We have established methods of targeting a sufficient number of 10B atoms on human melanoma cells to allow selective destruction of the cancer cells by thermal neutron irradiation. Thermal neutron capture therapy (NCT)1-3 requires the presence of at least 10(9) 10B atoms on each target cell for specific killing of that cell without injuring normal tissues. In order to accumulate an adequate number of 10B atoms on target cells, we first created an effective compound containing 12 atoms of 10B per molecule (10B12-chlorpromazine) and 10B-dopa analogue (10B1-paraboronophenylalanine). In the present study, about three molecules of our newly synthesized 10B12-compound were conjugated to an avidin molecule. The resulting 10B38.5-avidin compound can be specifically directed to human melanoma cells by biotinated monoclonal antibodies (MAbs) specific for the cells. We were able to accumulate 2.6 x 10(8) 10B atoms on a melanoma cell using this method. Cultured human melanoma cells treated with 10B-avidin-biotin-MAb (10B-AB-MAb) were selectively damaged by thermal neutron irradiation in vitro. This is the first study to indicate that thermal neutrons selectively damage target cells boronated by MAbs.

Animals↗

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New thermal neutron capture therapy for malignant melanoma: melanogenesis-seeking 10B molecule-melanoma cell interaction from in vitro to first clinical trial.

Human melanoma regression by single thermal neutron capture therapy (NCT) using melanoma-seeking 10B compounds has been achieved. Since 1972, the aim of my team has been to synthesize tumor-seeking 10B-compounds possessing selective affinity for specific metabolic activity of the target cancer cells. Once the melanoma takes up these 10B compounds, thermal neutrons, which cause insignificant cell damage, are easily absorbed by nonradioactive 10B, inducing the 10B(n, alpha)7Li reaction and releasing the high LET particles to 14 mu melanoma cell diameter, destroying the tumor without damaging surrounding tissue. Radiobiological and preclinical studies culminated in the first successful human NCT treatment, with no recurrence of the treated melanoma since July, 1987.

Animals↗

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Specific killing of human melanoma cells with an efficient 10B-compound on monoclonal antibodies.

We previously established methods which have enabled us to target a sufficient number of 10B atoms on human melanoma cells to destroy them by thermal neutron irradiation. Monoclonal antibodies were here used as vector of 10B atoms on the target cell. Thermal neutrons require at least 10(9) 10B atoms to destroy the cell. In order to accumulate an adequate number of 10B atoms on target cells, our first approach was to make an effective compound that contains 12 atoms of 10B in a molecule. The second step was to conjugate the compound with an avidin molecule (10B12-avidin). One molecule of the 10B12-avidin carries about 30 atoms of 10B. This 10B12-avidin can be specifically targeted on human melanoma cells by biotinated monoclonal antibodies specific for the cells. Furthermore, the number of 10B atoms on target cells can be augmented by a hapten-antihapten monoclonal antibody system. The cultured human melanoma cells treated with these methods were damaged by thermal neutron irradiation. This is the first study that indicates thermal neutrons do injure target cells boronated by monoclonal antibodies.

Antibodies, Monoclonal↗

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A trial to improve the analysis of boron in biological materials.

For the spectrophotometric determination of boron in biological materials, two decomposition methods were used. One is the alkali fusion method, and the other is the acid decomposition method using a uniseal decomposition vessel. A long-range calibration curve for the former method was obtained by using an ultrapure sulfuric acid. The alkali fusion method was applied to samples into which paraboronophenylalanine (BPA) or its hydrochloric salt (BPA.HCl) had been injected. By the acid decomposition method using a uniseal vessel, 50 mg dried liver was digested by a mixed solution of 1 ml conc. sulfuric acid and 6 ml 30% hydrogen peroxide. A problem exists, however, in that the hydrogen peroxide remaining in the decomposition solution must be eliminated without loss of boron.

Alkalies↗

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Neutron irradiation of human melanoma cells.

The biological characteristics and in vitro radiosensitivity of melanoma cells to thermal neutrons were investigated as a guide to the effectiveness of boron neutron capture therapy. Plateau phase cultures of three human malignant melanoma-established cell lines were examined for cell density at confluence, doubling time, cell cycle parameters, chromosome constitution, and melanin content. Cell survival dose-response curves, for cells preincubated in the presence or absence of p-boronophenylalanine. HCl (10B1-BPA), were measured over the dose range 0.6-8.0 Gy (N + gamma). The neutron fluence rate was 2.6 x 10(9) n/cm2/s and the total dose rate 3.7 Gy/h (31% gamma). Considerable differences were observed in the morphology and cellular properties of the cell lines. Two cell lines (96E and 96L) were amelanotic, and one was melanotic (418). An enhanced killing for neutron irradiation was found only for the melanotic cells after 20 h preincubation with 10 micrograms/ml 10B1-BPA. In view of the doubling times of the cell lines of about 23 h (96E and 96L) or of 36 h (418), it seems likely that an increased boron uptake, and hence increased radiosensitivity, might result if the preincubation period with 10B1-BPA is extended to several hours longer than the respective cell cycle times.

Boron Compounds↗

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Relative biological effectiveness (RBE) of thermal neutron capture therapy of cultured B-16 melanoma cells preincubated with 10B-paraboronophenylalanine.

An experimental study of the relative biological effectiveness (RBE) of thermal neutron capture therapy (TNCT) for melanoma cell inactivation using 10B1-paraboronophenylalanine (10B1-BPA) was carried out to demonstrate a high therapeutic effect of TNCT, compared with that of fast neutron. Cells preincubated with or without 10B1-BPA at a concentration of 50 micrograms/ml for 20 h were irradiated with 60Co gamma-ray, fast neutron or thermal neutron. The absorbed dose of the cells from thermal neutron was calculated by Kobayashi's model. The D0 value of fast neutron was 1.07 Gy, and the D0S of thermal neutron radiation with or without preincubation of the cells with 10B1-BPA were 0.46 Gy or 0.67 Gy, respectively. The RBEs of fast neutron, thermal neutron beams, and neutron capture therapy relative to 60Co gamma-ray were calculated as 2.78, 4.18, and 6.15 at 0.1 surviving fraction, respectively. These results indicate radiologically that thermal neutron capture therapy using 10B1-BPA is an excellent radiation therapy for malignant melanoma.

Animals↗

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Review: biological effectiveness of thermal neutrons and 10B(n,alpha)7Li reaction on cultured cells.

There are only a few reports on the relative biological effectiveness (RBE) of thermal neutrons and 10B(n,alpha)7Li reactions either in vitro or in vivo. The data in this paper summarize almost all previously published in vitro data. Because only a few reactors are available for biomedical purposes, it is difficult to make a comparison of data from experiments using the same kind of radiation, and also to make a comparison of data from experiments using the different kinds of radiations. However, it is indispensable for boron neutron capture therapy to make a radiobiological analysis. More intensive study, including repair process and oxygen effect, is necessary for establishing the fundamental basis of the clinical application of boron neutron capture therapy.

Animals↗

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Use of nude mice in experimental neutron capture therapy with 10B-BPA.

Mouse B16 melanoma allografts in nude mice were successfully treated by thermal neutron irradiation after IP injection of 10B-paraboronophenylalanine hydrochloride. The tumor growth was significantly suppressed for 4 weeks after irradiation, compared with animals given neutron irradiation alone. Tumor-bearing nude mice were shown to be useful for evaluating the treatment for melanoma.

Animals↗

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Morphological changes in human melanoma cells following irradiation with thermal neutrons.

Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.

Boron Compounds↗