% after 3 days of incubation, was 5.9 × 10–3 M BGPA, which is similar to that of 6.4 × 10–3 M for BPA. The amide bond of BGPA is free from enzymatic attack, since it is protected from hydrolysis by the presence of a boron atom at the α-carbon position of glycine. These results suggest promise for the use of this agent for BNCT of malignant brain tumors. Further preclinical studies of BGPA are warranted, since BGPA has advantages over both BPA and BSH.

Electroporation increases the effect of borocaptate (10B-BSH) in neutron capture therapy

PURPOSE The cell membrane permeability of borocaptate (10B-BSH) and its extent of accumulation in cells are controversial. This study was performed to elucidate these points. METHODS AND MATERIALS Two different treatments were applied to SCCVII tumor cells. The first group of tumor cells was incubated in culture medium with 10B-BSH or 10B-enriched boric acid, and was exposed to neutrons from the heavy water facility of the Kyoto University Reactor (KUR). More than 99% of neutrons were thermal neutrons at flux base. The second group was pretreated by electroporation in combination with 10B-BSH, and thereafter the cells were irradiated with neutrons. The cell killing effects of boron neutron capture therapy (BNCT) using BSH were investigated by colony formation assay. RESULTS Surviving cell fraction decreased exponentially with neutron fluence, and addition of BSH significantly enhanced the cell killing effect of neutron capture therapy (NCT) depending on 10B concentration. The effect of BSH-BNCT also increased with preincubation time of cells in the medium containing BSH. The electroporation of cells with BSH at 10 ppm 10B markedly enhanced BSH-BNCT effects in comparison with that of preincubation alone. The effect of BSH-BNCT with electroporation was equal to that of BNCT using 10B-boric acid at a same 10B concentration (10 ppm). CONCLUSIONS BSH is suggested to penetrate the cells slowly and remained after washing. Electroporation can introduce BSH into the cells very efficiently, and BSH stays in the cells and is not lost by washing. Therefore, if electroporation is applied to tumors after BSH injection, 10B remains in tumors but is cleared from normal tissues, and selective accumulation of 10B in tumors will be achieved after an adequate waiting time.