Synthesis and evaluation as MRI probe of the trifluoromethylated p-boronophenylalanine and its alcohol derivative

Boron-neutron capture therapy (BNCT) and magnetic resonance imaging (MRI) are quite attractive techniques for treatment and diagnosis of cancer, respectively. In order to develop practical tools for BNCT and MRI, novel compounds containing both the trifluoromethyl group and 10B atom in a single molecule were designed. In the present study, p-boronophenylalanine and p-boronophenylalaninol with the trifluoromethyl group were synthesized, and 19F NMR measurements of these compounds were carried out.     

Biological evaluation of fluorinated p-boronophenylalanine derivatives as a boron carrier

Boron neutron capture therapy (BNCT) and magnetic resonance imaging (MRI) are quite attractive techniques for treatment and diagnosis of cancer, respectively. In order to develop practical materials utilizing both for BNCT and MRI, fluorinated p-boronophenylalanines and their alcohol derivatives had already been designed and synthesized. In the present paper the cytotoxicity, the incorporated amount into cancer cells, and the tumor cell killing effects of these compounds were elucidated to evaluate their usefulness as a boron carrier.     

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.     

Analysis of boron distribution in vivo for boron neutron capture therapy using two different boron compounds by secondary ion mass spectrometry

The efficiency of boron neutron capture therapy (BNCT) for malignant gliomas depends on the selective and absolute accumulation of (10)B atoms in tumor tissues. Only two boron compounds, BPA and BSH, currently can be used clinically. However, the detailed distributions of these compounds have not been determined. Here we used secondary ion mass spectrometry (SIMS) to determine the histological distribution of (10)B atoms derived from the boron compounds BSH and BPA. C6 tumor-bearing rats were given 500 mg/kg of BPA or 100 mg/kg of BSH intraperitoneally; 2.5 h later, their brains were sectioned and subjected to SIMS. In the main tumor mass, BPA accumulated heterogeneously, while BSH accumulated homogeneously. In the peritumoral area, both BPA and BSH accumulated measurably. Interestingly, in this area, BSH accumulated distinctively in a diffuse manner even 800 microm distant from the interface between the main tumor and normal brain. In the contralateral brain, BPA accumulated measurably, while BSH did not. In conclusion, both BPA and BSH each have advantages and disadvantages. These compounds are considered to be essential as boron delivery agents independently for clinical BNCT. There is some rationale for the simultaneous use of both compounds in clinical BNCT for malignant gliomas.     

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.     

Design and synthesis of boron containing monosaccharides by the hydroboration of d-glucal for use in boron neutron capture therapy (BNCT)

Boron neutron capture therapy (BNCT) is one of the radiotherapies that involves the use of boron-containing compounds for the treatment of cancer. Boron-10 (¹⁰B) containing compounds that can accumulate in tumor tissue are expected to be suitable agents for BNCT. We report herein on the design and synthesis of some new BNCT agents based on a d-glucose scaffold, since glycoconjugation has been recognized as a useful strategy for the specific targeting of tumors. To introduce a boryl group into a d-glucose scaffold, we focused on the hydroboration of d-glucal derivatives, which have a double bond between the C1 and C2 positions. It was hypothesized that a C–B bond could be introduced at the C2 position of d-glucose by the hydroboration of d-glucal derivatives and that the products could be stabilized by conversion to the corresponding boronic acid ester. To test this hypothesis, we prepared some 2-boryl-1,2-dideoxy-d-glucose derivatives as boron carriers and evaluated their cytotoxicity and cellular uptake activity to cancer cells, especially under hypoxic conditions.     

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.     

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.     

A convenient method for the computer-aided molecular design of carborane containing compounds

Computer-aided molecular design (CAMD) of carborane containing compounds is of growing interest for scientists involved in boron neutron capture therapy (BNCT) and other pharmaceutical applications. However, the complex organo-metallic structures of carboranes pose difficulties in modeling and docking of these structures. This is the first report of a new strategy for modeling and docking of carborane containing molecules with the readily available software packages HyperChem, SYBYL and FlexX. It is intended as a guide for boron chemists interested in using CAMD of carborane containing agents for medical applications such as BNCT.     

Boron neutron capture therapy (BNCT) for the treatment of spontaneous nasal planum squamous cell carcinoma in felines

Recently, Boron neutron capture therapy (BNCT) was successfully applied to treat experimental squamous cell carcinomas (SCC) of the hamster cheek pouch mucosa, with no damage to normal tissue. It was also shown that treating spontaneous nasal planum SCC in terminal feline patients with low dose BNCT is safe and feasible. In an extension of this work, the present study aimed at evaluation of the response of tumor and dose-limiting normal tissues to potentially therapeutic BNCT doses. Biodistribution studies with ¹⁰B-boronophenylalanine (BPA enriched in ¹⁰B) as a ¹⁰B carrier were performed on three felines that showed advanced nasal planum SCC without any standard therapeutic option. Following the biodistribution studies, BNCT mediated by ¹⁰BPA was done using the thermalized epithermal neutron beam at the RA-6 Nuclear Reactor. Follow-up included clinical evaluation, assessment of macroscopic tumor and normal tissue response and biopsies for histopathological analysis. The treated animals did not show any apparent radiation-induced toxicity. All three animals exhibited partial tumor control and an improvement in clinical condition. Enhanced therapeutic efficacy was associated with a high ¹⁰B content of the tumor and a small tumor size. BNCT is therefore believed to be potentially effective in the treatment of spontaneous SCC. However, improvement in targeting ¹⁰B into all tumor cells and delivering a sufficient dose at a greater depth are still required for the treatment of deep-seated, large tumors. Future studies are needed to evaluate the potential efficacy of the dual mode cellular (e.g. BPA-BNCT) and vascular (e.g. GB-10-BNCT) targeting protocol in a preclinical scenario, employing combinations of ¹⁰B compounds with different properties and complementary uptake mechanisms.     

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) in Finland: Technological and physical prospects after 20 years of experiences

Boron Neutron Capture Therapy (BNCT) is a binary radiotherapy method developed to treat patients with certain malignant tumours. To date, over 300 treatments have been carried out at the Finnish BNCT facility in various on-going and past clinical trials. In this technical review, we discuss our research work in the field of medical physics to form the groundwork for the Finnish BNCT patient treatments, as well as the possibilities to further develop and optimize the method in the future. Accordingly, the following aspects are described: neutron sources, beam dosimetry, treatment planning, boron imaging and determination, and finally the possibilities to detect the efficacy and effects of BNCT on patients.     

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.     

"Sequential" boron neutron capture therapy (BNCT): a novel approach to BNCT for the treatment of oral cancer in the hamster cheek pouch model

In the present study the therapeutic effect and potential toxicity of the novel "Sequential" boron neutron capture therapy (Seq-BNCT) for the treatment of oral cancer was evaluated in the hamster cheek pouch model at the RA-3 Nuclear Reactor. Two groups of animals were treated with "Sequential" BNCT, i.e., BNCT mediated by boronophenylalanine (BPA) followed by BNCT mediated by sodium decahydrodecaborate (GB-10) either 24 h (Seq-24h-BNCT) or 48 h (Seq-48h-BNCT) later. In an additional group of animals, BPA and GB-10 were administered concomitantly [(BPA + GB-10)-BNCT]. The single-application BNCT was to the same total physical tumor dose as the "Sequential" BNCT treatments. At 28 days post-treatment, Seq-24h-BNCT and Seq-48h-BNCT induced, respectively, overall tumor responses of 95 ± 2% and 91 ± 3%, with no statistically significant differences between protocols. Overall response for the single treatment with (BPA + GB-10)-BNCT was 75 ± 5%, significantly lower than for Seq-BNCT. Both Seq-BNCT protocols and (BPA + GB-10)-BNCT induced reversible mucositis in the dose-limiting precancerous tissue around treated tumors, reaching Grade 3/4 mucositis in 47 ± 12% and 60 ± 22% of the animals, respectively. No normal tissue toxicity was associated with tumor response for any of the protocols. "Sequential" BNCT enhanced tumor response without an increase in mucositis in dose-limiting precancerous tissue.     

Noninvasive quantitative in vivo mapping and metabolism of boronophenylalanine (BPA) by nuclear magnetic resonance (NMR) spectroscopy and imaging

10B-enriched L-p-boronophenylalanine (BPA) is one of the compounds used in boron neutron capture therapy (BNCT). In this study, several variations of nuclear magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) were applied to investigate the uptake, clearance and metabolism of the BPA-fructose complex (BPA-F) in normal mouse kidneys, rat oligodendroglioma xenografts, and rat blood. Localized 1H MRS was capable of following the uptake and clearance of BPA-F in mouse kidneys with temporal resolution of a few minutes, while 1H MRSI was used to image the BPA distribution in the kidney with a spatial resolution of 9 mm3. The results also revealed significant dissociation of the BPA-F complex to free BPA. This finding was corroborated by 1H and 11B NMR spectroscopy of rat blood samples as well as of tumor samples excised from mice after i.v. injection of BPA-F. This investigation demonstrates the feasibility of using 1H MRS and MRSI to follow the distribution of BPA in vivo, using NMR techniques specifically designed to optimize BPA detection. The implementation of such procedures could significantly improve the clinical efficacy of BNCT.     

Boron determination in liver tissue by combining quantitative neutron capture radiography (QNCR) and histological analysis for BNCT treatment planning at the TRIGA Mainz

The typical primary malignancies of the liver are hepatocellular carcinoma and cholangiocarcinoma, whereas colorectal liver metastases are the most frequently occurring secondary tumors. In many cases, only palliative treatment is possible. Boron neutron capture therapy (BNCT) represents a technique that potentially destroys tumor tissue selectively by use of externally induced, locally confined secondary particle irradiation. In 2001 and 2003, BNCT was applied to two patients with colorectal liver metastases in Pavia, Italy. To scrutinize the rationale of BNCT, a clinical pilot study on patients with colorectal liver metastases was carried out at the University of Mainz. The distribution of the (10)B carrier (p-borono-phenylalanine) in the liver and its uptake in cancerous and tumor-free tissue were determined, focusing on a potential correlation between the uptake of p-borono-phenylalanine and the biological characteristics of cancerous tissue. Samples were analyzed using quantitative neutron capture radiography of cryosections combined with histological analysis. Methodological aspects of the combination of these techniques and results from four patients enrolled in the study are presented that indicate that the uptake of p-borono-phenylalanine strongly depends on the metabolic activity of cells.     

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.     

Enantioselective synthesis and biological evaluation of 5-o-carboranyl pyrimidine nucleosides

Base-modified carborane-containing nucleosides such as 5-o-carboranyl-2'-deoxyuridine (CDU) when combined with neutrons have potential for the treatment of certain malignancies. Lack of toxicity in various cells, high accumulation in cancer cells and intracellular phosphorylation are desirable characteristics for modified nucleosides used in boron neutron capture therapy (BNCT) for brain tumors and other malignancies. The aim of this work was to synthesize the two beta-enantiomers of several 5-o-carboranyl-containing nucleosides. These derivatives may possess favorable properties such as high lipophilicity, high transportability, the ability to be phosphorylated, and resistance to catabolism. Beta-isomers of 2',3'-dihydroxynucleosides and analogues containing a heteroatom in the sugar moiety were also synthesized. Carboranyl pyrimidine nucleosides were prepared either from the parent beta-D-nucleoside, beta-L-nucleoside, or by a coupling reaction. The dioxolane derivative 7 was prepared by a coupling reaction between protected 5-o-carboranyluracil (8, CU) and the corresponding protected heterocycle. Specific catalysts were used during the N-glycosylation process to favor the formation of the beta-isomer. Biological evaluation of these new chiral 5-o-carboranyl pyrimidine derivatives indicated that most of these compounds have low toxicity in a variety of normal and malignant cells and achieved high cellular levels in a lymphoblastoid cell line. Increasing the number of hydroxyl groups on the sugar moiety decreased the cellular accumulation and serum binding to different extents. Five compounds were identified for further biological evaluation as potential agents for BNCT.     

In vitro studies of DNA damage and repair mechanisms induced by BNCT in a poorly differentiated thyroid carcinoma cell line

Boron neutron capture therapy (BNCT) for aggressive tumors is based on nuclear reaction [¹⁰B (n, α) ⁷Li]. Previously, we demonstrated that BNCT could be applied for the treatment of undifferentiated thyroid carcinoma. The aim of the present study was to describe the DNA damage pattern and the repair pathways that are activated by BNCT in thyroid cells. We analyzed γH2AX foci and the expression of Ku70, Rad51 and Rad54, main effector enzymes of non-homologous end joining (NHEJ) and homologous recombination repair (HRR) pathways, respectively, in thyroid follicular carcinoma cells. The studied groups were: (1) C [no irradiation], (2) gamma [⁶⁰Co source], (3) N [neutron beam alone], (4) BNCT [neutron beam plus 10 µg ¹⁰B/ml of boronphenylalanine (¹⁰BPA)]. The total absorbed dose was always 3 Gy. The results showed that the number of nuclear γH2AX foci was higher in the gamma group than in the N and BNCT groups (30 min–24 h) (p?<?0.001). However, the focus size was significantly larger in BNCT compared to other groups (p?<?0.01). The analysis of repair enzymes showed a significant increase in Rad51 and Rad54 mRNA at 4 and 6 h, respectively; in both N and BNCT groups and the expression of Ku70 did not show significant differences between groups. These findings are consistent with an activation of HRR mechanism in thyroid cells. A melanoma cell line showed different DNA damage pattern and activation of both repair pathways. These results will allow us to evaluate different blocking points, to potentiate the damage induced by BNCT.     

Towards new boron carriers for boron neutron capture therapy: metallacarboranes and their nucleoside conjugates

Thymidine conjugates containing metallacarborane, {8-[5-(N(3)-thymidine)-3-oxa-pentoxy]-3-cobalt bis(1,2-dicarbollide)}- (5) and {8-[5-(O(4)-thymidine)-3-oxa-pentoxy]-3-cobalt bis(1,2-dicarbollide)}- (6) ions and several simple [3-cobalt bis(1,2-dicarbollide)]- ion (1) derivatives have been studied as potential boron carriers for BNCT. Compound 6 and some nonnucleoside derivatives of 1 were not toxic above 100 microM. The partition coefficient for both metallacarborane bearing thymidine conjugates 5 and 6 was more than 500 times higher than that of unmodified nucleoside. The cellular uptake studies showed accumulation of compounds 6 in V79 Chinese hamster cells but not of compound 5. The low toxicity of conjugate type of 6 together with its high partition coefficient suggest that judicially designed derivatives of metallacarboranes can be considered as potential boron carriers for BNCT.