A Novel Boron Lipid to Modify Liposomal Surfaces for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a cancer treatment with clinically demonstrated efficacy using boronophenylalanine (BPA) and sodium mercaptododecaborate (BSH). However, tumor tissue selectivity of BSH and retention of BPA in tumor cells is a constant problem. To ensure boron accumulation and retention in tumor tissues, we designed a novel polyethylene glycol (PEG)-based boron-containing lipid (PBL) and examined the potency of delivery of boron using novel PBL-containing liposomes, facilitated by the enhanced permeability and retention (EPR) effect. PBL was synthesized by the reaction of distearoylphosphoethanolamine and BSH linked by PEG with Michael addition while liposomes modified using PBL were prepared from the mixed lipid at a constant molar ratio. In this manner, novel boron liposomes featuring BSH in the liposomal surfaces, instead of being encapsulated in the inner aqueous phase or incorporated in the lipid bilayer membrane, were prepared. These PBL liposomes also carry additional payload capacity for more boron compounds (or anticancer agents) in their inner aqueous phase. The findings demonstrated that PBL liposomes are promising candidates to effect suitable boron accumulation for BNCT.

Optimization of preparation methods for high loading content and high encapsulation efficiency of BSH into liposomes.

To optimize the preparation methods for liposomes encapsulating mercaptoundecahydrododecaborate (BSH), we examined BSH and lipid concentrations that increased the boron content in liposomes. We improved the BSH encapsulation efficiency and boron content of the liposomes from 4.2 to 45.9 % and 9.5-54.3 µg, respectively, by changing the lipid concentration from 10 to 150 mg/mL. Notably, the boron content increased significantly from 26.2 µg to 326.3 µg at a constant lipid concentration of 30 mg/mL with increased BSH concentrations.

Boron analysis and imaging of cells with 2-hr BPA exposure by using micro-proton particle-induced gamma-ray emission (PIGE).

Particle-induced gamma-ray emission (PIGE), which measures prompt gamma rays at 428 keV from 10B (p, p'γ) 7Be, was used to confirm the boron distribution within 2hr-BPA-exposed cells. Distribution images of potassium, phosphate, and boron and the whole spectrum showed the ratios of boron counts to total (%) as follows: control group: 1.35 ± 0.073%; 2hr boron exposure group: 2.33 ± 0.35%; and boron exposure/wash group: 1.58 ± 0.095%. Micro-beam PIXE/PIGE can be a promising tool for visualization of intracellular Boron.

Difference in BPA uptake between glioma stem-like cells and their cancerous cells.

Tumor sphere-forming (TS) glioma stem cells and cancerous TS cells were analyzed in vivo and in vitro. The boron concentration in murine TS tumors was higher than normal tissue. The boron concentration at 24 h was 0.80 ± 0.09 µg/107 in the TS cells, and 1.08 ± 0.08 µg/107 in the cancerous cells. The LAT-1 amino-acid transporter positive rate was 35.4% in the TS cells and 100% in the cancerous cells. These results suggested the relation between LAT-1 expression and boronophenylalanine concentration in vitro.

Evaluation of a Novel Boron-Containing α-D-Mannopyranoside for BNCT.

Boron neutron capture therapy (BNCT) is a unique anticancer technology that has demonstrated its efficacy in numerous phase I/II clinical trials with boronophenylalanine (BPA) and sodium borocaptate (BSH) used as 10B delivery agents. However, continuous drug administration at high concentrations is needed to maintain sufficient 10B concentration within tumors. To address the issue of 10B accumulation and retention in tumor tissue, we developed MMT1242, a novel boron-containing α-d-mannopyranoside. We evaluated the uptake, intracellular distribution, and retention of MMT1242 in cultured cells and analyzed biodistribution, tumor-to-normal tissue ratio and toxicity in vivo. Fluorescence imaging using nitrobenzoxadiazole (NBD)-labeled MMT1242 and inductively coupled mass spectrometry (ICP-MS) were performed. The effectiveness of BNCT using MMT1242 was assessed in animal irradiation studies at the Kyoto University Research Reactor. MMT1242 showed a high uptake and broad intracellular distribution in vitro, longer tumor retention compared to BSH and BPA, and adequate tumor-to-normal tissue accumulation ratio and low toxicity in vivo. A neutron irradiation study with MMT1242 in a subcutaneous murine tumor model revealed a significant tumor inhibiting effect if injected 24 h before irradiation. We therefore report that 10B-MMT1242 is a candidate for further clinical BNCT studies.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage.

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTß), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Sodium Bicarbonate Facilitates Hemostasis in the Presence of Cerebrospinal Fluid Through Amplification of Platelet Aggregation.

BACKGROUND: Appropriate hemostasis is essential for clear visualization of the neural structures and cleavage planes. It is also essential for avoiding heat-induced injury, minimizing blood loss, and reducing operative time. OBJECTIVE: To determine the role of cerebrospinal fluid (CSF) in platelet-dependent hemostasis during neurosurgery. METHODS: The amplification of aggregation, activation of integrin αIIbß3, intrinsic and extrinsic coagulation pathways, and activation of signaling cascades in platelets were evaluated. For comparison, various concentrations of a commercially available artificial CSF solution (aCSF), an artificial CSF solution prepared by the authors, and normal saline (NS) were used. Differences between aCSF and NS in obtaining in vivo hemostasis were assessed by measuring the tail vein bleeding time in C57BL/6N mice. RESULTS: Platelet aggregation was directly amplified by the addition of aCSF through increased activation of integrin αIIbß3, phosphatidylserine exposure, and P-selectin expression. However, the prothrombin time and activated partial thromboplastin time were not primarily related to coagulation activity with the addition of aCSF. Activation of Src kinase was related to platelet activation by aCSF. The elimination of sodium bicarbonate from aCSF and the addition of the selective inhibitor of the HCO3/Cl exchanger, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt, significantly inhibited platelet aggregation. The bleeding time in aCSF-treated mice was significantly shorter than that in NS-treated mice. CONCLUSION: Sodium bicarbonate facilitates hemostasis through the amplification of platelet aggregation function. The existence of CSF and irrigation with aCSF provide better conditions for physiological hemostasis and they have the potential of improving hemostasis by bipolar coagulation or with irrigation during neuroendoscopic procedures.

Boron analysis for neutron capture therapy using particle-induced gamma-ray emission.

The neutron source of BNCT is currently changing from reactor to accelerator, but peripheral facilities such as a dose-planning system and blood boron analysis have still not been established. To evaluate the potential application of particle-induced gamma-ray emission (PIGE) for boron measurement in clinical boron neutron capture therapy, boronophenylalanine dissolved within a cell culture medium was measured using PIGE. PIGE detected 18 µgB/mL f-BPA in the culture medium, and all measurements of any given sample were taken within 20 min. Two hours of f-BPA exposure was required to create a boron distribution image. However, even though boron remained in the cells, the boron on the cell membrane could not be distinguished from the boron in the cytoplasm.

Intracellular boron accumulation in CHO-K1 cells using amino acid transport control.

BPA used in BNCT has a similar structure to some essential amino acids and is transported into tumor cells by amino acid transport systems. Previous study groups have tried various techniques of loading BPA to increase intracellular boron concentration. CHO-K1 cells demonstrate system L (LAT1) activity and are suitable for specifying the transport system of a neutral amino acid. In this study, we examined the intracellular accumulation of boron in CHO-K1 cells by amino acid transport control, which involves co-loading with L-type amino acid esters. Intracellular boron accumulation in CHO-K1 cells showed the greatest increased upon co-loading 1.0mM BPA, with 1.0mM l-Tyr-O-Et and incubating for 60min. This increase is caused by activation of a system L amino acid exchanger between BPA and l-Tyr. The amino acid esters are metabolized to amino acids by intracellular hydrolytic enzymes that increase the concentrations of intracellular amino acids and stimulate exchange transportation. We expect that this amino acid transport control will be useful for enhancing intracellular boron accumulation.

The optimization of fluorescence imaging of brain tumor tissue differentiated from brain edema--in vivo kinetic study of 5-aminolevulinic acid and talaporfin sodium.

OBJECTIVE: We aimed to clarify the optimal timing for the fluorescence imaging of brain tumor tissue differentiated from brain edema after the administration of photosensitizers. METHODS: We have performed an in vivo study of the kinetics of 5-aminolevulinic acid (5-ALA) in comparison with talaporfin sodium using the rat brain tumor model and rat vasogenic edema model produced by cold injury. The in vivo kinetics of 5-ALA and talaporfin sodium in brain tumor model and the vasogenic edema model was determined by a fluorescence macroscope and a microplate reader. RESULTS: The in vivo kinetic study of 5-ALA showed mild fluorescence intensity of protoporphyrin IX (PpIX) in brain tumor differentiated from vasogenic edema. The mean lesion-to-normal-brain ratio (L/N ratio) in the group of brain tumor model 2h after the administration of 5-ALA was 7.78+/-4.61, which was significantly higher (P<0.01) than that of the vasogenic edema 2h after the administration of 5-ALA (2.75+/-1.12). In vivo kinetic study of talaporfin sodium showed high fluorescence intensity and retention in brain tumor differentiated from vasogenic edema. The mean L/N ratio of the fluorescence intensity in the group of brain tumor model 12h after the administration of talaporfin sodium was 23.1+/-11.9, which was significantly higher (P<0.01) than that of the vasogenic edema 12h after the administration (8.93+/-8.03). CONCLUSIONS: The optimization of fluorescence imaging of brain tumors differentiated from brain edema is possible in the case of 5-ALA within 6h, and also possible in the case of talaporfin sodium beyond 12h.

Combined use of sodium borocaptate and buthionine sulfoximine in boron neutron capture therapy enhanced tissue boron uptake and delayed tumor growth in a rat subcutaneous tumor model.

We have previously reported that buthionine sulfoximine (BSO) enhances sodium borocaptate (BSH) uptake by down regulating glutathione (GSH) synthesis in cultured cells. This study investigated the influence of BSO on tissue BSH uptake in vivo and the efficacy of BSH-BSO-mediated boron neutron capture therapy (BNCT) on tumor growth using a Fisher-344 rat subcutaneous tumor model. With BSO supplementation, boron uptake in subcutaneous tumor, blood, skin, muscle, liver, and kidney was significantly enhanced and maintained for 12h. Tumor growth was significantly delayed by using BSO. With further improvement in experimental conditions, radiation exposure time, together with radiation damage to normal tissues, could be reduced.

Simple new method for making a rat carotid artery post-angioplasty stenosis model.

A simple new method for making a rat post-angioplasty stenosis model was developed using a single-lumen compliant balloon catheter/guidewire system and no special diet. This technique was applied to 10 9-week-old Wistar rats fed a normal diet. The catheter/guidewire system was inserted from the external carotid artery and advanced retrogradely into the common carotid artery. The balloon was inflated six times with rotation through 60 degrees . After angioplasty, the external carotid artery was ligated. Homogeneous stenoses due to intimal hyperplasia were demonstrated in cross sections of all cases. This simple and gentle method allows an easily reproducible post-angioplasty stenosis model.

Enhancement of sodium borocaptate (BSH) uptake by tumor cells induced by glutathione depletion and its radiobiological effect.

Sodium borocaptate (BSH) is widely used for boron neutron capture therapy (BNCT) of brain tumors. However, the mechanism of uptake by the tumor remains unclear. We investigated the sulfhydryl moiety of this compound. Down regulation of glutathione (GSH) by buthionine sulfoximine in cultured cells resulted in increase of BSH uptake (7.9-36.5%) compared to the control group and consequently the cytocidal effect of neutron irradiation also increased. On the other hand, the radiation caused damage by gamma-ray irradiation was suppressed when BSH uptake increased. These findings suggested that modulation of GSH enhanced the effect of B (n, alpha) reaction and the protective effect of secondary gamma-ray in BNCT.

In vivo gadolinium neutron capture therapy using a potentially effective compound (Gd-BOPTA).

BACKGROUND: In a previous study, we found that Gadobenate dimeglumine (Gd-BOPTA) resulted in a significantly greater Gd uptake by brain tumor tissue than Gadopentate dimeglumine (Gd-DTPA). Therefore, we investigated whether Gd-BOPTA is an efficient agent for neutron capture therapy (NCT). MATERIALS AND METHODS: Four groups of Fisher344 rats (control, neutron (n), n+ Gd-DTPA, n+ Gd-BOPTA) were subcutaneously injected 9L gliosarcoma cells in both hind legs. Gd-BOPTA and Gd-DTPA (0.05 mmol/g tumor weight) were injected directly into the tumor. At the peak of Gd uptake, thermal neutron irradiation was applied. RESULTS: Two Gd+ groups showed pronounced tumor growth delay as compared with the control and neutron groups (p = 0.0053, 0.0064, respectively). Furthermore, the BOPTA group showed significantly prolonged delay of tumor growth as compared to the DTPA group (p = 0.033). CONCLUSION: This is the first report of Gd-NCT to demonstrate that Gd-BOPTA serve as an effective compound for NCT. Better cytocidal effects of Gd-BOPTA warrant further investigation of subcellular Gd distribution.

Comparison of gadobenate dimeglumine and gadopentetate dimeglumine: a study of MR imaging and inductively coupled plasma atomic emission spectroscopy in rat brain tumors.

BACKGROUND AND PURPOSE: After the advent of extracellular contrast media, hepatobiliary-specific gadolinium chelates were developed to improve the diagnostic value of MR imaging of the liver. Gadobenate dimeglumine (Gd-BOPTA) is a new paramagnetic contrast agent with partial biliary excretion that produces prolonged enhancement of liver parenchyma on T1-weighted images. However, whether Gd-BOPTA is useful as a contrast agent in central nervous system disease, particularly in brain tumors, is unclear. METHODS: The behavior of Gd-BOPTA as a brain tumor-selective contrast agent was compared with that of gadopentetate dimeglumine (Gd-DTPA), an MR contrast agent used in central nervous system disease, in a common dose of 0.1 mmol/kg. An MR imaging study of these two contrast agents was performed, and tissue concentrations were measured with inductively coupled plasma atomic emission spectroscopy (ICP-AES). RESULTS: Gd-BOPTA showed better MR imaging enhancement in brain tumors than did Gd-DTPA at every time course until 2 hours after administration and no enhancement in peritumoral tissue and normal brain. Corresponding results with ICP-AES showed significantly greater uptake of Gd-BOPTA in tumor samples than that in peritumoral tissue and normal brain 5 minutes after administration. Gadolinium was retained for a longer time in brain tumors when Gd-BOPTA rather than Gd-DTPA was administered. CONCLUSION: Gd-BOPTA is a useful contrast agent for MR imaging in brain tumors and possibly an effective absorption agent for neutron capture therapy.