A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron‐containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein‐coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor‐directed boron uptake. In this study, we present the generation of short, boron‐rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super‐agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta‐carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron‐loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.     

Hyaluronan as carrier of carboranes for tumor targeting in boron neutron capture therapy

Boron neutron capture therapy (BNCT) represents a promising approach for tumor therapy. A critical requirement for BNCT is tumor targeting, a goal that is currently addressed with the development of low and high molecular weight agents capable of interacting with receptors expressed by cancer cells. Here, we describe a new bioconjugate (HApCB) composed by n-propyl carborane linked to hyaluronan (HA) via an ester linkage for a degree of substitution of approximately 30%, leading to a water-soluble derivative. The structure and main physicochemical characteristics of the new HA derivative were determined by means of Fourier transform infrared, fluorescence, and 1H, 13C, and 10B NMR analysis and are herein reported in detail. As HA is recognized by the CD44 antigen, densely populating the surface of many tumor cells, HApCB is expected to deliver boron atoms from the locally released carborane cages directly to target cells for antitumor application in BNCT. In vitro biological experiments showed that HApCB was not toxic for a variety of human tumor cells of different histotypes, specifically interacted with CD44 as the native unconjugated HA, and underwent uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach. Thus, HApCB may be regarded as a promising new BNCT agent for specific targeting of cancer cells overexpressing the CD44 receptor.     

Xylose as a carrier for boron containing compounds

A xylosylated carborane was synthesized by standard carbohydrate methodology and tested on normal HFL-1 cells as well as transformed T24 cells. The xylosylated carborane initiated glycosaminoglycan (GAG) synthesis in both cell lines and treatment with the carborane gave a pronounced translocation of proteoglycans to the nuclei of T24 cells. However, most of the boron-containing compounds were secreted to the medium. We conclude that xylosides carrying carboranes are not suitable for boron neutron capture therapy (BNCT) for T24 cells. However, the uptake of boron-containing xyloside, the GAG priming capacity, and the nuclear translocation of glypican-1 make this xyloside a candidate for further investigation for selectivity toward other tumor cell lines.     

Carborane-conjugated 2-quinolinecarboxamide ligands of the translocator protein for boron neutron capture therapy

Potential boron neutron capture therapy (BNCT) agents have been designed on the basis of the evidence about translocator protein (TSPO) overexpression on the outer mitochondrial membrane of tumor cells. The structure of the first TSPO ligand bearing a carborane cage (compound 2d) has been modified in order to find a suitable candidate for in vivo studies. The designed compounds were synthesized and evaluated for their potential interaction with TSPO and tumor cells. In vitro biological evaluation showed in the case of fluoromethyl derivative 4b a nanomolar TSPO affinity very similar to that of 2d, a significantly lower cytotoxicity, and a slightly superior performance as boron carrier toward breast cancer cells. Moreover, compound 4b could be used as a 1?F magnetic resonance imaging (MRI) agent as well as labeled with 11C or 1?F to obtain positron emission tomography (PET) radiotracers in order to apply the "see and treat" strategy in BNCT.     

Synthesis of ethyleneoxide modified 3-carboranyl thymidine analogues and evaluation of their biochemical, physicochemical, and structural properties

Eleven 3-carboranyl thymidine analogues (3CTAs) containing highly hydrophilic and flexible ethyleneoxide moieties were synthesized as potential agents for boron neutron capture therapy (BNCT) and their biochemical and physicochemical properties were evaluated. Based on specific structural features, this library of 3CTAs was divided into three subgroups. The first group contained 3CTAs with 1-4 ethyleneoxide units between the thymidine (Thd) scaffold and a carborane cluster. The second group of 3CTAs contained a pentylene spacer between Thd and the carborane and 2-4 ethyleneoxide units additionally attached to the carborane cluster. The third group contained three 3CTAs all with pentylene spacers and four ethylene units but with different carborane cages. The ethyleneoxide modified 3CTAs were good substrates of thymidine kinase 1 (TK1) and poor substrates of human mitochondrial thymidine kinase 2 (TK2) as determined in phosphoryl transfer assays. In the first group of 3CTAs, all the compounds were efficiently phosphorylated regardless of varying spacer lengths (37-42% of the activity of Thd). The second group of 3CTAs was less effectively phosphorylated (17-26% of the activity of Thd) probably due to a less favorable sterical orientation of Thd within the active site of TK1 and/or an increased lipophilicity compared with the first group. In the third group of structural isomers, no significant differences in phosphorylation rates were observed (17-25%). A structure-function hypothesis explaining these results is presented.     

Synthesis and in vitro evaluation of thiododecaborated α, α- cycloalkylamino acids for the treatment of malignant brain tumors by boron neutron capture therapy

Boron-neutron capture therapy (BNCT) is an attractive technique for cancer treatment. As such, α, α-cycloalkyl amino acids containing thiododecaborate ([B₁₂H₁₁]^2?-S-) units were designed and synthesized as novel boron delivery agents for BNCT. In the present study, new thiododecaborate α, α-cycloalkyl amino acids were synthesized, and biological evaluation of the boron compounds as boron carrier for BNCT was carried out.     

Towards improved boron neutron capture therapy agents: evaluation of in vitro cellular uptake of a glutamine-functionalized carborane

Abstract  Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. One drawback is the large uptake by the liver causing a decrease of its availability at the tumor region as well as bringing about toxicity problems. A novel carborane-based compound containing a boron payload very similar to that of BSH has been synthesized and tested on rat glioma (C6) cells, hepatoma tissue culture (HTC) cells, and hepatocytes. The newly synthesized system consists of an o-carborane unit (C₂B₁₀H₁₁, o-CB) conjugated to a glutamine residue through a proper spacer, namely, o-CB-Gln. As compared with BSH, it showed the same uptake by C6 cells, but a 50% decrease in uptake by HTC cells and an 80% decrease in uptake by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to that of BSH accompanied by a much lower liver toxicity. Graphical Abstract  A novel carborane based compound, consisting in an o-carborane unit (C2B10H11, o-CB) conjugated to a glutamine residue through a proper spacer (namely o-CB-Gln) has been synthesized, characterized and tested on rat glioma (C6), hepatoma (HTC) and hepatocytes. As compared to sodium borocaptate (BSH), widely used for boron neutron capture therapy (BNCT) of brain tumors, the newly synthesized system showed the same uptake by C6 cells, but a 50% decrease by HTC and 80% decrease by healthy hepatocytes. On this basis o-CB-Gln appears an interesting candidate for BNCT of brain tumors as it is expected to have a therapeutic index analogous to BSH accompanied by a much lower liver toxicity.      

Folate receptor-mediated boron-10 containing carbon nanoparticles as potential delivery vehicles for boron neutron capture therapy of nonfunctional pituitary adenomas

Invasive nonfunctional pituitary adenomas (NFPAs) are difficult to completely resect and often develop tumor recurrence after initial surgery. Currently, no medications are clinically effective in the control of NFPA. Although radiation therapy and radiosurgery are useful to prevent tumor regrowth, they are frequently withheld because of severe complications. Boron neutron capture therapy (BNCT) is a binary radiotherapy that selectively and maximally damages tumor cells without harming the surrounding normal tissue. Folate receptor (FR)-targeted boron-10 containing carbon nanoparticles is a novel boron delivery agent that can be selectively taken up by FR-expressing cells via FR-mediated endocytosis. In this study, FR-targeted boron-10 containing carbon nanoparticles were selectively taken up by NFPAs cells expressing FR but not other types of non-FR expressing pituitary adenomas. After incubation with boron-10 containing carbon nanoparticles and following irradiation with thermal neutrons, the cell viability of NFPAs was significantly decreased, while apoptotic cells were simultaneously increased. However, cells administered the same dose of FR-targeted boron-10 containing carbon nanoparticles without neutron irradiation or received the same neutron irradiation alone did not show significant decrease in cell viability or increase in apoptotic cells. The expression of Bcl-2 was down-regulated and the expression of Bax was up-regulated in NFPAs after treatment with FR-mediated BNCT. In conclusion, FR-targeted boron-10 containing carbon nanoparticles may be an ideal delivery system of boron to NFPAs cells for BNCT. Furthermore, our study also provides a novel insight into therapeutic strategies for invasive NFPA refractory to conventional therapy, while exploring these new applications of BNCT for tumors, especially benign tumors.     

Synthesis and biological evaluation of 3'-carboranyl thymidine analogues

Boron neutron capture therapy (BNCT) is a chemoradio-therapeutic method for the treatment of cancer. It depends on the selective targeting of tumor cells by boron-containing compounds. One category of BNCT agents with potential to selectively target tumor cells may be thymidine derivatives substituted at the 3'-position with appropriate boron moieties. Thus, several thymidine analogues were synthesized with a carborane cluster bound to the 3'-position either through an ether or a carbon linkage. The latter are the first reported carborane-containing nucleosides in which the carboranyl entity is directly linked to the carbohydrate portion of the nucleoside by a carbon-carbon bond. Low but significant phosphorylation rates in the range of 0.18% that of thymidine were observed for the carbon-linked 3'-carboranyl thymidine analogues in phosphoryl transfer assays using recombinant preparations of thymidine kinases 1 (TK1) and thymidine kinases 2 (TK2). Some of the ether-linked 3'-carboranyl thymidine analogues appeared to be slightly unstable under acidic as well as phosphoryl transfer assay conditions and were, if at all, poor substrates for TK1.     

Receptor-targeted liposomal delivery of boron-containing cholesterol mimics for boron neutron capture therapy (BNCT)

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.     

closo-borane conjugated regulatory peptides retain high biological affinity: synthesis of closo-borane conjugated Tyr(3)-octreotate derivatives for BNCT

Despite the improvements in cancer therapy during the past years, high-grade gliomas and many other types of cancer are still extremely resistant to current forms of therapy. Boron neutron capture therapy (BNCT) provides a promising way to destroy cancer cells without damaging healthy tissue. However, BNCT in practice is still limited due to the lack of boron-containing compounds that selectively deliver boron to cancer cells. Since many neuroendocrine tumors show an overexpression of the somatostatin receptor, it was our aim to synthesize compounds that contain a large number of boron atoms and still show high affinity toward this transmembrane receptor. The synthetic peptide Tyr (3)-octreotate (TATE) was chosen as a high-affinity and internalizing tumor targeting vector (TTV). Novel boron cluster compounds, containing 10 or 20 boron atoms, were coupled to the N-terminus of TATE. The obtained affinity data demonstrate that the use of a spacer between TATE and the closo-borane moiety is the option to avoid a loss of biological affinity of closo-borane conjugated TATE. For the first time, it was shown that closo-borane conjugated regulatory peptides retain high biological affinity and selectivity toward their transmembrane tumor receptors. The results obtained and the improvement of spacer and boron building block chemistry may stimulate new directions for BNCT.     

Boron Containing Pyrimidines,Nucleosides, and Oligonucleotides for Neutron Capture Therapy

Abstract

The synthesis and encouraging biological findings with boron-containing nucleosides, such as 5-dihydroxyboryl-2′-deoxyuridine, which could be used for boron neutron capture therapy (BNCT) for the treatment of various malignancies, has provided momentum to synthesize several boron containing nucleosides and oligomers. BNCT is based on the property of the non-radioactive boron-10 isotope to capture low energy neutrons, thereby producing a localized cell-destroying nuclear reaction. Thus, irradiation of tumor cells with neutrons, following incorporation of the boronated nucleoside, would result in the destruction of tumor tissue only. Intracellular phosphorylation by nucleoside kinases, and/or incorporation into the cancer cell DNA as a false nucleotide precursor, followed by irradiation by neutrons, would lead primarily to tumor cell death. The synthetic and biological approaches for boronated pyrimidines, nucleosides, and oligonucleotides for BNCT are reviewed.     

Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.     

Synthesis and structure–activity relationship of p-carborane-based non-secosteroidal vitamin D analogs

1α,25-Dihydroxyvitamin D₃ [1α,25(OH)₂D₃: 1] is a specific modulator of nuclear vitamin D receptor (VDR), and novel vitamin D analogs are therapeutic candidates for multiple clinical applications. We recently developed non-secosteroidal VDR agonists bearing a p-carborane cage (a carbon-containing boron cluster) as a hydrophobic core structure. These carborane derivatives are structurally quite different from classical secosteroidal vitamin D analogs. Here, we report systematic synthesis and activity evaluation of carborane-based non-secosteroidal vitamin D analogs. The structure–activity relationships of carborane derivatives are different from those of secosteroidal vitamin D derivatives, and in particular, the length and the substituent position of the dihydroxylated side chain are rather flexible in carborane derivatives. The structure–activity relationships presented here should be helpful in development of non-secosteroidal vitamin D analogs for clinical applications.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Boron neutron capture therapy (BNCT) for the treatment of liver metastases: biodistribution studies of boron compounds in an experimental model

We previously demonstrated the therapeutic efficacy of different boron neutron capture therapy (BNCT) protocols in an experimental model of oral cancer. BNCT is based on the selective accumulation of (10)B carriers in a tumor followed by neutron irradiation. Within the context of exploring the potential therapeutic efficacy of BNCT for the treatment of liver metastases, the aim of the present study was to perform boron biodistribution studies in an experimental model of liver metastases in rats. Different boron compounds and administration conditions were assayed to determine which administration protocols would potentially be therapeutically useful in in vivo BNCT studies at the RA-3 nuclear reactor. A total of 70 BDIX rats were inoculated in the liver with syngeneic colon cancer cells DHD/K12/TRb to induce the development of subcapsular tumor nodules. Fourteen days post-inoculation, the animals were used for biodistribution studies. We evaluated a total of 11 administration protocols for the boron compounds boronophenylalanine (BPA) and GB-10 (Na(2)(10)B(10)H(10)), alone or combined at different dose levels and employing different administration routes. Tumor, normal tissue, and blood samples were processed for boron measurement by atomic emission spectroscopy. Six protocols proved potentially useful for BNCT studies in terms of absolute boron concentration in tumor and preferential uptake of boron by tumor tissue. Boron concentration values in tumor and normal tissues in the liver metastases model show it would be feasible to reach therapeutic BNCT doses in tumor without exceeding radiotolerance in normal tissue at the thermal neutron facility at RA-3.     

In vitro and in vivo studies of boron neutron capture therapy: boron uptake/washout and cell death

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on thermal-neutron irradiation of cells enriched with (10)B, which produces α particles and (7)Li ions of short range and high biological effectiveness. The selective uptake of boron by tumor cells is a crucial issue for BNCT, and studies of boron uptake and washout associated with cell survival studies can be of great help in developing clinical applications. In this work, boron uptake and washout were characterized both in vitro for the DHDK12TRb (DHD) rat colon carcinoma cell line and in vivo using rats bearing liver metastases from DHD cells. Despite a remarkable uptake, a large boron release was observed after removal of the boron-enriched medium from in vitro cell cultures. However, analysis of boron washout after rat liver perfusion in vivo did not show a significant boron release, suggesting that organ perfusion does not limit the therapeutic effectiveness of the treatment. The survival of boron-loaded cells exposed to thermal neutrons was also assessed; the results indicated that the removal of extracellular boron does not limit treatment effectiveness if adequate amounts of boron are delivered and if the cells are kept at low temperature. Cell survival was also investigated theoretically using a mechanistic model/Monte Carlo code originally developed for radiation-induced chromosome aberrations and extended here to cell death; good agreement between simulation outcomes and experimental data was obtained.     

Inborn errors in metabolism and 4-boronophenylalanine-fructose-based boron neutron capture therapy

Infusions of boronophenylalanine-fructose complex (BPA-F), at doses up to 900 mg/kg of BPA and 860 mg/kg of fructose, have been used to deliver boron to cancer tissue for boron neutron capture therapy (BNCT). In patients with phenylketonuria (PKU), phenylalanine accumulates, which is harmful in the long run. PKU has been an exclusion criterion for BPA-F-mediated BNCT. Fructose is harmful to individuals with hereditary fructose intolerance (HFI) in amounts currently used in BNCT. The harmful effects are mediated through induction of hypoglycemia and acidosis, which may lead to irreversible organ damage or even death. Consequently, HFI should be added as an exclusion criterion for BNCT if fructose-containing solutions are used in boron carriers. Non-HFI subjects may also develop symptoms, such as gastrointestinal pain, if the fructose infusion rate is high. We therefore recommend monitoring of glucose levels and correcting possible hypoglycemia promptly. Except for some populations with extremely low PKU prevalence, HFI and PKU prevalences are similar, approximately 1 or 2 per 20,000.     

Boron uptake in normal melanocytes and melanoma cells and boron biodistribution study in mice bearing B16F10 melanoma for boron neutron capture therapy

Information on (10)B distribution in normal tissues is crucial to any further development of boron neutron capture therapy (BNCT). The goal of this study was to investigate the in vitro and in vivo boron biodistribution in B16F10 murine melanoma and normal tissues as a model for human melanoma treatment by a simple and rapid colorimetric method, which was validated by HR-ICP-MS. The B16F10 melanoma cell line showed higher melanin content than human melanocytes, demonstrating a greater potential for boronophenylalanine uptake. The melanocytes showed a moderate viability decrease in the first few minutes after BNCT application, stabilizing after 75 min, whereas the B16F10 melanoma showed the greatest intracellular boron concentration at 150 min after application, indicating a different boron uptake of melanoma cells compared to normal melanocytes. Moreover, at this time, the increase in boron uptake in melanoma cells was approximately 1.6 times higher than that in normal melanocytes. The (10)B concentration in the blood of mice bearing B16F10 melanoma increased until 90 min after BNCT application and then decreased after 120 min, and remained low until the 240th minute. On the other hand, the (10)B concentration in tumors was increased from 90 min and maximal at 150 min after application, thus confirming the in vitro results. Therefore, the present in vitro and in vivo study of (10)B uptake in normal and tumor cells revealed important data that could enable BNCT to be possibly used as a treatment for melanoma, a chemoresistant cancer associated with high mortality.     

Effect of boron neutron capture therapy for melanotic and amelanotic melanoma transplanted into mouse brain

In order to develop a protocol to treat brain metastatic melanoma using our 10B-p-boronophenylalanine (BPA) boron neutron capture therapy (BNCT), we initiated the following studies (i), Comparative analyses of boron biodistribution between melanoma proliferating in the brain and skin among melanotic and amelanotic types, and (ii) Therapeutic evaluation of BPA-BNCT for brain melanoma models of both types, using survival times. Our present data have revealed that boron concentration in melanoma proliferating in the brain, the major prerequisite for successful BNCT, showed a positive correlation to melanin synthesizing activity in the same way as melanoma proliferating in skin. Further, the boron concentration ratio of melanoma to normal surrounding tissue for brain melanoma models was considerably higher than that for subcutaneous (s.c.) ones because of the existence of the blood-brain barrier (BBB). Additionally, from analyses of median and mean survival times following BNCT using low, middle, and high neutron doses, the therapeutic effect of BNCT for the amelanotic A1059 melanoma appeared at first glance to be higher than that for the highly BPA attracting and highly relative biological effect equivalent dose obtaining B15b melanoma. As the survival time was dependent on both regression and regrowth curves, and because the brain melanoma model in small animals made it difficult to evaluate these curves separately, we further examined the in vivo growth curve of both types of melanomas following implantation in s.c. tissue. The melanotic B15b melanoma was indeed found to possess much higher growth rate as compared with that of the amelanotic A1059 melanoma. The significance of boron biodistribution studies and BNCT survival curve analyses in forming an effective clinical protocol for individual human cases of melanoma brain metastasis is discussed.