Altered modified boron carbide particles and neutron capture therapy agent comprising same

Abstract

The present invention provides a neutron capture therapy agent with a superior therapeutic effect.　The present invention provides modified boron carbide nanoparticle powder with a suitable particle diameter for polyglycerol-functionalized boron carbide, capable of producing a boron neutron capture therapy (BNCT) agent that exhibits high accumulation efficiency in tumors and excellent therapeutic efficacy, with the surface of the boron carbide nanoparticles not coated with graphite.

Description

Modified boron carbide particles and neutron capture therapy agents containing same - Patents.com

[0001] This patent application claims priority under the Paris Convention and the benefit of Japanese Patent Application No. 2024-105335 (filed June 28, 2024) and Japanese Patent Application No. 2025-059382 (filed March 31, 2025), and priority under Article 41 of the Japanese Patent Act, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to modified boron carbide particle powders and neutron capture therapy drugs containing the same, and more particularly to specifically modified boron carbide particle powders in which at least a portion of the surface of the boron carbide particles is modified with polyglycerol, and neutron capture therapy drugs containing the same.

[0003] Boron Neutron Capture Therapy (BNCT) is a therapy that combines neutrons with a stable isotope of boron ( １０ BNCT utilizes neutrons (B) and the particle beam generated by their nuclear reactions, enabling non-invasive cancer treatment with minimal side effects (Non-Patent Document 1). With the recent spread of hospital-installed neutron beam generators, insurance coverage began in May 2020, and BNCT is becoming a more familiar cancer treatment method (Non-Patent Documents 2 and 3).

[0004] Two types of agents have been used for BNCT to date: BSH (mercaptoundecahydrododecaborate), a cage-like cluster of boron, and BPA (p-boronophenylalanine), in which boron has been introduced into phenylalanine (Non-Patent Documents 4 and 5).

[0005] BSH has the problem of low tumor selectivity, while BPA has the problem of low water solubility. Because of BPA's low water solubility, tens of grams of BPA are currently administered by intravenous infusion over several hours. Furthermore, BPA is known to be taken up by cancer cells via LAT1 (L-amino acid transporter 1), which limits the types of cancers to which it can be applied. Therefore, for the future development of BNCT, there is a strong demand for the development of new boron formulations designed from an entirely new perspective.

[0006] In order to improve the therapeutic efficiency of BNCT, it is necessary to provide a high concentration of boron in tumor cells. However, when boron is oxidized, it becomes boric acid, which becomes toxic. Therefore, when used as a boron preparation for BNCT, it is preferable that it is chemically stable. In this context, the use of boron carbide nanoparticles in BNCT has been proposed. For example, Patent Document 1 discloses the following: １０ Disclosed is boron carbide nanoparticles containing 10 atomic % or more of boron in order to significantly increase the number of B atoms, the nanoparticles having an average particle size of 1 to 500 nm, an oxygen content of 5 mol % or less, and a coating layer of graphite into which carboxy groups have been introduced on the surface of the nanoparticles.

[0007] The present inventors discovered that modified boron carbide particles, in which at least a portion of the surface of the boron carbide particles is modified with polyglycerol by reacting the boron carbide particles with glycidol, are suitable for use in boron neutron capture therapy because they are essentially less toxic, can be administered in a shorter time, can accumulate boron at higher concentrations within tumors, and can persist in tumors for a long period of time while being rapidly removed from the blood, and have therefore filed a patent application (Patent Document 2).

[0008] Japanese Patent No. 5168690 Japanese Patent Application Laid-Open No. 2023-018447

[0009] MA Dymova, SY Taskaev, VA Richter, EV Kuligina, Cancer Commun. 2020, 40, 406.M. Suzuki, Int. J. Clin. Oncol. 2020, 25, 43.RF Barth, JC Grecula, Appl. Radiat. Isot. 2020, 160, 109029.Haritz,D., et al., Int. J. Radiat. Oncol. Biol. Phys., 28 1175-1181(1994).Ryynanen, PM, et al., Int. J. Radiat. Oncol. Biol. Phys., 48,1145-1154(2000).Y. Ishikawa, Q. Feng, N. Koshizaki, Appl. Phys. A 2010, 99, 797.T. Kobayashi, K. Kanda, Nucl. Instrum. Methods 1983, 204, 525.

[0010] The present inventors have conducted extensive research to create a boron neutron capture therapy therapeutic agent with superior therapeutic effects.

[0011] As a result, the inventors have developed polyglycerolized boron carbide (B ４ By focusing on the particle size of polyglycerolized boron carbide (C-PG) particles and controlling the particle size appropriately, the present inventors discovered a suitable particle size of polyglycerolized boron carbide that can lead to a boron neutron capture therapy therapeutic agent with good pharmacokinetics, high tumor accumulation efficiency due to the EPR (Enhanced Permeation and Retention Effect), and excellent therapeutic effects, and completed the present invention.

[0012] This specification includes the following aspects. <Modified boron carbide particle powder> [1] A powder of modified boron carbide nanoparticles, in which a portion of the surface of the boron carbide particles is modified with polyglycerol, wherein the surface of the boron carbide nanoparticles is not coated with graphite. [2] The modified boron carbide nanoparticle powder according to [1], in which, in a particle size distribution based on the number of the modified boron carbide nanoparticle powder measured in pure water by dynamic light scattering, the proportion of particles having a particle diameter of 43 to 61 nm is 35% or more relative to the total nanoparticle powder. [3] The modified boron carbide nanoparticle powder according to [1] or [2], in which the proportion of particles having a particle diameter of 43 to 61 nm is 45% or more relative to the total nanoparticle powder. [4] The modified boron carbide nanoparticle powder according to any of [1] to [3], in which the zeta potential in PBS (phosphate buffered saline) is -45 to -30 mV. [5] The modified boron carbide nanoparticle powder according to any one of [1] to [4], wherein the polyglycerol content is 15 to 60% by weight, preferably 20 to 55% by weight, and more preferably 30 to 55% by weight, based on the modified boron carbide nanoparticles (100% by weight). [6] The modified boron carbide nanoparticle powder according to any one of [1] to [5], wherein the number-based median diameter of the modified boron carbide nanoparticle powder is 30 to 66 nm. [7] The modified boron carbide nanoparticle powder according to [6], wherein the number-based median diameter of the modified boron carbide nanoparticle powder measured in pure water by dynamic light scattering is 40 to 66 nm. [8] The modified boron carbide nanoparticle powder according to [7], wherein the number-based median diameter of the modified boron carbide nanoparticle powder measured in pure water by dynamic light scattering is 45 to 55 nm. [9] The modified boron carbide nanoparticle powder according to any one of [1] to [8], wherein the number-based median diameter of the boron carbide nanoparticles measured in pure water by dynamic light scattering is 30 to 55 nm.

[10] The modified boron carbide nanoparticle powder according to any one of [1] to [9], wherein the boron carbide nanoparticles have a non-spherical shape.

[0013] <Drug for neutron capture therapy>

[11] A drug for neutron capture therapy comprising the modified boron carbide nanoparticle powder according to any one of [1] to

[10] .

[12] A drug for neutron capture therapy according to

[11] , which has an antitumor effect.

[13] A drug for neutron capture therapy according to

[11] or

[12] , which has high tumor retention.

[14] A drug for neutron capture therapy according to any one of

[11] to

[13] , which exerts an antitumor effect by injection.

[15] A drug for neutron capture therapy according to any one of

[11] to

[14] , which is administered by injection as a one-shot intravenous injection or a bolus intravenous injection.

[16] A drug for neutron capture therapy according to any one of

[11] to

[15] , which has tumor retention such that a boron concentration of 60 ppm or more can be maintained for three days after a single intravenous injection.

[17] The neutron capture therapy agent according to any one of

[11] to

[16] , which exhibits high tumor retention by a single one-shot intravenous injection or bolus intravenous injection and allows repeated irradiation with neutron beams.

[18] The neutron capture therapy agent according to any one of

[11] to

[17] , which induces an immunostimulating effect (cancer immunity) by neutron beam irradiation.

[19] The neutron capture therapy agent according to

[18] , which exhibits an additional antitumor effect due to the immunostimulating effect induced by neutron beam irradiation.

[20] The neutron capture therapy agent according to

[19] , which exhibits an abscopal effect due to the immunostimulating effect induced by neutron beam irradiation.

[21] The neutron capture therapy agent according to any one of

[11] to

[20] , which is used in combination with an immune checkpoint inhibitor.

[0014] <Neutron Capture Therapy> [11-2] Neutron capture therapy, comprising administering the modified boron carbide nanoparticle powder according to any one of [1] to

[10] to a cancer patient in need thereof. [12-2] Neutron capture therapy according to [11-2], in which the modified boron carbide nanoparticle powder according to any one of [1] to

[10] , which has an antitumor effect, is administered. [13-2] Neutron capture therapy according to [11-2] or [12-2], in which the modified boron carbide nanoparticle powder according to any one of [1] to

[10] , which has high tumor retention, is administered. [14-2] Neutron capture therapy according to any one of [11-2] to [13-2], which exerts an antitumor effect by injection. [15-2] Neutron capture therapy according to any one of [11-2] to [14-2], in which the injection is a one-shot intravenous injection or a bolus intravenous injection. [16-2] Neutron capture therapy according to any one of [11-2] to [15-2], which has a tumor retention of 60 ppm or more of boron for three days after a single intravenous injection. [17-2] Neutron capture therapy according to any one of [11-2] to [16-2], which allows repeated irradiation of neutron beams after a single one-shot intravenous injection or bolus intravenous injection. [18-2] Neutron capture therapy according to any one of [11-2] to [17-2], which induces an immunostimulatory effect by neutron beam irradiation. [19-2] Neutron capture therapy according to [18-2], which exerts an additional antitumor effect due to the immunostimulatory effect. [20-2] Neutron capture therapy according to [19-2], which exerts an abscopal effect due to the immunostimulatory effect. [21-2] The neutron capture therapy according to any one of [11-2] to [20-2], used in combination with an immune checkpoint inhibitor.

[0015] <Modified boron carbide nanoparticle powder for neutron capture therapy> [11-3] The modified boron carbide nanoparticle powder according to any one of [1] to

[10] , for use in neutron capture therapy. [12-3] The modified boron carbide nanoparticle powder according to [11-3], which exerts an antitumor effect. [13-3] The modified boron carbide nanoparticle powder according to [11-3] or [12-3], which has high tumor retention. [14-3] The modified boron carbide nanoparticle powder according to any one of [11-3] to [13-3], which exerts an antitumor effect by injection. [15-3] The modified boron carbide nanoparticle powder according to any one of [11-3] to [14-3], which is administered by injection as a one-shot intravenous injection or a bolus intravenous injection. [16-3] The modified boron carbide nanoparticle powder according to any one of [11-3] to [15-3], which has a tumor retention of 60 ppm or more of boron concentration for 3 days after a single intravenous injection. [17-3] The modified boron carbide nanoparticle powder according to any one of [11-3] to [16-3], which can be repeatedly irradiated with neutron beams after a single one-shot intravenous injection or bolus intravenous injection. [18-3] The modified boron carbide nanoparticle powder according to any one of [11-3] to [17-3], which induces an immunostimulatory effect by neutron beam irradiation. [19-3] The modified boron carbide nanoparticle powder according to [18-3], which exerts an additional antitumor effect due to the immunostimulatory effect. [20-3] The modified boron carbide nanoparticle powder according to [19-3], which exerts an abscopal effect due to the immunostimulatory effect. [21-3] The modified boron carbide nanoparticle powder according to any one of [11-3] to [20-3], which is used in combination with an immune checkpoint inhibitor.

[0016] <Drug that induces immunostimulatory effect>

[21] A drug that induces immunostimulatory effect in a subject who has been irradiated with neutron beams, the drug containing as an active ingredient a drug for neutron capture therapy according to any one of

[11] to

[20] .

[0017] <Method for inducing immunostimulatory effect> [21-2] A method for inducing immunostimulatory effect, comprising administering a neutron capture therapy drug described in any of

[11] to

[20] to a cancer patient in need thereof, and irradiating the patient with neutron beams.

[0018] <Modified boron carbide nanoparticle powder for inducing immunopotentiating effect> [21-3] The pharmaceutical agent for neutron capture therapy according to any one of

[11] to

[20] , for inducing immunopotentiating effect.

[0019] <Production method>

[22] A method for producing the modified boron carbide nanoparticle powder according to any one of [1] to

[10] , comprising: (1) converting boron oxide together with magnesium and graphite into boron carbide nanoparticles by a mechanochemical reaction; and (2) reacting the obtained nano-sized boron carbide nanoparticles with glycidol to produce modified boron carbide nanoparticle powder having surfaces modified with polyglycerol.

[23] The production method according to

[22] , wherein the mechanochemical reaction is a grinding treatment using a ball mill.

[0020] The modified boron carbide particle powder according to an embodiment of the present invention comprises boron carbide particles, at least some of which have at least a portion of their surfaces modified with polyglycerol. Therefore, the modified boron carbide particle powder according to an embodiment of the present invention exhibits inherently low toxicity, shorter administration time (higher water solubility or water dispersibility), higher boron accumulation within tumors, and long-term persistence within tumors while rapid removal from the blood, making it suitable for use in boron neutron capture therapy.

[0021] Figure 1 shows a transmission electron microscope (TEM) image of spherical boron carbide (B4C) submicron particles with a graphite layer, as shown in FIG. 3 of Y. Ishikawa, Y. Shimizu, T. Sasaki, and N. Koshizaki, Appl. Phys. Lett. 91, 161110 (2007). (a) TEM image of the particle obtained by laser irradiation in ethyl acetate, (b) electron diffraction pattern (graphite) of a selected area in image (a), and (c) high-resolution TEM image of the particle surface (graphite layer). Figure 2 shows the particle produced by the method reported in Y. Ishikawa, Y. Shimizu, T. Sasaki, and N. Koshizaki, Appl. Phys. Lett. 91, 161110 (2007). 10 3 is a graph showing the powder X-ray diffraction (XRD) profile of BC nanoparticles. This XRD profile is shown in FIG. S2a of Y. Wang, G. Reina, HG Kang, X. Chen, Y. Zou, Y. Ishikawa, M. Suzuki, N. Komatsu, Small, 18, 2204044 (2022). (Intensity: Intensity, Degree: °, Graphite: Graphite) FIG. 3 shows the powder X-ray diffraction (XRD) profile of BC nanoparticles (top: 10 4 is a graph showing the results of Fourier transform infrared spectroscopy (FTIR) of boron carbide nanoparticles (B4C(50), bottom: B4C(50)) with a median diameter of 50 nm produced in Example 1-1. 10 5 is a graph showing the powder X-ray diffraction (XRD) profile of modified boron carbide nanoparticles (B4C(50)) prepared in Example 1-2. 10 B4C-PG ( 10 6 is a graph showing the results of Fourier transform infrared spectroscopy (FTIR) of boron carbide nanoparticles (B4C(50)-PG) with a median diameter of 50 nm produced in Example 1-1. 107 is a graph showing the particle size distribution based on the number standard measured by dynamic light scattering (DLS) for modified boron carbide nanoparticles (B4C(50)) in pure water. 10 B4C-PG ( 10 8 is a graph showing the particle size distribution based on the number standard measured by DLS in pure water for modified boron carbide nanoparticles (BC(50)-PG). 10 B4C-PG ( 10 9 is a graph showing the results of thermogravimetric analysis (TGA) of boron carbide nanoparticles (B4C(50)-PG) with a median diameter of 50 nm produced in Example 1-1. 10 B4C(50)) and modified boron carbide nanoparticles prepared in Example 1-2 10 B4C-PG ( 10 10 shows the results of measuring the zeta potential of boron carbide nanoparticles (BC(50)-PG) in phosphate buffered saline (PBS). 11 shows the results of measuring the zeta potential of boron carbide nanoparticles (BC(50)-PG) in PBS (phosphate buffered saline). 12 shows the results of measuring the zeta potential of boron carbide nanoparticles (BC(50)-PG) in PBS (phosphate buffered saline). 13 shows the results of measuring the zeta potential of boron carbide nanoparticles (BC(50)-PG) in PBS (phosphate buffered saline). 14 shows the results of measuring the zeta potential of boron carbide nanoparticles (BC(50)-PG) in PBS (phosphate buffered saline). 15 shows the results of measuring the zeta potential of boron carbide nanoparticles (BC(50)-PG) in PBS (phosphate buffered saline). 16 10 B4C(35), b) 10 B4C(50), c) 10 B4C(80), and d) 10 Scanning electron microscope (SEM) photographs of B4C(110). The scale bar is 1.00 μm. Figure 11 shows the results of the CT scan of various sizes using tumor-bearing mice. 10 B4C-PG ( 10 B4C(35)-PG, 10 B4C(50)-PG, 10 B4C(80)-PG, and 10 Figure 12 shows a schematic diagram of the experimental procedure for evaluating the efficacy of various sizes of PGs using tumor-bearing mice. 10 B4C-PG ( 10 B4C(35)-PG, 10 B4C(50)-PG, 10 B4C(80)-PG, and 10This is a graph showing the time course of the relative tumor volume in the efficacy evaluation using B4C(110)-PG. PBS and N represent the administration of PBS (phosphate buffered saline) and neutron irradiation, respectively. In the figure, the black and white stars represent the 10 B4C(50)-PG + N, and 10 The results for B4C(35)-PG + N are shown in Figure 13. 10 B4C-PG ( 10 B4C(35)-PG, 10 B4C(50)-PG, 10 B4C(62)-PG, 10 B4C(80)-PG, and 10 11 is a graph showing the correlation between the proportion of particle sizes of 43 nm to 61 nm in B4C(110)-PG (right vertical axis, Table 11) and the proportion of mice that achieved complete remission in Test Example 1 (FIG. 12) (remission rate (%), left vertical axis). 10 The remission rate of B4C(62)-PG is based on the experimental results of Patent Document 2 and a non-patent paper (Y. Wang, G. Reina, HG Kang, X. Chen, Y. Zou, Y. Ishikawa, M. Suzuki, N. Komatsu, Small, 18, 2204044 (2022)). 10 The "BC-PG core size" refers to the core size of the unmodified boron carbide nanoparticles. 10 The particle size of B4C. 10 B4C-PG ( 10 B4C(35)-PG, 10 B4C(50)-PG, 10 B4C(62)-PG, 10 B4C(62)-PG, 10 B4C(80)-PG, and 10 15 is a graph showing the zeta potential of BC(110)-PG in pure water. 10 B4C-PG ( 10 B4C(35)-PG, 10 B4C(50)-PG, 10 B4C(62)-PG, 10 B4C(80)-PG, and 1014 is a graph showing the correlation between the zeta potential of B4C(110)-PG in pure water (right vertical axis, FIG. 14) and the percentage of mice that achieved complete remission (remission rate (%), left vertical axis) in Test Example 1 (FIG. 12). 10 The remission rate of B4C(62)-PG is based on the experimental results of Patent Document 2 and a non-patent document (Y. Wang, G. Reina, H.G. Kang, X. Chen, Y. Zou, Y. Ishikawa, M. Suzuki, N. Komatsu, Small, 18, 2204044 (2022)). Figure 16 is a schematic diagram of the experimental procedure for tumor cell reimplantation into cured mice. BALB / c represents the mouse strain used in the experiment, and CT26, 4T1, and Meth-A represent mouse colon cancer, breast cancer, and fibrosarcoma cells, respectively. Figure 17 is a graph comparing the time course of tumor volume in cured mice with that in untreated mice after reimplantation of CT26 tumor cells. Figure 18 shows the results of immunohistochemical staining of fixed spleens and lymph nodes of untreated (naive) and cured mice at 155 days (see Figure 16). 10 Figure 20 shows a schematic diagram of the experimental procedure for evaluating the efficacy of B4C(50)-PG in combination with immune checkpoint inhibitors (anti-PD-1 antibody, α-PD-1). 10 Figure 21 is a schematic diagram showing the experimental procedure for evaluating the efficacy of B4C(50)-PG in combination with an immune checkpoint inhibitor (anti-PD-1 antibody, α-PD-1) over time. PBS and N represent administration of PBS (phosphate-buffered saline) and neutron irradiation, respectively. Figure 21 is a schematic diagram showing the experimental procedure for evaluating the efficacy of B4C(50)-PG in combination with an immune checkpoint inhibitor (anti-PD-1 antibody, α-PD-1) over time. Figure 22 is a graph showing the time course of tumor size in tumor-bearing mice engrafted with 4T1, Meth-A, B16-F10, or LLC tumor cells. a) 4T1 tumor cells, b) Meth-A tumor cells, c) B16-F10 tumor cells, d) LLC tumor cells. Figure 23 shows the results of the treatment of CT26 tumor-bearing mice.10 Figure 24 is a schematic diagram showing the experimental procedure for evaluating the efficacy of B4C(50)-PG after two neutron beam irradiations following the introduction of half the dose of B4C(50)-PG used in Test Example 1. Figure 24 is a graph showing a) the change in tumor size over time and b) the change in survival rate over time after two neutron beam irradiations in CT26 tumor-bearing mice. Figure 25 is a schematic diagram showing the experimental procedure for evaluating the presence or absence of an abscopal effect in tumor-bearing mice with 4T1 tumor cells engrafted in the right foot and back. Figure 26 is a graph showing the change in tumor size over time in a) the right foot and b) the back after neutron beam irradiation only in the right foot of mice (n=3) implanted with 4T1 cells in both the right foot and back. Figure 27 is a graph showing the results of immunostaining tumor tissue sections from mice (Figure 25) after 21 days, comparing the expression levels of CD8+ T cells and CD4+ T cells in the right foot and back. a) Expression levels of CD8+ T cells in the right leg, b) CD8+ T cells in the back, c) CD4+ T cells in the right leg, and d) CD4+ T cells in the back. The horizontal axes of a) to d) represent, from the left, PBS, 10 B4C-PG, N and 10 BC-PG + N. Figure 28 shows 10 Figure 29 is a schematic diagram of the experimental procedure for evaluating cancer treatment using a combination of B4C-PG-BNCT and an anti-PD-1 antibody. 10 Graphs showing the changes over time in a) tumor size in CT26 tumor-bearing mice, b) survival rate of CT26 tumor-bearing mice, c) tumor size in 4T1 tumor-bearing mice, and d) tumor size in B16-F10 tumor-bearing mice during cancer treatment using a combination of B4C-PG-BNCT and an anti-PD-1 antibody.

[0022] <Modified boron carbide particle powder> In one embodiment, the present specification provides a powder of modified boron carbide nanoparticles in which a portion of the surface of the boron carbide particles is modified with polyglycerol, and the surface of the boron carbide nanoparticles is not coated with graphite. In this specification, the term "particle powder" refers to an aggregate of particles (powder or multiple particles) and may also be simply referred to as "particles." In this specification, "boron carbide particles" refers to an inorganic material composed of carbon and boron, and the composition formula thereof is generally "B４ The ratio of boron to carbon is described as "C" and is not particularly limited as long as the modified boron carbide particle powder of the present invention can be obtained. In other words, the ratio of boron to carbon may not be strictly 4:1.

[0023] The boron in boron carbide is mainly １０ B and １１ The boron in the particles, which consist of B and are derived from natural boron carbide, is １１ B is approximately 80.1%, １０ As long as the modified boron carbide particle powder and the neutron capture therapy drug containing the same that are the object of the present invention can be obtained, natural boron carbide can be used as the boron carbide, and the amount of boron in the boron carbide can be adjusted. １０ The B content may be, for example, about 19.9%. １０ The content of B is preferably 50% or more, more preferably 75% or more, even more preferably 90% or more, and even more preferably 98% or more. １０ When the B content is 50% or more, when used for boron neutron capture therapy, １０ Since the content of B is higher, the neutron capture ability is superior, and boron neutron capture therapy can be performed more efficiently.

[0024] In the present invention, the boron carbide particles have a portion of their surface modified with polyglycerol. In this specification, "polyglycerol" refers to a substance having a large number of "glycerol (or glycerin)" units and a large number of hydroxyl groups, and is not particularly limited as long as the modified boron carbide particle powder intended by the present invention can be obtained. Because the boron carbide particles have a portion of their surface modified with polyglycerol and are composed only of main group elements, they are inherently low-toxic, more water-soluble or water-dispersible, can accumulate boron at a higher concentration in tumors, can persist in tumors for a long time, yet can be rapidly removed from the blood, and are therefore suitable for use in boron neutron capture therapy.

[0025] In the present invention, the term "modified" in "a powder of modified boron carbide nanoparticles in which a portion of the surface of the boron carbide particles is modified with polyglycerol" refers to a form in which polyglycerol is covalently bonded (molecularly crosslinked) to hydroxyl or carboxyl groups present on the surface of the boron carbide particles. For example, polyglycerol may be synthesized in advance, and a desired molecular weight fraction may be separated and purified, followed by covalent bonding to hydroxyl or carboxyl groups on the surface of the boron carbide particles by a chemical reaction. Alternatively, polyglycerol may be covalently bonded (molecularly crosslinked) to the boron carbide nanoparticles by ring-opening polymerization of glycidol.

[0026] An example of the "glycerol (or glycerin)" unit is the chemical structure shown in the following chemical formula (I).

[0027] Chemical formula 1: [-CH ２ -CH(CH ２ OH)-O-] [-O-CH ２ -CH(CH ２ OH)-], and [-CH ２ -CH(OH)-CH ２ -O-]

[0028] Polyglycerol may contain other units, such as, but not necessarily, ethyleneoxy, propyleneoxy, etc., as long as the modified boron carbide particle powder of the present invention is obtained. Polyglycerol may also contain other substances, such as, but not necessarily, hydrophilic substances, such as, but not necessarily, polyethylene glycol, polypropylene glycol, and polyethylene glycol / polypropylene glycol copolymers, as long as the modified boron carbide particle powder of the present invention is obtained.

[0029] The boron carbide particles described in Patent Document 1 (Japanese Patent No. 5168690) are characterized in that their surfaces are provided with a coating layer of graphite into which carboxy groups have been introduced (see the examples of the patent, etc.). Furthermore, the boron carbide particles described in Patent Document 2 (Japanese Patent Laid-Open No. 2023-018447) also have a coating layer of graphite. In Example 1 described in paragraph

[0041] of Patent Document 2, it is stated that "a graphite coating layer is formed on the surface of the boron carbide particles in ethyl acetate." １０ From B, using pulsed laser melting method, １０ B ４ C is synthesized, and １０ B ４ By centrifuging C. １０ B ４ C nanoparticles were separated and boron carbide ( １０ B ４C) nanoparticles were obtained (see Non-Patent Document 6). The cited Non-Patent Document 6 is Y. Ishikawa, Q. Feng, and N. Koshizaki, Appl. Phys. A 2010, 99, 797, which states in its introduction, "We previously reported the preparation of spherical boron carbide (B4C) submicron particles by irradiating boron nanoparticles (50-100 nm) dispersed in ethyl acetate with a relatively weak laser beam and utilizing the reaction between the boron particles and the carbon component in the ethyl acetate

[0011] ." The cited Reference 11 is Y. Ishikawa, Y. Shimizu, T. Sasaki, and N. Koshizaki, Appl. Phys. Lett. 91, 161110 (2007). Figure 3 of this document clearly shows the presence of graphite layers in the particles obtained (Figure 1). The XRD profile of the boron carbide particles described in Patent Document 2 (JP 2023-018447 A) shown in Figure 2 is also shown in FIG. S2 of Y. Wang, G. Reina, H.G. Kang, X. Chen, Y. Zou, Y. Ishikawa, M. Suzuki, N. Komatsu, Small, 18, 2204044 (2022), in which the peak at 26.4° indicates the presence of a graphite layer. Therefore, it is clear that the modified boron carbide particles described in Patent Document 2 (JP 2023-018447 A), at least a portion of whose surface is modified with polyglycerol, also have a graphite coating layer.

[0030] In one embodiment, the present invention relates to modified boron carbide nanoparticles of the present invention, in which the proportion of particles having a particle diameter of 43 to 61 nm is 35% or more, preferably 45% or more, of the total nanoparticle powder in a particle size distribution based on the number of particles of the modified boron carbide nanoparticles measured in pure water by dynamic light scattering (DLS) (see Figure 13).

[0031] In general, particle size has a significant effect on powder performance, and in the field of nanomedicine in particular, it is known to affect the accumulation efficiency in tumors as an EPR effect.

[0032] In addition, Patent Document 2 describes that the particle diameter of the polyglycerol-modified boron carbide particles is 10 to 100 nm.

[0033] In another embodiment, the present invention relates to modified boron carbide nanoparticles of the present invention, which have a zeta potential in pure water of -45 to -30 mV, preferably -40 to -35 mV, and more preferably -39 to -33 mV (see Figure 14). Test examples have shown that a zeta potential of -42 to -30 mV or -39 to -33 mV improves the stability of the modified boron carbide nanoparticles of the present invention in the blood and enhances their accumulation in tumors. As a result, it has been concluded that a stronger anticancer effect can be expected from boron neutron capture therapy (see Figure 15).

[0034] In yet another embodiment, the present invention relates to the modified boron carbide nanoparticle powder of the present invention, in which the polyglycerol content is 15 to 60% by weight, based on the modified boron carbide nanoparticles (100% by weight) (see Table 8). The modified boron carbide nanoparticle powder of the present invention preferably has a polyglycerol content of 15 to 55% by weight, more preferably 20 to 55% by weight, and even more preferably 30 to 55% by weight.

[0035] In addition, the polyglycerol content of the polyglycerol-modified boron carbide particles in Patent Document 2 is described as 59 to 77 mass % in the examples.

[0036] In yet another embodiment, the present invention relates to modified boron carbide nanoparticles of the present invention, wherein the modified boron carbide nanoparticles have a number-based median diameter of 30 to 66 nm, as measured in pure water by dynamic light scattering. The median diameter is an index indicating the particle diameter at which the number of larger particles and the number of smaller particles are half and half, respectively. In some embodiments of the present invention, the number-based median diameters of the modified boron carbide nanoparticles, as measured in pure water by dynamic light scattering, include 32 to 52 nm, 40 to 66 nm, 56 to 66 nm, and 57 to 63 nm. For reference, five types of modified boron carbide nanoparticles produced by the present inventors are shown in Table 1. 10 As B4C-PG, 10 B4C(35)-PG, 10 B4C(50)-PG, 10 B4C(62)-PG, 10 B4C(80)-PG, and 10 The median diameter of each B4C(110)-PG is shown in Table 1. In light of the efficacy shown in Test Example 1, a median diameter of 40 to 66 nm is preferred, and 56 to 66 nm is more preferred. In addition, the median diameter of five types of B4C(110)-PG measured in pure water by dynamic light scattering method was 10 The median diameter of B4C based on the number of particles is shown in Table 2. In light of the efficacy shown in Test Example 1, 10 The median diameter of B4C is preferably 30 to 55 nm, more preferably 45 to 55 nm (see FIG. 13). Table 2

[0037] In addition, the median diameter of the polyglycerol-modified boron carbide particles in Patent Document 2 is described in the examples as being 72.5 to 94.7 nm.

[0038] In yet another embodiment, the present invention relates to boron carbide nanoparticles of the present invention having a non-spherical shape (FIG. 10). This shape is clearly different from the spherical shape shown in FIG. 1 of JP 2023-018447 A. While the submerged laser method of Patent Documents 1 and 2 is used, the present invention uses a ball mill method. Therefore, this difference in shape is thought to be due to the different manufacturing methods of the boron carbide nanoparticles.

[0039] <Production Method> In another aspect, the present invention relates to a method for producing the modified boron carbide nanoparticle powder of the present invention, comprising: (1) converting boron oxide together with magnesium and graphite into boron carbide nanoparticles by a mechanochemical reaction; and (2) reacting the obtained nano-sized boron carbide nanoparticles with glycidol to produce modified boron carbide nanoparticle powder having surfaces modified with polyglycerol. Preferably, in the production method of the present invention, the mechanochemical reaction is a pulverization treatment using a ball mill.

[0040] Traditionally, boron carbide nanoparticles have been synthesized by various methods, as shown in Table 3. Table 3. Main synthesis methods for boron carbide particles* *Master thesis “PRODUCTION AND CHARACTERIZATION OF BORON CARBIDE POWDER BY MECHANOCHEMICAL METHOD” by B. BUEYUEKLUEOGLU (Middle East Technical University) JANUARY 2023

[0041] However, most of the above methods have problems such as the need for high temperatures or multiple steps, or difficulty in large-scale synthesis.

[0042] The synthesis method using ball milling (included in the mechanical alloying category in Table 1) has the potential to solve all of these problems at once. Therefore, the synthesis of boron carbide nanoparticles using ball milling, as shown in Table 4, has also been investigated. Table 4: Synthesis method of boron carbide nanoparticles using ball milling

[0043] As described above, boron carbide nanoparticles of various sizes have been synthesized from the same type of raw material; however, no synthesis method has been reported to date that allows for control of the median diameter (D50) of boron carbide nanoparticles with a relatively narrow particle size distribution.

[0044] Meanwhile, the inventors of the present invention have succeeded in controlling the median diameter (D50) of boron carbide nanoparticles, which have a relatively narrow particle size distribution, by pretreating the raw material boron oxide (BO) with ball milling to reduce the particle size. This BO pretreatment was carried out based on the idea that reducing the particle size of BO and increasing its specific surface area would make it easier to reduce, thereby increasing the yield of the target boron carbide nanoparticles. However, this resulted in an unexpected result: particle size control. In other words, particle size can be controlled by selecting the ball milling conditions.

[0045] As long as boron carbide particles and glycidol can actually react with each other and at least a portion of the boron carbide particles can be modified with polyglycerol, the reaction method is not particularly limited. Examples of the reaction method include ultrasonic irradiation, using an epoxy ring polymerization catalyst, appropriate heating, UV light irradiation, using a solvent, and adding glycidol dropwise. For example, reaction conditions such as reaction time, reaction temperature, and reaction concentration can be appropriately selected.

[0046] In the method for producing modified boron carbide particles according to an embodiment of the present invention, the method for producing the boron carbide particles by reacting with glycidol is not particularly limited, as long as the desired modified boron carbide particles can be obtained. For example, the boron carbide particles can be produced using pulsed laser melting, chemical vapor deposition, arc discharge, coprecipitation (liquid phase synthesis), ball mill (solid phase synthesis), or the like. The boron carbide particles may be used after adjusting their particle size, and the particle size can be adjusted using, for example, elutriation, chromatography, centrifugation, or the like.

[0047] The method for producing modified boron carbide particles according to an embodiment of the present invention can be used as is as a method for producing boron carbide particle powder containing the above-described boron carbide particles, in which at least some of the boron carbide particles have at least a portion of their surfaces modified with polyglycerol. That is, the present specification can provide a boron carbide particle powder containing boron carbide particles, in which at least some of the boron carbide particles have at least a portion of their surfaces modified with polyglycerol, and a method for producing modified boron carbide particles, the method comprising: reacting the boron carbide particles with glycidol. The above descriptions can be referenced for the method for reacting boron carbide particles with glycidol, the reaction conditions, the method for producing boron carbide particles, and so forth.

[0048] The modified boron carbide particles of the present invention can also be produced by a surface modification method in which polyglycerol is synthesized in advance, a desired molecular weight fraction is separated and purified, and then the desired molecular weight fraction is covalently bonded to the surface of the boron carbide particles by a chemical reaction.

[0049] <Drug for Neutron Capture Therapy> In yet another aspect, the present invention relates to a drug for neutron capture therapy, comprising the modified boron carbide nanoparticle powder of the present invention. Boron neutron capture therapy (BNCT) is a promising method for treating malignant tumors with almost no side effects. However, various boron preparations reported to date have required large doses for effective tumor treatment. In the present invention, boron carbide ( 10 We synthesized nanoparticles (BC) with a diameter of 50 nm coated with the hydrophilic polymer polyglycerol (PG). 10 B4C(50)-PG nanoparticles showed the highest intratumoral biodistribution in tumor-bearing mice. 10 The B concentration was 12 mg / kg, and a single dose of neutron irradiation induced tumor remission in the mouse colon cancer cell line CT26. Notably, a lower dose (6 mg / kg) 10 B4C(50)-PG nanoparticles, administered with two doses of neutron beam radiation or in combination with an anti-PD-1 antibody, similarly induced tumor remission.

[0050] Furthermore, when CT26 or 4T1 (mouse breast cancer cell lines) were re-implanted into the mice that had achieved remission, most of the mice failed to engraft. This is thought to be due to long-term, non-specific immune memory. Compared to previously reported boron preparations for BNCT, 10 B4C(50)-PG showed excellent therapeutic efficacy with the lowest dose, highest delivery efficiency, and minimal toxicity, making it promising for clinical application.

[0051] The modified boron carbide nanoparticles that can be contained in the neutron capture therapy drug of the present invention have a mean particle size of 150 nm or more, and therefore exhibit low tumor accumulation and tumor retention, failing to exhibit significant antitumor effects. The modified boron carbide nanoparticles of the present invention, when their particle size is controlled so that the proportion of particles with a particle size of 43 to 61 nm is 35% or more, preferably 45% or more, of the total nanoparticle powder, not only exhibit significant antitumor effects due to high tumor accumulation and tumor retention, but also exhibit additional antitumor effects due to immunostimulation. "Antitumor effect" generally refers to the ability to treat, manage, and / or prevent cancer.

[0052] The following properties are considered to be required for drugs for boron neutron capture therapy: (1) inherently low toxicity, (2) the ability to be administered in a shorter time (higher water solubility or water dispersibility), and (3) the ability to accumulate boron at higher concentrations in tumors (higher boron concentration in tumor cells). １０ (4) be able to persist in the tumor for a long time but be rapidly removed from the blood (e.g., the B concentration in the tumor is more preferably 20 ppm (wt / wt) or more); １０ B concentration and blood １０ The ratio to the B concentration (T / B ratio) is more preferably 3 or more).

[0053] The neutron capture therapy drug containing the modified boron carbide particle powder of the present invention may have various forms, and the form is not particularly limited, as long as it can be used for neutron capture therapy. Examples of the form include a suspension in water, a dispersion in water, a suspension in phosphate buffered saline, a dispersion in phosphate buffered saline, other suspensions and dispersions, a solid, a gel, etc.

[0054] The neutron capture therapy drug may contain various additives in addition to the modified boron carbide particles according to an embodiment of the present invention, as long as the drug can be used for neutron capture therapy. The additives are not particularly limited. Examples of such additives include anticancer drugs, photosensitizers, solubilizing aids, surfactants, lipids, carbohydrates, proteins, polymers, small molecules, and biomaterials.

[0055] The neutron capture therapy drug preferably contains 0.1 to 100 mass %, more preferably 1 to 80 mass %, even more preferably 5 to 60 mass %, and even more preferably 10 to 50 mass % of the modified boron carbide particle powder of an embodiment of the present invention. When the drug contains 0.1 to 100 mass % of the modified boron carbide particle powder of an embodiment of the present invention, the drug can be injected in a more stable dispersed state. When the drug contains 5 to 60 mass %, the drug can be injected in a more stable dispersed state and other functions can be added by including various additives such as those described above. When the drug contains 10 to 50 mass %, the drug can be injected in a more stable dispersed state and other functions can be added and improved by including various additives such as those described above, resulting in an excellent balance of these.

[0056] The neutron capture therapy drug according to an embodiment of the present invention has the advantageous effects of being inherently less toxic, being able to be administered in a shorter time (having better water solubility or dispersibility in water), being able to accumulate boron at a higher concentration in the tumor, and being able to remain in the tumor for a long period of time while being quickly removed from the blood.

[0057] The neutron capture therapy drug according to an embodiment of the present invention comprises: １０The B concentration may be, for example, 0.1 mg / mL or more, preferably 0.5 mg / mL or more, more preferably 1 mg / mL or more, even more preferably 2 mg / mL or more, and even more preferably 5 mg / mL or more. １０ It can be used as a drug with a relatively high concentration of B. Therefore, the administration time can be shortened.

[0058] The neutron capture therapy drug of the embodiment of the present invention may contain, for example, 0.01 mg [ １０ B] / kg or more, and 0.1 mg [ １０ B] / kg or more, and 0.5 mg [ １０ B] / kg or more, and 1 mg [ １０ It is more preferable to use it at a dosage of 100 mg / kg or more.

[0059] The neutron capture therapy drug of the embodiment of the present invention can accumulate boron at a higher concentration in tumors, and １０ The B concentration (wt / wt) 24 hours after drug injection can be, for example, 1 ppm or more, preferably 3 ppm or more, more preferably 6 ppm or more, even more preferably 10 ppm or more, and even more preferably 20 ppm or more. １０ The B concentration can be measured by the method described in the Examples.

[0060] The neutron capture therapy drugs of the present invention can persist in tumors for a long time, but can be rapidly cleared from the blood, resulting in tumor growth. １０ B concentration (T) and blood １０ The ratio to the B concentration (B) (T / B ratio) 24 hours after drug injection is, for example, 0.5 or more, preferably 1 or more, more preferably 2 or more, even more preferably 3 or more, and even more preferably 4 or more.

[0061] In yet another aspect, the present invention relates to a combination therapy in which a neutron capture therapy drug containing the modified boron carbide nanoparticle powder of the present invention is administered simultaneously or sequentially with an immune checkpoint inhibitor. Possible immune checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti-KIR antibodies, and anti-PD-1 / CTLA-4 bispecific antibodies. The neutron capture therapy provided by the present invention can provide a synergistic effect on the effects of conventional cancer immunotherapy.

[0062] In one embodiment of the present invention, as demonstrated in Test Example 1 (FIG. 12) and Test Example 6 (FIG. 22), a significant tumor growth inhibitory effect was confirmed against various mouse-derived tumors, including CT26 (mouse colon cancer), 4T1 (mouse breast cancer), Meth-A (mouse skin sarcoma), B16-F10 (mouse malignant melanoma), and LLC (mouse lung cancer). These results suggest that, compared with clinically used BPA (p-boronophenylalanine), this compound is effective against a wider variety of carcinomas, regardless of the transporters expressed in tumor cells.

[0063] In one embodiment of the present invention, as demonstrated in Test Example 7 (Figure 24), 10 In a drug efficacy test using half the usual amount of B4C(50)-PG, two doses with a one-week interval were found to be more effective than a single dose of neutron radiation. 10 This is due to the high tumor retention of BC(50)-PG, a feature that significantly differs from other boron preparations, including BPA. Furthermore, Test Example 1 demonstrated that high boron concentrations were maintained for three days, providing direct evidence of the tumor retention of the present invention (Table 10). A single intravenous injection demonstrated high tumor retention of boron concentrations of 60 ppm or more even three days later.

[0064] Conventional BNCT using BPA is known under the generic name Borofalan ( 10B) It is marketed under the trade name Steboronine for Intravenous Infusion 9000 mg / 300 mL. The dosage and administration are as follows: Usually, adults are given Borofaran ( 10 B), 500 mg / kg is administered intravenously over a period of three hours. The infusion rate is 200 mg / kg / h for the first two hours and 100 mg / kg / h for the remaining hour. Two hours after the infusion begins, the lesion is irradiated once with neutrons (approximately 30 minutes to one hour), and the infusion is terminated at the same time as the neutron irradiation is completed (for example, in the case of a 60 kg patient, 800 mL of steboronine is administered intravenously over two hours, followed by 200 mL in the remaining hour). During the infusion, boron ( １０ B) The concentration is maintained at approximately 20 ppm, but decreases and disappears once the infusion is completed. In other words, conventional BPA can only irradiate a tumor site with neutron beams once during a single intravenous infusion. Because the collimator opening of the irradiation device is typically between 100 and 150 mm in diameter, a single neutron beam irradiation can cover a tumor of that size. However, for tumors that have spread widely or are larger, multiple neutron beam irradiations are required to cover the entire tumor. Therefore, conventional BPA is difficult to treat and cannot adequately treat such large tumors. Repeated irradiation requires a three-hour intravenous infusion followed by a single neutron beam irradiation, with intervals of several days to several weeks. This requires prolonged confinement of the patient and exposure to large amounts of drug, placing a significant burden on the patient.

[0065] On the other hand, as described above, the modified boron carbide nanoparticle powder of the present invention, when administered intravenously in a single small amount, remains at the tumor site at a boron concentration of 60 ppm or more for at least three days, as shown in Table 10. Therefore, while the drug remains at the tumor site, neutron beam irradiation can be repeated without additional drug administration, allowing for thorough tumor destruction. Again, while conventional BNCT using BPA is administered by continuous infusion over a three-hour period, the modified boron carbide nanoparticle powder of the present invention can exert its effects with a single rapid intravenous injection (one-shot intravenous injection, bolus injection). Furthermore, due to its long retention at the tumor site, neutron beam irradiation (fractionated irradiation) can be performed repeatedly at the lesion site over several days after a single intravenous injection. These are significant advantages in terms of reducing the burden on the patient. Furthermore, the dosage of the previous formulation is 500 mg / kg administered intravenously in a single infusion, which is extremely high, at approximately 30 g based on a human weight of 60 kg. On the other hand, the present invention is expected to achieve antitumor effects at a dose of approximately 70-100 mg, calculated based on a 60 kg human body weight (body surface area). A reduced dose is a major advantage, leading to fewer side effects, a lighter burden on patients, and lower costs.

[0066] In summary, the features of the neutron capture therapy drug of the present invention are its high tumor concentration of 60 ppm or more and its ability to maintain that high concentration for a long period of time, for example, three days or more.The existing drug BPA (steboronine) is thought to have a blood concentration of about 20 ppm and a tumor concentration of about two to three times that.This concentration is maintained during intravenous infusion, but once the infusion is terminated, the blood and tumor concentrations rapidly decrease.

[0067] Furthermore, in one embodiment of the present invention, as demonstrated in Test Example 8 (Fig. 26), an abscopal effect was observed, which is rarely seen in conventional radiation therapy. The abscopal effect is a phenomenon in which, when performing radiation therapy on a tumor, the growth of not only the irradiated malignant tumor but also a distant malignant tumor that has not been irradiated is suppressed. This is believed to be useful in actual cancer treatment, both for shrinking metastatic cancer and for curing it completely. 10This indicates the possibility that BNCT using B4C(50)-PG may be effective. In recent years, many academic papers have reported that the abscopal effect was observed in the combination of immunotherapy and radiation therapy, but there have been few cases in which the abscopal effect was observed reproducibly with radiation therapy alone, which is a local therapy. On the other hand, as shown in Test Example 8 (Figure 27), the present invention 10 In the B4C(50)-PG + N group, CD4 + T cells, CD8 + T cells are more abundant than in the multi-group, 10 It is clear that B4C(50)-PG-BNCT clearly activates the immune system, and is therefore expected to exert a reproducible abscopal effect.

[0068] Based on the above, in the present invention, neutron irradiation of cancer tissue in which the modified boron carbide nanoparticle powder of the present invention has accumulated destroys cancer cells, produces cancer antigens, and activates immune cells present in the body, i.e., exerts an immunopotentiating effect. Furthermore, the immune system recognizes the cancer antigens and induces a cancer immunity effect that attacks and kills cancer cells. That is, the immunopotentiating effect induced after neutron irradiation exerts the additional antitumor effect of the present invention. In this way, the neutron capture therapy drug of the present invention exerts an immunopotentiating effect with good reproducibility after neutron irradiation, thereby obtaining a cancer immunity effect. In another embodiment of the present invention, the tumor immunity potentiating effect after neutron irradiation allows a reproducible abscopal effect to be obtained. Thus, as one embodiment of the present invention, there is provided a neutron capture therapy drug of the present invention that can exert an immunopotentiating effect (cancer immunity effect) after neutron irradiation. Here, "additional antitumor effect" means, for example, that in addition to the damage to the DNA of cancer cells caused by alpha rays generated by BNCT, an antitumor effect due to immunity is observed.

[0069] In another aspect of such an embodiment, the present invention provides a drug for inducing an immunostimulatory effect in a cancer patient who will receive neutron beam irradiation, i.e., a cancer patient who is scheduled to receive neutron beam irradiation, the drug containing the neutron capture therapy drug of the present invention as an active ingredient.

[0070] The present invention will be specifically and in detail explained below with reference to examples and comparative examples, but these examples are merely one embodiment of the present invention, and the present invention is not limited by these examples. In the description of the examples, unless otherwise specified, parts by weight and percent by weight are based on the parts not taking into account the solvent.

[0071] Example 1 Boron carbide nanoparticles ( 10 B4C) by polyglycerol (PG) modification 10 Synthesis of BC-PG 1-1: Boron carbide nanoparticles with a median diameter of 50 nm ( 10 Synthesis of boron oxide (BC(50)) 10 B2O3) was prepared by heating boric acid (H3 10 BO3) was dehydrated at 130 °C for 40 minutes and then heated at 330 °C for 80 minutes. Then, it was milled in a planetary ball mill (Pulverisette 7, Fritsch) under the following conditions: 10 B4C was synthesized.

[0072] First, 10 mm stainless steel balls (approximately 20 g, 304 stainless steel, Ohashi Steel Ball Co., Ltd., Japan) and 10 wt% (ball powder ratio = 10) magnesium (approximately 2 g) were placed in a 12 mL stainless steel container (304 stainless steel milling vial, Mitr Instrument Equipment Co. Ltd., China), and ball milled at 800 rpm for 30 minutes. 10 For B2O3, a 12 mL container was filled with 10 mm new balls (approximately 20 g) and 5 wt. % (ball powder ratio = 20) of the above-prepared B2O3. 10 B2O3 (approximately 1 g) was added and pretreated by ball milling at 800 rpm for 20 minutes.

[0073] Next, new balls of 10 mm and 5 mm (mass ratio of the two was 4, total weight 60 g) were placed in a 45 mL stainless steel container, and the balls were further pretreated in a ball mill. 10 B2O3 (0.90 g) was mixed with magnesium (1.00 g) and graphite (0.089 g), which had also been ball-milled previously. 10 The amounts of the sintered materials were approximately 1.1 times and 0.1 times the mass of B2O3. 10 The total weight of B2O3, magnesium, and graphite (approximately 2 g) was 3.3 wt% of the balls (ball-powder ratio = 30). This mixture was ball-milled at 800 rpm for 8 hours. After ball-milling, NaCl (5:1 mass ratio of NaCl to total powder) was added, and the mixture was ball-milled for another hour at 800 rpm.

[0074] Finally, the powder in the stainless steel container of the ball mill was transferred to a glass flask, and 15% hydrochloric acid (40 mL) was added. The mixture was stirred with a magnetic stirrer at 90°C for 3 hours. The dispersion was transferred to a centrifuge tube and centrifuged at 22,260g and 25°C for 50 minutes. After removing the supernatant, Milli-Q water was added to the precipitate, which was then redispersed. The mixture was then centrifuged at 50,400g and 25°C for 30 minutes. The dispersion / centrifugation process was repeated several times until the pH of the supernatant reached 7, yielding boron carbide nanoparticles ( 10 The boron carbide nanoparticles (BC(50)) were washed. Yield: 0.11 g (33%). 10 B4C(50)) was analyzed by Fourier transform infrared spectroscopy (FTIR) to determine the structure of the nanoparticles.

[0075] The IR measurement was carried out using an IR Prestige-21 (trade name) manufactured by Shimadzu Corporation. －１ 4 cm in the wave number range －１ The FTIR spectrum was obtained by collecting 32 scans at a resolution of 100 kHz. The results are shown in Figure 3. The IR spectrum showed that boron carbide nanoparticles ( 10 It was shown that hydroxy groups exist on the surface of B4C(50). 10The absorption of B4C is shifted to the higher wavenumber side compared to that of B4C. 10 Compared to B4C, 10 It can be seen that the proportion of B is high.

[0076] In addition, boron carbide nanoparticles ( 10 The powder X-ray diffraction (XRD) profile of B4C(50) was measured using a Rint2200 (Rigaku Corporation). The results are shown in Figure 4.

[0077] On the other hand, boron carbide nanoparticles ( 10 As shown in Figure 2, the powder X-ray diffraction profile of boron carbide nanoparticles (BCC) shows a graphite-derived peak at 26.4°. 10 It has been shown that the surface of boron carbide nanoparticles (B4C) is covered with graphite (Yoshie Ishikawa et al., APPLIED PHYSICS LETTERS 91, 161110 (2007)). However, the surface of boron carbide nanoparticles ( 10 This is not seen in the powder X-ray diffraction (XRD) profile of B4C(50) in Figure 4, a major difference between the two.

[0078] 1-2: Polyglycerol modified 10 B4C-PG ( 10 Synthesis of B4C(50)-PG nanoparticles The nanoparticles synthesized in Example 1-1 were prepared according to the method described in Patent Document 2 (JP 2023-018447 A) or the literature (L. Zhao, T. Takimoto, M. Ito, N. Kitagawa, T. Kimura, N. Komatsu, Angew. Chem. Int. Ed. 2011, 50, 1388). 10 50 mg of B4C(50) was polyglycerol-modified using 5 g of glycidol. 10 B4C-PG ( 10 The infrared absorption (IR) spectrum of the B4C(50)-PG nanoparticles is shown in Figure 5.

[0079] 1-3: 10B4C(50) and 10 Measurement of particle size of B4C(50)-PG by dynamic light scattering a) 10 Particle size of B4C(50): 10 The cumulative particle size (nm) (%) of B4C(50) in pure water was determined by dynamic light scattering based on the number standard using a Nanotrac UPA-UT151 system (Microtrac, Inc.). The results are shown in Table 5 and Figure 6.

[0080] Table 5

[0081] b) 10 Particle size of B4C(50)-PG synthesized in Example 1-2 10 The particle size of B4C(50)-PG was measured in pure water by dynamic light scattering (DLS). The results (number basis) are shown in Table 6 and Figure 7.

[0082] Table 6 10 The median diameter (D50) of B4C(50)-PG was 60.7 nm. 10 The particle size distribution of B4C(50)-PG did not change over a week, indicating the high colloidal stability of these dispersions.

[0083] c) 10 Particle size distribution of B4C(50)-PG 10 The cumulative particle size (nm) (%) of B4C(50)-PG in pure water was determined by dynamic light scattering based on the number standard using a Nanotrac UPA-UT151 system (Microtrac, Inc.). The results are shown in Table 7.

[0084] Table 7

[0085] ​Table 7 shows that the abundance ratio of particles having a particle diameter (nm) of 43 to 60.8 nm was 46.55 (50.19-3.64)% of all particles. From the activity test in a mouse tumor model shown in the following Test Example 1, it was found that good antitumor activity could be obtained if the abundance ratio of particles having a particle diameter (nm) of 43 to 60.8 nm was 35% or more. Details are explained in Test Example 4.

[0086] 1-4: 10 Content of polyglycerol modification of B4C(50)-PG nanoparticles a) Thickness of polyglycerol modification layer: 10 B4C(50) and 10 From a comparison of D50 (median diameter) of B4C(50)-PG in pure water, 10 The thickness of the PG layer on the B4C(50) surface was shown to be 5.5 nm. b) Content of the polyglycerol-modified layer: 10 The polyglycerol content of B4C(50)-PG was determined to be 36.6 wt% by heat flow analysis (TGA) under a nitrogen atmosphere, as shown in Figure 8. TGA was performed using a Q50 analyzer (TA Instruments) at a heating rate of 20 °C / min under a nitrogen stream.

[0087] 1-5: 10 Zeta potential of B4C(50)-PG nanoparticles: The zeta potential is 10 B4C(50) or 10 The PBS dispersion of B4C(50)-PG was placed in a cell and measured 20 times using a Zetasizer Nano Series (Malvern Instruments, UK). The average values ​​are shown in Figure 9. As shown in Figure 9, the B4C(50)-PG in PBS 10 The zeta potential of B4C(50) was −25.0 mV, but after PG modification, 10 The zeta potential of B4C(50)-PG in PBS was -4.5 mV, and the absolute value of the potential was significantly smaller. 10 This indicates that the hydroxyl groups (-B(OH)2) on the surface of the B4C(50) nanoparticles reacted with glycidol to form boronate esters, which changed the acidic state to closer to neutral.10 This is thought to suppress the formation of a protein corona on the B4C(50)-PG surface, and as a result, a high stealth effect was expected to avoid uptake by macrophages.

[0088] 1-6: 10 Polyglycerol content of B4C-PG nanoparticles Described in JP 2023-018447 10 Polyglycerol layer content (wt%) of B4C(62)-PG and other 10 The results for B4C-PG are summarized in Table 8. 10 The content of graphite in B4C(62)-PG differs significantly from that in other materials. This is thought to be due to differences in the presence or absence of graphite layers and their shape, which are caused by differences in synthesis methods. Table 8

[0089] Similarly, 10 B4C was modified with polyglycerol to prepare the following modified nanoparticles: Table 9

[0090] Test Example 1: In a mouse tumor model 10 Optimization of the antitumor effect and particle size of B4C(50)-PG Objectives Using tumor-bearing mice, 10 To clarify the difference in anti-cancer activity due to differences in particle size of B4C-PG, approximately 1 × 10 B4C-PG particles suspended in PBS (100 μL) were administered to 7- to 9-week-old BALB / c and C57BL / 6 mice (female). 6 CT26 tumor cells were subcutaneously implanted into the right leg. The tumor volume of each mouse was calculated according to the following formula and statistically analyzed: tumor volume = (long axis) x (short axis). 2 / 2

[0091] Produced in Example 1 10 The therapeutic effect of B4C(50)-PG on tumors was evaluated by the following experimental method (FIG. 11).

[0092] Specifically, CT26 tumor cells were transplanted into BALB / c mice (female). 10 A 200 μL suspension of B4C(50)-PG (12 mg / kg) in phosphate-buffered saline (PBS) was administered via the tail vein. Two days later, neutrons were injected at 5×109 cm -2 s -1 The tumors were irradiated for 12 minutes at a flux of 1000 Hz, and changes in tumor size were monitored for 22 days. 10 The mice were then administered 200 μL of PBS alone (PBS group, n = 5), referred to as the "B4C(50)-PG + N group" (n = 5). 10 200 μL of B4C(50)-PG (12 mg / kg) dispersion in PBS (phosphate buffered saline) was administered. 10 B4C(50)-PG group (n = 5), and 200 μL of PBS was administered. Two days later, neutrons were administered at a dose of 5 × 10 9 cm -2 s -1 The "PBS + N group" (n = 5), which was irradiated for 12 minutes at a flux of 1000 Hz, was also used as a control, and changes in tumor size were monitored for 22 days.

[0093] Median diameters of 35, 80, and 110 nm other than 50 nm 10 B4C was chemically modified with polyglycerol 10 B4C(35)-PG, 10 B4C(80)-PG, and 10 B4C(110)-PG was also evaluated using the same experimental method.

[0094] Results and Discussion The results are shown in Figure 12. First, the control (PBS group, 10 B4C-PG group, PBS + N group) 10 In the B4C-PG + N group, tumor growth was more significantly suppressed. 10 Even within the B4C-PG + N group, the efficacy varied significantly depending on the size. First, the 35 and 50 nm sizes showed greater tumor growth suppression effects than the 80 and 110 nm sizes. 10 In the B4C(50)-PG + N group, CR (complete remission) was 80%; 10 In the B4C(35)-PG + N group, CR was 60%; 10 The B4C(50)-PG + N group showed the highest efficacy.

[0095] next,10 B4C(50)-PG, and 10 B4C(35)-PG, 10 B4C(80)-PG and 10 Regarding B4C(110)-PG, 10 To clarify the size dependency of the efficacy of B4C-PG + N group, each PBS dispersion (12 mg / kg) was administered to tumor-bearing mice (n = 3) prepared by the above method, and the tumor size was measured at 24, 48, and 72 hours after administration. 10 The B concentration in the tumor tissue was measured by prompt gamma ray analysis. The results are shown in Table 10. 10 The B concentration is shown along with the calculated %ID (injection dose).

[0096] Table 10

[0097] From the results in Table 10, the boron concentration in the tumor increased over time between 24 and 72 hours, and the %ID remained at almost the same level, indicating high tumor retention. The results in Table 10 are clearly correlated with the efficacy shown in Figure 12. First, the results of the high efficacy were 10 B4C(35)-PG and 10 B4C(50)-PG is 10 B4C(80)-PG and 10 B4C(110)-Higher than PG 10 B concentration and %ID were confirmed. Furthermore, at each time, 10 B4C(50)-PG is 10 Compared to B4C(35)-PG, this shows a slightly higher value, which also shows a correlation with the efficacy shown in Figure 12. Overall, it has the highest tumor accumulation ability. 10 It can be concluded that B4C(50)-PG showed the highest efficacy (% remission rate) (see Figure 13). The remission rate (%) represents the percentage of mice that achieved complete remission. 10 This is the value divided by the number of mice in the B4C-PG + N group. For example, 10 In the B4C(50)-PG + N group, four out of five tumor-bearing mice achieved complete remission, resulting in a remission rate of 80%.

[0098] Test Example 2: Test of re-implantation of tumor cells into mice in complete remission (cancer recurrence model) Objective: To clarify the ability to suppress cancer recurrence by re-implanting tumor cells into mice that have once achieved complete remission and observing whether they will take root. Method: An outline of the experimental procedure is shown in Figure 16. First, the same procedure as in Test Example 1 was performed. 10 In an in vivo efficacy study using B4C(50)-PG, CT26 cells (1 × 10 cells) suspended in PBS were injected into the left flank of BALB / c mice (n = 6) that had achieved complete remission of tumors in the right flank. 6 As control mice (n = 6) of the same age but with no history of tumor cell transplantation, CT26 cells were subcutaneously transplanted in the same way as the previous mice, and their growth was followed over time.

[0099] Results and Discussion The results are shown in Figure 17. Figure 17 shows that tumors grew rapidly in untreated mice, whereas no tumor cells engrafted in mice that achieved complete remission. This is thought to be due to the marked activation of the immune system in the mice that achieved complete remission.

[0100] Interestingly, when the heterologous 4T1 tumor cells were subcutaneously injected (reimplanted) into the left flank of mice (n = 6) that had achieved complete remission from CT26, four mice showed no sign of engraftment. Furthermore, when the heterologous Meth-A tumor cells were subcutaneously injected (reimplanted) into the right flank of the mice (n = 6) that had not engrafted with CT26 cells (Figure 17), none of the mice showed any sign of engraftment. These results demonstrate that mice that have achieved complete remission have a significant ability to suppress cancer recurrence, not only against homologous tumor cells but also against heterologous tumor cells.

[0101] Test Example 3: Immunohistochemical Staining of Spleen and Lymph Nodes Spleens and lymph nodes were removed from untreated mice and mice in complete remission obtained in Test Example 2 on day 155 (Day 155 in Figure 16). The collected spleen and lymph node cells were subjected to immunohistochemical staining. The results are shown in Figure 18. Figure 18 shows that both CD4-positive T cells and CD8-positive T cells were expressed at higher levels in mice in complete remission than in untreated mice.

[0102] Test Example 4: Examination of preferred particles. The high efficacy demonstrated in Test Example 1 (Fig. 12) was observed. 10 B4C(35)-PG and 10 The particle size distribution of B4C(50)-PG was measured in pure water using dynamic light scattering based on the number of particles. The results are shown in Table 11. Table 11 shows the cumulative percentage of particles in the size range of 30.4-818 nm. 10 B4C(35)-PG and 10 In addition to B4C(50)-PG, it has a larger particle size 10 B4C(62)-PG, 10 B4C(80)-PG, 10 The results for B4C(110)-PG are also shown for comparison.

[0103] The values ​​shown at the bottom of Table 11 are for five types 10 The percentages of particle sizes of 43 nm to 60.8 nm (shown in separate boxes in Table 11) for each of the B4C-PGs are shown below. 10 B4C(50)-PG was synthesized by the method of Example 1-1, and 10 B4C(35)-PG, 10 B4C(80)-PG, 10 B4C(110)-PG is 10 The particle size was controlled by changing the ball milling time of B2O3 and synthesis was carried out. 10The synthesis method of B4C(62)-PG is described in Patent Document 2 (JP 2023-018447 A) and in Y. Wang, G. Reina, HG Kang, X. Chen, Y. Zou, Y. Ishikawa, M. Suzuki, N. Komatsu, Small, 18, 2204044 (2022). 10 The median diameter of B4C in pure water is 62 nm.

[0104] Table 11

[0105] The five types in Table 11 10 The correlation between the proportion of particles between 43 nm and 60.8 nm in B4C-PG and the proportion of mice that achieved complete remission (remission rate (%)) in Test Example 1 (Figure 12) was investigated. The results are shown in Figure 13. The proportion of particles between 43 nm and 60.8 nm was relatively high, at approximately 35% or more. 10 B4C(35)-PG and 10 In B4C(50)-PG, a relatively high remission rate of over 60% was achieved, while the proportion of particles between 43 nm and 60.8 nm was relatively low at less than 25%. 10 B4C(62)-PG, 10 B4C(80)-PG and 10 A relatively low remission rate of less than 40% was observed in B4C(110)-PG. 10 It can be concluded that a higher anti-cancer effect from boron neutron capture therapy can be expected when the B4C-PG ratio is 35% or higher (equivalent to "34.83%" in the table), and preferably 45% or higher (equivalent to "46.55%" in the table).

[0106] In addition, the above five types 10 The zeta potential of B4C-PG in pure water is shown in Figure 14. The correlation between the zeta potential shown in Figure 14 and the remission rate (%) was also investigated. The results are shown in Figure 15. The zeta potential was relatively high at -35 to -40 mV. 10 B4C(35)-PG, 10 B4C(50)-PG showed a relatively high remission rate of over 60%, but a relatively low zeta potential of -47 to -52 mV.10 B4C(62)-PG, 10 B4C(80)-PG, 10 A relatively low remission rate of less than 40% was observed for B4C(110)-PG. Therefore, we conclude that a higher anticancer effect of boron neutron capture therapy can be expected when the zeta potential is between -30 and -42 mV, preferably between -33 and -39 mV.

[0107] Test Example 5: Combination test with immune checkpoint inhibitor (anti-PD1 antibody) Objective: To confirm that the cancer treatment effects of BNCT are synergistically enhanced when combined with cancer immunotherapy.

[0108] Method: The same experiment as in the in vivo efficacy test in Figure 11 was performed using half the dose (6 mg / kg). 10 The experiment was conducted using B4C(50)-PG (Example 1-2). 10 B4C(50)-PG group, PBS+N group, 10 The B4C(50)-PG + N group (all n = 6) received intraperitoneal injections of 200 μg of anti-PD-1 antibody (α-PD-1) every 3-4 days starting immediately after neutron irradiation ( 10 Mice were treated with either B4C(50)-PG + N + α-PD-1 (n = 6) or α-PD-1 alone (α-PD-1 group) (n = 5) (Figure 19), and the relative tumor volume was monitored for 25 days.

[0109] Results and Discussion The results are shown in Figure 20. First, the control (PBS group, 10 B4C(50)-PG group, α-PD-1 group, PBS+N group) 10 B4C(50)-PG + N group and 10 The B4C(50)-PG + N + α-PD-1 group showed a more significant inhibition of tumor growth. In particular, α-PD-1 alone did not show any significant efficacy. 10 The dose of B4C(50)-PG was halved, 10 The efficacy of the B4C(50)-PG + N group was also reduced compared to the results shown in Figure 12. 10The efficacy of B4C(50)-PG + N + α-PD-1 was significantly improved, demonstrating that BNCT and cancer immunotherapy synergistically enhance each other's effectiveness.

[0110] Test Example 6: In tumor-bearing mice engrafted with 4T1, Meth-A, B16-F10 or LLC tumor cells other than CT26 10 6-1: Efficacy test of B4C(50)-PG According to the procedure shown in Figure 21, female BALB / c mice (n = 3) with 4T1 were treated with PBS or 10 B4C(50)-PG was administered at 12 mg / kg (mouse), i.e., 5.1 mg [ 10 B] / kg (mouse) was administered via the tail vein on day 0, and 5 × 10 9 cm -2 s -1 The average mouse weight on day -2 was 17 g, and the average tumor volume on day 0 was 97 mm. 3 Other tumor mouse models were also investigated using Meth-A female BALB / c mice (PBS and 10 n = 4 in the B4C(50)-PG group, n = 5 in the other groups), B16-F10 female C57BL / 6 mice ( 10 n = 15 for the B4C(50)-PG+N group, n = 6 for the other groups), and C57BL / 6 mice with LLC ( 10 The BNCT experiment was performed under the same conditions as in Test Example 1, except for the B4C(50)-PG+N group (n = 15) and the other groups (n = 6). The average body weights of mice on day -2 and tumor volumes on day 0 were 17, 20, 20, and 19 g and 97, 154, 94, and 71 mm, respectively, in the 4T1, Meth-A, B16-F10, and LLC tumor models. 3 It was.

[0111] 6-2: Results Using four types of tumor models, 4T1, B16-F10, LLC, and Meth-A, BNCT was performed under the same conditions as in Test Example 1 according to the method in 6-1, and the antitumor effect was evaluated. 10 Tumor growth was inhibited in the B4C(50)-PG + N group, but the effects were different.10 All Meth-A mice in the B4C(50)-PG+N group achieved remission (100% remission rate, Fig. 22b), whereas the remission rates in the other models were 33% or less (Fig. 22a, c, d).

[0112] Test Example 7: Two-time neutron irradiation of CT26 tumor-bearing mice 10 7. Efficacy test using B4C(50)-PG 7-1 Method According to the procedure shown in Figure 23, female BALB / c mice (n = 5) bearing CT26 tumors were treated with PBS or 10 B4C(50)-PG was administered at 6.0 mg / kg (mouse), half the dose used in Test Example 1 (2.5 mg [ 10 B] / kg (mouse) was administered via the tail vein, and 5 × 10 9 cm -2 s -1 The mean tumor volume on day 0 was 122 mm. 3 The average body weight on day -2 was 21 g.

[0113] 7-2 Results 10 Taking advantage of the high retention of B4C(50)-PG in tumors, neutron irradiation was performed twice at a one-week interval, as shown in Figure 23. As shown in Figure 24a, in the CT26 tumor model, half the dose of that in Test Example 1 was administered. 10 After introducing B4C(50)-PG, neutron irradiation was performed twice ( 10 B4C(50)-PG(Half) + N + N), and in the case of single irradiation under the same conditions ( 10 The tumor growth inhibitory effect was greater than that of B4C(50)-PG(Half) + N. The survival rate of tumor-bearing mice was also significantly improved with two doses of irradiation compared to a single dose (Fig. 24b).

[0114] Test Example 8: 4T1 tumor-bearing mice 10 Verification test of the abscopal effect using B4C(50)-PG 8-1 Method Following the procedure shown in Figure 25, 4T1 tumors (1.0 × 10 6 On days -6 and -2, tumor-bearing mice (n = 3) were transplanted into the right foot and back of female BALB / c mice.10 B4C(50)-PG was administered at 12 mg / kg (mouse) (5.1 mg [ 10 B] / kg (mouse)) was administered via the tail vein on day 0, and 5 × 10 9 cm －2 s －1 The tumors on the right leg were irradiated for 12 minutes with a neutron flux of 1000 Hz. The mean tumor volume on the right leg was 77 mm on day 0. 3 , back 30 mm 3 It was.

[0115] 8-2 Results 10 Although the tumor growth suppression in the right leg of the B4C(50)-PG+N group mice was slightly less than that of the mice with tumors implanted only in the right leg (Fig. 22a), a significant suppression effect was observed compared with the N group, which was irradiated only with neutrons (Fig. 26a). 10 The phenomenon of slight suppression of tumor growth in the N group compared to the B4C(50)-PG group can also be seen in Figure 22a, indicating that the tumor on the right leg was exposed to neutron radiation. On the other hand, the growth of the tumor on the back was also significantly suppressed compared to the control group (Figure 26b). Furthermore, the growth of the N group was significantly suppressed compared to the other control groups (PBS group, 10 This was similar to the B4C(50)-PG group, indicating that, unlike the right leg, the tumor was barely hit by the neutron beam. The actual neutron dose at the tumor site on the back was measured and was found to be 13%-22% of that at the tumor site on the right leg. From these results, it was concluded that almost no BNCT activity occurred in the tumor on the back, and therefore the tumor growth suppression effect on the back was due to the abscopal effect.

[0116] Next, to verify the abscopal effect, tumor tissue sections from mice 21 days later (Fig. 25) were immunostained to detect CD8 + T cells and CD4 + The expression levels of T cells were compared. The results are shown in Figure 27. In the figure, a) CD8 + T cells, b) CD8 on the back + T cells, c) CD4 in the right leg + T cells, and d) CD4 on the back +The horizontal axes of a) to d) are, from the left, PBS, 10 B4C(50)-PG, N and 10 B4C(50)-PG-PG + N. The tumor on the right leg that was irradiated with neutrons and the tumor on the back that was not directly irradiated with neutrons both showed CD8 + T cells, CD4 + It was found that T cells were highly expressed. 10 It was suggested that the immunostimulation by B4C(50)-PG in BNCT induces an abscopal effect.

[0117] Example 9 4 Tumor-bearing mice engrafted with CT26, 4T1, B16-F10 or LLC tumor cells were used. 10 9-1 Efficacy test of B4C(50)-PG and immune checkpoint inhibitors (anti-PD-1 antibody, α-PD-1) 9-1 Methods According to the procedure shown in Figure 28, female BALB / c mice (n = 6) bearing CT26 tumors were injected with PBS or 10 B4C(50)-PG was administered at 6.0 mg / kg (mouse) (2.5 mg [ 10 B] / kg (mouse)) was administered via the tail vein on day 0. 9 cm -2 s -1 The average mouse weight on day 0 was 21 g and the tumor volume was 114 mm. 3 Anti-PD-1 antibody (200 μg) was injected intraperitoneally into mice on days 0, 4, 7, and 11. BNCT experiments were also performed on other tumor models under similar conditions, except for 4T1 female BALB / c mice (n = 3) and B16-F10 female C57BL / 6 mice (anti-PD-1 group: n = 5, other groups: n = 6). The average mouse weight and tumor volume on day 0 were 17, 20 g, 99, and 90 mm for the 4T1 and B16-F10 tumor models, respectively. 3 Anti-PD-1 antibody (200 μg) was intraperitoneally injected into the 4T1 tumor model on days 0, 3, 7, and 10, and into the B16-F10 tumor model on days 3, 6, 9, and 12.

[0118] 9-2 Results Following the procedure shown in Figure 28, we first attempted to treat CT26 tumor-bearing mice with a combination of immunotherapy using BNCT and anti-PD-1 antibody (α-PD-1). The results showed that the treatment was effective at a dose of 6.0 mg / kg (mouse) (2.5 mg [ 10 B] / kg (mouse) 10 In the B4C(50)-PG (Half) + N + α-PD-1 group, 67% of CT26 mice achieved remission and survived for more than 300 days (Fig. 29a, b). 10 No remissions were observed in other groups, including the B4C(50)-PG (Half) + N group. In other models, including 4T1 and B16-F10, tumor eradication was not observed, but the combination of BNCT and α-PD-1 demonstrated a higher therapeutic effect than BNCT alone (Fig. 29c, d).

[0119] The modified boron carbide particle powder according to an embodiment of the present invention comprises boron carbide particles, at least some of which have at least a portion of their surfaces modified with polyglycerol. Therefore, a drug comprising the modified boron carbide particle powder according to an embodiment of the present invention exhibits advantageous effects such as inherently low toxicity, shorter administration time (higher water solubility or water dispersibility), higher boron accumulation in tumors, and long-term persistence in tumors while being rapidly removed from the blood, making it suitable for use in boron neutron capture therapy.

Claims

A powder of modified boron carbide nanoparticles, in which a portion of the surface of the boron carbide particles is modified with polyglycerol, and the surface of the boron carbide nanoparticles is not coated with graphite.

2. The modified boron carbide nanoparticle powder according to claim 1, wherein a particle size distribution based on the number of particles of the modified boron carbide nanoparticle powder measured in pure water by dynamic light scattering method contains particles having a particle diameter of 43 to 61 nm in an amount of 35% or more of the total nanoparticle powder.

3. The modified boron carbide nanoparticle powder according to claim 2, wherein the proportion of particles having a particle diameter of 43 to 61 nm is 45% or more of the total nanoparticle powder.

2. The modified boron carbide nanoparticle powder according to claim 1, which has a zeta potential in pure water of −45 to −30 mV. 　 The modified boron carbide nanoparticle powder according to claim 1, wherein the content of polyglycerol is 15 to 60 mass % based on the modified boron carbide nanoparticles (100 mass %).

2. The modified boron carbide nanoparticles according to claim 1, wherein the modified boron carbide nanoparticles have a number-based median diameter of 30 to 66 nm.

7. The modified boron carbide nanoparticle powder according to claim 6, wherein the modified boron carbide nanoparticle powder has a number-based median diameter of 40 to 66 nm as measured in pure water by dynamic light scattering.

8. The modified boron carbide nanoparticle powder according to claim 7, wherein the modified boron carbide nanoparticle powder has a number-based median diameter of 45 to 55 nm as measured in pure water by dynamic light scattering.

2. The modified boron carbide nanoparticle powder according to claim 1, wherein the boron carbide nanoparticles have a number-based median diameter of 30 to 55 nm as measured in pure water by dynamic light scattering.

10. The modified boron carbide nanoparticle powder of claim 1, wherein the boron carbide nanoparticles have a non-spherical shape. 　 A neutron capture therapy drug comprising the modified boron carbide nanoparticle powder of any one of claims 1 to 10. 　 The drug for neutron capture therapy according to claim 11, which has an antitumor effect. 　 The neutron capture therapy drug according to claim 12, which has high tumor retention. 　 The drug for neutron capture therapy according to claim 11, which exerts an antitumor effect when administered by injection. 　 The pharmaceutical composition for neutron capture therapy according to claim 14, wherein the injection is a one-shot intravenous injection or a bolus intravenous injection.

16. The drug for neutron capture therapy according to claim 15, which has high tumor retention, capable of maintaining a boron concentration of 60 ppm or more for three days after a single intravenous injection. 　 The drug for neutron capture therapy according to claim 11, which exhibits high tumor retention by a single one-shot intravenous injection or a bolus intravenous injection and allows repeated irradiation with neutron beams. 　 The drug for neutron capture therapy according to claim 11, which induces an immunopotentiating effect when irradiated with neutron rays. 　 The drug for neutron capture therapy according to claim 18, which exerts an additional antitumor effect due to the immunopotentiating effect induced by neutron irradiation.

20. The drug for neutron capture therapy according to claim 19, which exerts an abscopal effect by virtue of an immunopotentiating effect induced by neutron irradiation. 　 The neutron capture therapy drug according to claim 11 , used in combination with an immune checkpoint inhibitor. 　 A drug for neutron capture therapy that induces an immunopotentiating effect after neutron irradiation, comprising the drug for neutron capture therapy according to claim 11 as an active ingredient. 　 A method for producing modified boron carbide nanoparticle powder according to any one of claims 1 to 10, comprising the steps of: (1) Boron oxide is reacted with magnesium and graphite to form boron carbide nanoparticles by mechanochemical reaction; (2) The resulting nano-sized boron carbide nanoparticles are reacted with glycidol to produce modified boron carbide nanoparticle powders whose surfaces are modified with polyglycerol. 　 The method according to claim 23, wherein the mechanochemical reaction is a grinding treatment using a ball mill.

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