JP2023018447A - Modified boron carbide particles and neutron capture therapy agent containing the same - Google Patents

Abstract

To provide a new boron preparation.SOLUTION: A modified boron carbide particle powder contains boron carbide particles, wherein at least part of the surface of at least some of the boron carbide particles in the boron carbide particles is modified with polyglycerol. The polyglycerol-modified boron carbide particle powder is inherently less toxic and can be administered in a shorter time, and can be suitably used in boron neutron capture therapy applications.SELECTED DRAWING: None

Description

There is an application for the application of Article 30, Paragraph 2 of the Patent Act. https://kaken. nii. ac. jp/report/KAKENHI-PROJECT-17H02738/17H02738seika/ Website publication date: February 19, 2021 2. KURNS Progress Report 2019 Issued by the Institute for Integrated Radiation and Nuclear Science, Kyoto University (published by "Institute for Integrated Radiation and Nuclear Science", Kyoto University), page 20, 31P3-3 Publication date: August 2020

TECHNICAL FIELD The present invention relates to modified boron carbide particles and agents for neutron capture therapy containing the same, and more particularly, boron carbide particles in which at least a portion of the surface of the boron carbide particles is modified with polyglycerol and for neutron capture therapy containing the same. Regarding drugs.

Boron Neutron Capture Therapy (BNCT) is a non-invasive and side-effect-free cancer therapy based on a particle beam generated by a nuclear reaction between neutrons and boron ( ¹⁰ B) (e.g., non-patent literature 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 (see, for example, Non-Patent Documents 2 and 3). However, the boron preparations used for it still have insufficient dispersibility and tumor accumulation, etc., and the development of new boron preparations is required for the spread of BNCT.

As BNCT preparations, BSH (mercaptoundecahydrododecaborate), which is a cage-like cluster of boron, and BPA (p-boronophenylalanine), which is an amino acid (phenylalanine) introduced with boron, have been used (see Non-Patent Documents 4 and 5). ). The former (BSH) has the problem of low tumor selectivity, and the latter (BPA) has the problem of low water solubility. BPA is manufactured and sold by Stella Pharma Co., Ltd. under the trade name of borofaran, but due to its low water solubility, tens of grams of BPA are currently administered by infusion over several hours. In addition, it is known that BPA is taken up by cancer cells via LAT1 (L-amino acid transporter 1), and there is also the problem that applicable carcinomas are limited. Therefore, the development of new boron formulations designed from a completely new perspective is strongly required for the future development of BNCT.

In Patent Document 1, the surfaces of boron carbide particles having a particle size of 200 nm to 1000 nm are coated in this order with polylysine and polyglutamic acid, and then modified with transferrin. Disclosed are particles with selective uptake into tumor cells, and agents for neutron capture therapy comprising the particles, wherein the groups are bridging and bound to said transferrin. Patent Document 1 discloses that modification of boron carbide particles having the above-mentioned particle size with transferrin allows efficient incorporation into cancer cells (see 0009, 0013, 0027 to 0035, etc. of Patent Document 1).

Patent No. 6142994

M. A. Dymova, S. Y. Taskaev, V. A. Richter, E. V. Kuligina, Cancer Commun. 2020, 40, 406. M. Suzuki, Int. J. Clin. Oncol. 2020, 25, 43. R. F. Barth, J. C. Grecula, Appl. Radiat. Isot. 2020, 160, 109029. Haritz, D., et al., Int. J. Radiat. Oncol. Biol Phys., 28 1175-1181(1994). Ryynanen, P.M., 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.

New agents for boron neutron capture therapy are expected to have the following properties: (1) inherently low toxicity, (2) shorter administration time (more water-soluble or (3) the ability to accumulate boron at a higher concentration in tumors (the ¹⁰ B concentration in tumor cells is preferably 20 ppm (wt/wt) or more); and (4) It can persist in the tumor for a long time, but can be rapidly cleared from the blood (for example, the ratio of the ¹⁰ B concentration in the tumor to the ¹⁰ B concentration in the blood (T/B ratio) is more preferably 3 or more. preferable).

As a result of extensive studies, the present inventors have found that by reacting glycidol with boron carbide particles, modified boron carbide particles in which at least a portion of the surface of the boron carbide particles is modified with polyglycerol can be newly obtained. I found out. Furthermore, such modified boron carbide particles are inherently less toxic, can be administered more quickly, can accumulate higher concentrations of boron in tumors, can persist longer in tumors, but can be removed from the blood. The inventors have found that it is suitable for use in boron neutron capture therapy, such as being able to be removed rapidly, and have completed the present invention.

This specification includes the following forms.
1. A boron carbide particle powder comprising boron carbide particles, wherein at least a portion of the surface of at least a portion of the boron carbide particles among the boron carbide particles is modified with polyglycerol.
2. 2. The modified boron carbide particle powder according to 1 above, which has a median diameter of more than 0.5 nm and not more than 1000 nm.
3. 3. The modified boron carbide particle powder according to 1 or 2 above, wherein the polyglycerol content is 0.1% by mass or more and 99.9% by mass or less based on the modified boron carbide particles (100% by mass).
4. 4. The modified boron carbide particle powder according to any one of 1 to 3 above, wherein the average particle size of the boron carbide particles is less than 1000 nm.
5. 5. The modified boron carbide particle powder according to any one of 1 to 4 above, which has a spherical shape.
6. A medicament for neutron capture therapy comprising the modified boron carbide particle powder according to any one of 1 to 5 above.
7. A method for producing modified boron carbide particles having at least a portion of the surface modified with polyglycerol, comprising reacting the boron carbide particles with glycidol.
8. A method for producing modified boron carbide particles according to 7 above, comprising producing the boron carbide particles using a pulsed laser melting method.

The modified boron carbide particle powder of an embodiment of the present invention comprises boron carbide particles, and at least a portion of the surfaces of at least some of the boron carbide particles are modified with polyglycerol. Thus, the modified boron carbide particle powders of embodiments of the present invention are inherently less toxic, can be administered in a shorter period of time (more water-soluble or water-dispersible), and are more highly intratumoral. It has been shown that it can accumulate boron in concentrations, that it can persist in tumors for a long time, but that it can be rapidly cleared from the blood, etc., and can be suitably used for boron neutron capture therapy applications.

1 shows an SEM image of polyglycerol-unmodified boron carbide ( ¹⁰ B ₄ C) nanoparticles of Comparative Example 1. FIG. 2 shows the FTIR spectra of the ¹⁰ B ₄ C-PG nanoparticles of Example 1 and the ¹⁰ B ₄ C nanoparticles of Comparative Example 1. FIG. FIG. 3 shows the change in mass (TGA) versus temperature for the ¹⁰ B ₄ C-PG nanoparticles of Example 1 and the ¹⁰ B ₄ C nanoparticles of Comparative Example 1. FIG. FIG. 4 shows the concentration of ¹⁰ B ₄ C-PG nanoparticles of Example 3 in primary tissues, tumor and blood versus time from injection of ¹⁰ B ₄ C-PG nanoparticles. FIG. 5 shows the ¹⁰ B concentration in tumor (T), the ¹⁰ B concentration in blood (B), and the ¹⁰ B concentration in tumor (T) and blood for the ¹⁰ B ₄ C-PG nanoparticles of Example 3. The ratio of ¹⁰ B concentration in (B) (T/B ratio) is shown versus time from ¹⁰ B ₄ C-PG nanoparticle injection. 6 shows in vivo BCNT results (tumor size) for ¹⁰ B ₄ C-PG nanoparticles of Example 1. FIG. FIG. 7 shows the in vivo BCNT results (mouse body weight) for the ¹⁰ B ₄ C-PG nanoparticles of Example 1. FIG. FIG. 8 shows the ¹⁰ B concentration in tumor (T), the ^{10 B concentration in blood (B), and the 10 B} concentration in tumor (T) and blood for the ¹⁰ B ₄ C-PG nanoparticles of Example 1. The ratio of ¹⁰ B concentration in (B) (T/B ratio) is shown versus time from ¹⁰ B ₄ C-PG nanoparticle injection. FIG. 9 shows images of tumor tissue sections taken 24 hours or 48 hours after injection of ¹⁰ B ₄ C-PG nanoparticles of Example 1, observed using a transmission electron microscope (TEM). .

In one aspect, the present specification provides a boron carbide particle powder comprising boron carbide particles, wherein at least a portion of the surface of at least a portion of the boron carbide particles among the boron carbide particles is modified with polyglycerol. do. In the present specification, the term "particle powder" means an aggregate of particles (granules or a plurality of particles), and may be simply described as "particles".
As used herein, the term “boron carbide particles” refers to an inorganic material made of carbon and boron, the composition formula of which is generally described as B ₄ C, and the modified boron carbide particles targeted by the present invention. There are no particular restrictions as long as powder can be obtained.

The boron in boron carbide consists mainly of ^10B and ^11B , and the boron in particles derived from natural boron carbide contains about 80.1% ^11B and about 19.9% ^10B . Natural boron carbide can be used as the boron carbide, as long as the modified boron ^carbide particle powder and the neutron capture therapy agent containing the same can be obtained. The B content may be, for example, about 19.9%. Regarding boron in boron carbide, the content of ¹⁰ B is preferably 50% or more, more preferably 75% or more, still more preferably 90% or more, and 98% or more. Even more preferred. When the content of ¹⁰ B is 50% or more, when used for boron neutron capture therapy, the higher the content of ¹⁰ B, the better the neutron capture ability and the more efficient boron neutron capture therapy. can be done.

The average particle diameter of the boron carbide particles is, for example, less than 1000 nm, less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, preferably less than 500 nm, preferably less than 400 nm. Preferably, it is less than 300 nm, more preferably less than 250 nm, even more preferably less than 200 nm, even more preferably less than 150 nm, even more preferably less than 100 nm.

The average particle size of the boron carbide particles may be, for example, less than 0.5 nm, may be 0.5 nm or more, preferably 1.0 nm or more, preferably 1.5 nm or more, and 2.0 nm. It is preferably 2.5 nm or more, more preferably 3.0 nm or more, more preferably 3.5 nm or more, and even more preferably 4.0 nm or more. , more preferably 5.0 nm or more, more preferably 6.0 nm or more, still more preferably 7.0 nm or more, still more preferably 8.0 nm or more, and 10 nm or more is even more preferred.

The average particle diameter of the boron carbide particles is, for example, 0.5 nm or more and less than 1000 nm, preferably 1.0 nm or more and less than 500 nm, more preferably 2.5 nm or more and less than 300 nm, and 5.0 nm or more and 200 nm. It is more preferably less than 5.0 nm or more and less than 150 nm, and even more preferably 10 nm or more and less than 100 nm.
When the average particle diameter of the boron carbide particles is less than 1000 nm, it is less toxic and more water-soluble or water-dispersible. When it is 2.5 nm or more and less than 300 nm, it is less toxic and more water-soluble. or a good balance between good water dispersibility, relatively longer residence time in the blood, and easy uptake by tumor tissue.
The average particle size of boron carbide particles can be measured, for example, by SEM observation as described in the Examples. More specifically, for 100 particles in the SEM image, the major axis and minor axis were measured to obtain the average value of the major axis and minor axis. Furthermore, an average particle size was obtained by averaging the average value of the major axis and the average value of the minor axis.

At least part of the surface of the boron carbide particles is modified with polyglycerol.
As used herein, the term "polyglycerol" refers to a substance having a large number of "glycerol (or glycerin)" units and a large number of hydroxyl groups, and the modified boron carbide particle powder targeted by the present invention can be obtained. There is no particular limit as long as Boron carbide particles are inherently less toxic because at least a portion of their surface is modified with polyglycerol, are more water soluble or dispersible, and accumulate boron at higher concentrations within tumors. The ability to persist in tumors for a long period of time yet be rapidly cleared from the blood makes it suitable for use in boron neutron capture therapy applications.

As a "glycerol (or glycerin)" unit, for example, the chemical structure shown in the following chemical formula (I) can be exemplified.

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-]

The polyglycerol can contain other units, such as ethyleneoxy, propyleneoxy, etc., as long as the modified boron carbide particle powder targeted by the present invention can be obtained, but need not necessarily contain it. . In addition, polyglycerol can contain other substances as long as the modified boron carbide particle powder aimed at by the present invention can be obtained, and such substances preferably have hydrophilic properties, such as polyethylene glycol, Polypropylene glycol, polyethylene glycol/polypropylene glycol copolymers, etc. can be exemplified, but need not necessarily be included.

The content of polyglycerol is preferably 0.1% by mass or more and 99.9% by mass or less, and 1% by mass or more and 99% by mass or less based on the modified boron carbide particle powder (100% by mass). More preferably, it is 5% by mass or more and 90% by mass or less, even more preferably 7% by mass or more and 85% by mass or less, and even more preferably 10% by mass or more and 80% by mass or less. preferable.
When the amount of polyglycerol is 0.1% by mass or more and 99.9% by mass or less based on the modified boron carbide particle powder (100% by mass), the modified boron carbide particle powder has appropriate water solubility. It can have the advantage of having a size.

The median diameter of the modified boron carbide particles of the embodiment of the present invention is, for example, preferably 1000 nm or less, preferably 900 nm or less, preferably 800 nm or less, preferably 700 nm or less, and 600 nm. It is preferably 500 nm or less, more preferably 400 nm or less, still more preferably 300 nm or less, even more preferably 250 nm or less, and further preferably 200 nm or less. It is preferably 150 nm or less, even more preferably 100 nm or less.

The median diameter of the modified boron carbide particles is, for example, preferably larger than 0.5 nm, more preferably larger than 1.0 nm, more preferably larger than 1.5 nm, more preferably larger than 2.0 nm, more preferably greater than 2.5 nm, more preferably greater than 3.0 nm, more preferably greater than 3.5 nm, even more preferably greater than 4.0 nm, even more preferably greater than 5.0 nm , is even more preferably greater than 6.0 nm, even more preferably greater than 7.0 nm, even more preferably greater than 8.0 nm, and even more preferably greater than 10 nm.

The median diameter of the modified boron carbide particles of the embodiment of the present invention is preferably greater than 0.5 nm and no greater than 1000 nm, more preferably greater than 1.0 nm and no greater than 500 nm, and greater than 2.5 nm and no greater than 300 nm. more preferably greater than 5.0 nm and 200 nm or less, even more preferably greater than 5.0 nm and 150 nm or less, and even more preferably greater than 10 nm and 100 nm or less.
When the median diameter of the modified boron carbide particles is greater than 0.5 nm and 1000 nm or less, the modified boron carbide particles are less toxic, more excellent in water solubility or water dispersibility, and have a median diameter of greater than 2.5 nm and 300 nm or less. In this case, there is a good balance between lower toxicity, better water solubility or water dispersibility, and easier uptake by the tumor tissue of the modified boron carbide particle powder.
The median size of modified boron carbide particles can be measured, for example, using dynamic light scattering (DLS) as described in the Examples.

The modified boron carbide particles of embodiments of the present invention preferably have a spherical shape. The shape can be observed using TEM, for example, as described in the Examples. When the modified boron carbide particles have a spherical shape, the surface chemical modification is more uniformly performed, and advantageous effects such as better water solubility or water dispersibility are exhibited.
The ratio of the length of the longest axis (major axis) to the length of the shortest axis (minor axis) of the particles in the TEM image (major axis length/minor axis length) is preferably 10 or less, It is more preferably 5 or less, still more preferably 3 or less, and even more preferably 2 or less. When the ratio (major axis length/minor axis length) is 10 or less, the surface chemical modification is performed more uniformly and the water solubility or water dispersibility is more excellent. The surface chemical modification is performed more homogeneously, and an advantageous effect of more excellent water-solubility or water-dispersibility is exhibited.

In another aspect, the present invention can provide a neutron capture therapy agent comprising the modified boron carbide particle powders of the present embodiments. The drug can have various forms as long as it can be used for neutron capture therapy, and the form is not particularly limited. Examples of such forms include suspensions in water, dispersions in water, suspensions in phosphate-buffered saline, dispersions in phosphate-buffered saline, other suspensions and dispersions, solids, gels, and the like. can be done.

As long as it can be used for neutron capture therapy, the drug can contain various additives and the like in addition to the modified boron carbide particle powder of the embodiment of the present invention, and the additives are particularly limited. no. Examples of such additives include anticancer agents, photosensitizers, solubilizing aids, surfactants, lipids, carbohydrates, proteins, polymers, biomaterials, and the like.

The drug preferably contains 0.1 to 100% by mass, more preferably 1 to 80% by mass, and even more preferably 5 to 60% by mass of the modified boron carbide particle powder of the embodiment of the present invention. , 10 to 50 mass %. The drug can be injected in a more stable dispersion state when the modified boron carbide particle powder of the embodiment of the present invention is contained in an amount of 0.1 to 100% by mass. In addition to being able to be injected in a stable dispersed state, other functions can be added by including various additives as described above, and when 10 to 50% by mass, the drug can be injected in a more stable dispersed state; By including various additives as described above, other functions can be added and improved, and the balance between these can be improved.

The neutron capture therapeutic agent of the present invention is inherently less toxic, can be administered in a shorter period of time (more water-soluble or water-dispersible), and has a higher concentration of boron in the tumor. can accumulate, can be retained in tumors for a long time, but can be rapidly cleared from the blood.

The agent for neutron capture therapy according to the embodiment of the present invention has a ¹⁰ B concentration of, for example, 0.1 mg/mL or higher, preferably 0.5 mg/mL or higher, and 1 mg/mL or higher. More preferably, it is 2 mg/mL or more, and even more preferably 5 mg/mL or more. Since the drug for neutron capture therapy of the embodiment of the present invention is superior in dispersibility, it can be used as a drug with a relatively high concentration of ¹⁰ B. Therefore, administration time can be shortened.

Neutron capture therapy agents of embodiments of the present invention can be used, for example, at dosages of 0.01 mg [ ¹⁰ B]/kg or greater, and are used at dosages of 0.1 mg [ ¹⁰ B]/kg. Preferably, it is used at a dosage of 0.5 mg [ ¹⁰ B]/kg, more preferably at a dosage of 1 mg [ ¹⁰ B]/kg.

Neutron capture therapeutic agents of embodiments of the present invention are capable of accumulating boron to higher concentrations in tumors, with ¹⁰ B concentrations (wt/wt) in tumor cells 24 hours after drug injection, e.g. It is preferably 3 ppm or more, more preferably 6 ppm or more, still more preferably 10 ppm or more, and even more preferably 20 ppm or more. ¹⁰ B concentration in tumor cells can be measured by the method described in the Examples.

The neutron capture therapeutic agents of embodiments of the present invention can persist in tumors for long periods of time, but can be rapidly cleared from the blood, resulting in an increase in the ¹⁰ B concentration in tumors (T) to the ¹⁰ B concentration in blood (B). The ratio (T/B ratio) 24 hours after drug injection is, for example, 0.5 or more, preferably 1 or more, more preferably 2 or more, and even more preferably 3 or more. , 4 or more is even more preferred. The T/B ratio can be measured by the method described in Examples.

The method for producing the modified boron carbide particle powder of the embodiment of the present invention is not particularly limited as long as it can be produced.
In a preferred aspect, the present invention provides a method for producing modified boron carbide particles having at least a portion of the surface modified with polyglycerol, comprising reacting the boron carbide particles with glycidol. A reaction formula between the boron carbide particles and glycidol is schematically shown in the following chemical formula. Although only hydroxyl groups are described on the boron carbide particles of the chemical formula below, they may also have other groups such as carboxyl groups.

As long as the boron carbide particles and glycidol can actually be reacted and at least part of the boron carbide particles can be modified with polyglycerol, the reaction method is not particularly limited. For example, a method of irradiating ultrasonic waves, a method of using an epoxy ring polymerization catalyst, a method of appropriately heating, a method of irradiating with UV light, a method of using a solvent, and a method of dropping glycidol can be exemplified. . For example, reaction conditions such as reaction time, reaction temperature, and reaction concentration can be appropriately selected.

In the method for producing modified boron carbide particles of the embodiment of the present invention, the method for producing 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, boron carbide particles can be produced using a pulsed laser melting method, a chemical vapor deposition method, an arc discharge method, a coprecipitation (liquid phase synthesis) method, a ball mill (solid phase synthesis) method, or the like. A pulsed laser melting process is preferably used to produce the boron carbide particles.
Boron carbide particles may be used after adjusting the particle size. For example, the particle size can be adjusted using methods such as elutriation, chromatography, and centrifugation.

A method for producing modified boron carbide particles according to an embodiment of the present invention includes the above-described boron carbide particles as they are, and at least a portion of the surface of at least a portion of the boron carbide particles among the boron carbide particles is modified with polyglycerol. It can be used as a method for producing powdered boron carbide particles. That is, the present specification
A boron carbide particle powder containing boron carbide particles, wherein at least a part of the surface of at least a part of the boron carbide particles among the boron carbide particles is modified with polyglycerol, comprising reacting the boron carbide particles with glycidol. Accordingly, a method for producing modified boron carbide particles can be provided. The above description can be referred to for the reaction method between boron carbide particles and glycidol, reaction conditions, method for producing boron carbide particles, and the like.

The present invention can provide neutron capture therapy using the neutron capture therapy agents of the embodiments of the present invention.

EXAMPLES Hereinafter, the present invention will be described specifically and in detail with reference to examples and comparative examples, but these examples are merely one aspect of the present invention, and the present invention is not limited by these examples.
In addition, in the description of the examples, unless otherwise specified, parts by weight and weight % are based on the portion not considering the solvent.

Example 1. Preparation of Polyglycerol Modified Boron Carbide ( ¹⁰ B ₄ C-PG) Nanoparticles ¹⁰ B ₄ C was synthesized from ¹⁰ B in ethyl acetate using a pulsed laser melting method and the ¹⁰ B ₄ C was centrifuged. By doing so, the ¹⁰ B ₄ C nanoparticles were separated to obtain boron carbide ( ¹⁰ B ₄ C) nanoparticles (see Non-Patent Document 6). Centrifugation was performed using Avanti JE (trade name) manufactured by Beckman Coulter. As a result of SEM observation, spherical nanoparticles with an average particle size of about 60 nm were confirmed (see FIG. 1). SEM observation was performed using an S-4800 scanning electron microscope manufactured by Hitachi High-Technologies Corporation. The polyglycerol-unmodified boron carbide ( ¹⁰ B ₄ C) nanoparticles are also referred to as boron carbide nanoparticles of Comparative Example 1.

Glycidol (10 mL) per boron carbide ( ¹⁰ B ₄ C) nanoparticles (50 mg) was added to a round bottom flask, mixed, and sonicated in a bath for 15-30 minutes until the nanoparticles were evenly dispersed. Bath sonication was performed using a Branson 5800 (trade name) at 20°C. The mixture was then stirred at 140° C. for 2 hours to obtain a suspension. The resulting suspension was centrifuged at 50400 g for 30 minutes to obtain particles. The particles were washed 3-4 times with MiliQ water to remove the polyglycerol-modified boron carbide ( ¹⁰ B ₄ C-PG) nanoparticle of Example 1 until the supernatant was clear and transparent with no Tyndall phenomenon. Particles were obtained. The chemical reaction is schematically shown in the following chemical formula. Schematic representation of surface modification of ¹⁰ B ₄ C with hyperbranched polyglycerol (PG) via ring-opening polymerization of glycidol.

The dispersibility of the ¹⁰ B ₄ C-PG nanoparticles of Example 1 in water and phosphate-buffered saline was investigated. First, the dispersibility in water was evaluated by adding 15 mg of ¹⁰ B ₄ C-PG nanoparticles to 1.00 mL of water, irradiating with ultrasonic waves, allowing the mixture to stand for two days, and then confirming that no precipitate was formed. and the dispersibility in water was determined to be 15 mg/mL. Next, 0.11 mL of 10-fold concentrated phosphate-buffered saline solution was added to the aqueous dispersion, and after sonication, it was allowed to stand for 2 days. Dispersibility in water was determined to be 13 mg/mL.

The nanoparticles of Example 1 were analyzed using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and dynamic light scattering (DLS) to determine the structure of the nanoparticles, the amount of polyglycerol layer and hydrodynamic diameter were measured respectively.
The IR measurement was performed using IR Prestige-21 (trade name) manufactured by Shimadzu Corporation. The FTIR spectrum was obtained by collecting 32 scans at a resolution of 4 cm ^{−1 in the wavenumber range from 4000 to 500 cm −1 .}
2 shows the FTIR spectra of the ¹⁰ B ₄ C-PG nanoparticles of Example 1 and the ¹⁰ B ₄ C nanoparticles of Comparative Example 1. FIG. The ¹⁰ B ₄ C nanoparticles in the FTIR spectrum show peaks at 1130 cm ⁻¹ and 1612 cm ⁻¹ assigned to icosahedral B ₄ C and a peak around 1415 cm ⁻¹ for the ¹⁰ BO vibration on the surface of ¹⁰ B ₄ C. indicates When compared to B ₄ C containing ¹⁰ B and ¹¹ B (natural abundance of ¹⁹ % and 81%, _respectively ), the lower atomic weight of B in ¹⁰ B ₄ C than in B ₄ C results in all The peak is shifted to higher wavenumbers. This indicates that the nanoparticles used in Example ¹ are 10B enriched (enhanced). In the ^FTIR spectra of ¹⁰ B ₄ C ^{- PG} nanoparticles, poly A typical peak for glycerol appears. This indicates a polyglycerol coating _on the ^10B4C surface.

TGA was performed by heating from room temperature to 600° C. at a heating rate of 20° C./min in a nitrogen atmosphere using Q-50 analyzer manufactured by TA Instruments.
FIG. 3 shows the change in mass versus temperature for the ¹⁰ B ₄ C-PG nanoparticles of Example 1 and the ¹⁰ B ₄ C nanoparticles of Comparative Example 1. FIG. The polyglycerol layer in the ¹⁰ B ₄ C-PG nanoparticles of Example 1 was about 73% by weight.

DLS was measured five times on samples in number mode using a Microtrac Nanotrac UPA-UT151 system. The median diameter (d50) of ¹⁰ B ₄ C nanoparticles in water was 62.1 nm, while the median diameter of ¹⁰ B ₄ C-PG nanoparticles increased to 72.5 nm. From this, the thickness of the polyglycerol layer of the ¹⁰ B ₄ C-PG nanoparticles of Example 1 is considered to be 5.2 nm.

Preparation of Polyglycerol-Modified Boron Carbide ( ¹⁰ B ₄ C-PG) Nanoparticles of Examples 2-4 Using methods similar to those described in Example 1, the ¹⁰ B ₄ C- PG nanoparticles were produced. The results are summarized in Table 1 below together with the results of Example 1. Comparative Example 1 shows ¹⁰ B ₄ C nanoparticles obtained by removing the polyglycerol layer from the ¹⁰ B ₄ C-PG nanoparticles of Example 1.

Pharmacokinetics of Polyglycerol-Modified Boron Carbide ( ¹⁰ B ₄ C-PG) Nanoparticles From the viewpoint of application to in vivo BNCT, which will be described later, pharmacokinetic experiments on the above-mentioned ¹⁰ B ₄ C-PG nanoparticles were conducted as follows. rice field.
Animals and Tumor Models Female BALB/c mice, 8-11 weeks old, were housed in the KUR Animal Care Facility (Kumatori Campus, Kyoto University) with free access to water and standard food. All animal experiments were performed in accordance with standards approved by the Kyoto University Ethics Committee. Approximately 1×10 ⁶ CT26 cells suspended in PBS (100 μL) were implanted subcutaneously in the right paw of BALB/c mice 7-9 days prior to injection of the nanoformulation. Mice were kept pathogen-free, handled and housed according to the Science Counsel of Japan guidelines. All animal experiments were performed according to the standards of the Experimental Animal Center of Kyoto University.

Pharmacokinetic Experiments ¹⁰ B ₄ C-PG nanoparticles in PBS dispersion (200 μL) were injected into tumor-bearing BALB/c mice via the tail vein after the long axis of the tumor exceeded 10 mm. After a period of time, mice were euthanized and boron content in blood, tumors and organs (liver, spleen, kidney) was assessed. Samples were placed in Teflon tubes and the ¹⁰ B concentration of each sample was measured by a rapid gamma-ray microanalytical system to obtain ppm-order concentrations (μg [ ¹⁰ B]/g [biomaterial]). (See Non-Patent Document 7).

Pharmacokinetics of ¹⁰ B ₄ C-PG Nanoparticles of Example 3 A _{pharmacokinetic} study of ¹⁰ B ₄ C-PG nanoparticles of Example 3 was carried out by ^adding a PBS dispersion (200 μL) of A dose of 0.9 mg [ ¹⁰ B]/kg [mouse] was injected.
FIG. 4 shows the concentration of ¹⁰ B ₄ C-PG nanoparticles of Example 3 in primary tissues, tumor and blood versus time after injection of ¹⁰ B ₄ C-PG nanoparticles. ^{10 B concentrations in spleen, liver and blood were much higher in early stages than 10 B} concentrations in kidney and tumor. The ¹⁰ B concentrations in the spleen and liver remained nearly the same until 48 hours, but the tumor ¹⁰ B concentrations gradually increased, reaching a maximum (36.7 ppm) at 24 hours and reaching above 20 ppm by 48 hours. had maintained.
Figure 5 shows ^{10 B concentration in tumor (T), 10 B concentration in blood (B), and the ratio of 10 B concentration} in tumor (T) to ¹⁰ B concentration in blood (B) (T/ B ratio) is shown versus time from ¹⁰ B ₄ C-PG nanoparticle injection. In BNCT against cancer, it is more preferable that the concentration of ¹⁰ B in the tumor (T) is 20 ppm or more, and in FIG. 5, this condition was satisfied for a considerably long period of 18 to 48 hours.
It should be noted that the ¹⁰ B ₄ C-PG nanoparticles of Example 3 did not exhibit acute toxicity, as none of the mice (n=6) died over 48 hours.

While sufficient ¹⁰ B concentration levels for BNCT were maintained in tumors for 30 hours from 18 to 48 hours, ¹⁰ B concentrations in blood gradually decreased with a half-life of 7.3 hours. Therefore, the T/B ratio increased with time and reached 4.4 in 48 hours, which satisfied T/B=3 or more, which is more preferable in BNCT. As described above, from the time at which the maximum ¹⁰ B concentration ₍ ^36.7 ppm) in the tumor and the time at which the maximum T/B ratio are shown in FIG. Decided to shoot the line.

In vivo BNCT of polyglycerol-modified boron carbide ( ¹⁰ B ₄ C-PG) nanoparticles
In vivo BNCT of ¹⁰ B ₄ C-PG nanoparticles was performed as follows.
Mice were injected with ¹⁰ B ₄ C-PG nanoparticles using methods similar to those described in the pharmacokinetic studies. Twenty-four or forty-eight hours after injection of ¹⁰ B ₄ C-PG nanoparticles, mice were placed in an acrylic holder with a circular hole in the center and irradiated with neutrons. The acrylic holder was covered with a 5 mm thick thermoplastic plate containing 40 wt% ⁶ LiF (96% ⁶ Li) to block thermal neutrons. The thigh with the tumor (thigh) was stretched into the hole and exposed to neutrons at a fluence of 5×10 ⁹ cm ⁻² s ⁻¹ for 12 minutes or at a fluence of 6.5×10 ⁸ cm ⁻² s ⁻¹ . Irradiated for 60 minutes with a KUR (Kyoto University research reactor). The day on which neutrons were irradiated was defined as 0. Tumor volumes in all mice were measured serially by the same observer using the same calipers (vernier calipers) throughout the experiment. Tumor volumes were calculated and statistically analyzed according to Equation 1 below.

Calculation of Tumor Volume Tumor volume was calculated based on the following formula 1.
Formula 1: Volume = (major axis x single axis ² )/2
The same balance was used to measure the weight change of the mice.
When statistically analyzing tumor growth data, all data were normalized using Equation 2 below.
Equation 2: V _Relative = (Volume [Group x, Day n])/(Volume [Group x, Day 0])

Analysis of Pharmacokinetic Data Data analysis was performed by Graphpad Prism 9 (Graphpad Software, Inc., San Diego, Calif.). Blood concentration data were fitted using a one-phase decay model, which treats the concentration curve as the sum of exponential decreases. The attenuation of blood concentration of nanoparticles was approximated by Equation 3 below.
Formula 3: C _t = (C ₀ −plateau)×e ^−kt +plateau [t is time (h), _Ct is concentration (μg [ ¹⁰ B]/g [blood]), k is rate constant (h ^-1 ). ]

In vivo BNCT of ¹⁰ B ₄ C-PG nanoparticles of Example 1
Each mouse was intravenously injected with ¹⁰ B ₄ C-PG nanoparticles of Example 1 at a dosage of 34.3 mg [ ¹⁰ B]/kg [mouse]. Twenty-four hours or 48 hours after the injection of the ¹⁰ B ₄ C-PG nanoparticles, neutrons were irradiated at a fluence of 3.6×10 ¹² cm ⁻² . Tumor size and body weight were monitored for 29 days after neutron irradiation.
Concurrently in parallel, ¹⁰ B concentrations in tumor and ¹⁰ B concentration in blood were quantitated from injections of ¹⁰ B ₄ C-PG nanoparticles using methods similar to those described in the pharmacokinetic studies above. Measurements were taken after hours, 24 hours and 48 hours.

6 and 7 show the in vivo BCNT results for the ¹⁰ B ₄ C-PG nanoparticles of Example 1. FIG.
BCNT24h and BCNT48h in FIG. 6 were irradiated with neutrons 24 hours or 48 hours after injection of the ¹⁰ B ₄ C-PG nanoparticles of Example 1, respectively, and the relative tumor size versus the number of days from that time point. indicates The cold control was not injected with the ¹⁰ B ₄ C-PG nanoparticles of Example 1 and was not irradiated with neutrons, and the hot control was not injected with the ¹⁰ B ₄ C-PG nanoparticles of Example 1 but was irradiated with neutrons. Irradiate, injection control indicates the condition of injecting the ¹⁰ B ₄ C-PG nanoparticles of Example 1 but not neutron irradiation.
It can be understood by comparing the cold control and injection control with the hot control that neutron irradiation suppresses tumor growth. It can be seen that BNCT24h and BNCT48h suppress tumor size more efficiently than the hot control. BNCT24h and BNCT48h suppressed tumor size by 13% and 12%, respectively, after 29 days relative to cold controls. BNCT24h and BNCT48h suppressed tumor size by 20% and 19%, respectively, after 29 days relative to the hot control.

BCNT24h and BCNT48h in FIG. 7 are neutron-irradiated 24 hours or 48 hours after injection of the ¹⁰ B ₄ C-PG nanoparticles of Example 1, respectively, and the relative number of days since that time point. Indicates weight. Cold control, hot control and injection control represent the same experimental conditions as in FIG.
It can be understood from FIG. 7 that the weight change is suppressed by neutron irradiation. BNCT24h and BNCT48h were shown to suppress tumor growth more efficiently than the hot control, as in FIG.

FIG. 8 shows ¹⁰ B concentration in tumor (T) and blood (B) at 8, 24 and 48 hours after injection of ¹⁰ B ₄ C-PG nanoparticles of Example 1 into mice. and the ratio of ¹⁰ B concentration in tumor (T) to ¹⁰ B concentration in ^blood (B) (T/B ratio). The ¹⁰ B ₄ C-PG nanoparticles of Example 1 exhibited pharmacokinetics similar to the ¹⁰ B ₄ C-PG nanoparticles of Example 3 shown in FIG. ¹⁰ B concentrations in tumor (T) were above 20 ppm from 24 hours to 48 hours after injection. Furthermore, no mice (n=7) died over 48 hours, so it was concluded that the ¹⁰ BC-PG nanoparticles of Example 1 were not acutely toxic.

In vivo BNCT of ¹⁰ B ₄ C-PG nanoparticles of Example 2
Each mouse was intravenously injected with ¹⁰ B ₄ C-PG nanoparticles of Example 2 at a dosage of 27.9 mg [ ¹⁰ B]/kg [mouse]. Twenty-four hours or 48 hours after the injection of the ¹⁰ B ₄ C-PG nanoparticles, neutron irradiation was performed at a fluence of 2.3×10 ¹² cm ⁻² . Tumor size and body weight were monitored for 20 days after neutron irradiation.
Concurrently in parallel, ¹⁰ B concentrations in tumor and ¹⁰ B concentration in blood were quantitated from injections of ¹⁰ B ₄ C-PG nanoparticles using methods similar to those described in the pharmacokinetic studies above. Measurements were taken after hours, 24 hours and 48 hours.

Similar trends to those shown in FIGS. 6 and 7 were also observed in the in vivo BNCT of the ¹⁰ B ₄ C-PG nanoparticles of Example 2. Again, experiments were conducted under the same conditions as in Figures 6 and 7, including cold control, injection control, hot control, BNCT24 and BNCT48, and the hot control suppressed tumor size more than the cold control and injection control. BNCT24h and BNCT48h suppressed tumor size even more than the hot control. BNCT24h and BNCT48h suppressed tumor size by 52% and 39%, respectively, after 20 days relative to the cold control. BNCT24h and BNCT48h suppressed tumor size by 63% and 48%, respectively, after 20 days relative to the hot control.

TEM observation of tumors 24 hours and 48 hours after injection of ¹⁰ B ₄ C-PG nanoparticles of Example 1 into mice
To further understand BCNT with ¹⁰ B ₄ C-PG nanoparticles, sections of tumor tissue taken 24 or 48 hours after injection of the ¹⁰ B ₄ C-PG nanoparticles of Example 1 were examined in transmission mode. It was observed using an electron microscope (TEM).
Figure 9a shows a TEM image of a section of tumor tissue taken 24 hours after injection of ¹⁰ B ₄ C-PG nanoparticles of Example 1 and Figure 9b shows the ¹⁰ B ₄ C-PG nanoparticles of Example 1. Shown are TEM images of sections of tumor tissue taken 48 hours after injection of . Numerous particles were observed as black dots in both Figures 9a and b. This supports the accumulation of ¹⁰ B in tumor tissue confirmed by pharmacokinetic experiments.

The modified boron carbide particle powder of an embodiment of the present invention comprises boron carbide particles, and at least a portion of the surfaces of at least some of the boron carbide particles are modified with polyglycerol. Therefore, medicaments comprising the modified boron carbide particle powders of embodiments of the present invention are inherently less toxic, can be administered in a shorter period of time (better water solubility or water dispersibility), and are more effective against tumors. Boron can be favorably used for boron neutron capture therapy applications due to its advantageous effects such as the ability to accumulate higher concentrations of boron in the tumor, the ability to persist in tumors for a long time, but the ability to be rapidly cleared from the blood.

Claims (8)

A boron carbide particle powder comprising boron carbide particles, wherein at least a portion of the surface of at least a portion of the boron carbide particles among the boron carbide particles is modified with polyglycerol. 2. The modified boron carbide particle powder according to claim 1, which has a median diameter of more than 0.5 nm and less than or equal to 1000 nm. 3. The modified boron carbide particle powder according to claim 1, wherein the content of polyglycerol is 0.1% by mass or more and 99.9% by mass or less based on the modified boron carbide particles (100% by mass). The modified boron carbide particle powder according to any one of claims 1 to 3, wherein the boron carbide particles have an average particle size of less than 1000 nm. Modified boron carbide particle powder according to any one of claims 1 to 4, having a spherical shape. A medicament for neutron capture therapy comprising the modified boron carbide particle powder according to any one of claims 1-5. A method for producing modified boron carbide particles having at least a portion of the surface modified with polyglycerol, comprising reacting the boron carbide particles with glycidol. 8. A method for producing modified boron carbide particles according to claim 7, comprising using a pulsed laser melting process to produce the boron carbide particles.

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