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, (10)B 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 D(10) and D(0) 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 (10)B, 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.     

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.     

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 D ₁₀ and D ₀ 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.     

L-DOPA preloading increases the uptake of borophenylalanine in C6 glioma rat model: a new strategy to improve BNCT efficacy

PURPOSE: Boron neutron capture therapy (BNCT) is a radiotherapeutic modality based on (10)B(n,alpha)(7)Li reaction, for the treatment of malignant gliomas. One of the main limitations for BNCT effectiveness is the insufficient intake of (10)B nuclei in the tumor cells. This work was aimed at investigating the use of L-DOPA as a putative enhancer for (10)B-drug 4-dihydroxy-borylphenylalanine (BPA) uptake in the C6-glioma model. The investigation was first performed in vitro and then extended to the animal model. METHODS AND MATERIALS: BPA accumulation in C6-glioma cells was assessed using radiowave dielectric spectroscopy, with and without L-DOPA preloading. Two L-DOPA incubation times (2 and 4 hours) were investigated, and the corresponding effects on BPA accumulation were quantified. C6-glioma cells were also implanted in the brain of 32 rats, and tumor growth was monitored by magnetic resonance imaging. Rats were assigned to two experimental branches: (1) BPA administration; (2) BPA administration after pretreatment with L-DOPA. All animals were sacrificed, and assessments of BPA concentrations in tumor tissue, normal brain, and blood samples were performed using high-performance liquid chromatography. RESULTS: L-DOPA preloading induced a massive increase of BPA concentration in C6-glioma cells only after a 4-hour incubation. In the animal model, L-DOPA pretreatment produced a significantly higher accumulation of BPA in tumor tissue but not in normal brain and blood samples. CONCLUSIONS: This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT. L-DOPA preloading effect is discussed in terms of membrane transport mechanisms.     

Boron Neutron-Capture Therapy (BNCT) for Glioblastoma Multiforme (GBM) Using the Epithermal Neutron Beam at the Brookhaven National Laboratory

Objective: Boron neutron-capture therapy (BNCT) is a binary form of radiation therapy based on the nuclear reactions that occur when boron (¹⁰B) is exposed to thermal neutrons. Preclinical studies have demonstrated the therapeutic efficacy of p-boronophenylalanine (BPA)-based BNCT. The objectives of the Phase I/II trial were to study the feasibility and safety of single-fraction BNCT in patients with GBM.Materials and Methods: The trial design required (a) a BPA biodistribution study performed at the time of craniotomy; and (b) BNCT within ≈ 4 weeks of the biodistribution study. From September 1994 to July 1995, 10 patients were treated. For biodistribution, patients received a 2-hour intravenous (i.v.) infusion of BPA-fructose complex (BPA-F). Blood samples, taken during and after infusion, and multiple tissue samples collected during surgical debulking were analyzed for ¹⁰B concentration. For BNCT, all patients received a dose of 250 mg BPA/kg administered by a 2-hour i.v. infusion of BPA-F, followed by neutron beam irradiation at the Brookhaven Medical Research Reactor (BMRR). The average blood ¹⁰B concentrations measured before and during treatment were used to calculate the time of reactor irradiation that would deliver the prescribed dose.Results: ¹⁰B concentrations in specimens of scalp and tumor were higher than in blood by factors of ≈1.5 and ≈3.5, respectively. The ¹⁰B concentration in the normal brain was ≤ that in the blood; however, for purposes of estimating radiation doses to normal brain endothelium, it was always assumed to be equal to blood. BNCT doses are expressed as gray-equivalent (Gy-Eq), which is the sum of the various physical dose components multiplied to appropriate biologic effectiveness factors. The dose to a 1-cm³ volume where the thermal flux reached a maximum was 10.6 ± 0.3 Gy-Eq in 9 patients and 13.8 Gy-Eq in 1 patient. The minimum dose in tumor ranged from 20 to 32.3 Gy-Eq. The minimum dose in the target volume (tumor plus 2 cm margin) ranged from 7.8 to 16.2 Gy-Eq. Dose to scalp ranged from 10 to 16 Gy-Eq. All patients experienced in-field alopecia. No CNS toxicity attributed to BNCT was observed. The median time to local disease progression following BNCT was 6 months (range 2.7 to 9.0). The median time to local disease progression was longer in patients who received a higher tumor dose. The median survival time from diagnosis was 13.5 months.Conclusion: It is feasible to safely deliver a single fraction of BPA-based BNCT. At the dose prescribed, the patients did not experience any morbidity. To further evaluate the therapeutic efficacy of BNCT, a dose-escalation study delivering a minimum target volume dose of 17 Gy-Eq is in progress.     

The hamster cheek pouch as a model of oral cancer for boron neutron capture therapy studies: selective delivery of boron by boronophenylalanine.

Herein we propose and validate the hamster cheek pouch model of oral cancer for boron neutron capture therapy (BNCT) studies. This model serves to explore new applications of the technique, study the biology and radiobiology of BNCT, and assess the uptake of boron compounds and response of tumor, precancerous tissue, and clinically relevant normal tissues. These issues are central to evaluating and improving the therapeutic gain of BNCT. The success of BNCT is dependent on the absolute amount of boron in the tumor, and the tumor:blood and tumor:normal tissue boron concentration ratios. Within this context, biodistribution studies are pivotal. Tumors were induced in the hamsters with a carcinogenesis protocol that uses dimethyl-1,2-benzanthracene and mimics spontaneous tumor development in human oral mucosa. The animals were then used for biodistribution and pharmacokinetic studies of boronophenylalanine (BPA). Blood, tumor, precancerous pouch tissue surrounding tumor, normal pouch tissue, tongue, skin, cheek mucosa, palate mucosa, liver, and spleen, were sampled at 0-12 h after administration of 300 mg BPA/kg. The data reveal selective uptake of BPA by tumor tissue and, to a lesser degree, by precancerous tissue. Mean tumor boron concentration was 36.9 +/- 17.5 ppm at 3.5 h and the mean boron ratios were 2.4:1 for tumor:normal pouch tissue and 3.2:1 for tumor:blood. Higher doses of BPA (600 and 1200 mg BPA/kg) increased tumor uptake. Potentially therapeutic absolute boron concentrations, and tumor:normal tissue and tumor:blood ratios can be achieved in the hamster oral cancer model using BPA as the delivery agent.     

Optimization of boron neutron capture therapy for the treatment of undifferentiated thyroid cancer

PURPOSE: To analyze the possible increase in efficacy of boron neutron capture therapy (BNCT) for undifferentiated thyroid carcinoma (UTC) by using p-boronophenylalanine (BPA) plus 2,4-bis (alpha,beta-dihydroxyethyl)-deutero-porphyrin IX (BOPP) and BPA plus nicotinamide (NA) as a radiosensitizer of the BNCT reaction. METHODS AND MATERIALS: Nude mice were transplanted with a human UTC cell line (ARO), and after 15 days they were treated as follows: (1) control, (2) NCT (neutrons alone), (3) NCT plus NA (100 mg/kg body weight [bw]/day for 3 days), (4) BPA (350 mg/kg bw) + neutrons, (5) BPA + NA + neutrons, and (6) BPA + BOPP (60 mg/kg bw) + neutrons. The flux of the mixed (thermal + epithermal) neutron beam was 2.8 x 10(8) n/cm(2)/sec for 83.4 min. RESULTS: Neutrons alone or with NA caused some tumor growth delay, whereas in the BPA, BPA + NA, and BPA + BOPP groups a 100% halt of tumor growth was observed in all mice at 26 days after irradiation. When the initial tumor volume was 50 mm(3) or less, complete remission was found with BPA + NA (2 of 2 mice), BPA (1 of 4), and BPA + BOPP (7 of 7). After 90 days of complete regression, recurrence of the tumor was observed in BPA + NA (2 of 2) and BPA + BOPP (1 of 7). The determination of apoptosis in tumor samples by measurements of caspase-3 activity showed an increase in the BNCT (BPA + NA) group at 24 h (p < 0.05 vs. controls) and after the first week after irradiation in the three BNCT groups. Terminal transferase dUTP nick end labeling analysis confirmed these results. CONCLUSIONS: Although NA combined with BPA showed an increase of apoptosis at early times, only the group irradiated after the combined administration of BPA and BOPP showed a significantly improved therapeutic response.     

Delivery of (10)boron to oral squamous cell carcinoma using boronophenylalanine and borocaptate sodium for boron neutron capture therapy

Boron neutron capture therapy (BNCT) is a unique radiation therapy in which boron compounds are trapped into tumor cells. To determine the biodistribution of boronophenylalanine (BPA) in nude mice carrying oral squamous cell carcinoma (SCC), BPA was administered at a dose of 250 mg/kg body weight intraperitoneally. Two hours later, (10)B concentration in the tumor was 15.96 ppm and tumor/blood, tumor/tongue, tumor/skin and tumor/bone (10)B concentration ratios were 6.44, 4.19, 4.68 and 4.56, respectively. Two hours after the administration of borocaptate sodium (BSH) at a dose of 75 mg/kg body weight, (10)B concentration in the tumor was 3.61 ppm, and tumor/blood, tumor/tongue, tumor/skin and tumor/bone (10)B concentration ratios were 0.77, 1.05, 0.60 and 0.59, respectively. When cultured oral SCC cells were incubated with BPA or BSH for 2 h and then exposed to thermal neutrons, the proportion of survival cells that were capable of forming cell colonies decreased exponentially, depending on (10)B concentration. BPA-mediated BNCT was more efficient than BSH-mediated BNCT. Addition of boron compounds in the cell suspension during neutron irradiation enhanced the cell-killing effect of the neutrons. These results indicate that BPA is more selectively incorporated into human oral SCC as compared with normal oral tissues, and that both extra- and intra-cellular BPA contribute to the cell-killing effect of BNCT. BPA may be a useful boron carrier for BNCT in the treatment of advanced oral SCC.     

Cationized gelatin-HVJ envelope with sodium borocaptate improved the BNCT efficacy for liver tumors <Emphasis Type="Italic">in vivo</Emphasis>

Background

Boron neutron capture therapy (BNCT) is a cell-selective radiation therapy that uses the alpha particles and lithium nuclei produced by the boron neutron capture reaction. BNCT is a relatively safe tool for treating multiple or diffuse malignant tumors with little injury to normal tissue. The success or failure of BNCT depends upon the ¹⁰B compound accumulation within tumor cells and the proximity of the tumor cells to the body surface. To extend the therapeutic use of BNCT from surface tumors to visceral tumors will require ¹⁰B compounds that accumulate strongly in tumor cells without significant accumulation in normal cells, and an appropriate delivery method for deeper tissues.Hemagglutinating Virus of Japan Envelope (HVJ-E) is used as a vehicle for gene delivery because of its high ability to fuse with cells. However, its strong hemagglutination activity makes HVJ-E unsuitable for systemic administration.In this study, we developed a novel vector for ¹⁰B (sodium borocaptate: BSH) delivery using HVJ-E and cationized gelatin for treating multiple liver tumors with BNCT without severe adverse events.

Methods

We developed cationized gelatin conjugate HVJ-E combined with BSH (CG-HVJ-E-BSH), and evaluated its characteristics (toxicity, affinity for tumor cells, accumulation and retention in tumor cells, boron-carrying capacity to multiple liver tumors in vivo, and bio-distribution) and effectiveness in BNCT therapy in a murine model of multiple liver tumors.

Results

CG-HVJ-E reduced hemagglutination activity by half and was significantly less toxic in mice than HVJ-E. Higher ¹⁰B concentrations in murine osteosarcoma cells (LM8G5) were achieved with CG-HVJ-E-BSH than with BSH. When administered into mice bearing multiple LM8G5 liver tumors, the tumor/normal liver ratios of CG-HVJ-E-BSH were significantly higher than those of BSH for the first 48 hours (p < 0.05). In suppressing the spread of tumor cells in mice, BNCT treatment was as effective with CG-HVJ-E-BSH as with BSH containing a 35-fold higher ¹⁰B dose. Furthermore, CG-HVJ-E-BSH significantly increased the survival time of tumor-bearing mice compared to BSH at a comparable dosage of ¹⁰B.

Conclusion

CG-HVJ-E-BSH is a promising strategy for the BNCT treatment of visceral tumors without severe adverse events to surrounding normal tissues.

     

A critical assessment of boron target compounds for boron neutron capture therapy

Summary Boron neutron capture therapy (BNCT) has undergone dramatic developments since its inception by Locher in 1936 and the development of nuclear energy during World War II. The ensuing Cold War spawned the entirely new field of polyhedral borane chemistry, rapid advances in nuclear reactor technology and a corresponding increase in the number to reactors potentially available for BNCT. This effort has been largely oriented toward the eradication of glioblastoma multiforme (GBM) and melanoma with reduced interest in other types of malignancies. The design and synthesis of boron-10 target compounds needed for BNCT was not channeled to those types of compounds specifically required for GBM or melanoma. Consequently, a number of potentially useful boron agents are known which have not been biologically evaluated beyond a cursory examination and only three boron-10 enriched target species are approved for human use following their Investigational New Drug classification by the US Food and Drug Administration; BSH, BPA and GB-10. All ongoing clinical trials with GBM and melanoma are necessarily conducted with one of these three species and most often with BPA. The further development of BNCT is presently stalled by the absence of strong support for advanced compound evaluation and compound discovery driven by recent advances in biology and chemistry. A rigorous demonstration of BNCT efficacy surpassing that of currently available protocols has yet to be achieved. This article discusses the past history of compound development, contemporary problems such as compound classification and those problems which impede future advances. The latter include means for biological evaluation of new (and existing) boron target candidates at all stages of their development and the large-scale synthesis of boron target species for clinical trials and beyond. The future of BNCT is bright if latitude is given to the choice of clinical disease to be treated and if a recognized study demonstrating improved efficacy is completed. Eventually, BNCT in some form will be commercialized.     

Rat brain tumor models to assess the efficacy of boron neutron capture therapy: a critical evaluation

Summary Development of any therapeutic modality can be facilitated by the use of the appropriate animal models to assess its efficacy. This report primarily will focus on our studies using the F98 and 9L rat glioma models to evaluate the effectiveness of boron neutron capture therapy (BNCT) of brain tumors. Following intracerebral implantation the biological behavior of each tumor resembles that of human high grade gliomas in a number of ways. In both models, glioma cells were implanted intracerebrally into syngeneic Fischer rats and ∼10–14 days later BNCT was initiated at the Brookhaven National Laboratory Medical Research Reactor. Two low molecular weight (M _r<210 Da)¹⁰B-containing drugs, boronophenylalanine (BPA) and/or sodium borocaptate (BSH) were used as capture agents, either alone or in combination with each other. The 9L gliosarcoma, which has been difficult to cure by means of either chemo- or radiotherapy alone, was readily curable by BNCT. The best survival data were obtained using BPA at a dose of 1200 mg/kg (64.8 mg¹⁰B), administered intraperitoneally (i.p.), with a 100% survival rate at 8 months. In contrast, the F98 glioma has been refractory to all therapeutic modalities. Tumor bearing animals, which had received 500 mg/kg (27 mg¹⁰B) of BPA, or an equivalent amount of BSH i.v., had mean survival time (MST) of 37 and 33 days, respectively, compared to 29 days for irradiated controls. The best survival data with the F98 glioma model were obtained using BPA + BSH in combination, administered intra-arterially via the internal carotid artery (i.c.) with hyperosomotic mannitol induced blood-brain barrier disruption (BBB-D). The MST was 140 days with a cure rate of 25%, compared to a MST of 73 days with a 5% cure rate without BBB-D, and 41 days following i.v. administration of both drugs. A modest but significant increase in MST also was observed in rats that received intracarotid (i.c.) BPA in combination with Cereport (RMP-7), which produced a pharmacologically mediated opening of the BBB. Studies also have been carried out with the F98 glioma to determine whether an X-ray boost could enhance the efficacy of BNCT, and it was shown that there was a significant therapeutic gain. Finally, molecular targeting of the epidermal growth factor receptor (EGFR) has been investigated using F98 glioma cells, which had been transfected with the gene encoding EGFR and, intratumoral injection of boronated EGF as the delivery agent, followed by BNCT. These studies demonstrated that there was specific targeting of EGFR and provided proof of principle for the use of high molecular weight, receptor targeting-boron delivery agents. Finally, a xenograft model for melanoma metastatic to the brain has been developed using a human melanoma (MRA27), stereotactically implanted into the brains of nude rats, and these studies demonstrated that BNCT either cured or significantly prolonged the survival of tumor-bearing rats. It remains to be determined, which, if any, of these experimental approaches will be translated into clinical studies. Be that as it may, rat brain tumor models already have made a significant contribution to the design of clinical BNCT protocols, and should continue to do so in the future.     

Applicability of Combination with Tirapazamine in Boron Neutron Capture Therapy

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells. After injection of tirapazamine (TPZ), a bioreductive agent, combined with sodium borocaptate-¹⁰B (BSH) or dl-p -boronophenylalanine-¹⁰B (BPA) administration, the tumors were irradiated with thermal neutrons, and then isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined by means of immunofluorescence staining for BrdU, and that for total cells was obtained from tumors not pretreated with BrdU. Even when no ¹⁰B-compound was administered, TPZ increased the MN frequency of tumor cells including Q cells, resulting in reduction of the difference in MN frequency between total and Q cells, mainly by increasing the MN frequency of Q cells. TPZ increased the MN frequency of Q cells when combined with BPA administration, but TPZ showed no apparent effect on each cell population when combined with BSH. Namely, TPZ reduced the difference in MN frequency between total and Q cells caused by ¹⁰B-compound administration, especially when BPA was administered. From the viewpoint of the overall cell killing effect in boron neutron capture therapy (BNCT), combination with TPZ appeared to be useful in BPA-BNCT, but not in BSH-BNCT.     

Boron Neutron Capture Therapy: Effects of Split Dose and Overall Treatment Time

New clinical protocols are being developed that will entail the administration of considerably higher doses of the boron delivery agent boronophenylalanine (BPA) than those in current clinical use. Fractionation (2 or 4 fractions) of BPA mediated boron neutron capture therapy (BNCT) is also under consideration at some clinical centres. Given the considerably higher infusion volumes that will be entailed in the delivery of BPA in the new high dosage protocols, there will be a requirement to extend the gap between fractions to 2 or more days. In order to assess the effects of a 2 fraction protocol on the therapeutic efficacy of BPA mediated BNCT, a series of split dose irradiations (two equal fractions) were undertaken using the rat intracranially implanted 9L gliosarcoma model. A single dose exposure to BPA mediated BNCT of 3.0Gy resulted in long term survival levels of 50%. Survival levels increased to 71% and 77% with a 3 and 5 day gap between dose fractions (two equal fractions), respectively, using the same total dose. A further increase in the time interval between dose fractions to 7 days resulted in a reduction in survival to 36%. However, there was no significant difference between the single dose and the 3, 5 and 7 day survival data (P > 0.1) The difference between the 5 and 7 day split dose survival data was of border-line significance (P = 0.05). It is anticipated that mucositis, could become a potential problem in future BNCT clinical protocols involving higher doses, larger field sizes or multiple fields. The potential sparing of the oral mucosa, due to repopulation during the interval between the two fractions, was investigated using a series of split dose BPA mediated BNC irradiations. The ventral surface of the rat tongue was utilised as a model for oral mucosa. The ED₅₀ (50% incidence) values for the ulceration end point were 3.0±0.1,3.2±0.1,3.0±0.1 and 3.6±0.1Gy, for 3, 5, 7 and 9 day splits between doses, respectively. It is evident from this data that there were no significant changes in the ED₅₀ (p < 0.001) until the 9 day dose split, when the ED₅₀ value was 20% higher than the ED₅₀ value after a 7 day split. It was concluded that the two fraction BNCT protocol, with dose splits of up to 5 days, did not diminish the therapeutic response of the rat 9L gliosarcoma, when compared with a single dose BNCT protocol. Tolerance of the oral mucosa to BNC irradiation was not increased until there was a 9 day gap between fractions. However, the beneficial effects of dose sparing at this time interval between doses, would probably be counteracted by a reduction in the therapeutic effectiveness of the BNCT modality, due to repopulation of tumour clonogens between doses.     

A Critical Assessment of Boron Target Compounds for Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) has undergone dramatic developments since its inception by Locher in 1936 and the development of nuclear energy during World War II. The ensuing Cold War spawned the entirely new field of polyhedral borane chemistry, rapid advances in nuclear reactor technology and a corresponding increase in the number to reactors potentially available for BNCT. This effort has been largely oriented toward the eradication of glioblastoma multiforme (GBM) and melanoma with reduced interest in other types of malignancies. The design and synthesis of boron-10 target compounds needed for BNCT was not channeled to those types of compounds specifically required for GBM or melanoma. Consequently, a number of potentially useful boron agents are known which have not been biologically evaluated beyond a cursory examination and only three boron-10 enriched target species are approved for human use following their Investigational New Drug classification by the US Food and Drug Administration; BSH, BPA and GB-10. All ongoing clinical trials with GBM and melanoma are necessarily conducted with one of these three species and most often with BPA. The further development of BNCT is presently stalled by the absence of strong support for advanced compound evaluation and compound discovery driven by recent advances in biology and chemistry. A rigorous demonstration of BNCT efficacy surpassing that of currently available protocols has yet to be achieved. This article discusses the past history of compound development, contemporary problems such as compound classification and those problems which impede future advances. The latter include means for biological evaluation of new (and existing) boron target candidates at all stages of their development and the large-scale synthesis of boron target species for clinical trials and beyond. The future of BNCT is bright if latitude is given to the choice of clinical disease to be treated and if a recognized study demonstrating improved efficacy is completed. Eventually, BNCT in some form will be commercialized.     

Tumor-specific targeting of sodium borocaptate (BSH) to malignant glioma by transferrin-PEG liposomes: a modality for boron neutron capture therapy

Object Boron neutron capture therapy (BNCT) requires selective delivery of a high concentration of boron-10 (¹⁰B) to tumor tissue. To improve a drug delivery in BNCT, we devised transferrin-conjugated polyethylene-glycol liposome encapsulating sodium borocaptate (TF-PEG-BSH). Methods ¹⁰B concentrations of U87Δ human glioma cells from three boron delivery systems (BDS) (bare BSH, PEG-BSH, and TF-PEG-BSH) were analyzed in vitro by use of inductively coupled plasma-atomic emission spectrometry (ICP-AES). A colony-forming assay (CFA) was performed using this cell line with the three BDS and neutron irradiation. Subcellular localization of BSH in tumor cells was analyzed in vitro by immunocytochemistry using monoclonal antibodies raised for BSH. Brain tumor models were made and the ¹⁰B concentrations of the tumor, blood, and normal brain from each BDS were analyzed in vivo by use of ICP-AES. The tumor-to-blood and the tumor-to-normal brain ratios were evaluated. BNCT for the brain tumor models was performed and survival was analyzed. Results In CFA, TF-PEG-BSH showed the most prominent effects by neutron irradiation among the three BDS. TF-PEG-BSH showed highly selective and highly efficient ¹⁰B delivery in tumor tissue. The survival rate in the treatment experiment was best in the TF-PEG-BSH group. Immunocytochemically, TF-PEG-BSH delivered BSH efficiently not only into the cytoplasm but also into the nucleus. Conclusion TF-PEG-BSH is a potent BDS for BNCT not only in terms of delivering a high concentration of ¹⁰B into tumor tissue, but also the selective delivery of ¹⁰B into the tumor cells.     

Pharmacokinetic study of BSH and BPA in simultaneous use for BNCT

Summary In order to improve the effectiveness of boron neutron capture therapy (BNCT) for malignant gliomas, we examined the optimization of the administration of boron compounds in brain tumor animal model. We analyzed the concentration of boron atoms in intracranial C6 glioma -bearing rats using inductively coupled plasma atomic emission spectrometry. Each tumor-bearing rat received one of two different amounts of sodium borocaptate (BSH) and/or 500 mg/kg of boronophenylalanine (BPA) via intraperitoneal injection. We compared the boron concentrations of the tumor, the contralateral normal brain and the blood in rats of 3 different treatment groups (BSH alone, BPA alone and a combination of both BSH and BPA). Our results show that the tumor boron concentration increased much more than 30 μg/g by the coadministration of both compounds. Additionally, the blood boron concentration remained below 30 μg/g and the boron concentration in the normal brain was low (mean 4.7±1.1 μg/g). Even in comparison with the administration of BPA alone, coadministration of BPA and BSH shows an improved tumor/normal brain ratio of boron concentrations.     

Non‐invasive estimation of 10B‐4‐borono‐L‐phenylalanine‐derived boron concentration in tumors by PET using 4‐borono‐2‐18F‐fluoro‐phenylalanine

In boron neutron capture therapy (BNCT), ¹⁰B‐4‐borono‐L‐phenylalanine (BPA) is commonly used as a ¹⁰B carrier. PET using 4‐borono‐2‐¹⁸F‐fluoro‐phenylalanine (¹⁸F‐FBPA PET) has been performed to estimate boron concentration and predict the therapeutic effects of BNCT; however, the association between tumor uptake of ¹⁸F‐FBPA and boron concentration in tumors remains unclear. The present study investigated the transport mechanism of ¹⁸F‐FBPA and BPA, and evaluated the utility of ¹⁸F‐FBPA PET in predicting boron concentration in tumors. The transporter assay revealed that 2‐aminobicyclo‐(2.2.1)‐heptane‐2‐carboxylic acid, an inhibitor of the L‐type amino acid transporter, significantly inhibited ¹⁸F‐FBPA and ¹⁴C‐4‐borono‐L‐phenylalanine (¹⁴C‐BPA) uptake in FaDu and LN‐229 human cancer cells. ¹⁸F‐FBPA uptake strongly correlated with ¹⁴C‐BPA uptake in 7 human tumor cell lines (r = .93; P < .01). PET experiments demonstrated that tumor uptake of ¹⁸F‐FBPA was independent of the administration method, and uptake of ¹⁸F‐FBPA by bolus injection correlated well with BPA uptake by continuous intravenous infusion. The results of this study revealed that evaluating tumor uptake of ¹⁸F‐FBPA by PET was useful for estimating ¹⁰B concentration in tumors.     

Assessment of the Results from the Phase I/II Boron Neutron Capture Therapy Trials at the Brookhaven National Laboratory from a Clinician's Point of View

Boron neutron capture therapy (BNCT) represents a promising modality for a relatively selective radiation dose delivery to the tumor tissue. The key to effective BNCT of tumors such as glioblastoma multiforme (GBM) is the homogeneous preferential accumulation of ¹⁰B in the tumor, including the infiltrating GBM cells, as compared to that in the vital structures of the normal brain. Provided that sufficiently high tumor ¹⁰B concentration (10⁹ boron-10 atoms/cell) and an adequate thermal neutron fluence (10⁹ neutrons/cm²⁾ are achieved, it is the ratio of the ¹⁰B concentration in tumor cells to that in the normal brain cells and the blood that will largely determine the therapeutic gain of BNCT.     

Boron microlocalization in oral mucosal tissue: implications for boron neutron capture therapy

Clinical studies of the treatment of glioma and cutaneous melanoma using boron neutron capture therapy (BNCT) are currently taking place in the USA, Europe and Japan. New BNCT clinical facilities are under construction in Finland, Sweden, England and California. The observation of transient acute effects in the oral mucosa of a number of glioma patients involved in the American clinical trials, suggests that radiation damage of the oral mucosa could be a potential complication in future BNCT clinical protocols, involving higher doses and larger irradiation field sizes. The present investigation is the first to use a high resolution surface analytical technique to relate the microdistribution of boron-10 (10B) in the oral mucosa to the biological effectiveness of the 10B(n,alpha)7Li neutron capture reaction in this tissue. The two boron delivery agents used clinically in Europe/Japan and the USA, borocaptate sodium (BSH) and p-boronophenylalanine (BPA), respectively, were evaluated using a rat ventral tongue model. 10B concentrations in various regions of the tongue mucosa were estimated using ion microscopy. In the epithelium, levels of 10B were appreciably lower after the administration of BSH than was the case after BPA. The epithelium:blood 10B partition ratios were 0.2:1 and 1:1 for BSH and BPA respectively. The 10B content of the lamina propria was higher than that measured in the epithelium for both BSH and BPA. The difference was most marked for BSH, where 10B levels were a factor of six higher in the lamina propria than in the epithelium. The concentration of 10B was also measured in blood vessel walls where relatively low levels of accumulation of BSH, as compared with BPA, was demonstrated in blood vessel endothelial cells and muscle. Vessel wall:blood 10B partition ratios were 0.3:1 and 0.9:1 for BSH and BPA respectively. Evaluation of tongue mucosal response (ulceration) to BNC irradiation indicated a considerably reduced radiation sensitivity using BSH as the boron delivery agent relative to BPA. The compound biological effectiveness (CBE) factor for BSH was estimated at 0.29 +/- 0.02. This compares with a previously published CBE factor for BPA of 4.87 +/- 0.16. It was concluded that variations in the microdistribution profile of 10B, using the two boron delivery agents, had a significant effect on the response of oral mucosa to BNC irradiation. From a clinical perspective, based on the findings of the present study, it is probable that potential radiation-induced oral mucositis will be restricted to BNCT protocols involving BPA. However, a thorough high resolution analysis of 10B microdistribution in human oral mucosal tissue, using a technique such as ion microscopy, is a prerequisite for the use of experimentally derived CBE factors in clinical BNCT.     

Improvement of the tumor-suppressive effect of boron neutron capture therapy for amelanotic melanoma by intratumoral injection of the tyrosinase gene

Boron neutron capture therapy (BNCT) is successful when there is a sufficient (10)B concentration in tumor cells. In melanoma, (10)B-para-boronophenylalanine (BPA) accumulation is proportional to melanin-producing activity. This study was done to confirm enhancement of the tumor-suppressive effect of BNCT on amelanotic melanoma by intratumoral injection of the tyrosinase gene. D178 or FF amelanotic melanomas were implanted s.c. in Syrian hamsters. One group of D178- or FF-bearing hamsters (TD178 or TFF group) received intratumoral injections of pcDNA-Tyrs constructed as a tyrosinase expression plasmid. The other hamsters (pD178 and pFF groups) were injected with pUC119, and control hamsters (D178 and FF groups) only with transfection reagents. All the groups underwent immunofluorescence analysis of tyrosinase expression and BPA biodistribution studies. BNCT experiments were done at the Kyoto University Research Reactor. Tyrosinase expression increased in the tumors of the TD178 and TFF groups but remained the same in the pD178 and pFF groups. Tumor boron concentrations in the TD178 and TFF groups increased significantly (TD178: 49.7 +/- 12.6 versus D178: 27.2 +/- 4.9 microg/g, P < 0.0001; TFF: 30.7 +/- 6.6 versus FF: 13.0 +/- 4.7 microg/g, P < 0.0001). The BNCT tumor-suppressive effect was marked in the TD178 and TFF groups. In vivo transfection with the tyrosinase gene increased BPA accumulation in the tumors, the BNCT tumor-suppressive effect on amelanotic melanoma being significantly enhanced. These findings suggest a potential new clinical strategy for the treatment of amelanotic melanoma with BNCT.