JP7782825B2 - Modified boron carbide particles and neutron capture therapy drugs containing the same - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies. 1. 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 Kyoto University's Institute for Integrated Radiation and Nuclear Science), page 20, 31P3-3. Publication date: August 2020.

The present invention relates to modified boron carbide particles and a neutron capture therapy drug containing the same. More specifically, the present invention relates to boron carbide particles in which at least a portion of the surface of the boron carbide particles has been modified with polyglycerol, and a neutron capture therapy drug containing the same.

Boron Neutron Capture Therapy (BNCT) is a non-invasive cancer treatment with few side effects that uses particle beams generated by the nuclear reaction between neutrons and boron ( ¹⁰ B) (see, for example, Non-Patent Document 1). With the recent spread of hospital-based neutron beam generators, insurance coverage began in May 2020, and BNCT is becoming a more familiar cancer treatment (see, for example, Non-Patent Documents 2 and 3). However, the boron formulations used in BNCT still have insufficient dispersibility and tumor accumulation, and the development of new boron formulations is required for the widespread use of BNCT.

Two types of formulations have traditionally been used for BNCT: BSH (mercaptoundecahydrododecaborate), a boron cage cluster, and BPA (p-boronophenylalanine), an amino acid (phenylalanine) to which boron has been introduced (see Non-Patent Documents 4 and 5). The former (BSH) suffers from low tumor selectivity, while the latter (BPA) suffers from low water solubility. BPA is manufactured and sold by Stella Pharma Corporation under the trade name Borofalan, but due to its low water solubility, tens of grams of BPA are currently administered intravenously over several hours. Furthermore, BPA is known to be taken up by cancer cells via LAT1 (L-amino acid transporter 1), limiting the types of cancers to which it can be applied. Therefore, the future development of BNCT strongly demands the development of new boron formulations designed from an entirely new perspective.

Patent Document 1 discloses particles capable of selective uptake by tumor cells, in which the surfaces of boron carbide particles with particle sizes of 200 nm to 1000 nm are coated with polylysine and then polyglutamic acid, which are then modified with transferrin, with the polyglutamic acid and the transferrin being bound to each other via crosslinking via carboxyl groups of the polyglutamic acid, and a drug for neutron capture therapy comprising such particles. Patent Document 1 discloses that boron carbide particles of the above particle size, when modified with transferrin, are efficiently taken up by cancer cells (see paragraphs 0009, 0013, 0027-0035, etc. of Patent Document 1).

Patent No. 6142994 specification

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. Instrument. Methods 1983, 204, 525.

New drugs for boron neutron capture therapy are expected to be required to have the following properties: (1) inherently low toxicity, (2) ability to be administered in a shorter time (superior water solubility or water dispersibility), (3) ability to accumulate boron at a higher concentration in the tumor (preferably, the ^10B concentration in tumor cells is 20 ppm (wt/wt) or more), and (4) ability to persist in the tumor for a long period of time but be rapidly removed from the blood (for example, the ratio of the ^10B concentration in the tumor to the ^10B concentration in the blood (T/B ratio) is preferably 3 or more).

After extensive research, the inventors discovered that by reacting boron carbide particles with glycidol, modified boron carbide particles can be obtained, in which at least a portion of the surface of the boron carbide particles is modified with polyglycerol. Furthermore, they discovered that such modified boron carbide particles are suitable for boron neutron capture therapy because they are inherently 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 time while being rapidly removed from the blood, leading to the completion of the present invention.

This specification includes the following forms.
1. A boron carbide particle powder comprising boron carbide particles, at least a portion of the surface of at least some of the boron carbide particles being modified with polyglycerol.
2. The modified boron carbide particles according to item 1, having a median diameter of more than 0.5 nm and not more than 1000 nm.
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. The modified boron carbide particle powder according to any one of 1 to 3 above, wherein the boron carbide particles have an average particle size of less than 1000 nm.
5. The modified boron carbide particles according to any one of 1 to 4 above, which have a spherical shape.
6. A drug 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, at least a portion of the surface of which is modified with polyglycerol, comprising reacting boron carbide particles with glycidol.
8. A method for producing modified boron carbide particles according to claim 7, comprising producing the boron carbide particles using pulsed laser melting.

The modified boron carbide particle powder of an embodiment of the present invention comprises boron carbide particles, at least some of the surfaces of which are modified with polyglycerol. Therefore, the modified boron carbide particle powder of an embodiment of the present invention exhibits inherently low toxicity, can be administered in a shorter time (having better water solubility or water dispersibility), can accumulate boron at higher concentrations within tumors, can persist in tumors for a long time but can be rapidly removed from the blood, and is therefore suitable for use in boron neutron capture therapy.

FIG. 1 shows an SEM image of the 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. 3 shows the mass change (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. 4 shows the concentration of the ¹⁰ B ₄ C-PG nanoparticles of Example 3 in major tissues, tumor and blood, versus time from injection of the ¹⁰ B ₄ C-PG nanoparticles. FIG. 5 shows _the ¹⁰ B concentration in the tumor (T), the ¹⁰ B concentration in the blood (B), and the ratio of the ¹⁰ B concentration in the tumor (T) to the ¹⁰ B concentration in the blood (B) (T/B ratio) for the ¹⁰ B 4 C-PG nanoparticles of Example 3, relative to the time from injection of ^{the 10} B ₄ C-PG nanoparticles. FIG. 6 shows the in vivo BCNT results (tumor size) for the ¹⁰ B ₄ C-PG nanoparticles of Example 1. FIG. 7 shows the in vivo BCNT results (mouse body weight) for the ¹⁰ B ₄ C-PG nanoparticles of Example 1. FIG. 8 shows _the ¹⁰ B concentration in the tumor (T), the ¹⁰ B concentration in the blood (B), and the ratio of the ¹⁰ B concentration in the tumor (T) to the ¹⁰ B concentration in the blood (B) (T/B ratio) for the ¹⁰ B 4 C-PG nanoparticles of Example 1, relative to the time from injection of ^{the 10} B ₄ C-PG nanoparticles. FIG. 9 shows images of sections of tumor tissues taken 24 hours or 48 hours after injection of the 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, at least some of the boron carbide particles having at least a portion of the surface thereof modified with polyglycerol. Note that in this specification, the term "particle powder" refers to an aggregate of particles (a powder or a plurality of particles) and may also be simply referred to as "particles."
In this specification, "boron carbide particles" refers to an inorganic material made of carbon and boron, and its composition formula is generally expressed as _B4C . There are no particular limitations on the composition formula as long as it can provide the modified boron carbide particle powder that is the object of the present invention.

The boron in boron carbide is mainly composed of ¹⁰ B and ¹¹ B, and the boron in particles derived from natural boron carbide contains about 80.1% ¹¹ B and about 19.9% ¹⁰ B. Natural boron carbide can be used as the boron carbide 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, and ^{the 10} B content of the boron in the boron carbide may be, for example, about 19.9%. With respect to the boron in boron carbide, the ¹⁰ B content is preferably 50% or more, more preferably 75% or more, even more preferably 90% or more, and even more preferably 98% or more. When ^{the 10} B content is 50% or more, when used for boron neutron capture therapy, the higher ¹⁰ B content provides better neutron capture ability, allowing for more efficient boron neutron capture therapy.

The average particle size 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, more preferably less than 300 nm, more preferably less than 250 nm, even more preferably less than 200 nm, even more preferably less than 150 nm, and 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, or may be 0.5 nm or greater, preferably 1.0 nm or greater, preferably 1.5 nm or greater, preferably 2.0 nm or greater, more preferably 2.5 nm or greater, more preferably 3.0 nm or greater, more preferably 3.5 nm or greater, even more preferably 4.0 nm or greater, even more preferably 5.0 nm or greater, even more preferably 6.0 nm or greater, even more preferably 7.0 nm or greater, even more preferably 8.0 nm or greater, and even more preferably 10 nm or greater.

The average particle size 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, even more preferably 5.0 nm or more and less than 200 nm, even more preferably 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 size of the boron carbide particles is less than 1000 nm, the particles have lower toxicity and better water solubility or water dispersibility, and when the average particle size is 2.5 nm or more but less than 300 nm, the particles have an excellent balance of lower toxicity, better water solubility or water dispersibility, a relatively longer retention time in the blood, and easier uptake by tumor tissue.
The average particle size of the boron carbide particles can be measured, for example, by SEM observation as described in the Examples. More specifically, the major and minor diameters of 100 particles in the SEM image were measured to obtain the average values of the major and minor diameters. The average major and minor diameters were then averaged to obtain the average particle size.

The boron carbide particles have at least a portion of their surface 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 is not particularly limited as long as the modified boron carbide particle powder intended by the present invention can be obtained. Since the boron carbide particles have at least a portion of their surfaces modified with polyglycerol, they are essentially less toxic, more water-soluble or water-dispersible, can accumulate boron at higher concentrations within tumors, and can persist in tumors for a long time while being rapidly removed from the blood, making them suitable for use in boron neutron capture therapy.

An example of a "glycerol (or glycerin)" unit is the chemical structure shown in chemical formula (I) below.

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-]

Polyglycerol may contain other units, such as ethyleneoxy or propyleneoxy, as long as the modified boron carbide particle powder desired by the present invention can be obtained, but this does not necessarily have to be the case. Furthermore, polyglycerol may contain other substances, such as polyethylene glycol, polypropylene glycol, and polyethylene glycol/polypropylene glycol copolymers, as long as the modified boron carbide particle powder desired by the present invention can be obtained. Such substances are preferably hydrophilic, and examples thereof include polyethylene glycol, polypropylene glycol, and polyethylene glycol/polypropylene glycol copolymers, but this does not necessarily have to be the case.

The polyglycerol content is preferably 0.1% by mass or more and 99.9% by mass or less, more preferably 1% by mass or more and 99% by mass or less, even more preferably 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, based on the modified boron carbide particle powder (100% by mass).
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 can have the advantageous effect of having suitable water solubility and size.

The median diameter of the modified boron carbide particles according to an 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, preferably 600 nm or less, more preferably 500 nm or less, more preferably 400 nm or less, even more preferably 300 nm or less, even more preferably 250 nm or less, even more preferably 200 nm or less, even more preferably 150 nm or less, and even more preferably 100 nm or less.

The median diameter of the modified boron carbide particles is, for example, preferably greater than 0.5 nm, more preferably greater than 1.0 nm, more preferably greater than 1.5 nm, more preferably greater than 2.0 nm, even more preferably greater than 2.5 nm, even more preferably greater than 3.0 nm, even more preferably greater than 3.5 nm, even more preferably greater than 4.0 nm, even more preferably greater than 5.0 nm, 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 according to an embodiment of the present invention is preferably greater than 0.5 nm and less than or equal to 1000 nm, more preferably greater than 1.0 nm and less than or equal to 500 nm, even more preferably greater than 2.5 nm and less than or equal to 300 nm, even more preferably greater than 5.0 nm and less than or equal to 200 nm, even more preferably greater than 5.0 nm and less than or equal to 150 nm, and even more preferably greater than 10 nm and less than or equal to 100 nm.
When the median diameter of the modified boron carbide particle powder is greater than 0.5 nm and not greater than 1000 nm, the modified boron carbide particle powder has lower toxicity and better water solubility or water dispersibility, and when the median diameter is greater than 2.5 nm and not greater than 300 nm, the modified boron carbide particle powder has lower toxicity and better water solubility or water dispersibility, and the modified boron carbide particle powder is more easily taken up by tumor tissue, providing an excellent balance.
The median diameter of the 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 the present invention preferably have a spherical shape. The shape can be observed, for example, using a TEM as described in the Examples. When the modified boron carbide particles have a spherical shape, the surface chemical modification is more uniform, which advantageously results in better water solubility or water dispersibility.
The ratio of the length of the longest axis (major axis) to the length of the shortest axis (minor axis) of a particle in a TEM image (length of major axis/length of minor axis) is preferably 10 or less, more preferably 5 or less, even more preferably 3 or less, and even more preferably 2 or less. When the ratio (length of major axis/length of minor axis) is 10 or less, the surface chemical modification is more uniform and the water solubility or water dispersibility is better, and when the ratio is 3 or less, the surface chemical modification is even more uniform and the water solubility or water dispersibility is even better, which is an advantageous effect.

In another aspect, the present invention provides a drug for neutron capture therapy, comprising the modified boron carbide particle powder of an embodiment of the present invention. The drug can have various forms, and the form is not particularly limited, as long as it can be used in neutron capture therapy. Examples of forms 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.

As long as the drug can be used in neutron capture therapy, it can contain various additives in addition to the modified boron carbide particles of the present invention, and the additives are not particularly limited. Examples of such additives include anticancer drugs, photosensitizers, solubilizing agents, surfactants, lipids, carbohydrates, proteins, polymers, and biomaterials.

The drug preferably contains 0.1 to 100% by mass, more preferably 1 to 80% by mass, even more preferably 5 to 60% by mass, and even more preferably 10 to 50% by mass of the modified boron carbide particle powder of an embodiment of the present invention. When the drug contains 0.1 to 100% by 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% by 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% by 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.

The neutron capture therapy drug of 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), being able to accumulate boron at higher concentrations within tumors, and being able to remain in tumors for a long period of time while being rapidly removed from the blood.

The neutron capture therapy drug of the embodiment of the present invention may have a ^B concentration of, 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. The neutron capture therapy drug of the embodiment of the present invention has excellent dispersibility and can be used as a drug with a relatively ^high B concentration. Therefore, the administration time can be shortened.

The neutron capture therapy drug according to the embodiment of the present invention can be used, for example, at a dosage of 0.01 mg [ ¹⁰ B]/kg or more, preferably at a dosage of 0.1 mg [ ¹⁰ B]/kg, more preferably at a dosage of 0.5 mg [ ¹⁰ B]/kg, and even more preferably at a dosage of 1 mg [ ¹⁰ B]/kg.

The neutron capture therapy drug of the embodiment of the present invention can accumulate boron at a higher concentration in tumors, and ^the B concentration (wt/wt) in tumor cells 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. The ^B concentration in tumor cells can be measured by the method described in the Examples.

The neutron capture therapy drug of the embodiment of the present invention can persist in the tumor for a long period of time but can be rapidly removed from the blood, and the ratio of the ¹⁰ B concentration in the tumor (T) to the ¹⁰ B concentration in the blood (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. The T/B ratio can be measured by the method described in the Examples.

The method for producing the modified boron carbide particles according to the embodiment of the present invention is not particularly limited as long as it is possible to produce the particles.
In a preferred aspect, the present invention provides a method for producing modified boron carbide particles, at least a portion of whose surface is modified with polyglycerol, comprising reacting boron carbide particles with glycidol. The reaction between the boron carbide particles and glycidol is schematically shown in the following chemical formula. Although only hydroxyl groups are shown on the boron carbide particles in the following chemical formula, other groups such as carboxyl groups may also be present.

The reaction method between boron carbide particles and glycidol is not particularly limited, as long as it is actually possible to react and at least a portion of the boron carbide particles can be modified with polyglycerol. Examples of suitable methods include ultrasonic irradiation, using an epoxy ring polymerization catalyst, appropriate heating, UV light irradiation, using a solvent, and adding glycidol dropwise. Reaction conditions such as reaction time, reaction temperature, and reaction concentration can be selected as appropriate.

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), etc. It is preferable to produce the boron carbide particles using pulsed laser melting.
The boron carbide particles may be used after adjusting the particle size, and the particle size can be adjusted using, for example, elutriation, chromatography, centrifugation, or other methods.

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 a powder of boron carbide particles, which contains the above-described boron carbide particles, and in which at least a portion of the surface of at least some of the boron carbide particles is modified with polyglycerol.
The present invention provides a boron carbide particle powder containing boron carbide particles, at least some of the surfaces of which are modified with polyglycerol, and a method for producing modified boron carbide particles, the method including reacting boron carbide particles with glycidol. The above descriptions regarding the method for reacting boron carbide particles with glycidol, the reaction conditions, the method for producing boron carbide particles, etc. can be referenced.

The present invention can provide neutron capture therapy using a neutron capture therapy drug according to an embodiment of the present invention.

EXAMPLES Hereinafter, the present invention will be specifically and in detail explained using examples and comparative examples, but these examples are merely one embodiment of the present invention, and the present invention is not limited to these examples in any way.
In the examples, unless otherwise specified, parts by weight and percentages by weight are based on amounts excluding the solvent.

Example 1. Production of Polyglycerol-Modified Boron Carbide ( ¹⁰ B ₄ C-PG) Nanoparticles ¹⁰ B ₄ C was synthesized from ¹⁰ B in ethyl acetate using pulsed laser melting, and the ¹⁰ B ₄ C was centrifuged to separate the ¹⁰ B ₄ C nanoparticles, yielding boron carbide ( ¹⁰ B ₄ C) nanoparticles (see Non-Patent Document 6). Centrifugation was performed using an Avanti JE (trade name) manufactured by Beckman Coulter. SEM observation confirmed spherical nanoparticles with an average particle size of approximately 60 nm (see Figure 1). SEM observation was performed using an S-4800 scanning electron microscope manufactured by Hitachi High-Technologies Corporation. These polyglycerol-unmodified boron carbide ( ¹⁰ B ₄ C) nanoparticles are also referred to as the boron carbide nanoparticles of Comparative Example 1.

Glycidol ( ¹⁰ mL) per 50 mg of boron carbide ( _10B4C ) nanoparticles was added to a round-bottom flask and mixed. The mixture was then bath sonicated for 15 to 30 minutes until the nanoparticles were uniformly 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 50,400 xg for 30 minutes to obtain particles. The particles were washed 3 to 4 times with MiliQ water until the supernatant was clear and transparent without Tyndall behavior, yielding the _polyglycerol -modified boron carbide ( ^10B4C -PG) nanoparticles of Example 1. The chemical reaction is shown schematically in the following chemical formula: The surface modification of ^10B4C with hyperbranched polyglycerol (PG) via ring-opening polymerization of _glycidol is shown schematically below.

The dispersibility of the ^10B4C -PG nanoparticles of Example 1 in water and phosphate buffered saline was investigated. First, ¹⁵ mg of _10B4C -PG nanoparticles were added to 1.00 _mL of water, and after ultrasonic irradiation and standing for two days, it was confirmed that no precipitate had formed, and the dispersibility in water was determined to be 15 mg/mL. Next, 0.11 mL of 10x concentrated phosphate buffered saline was added to the aqueous dispersion, and after ultrasonic irradiation and standing for two days, it was confirmed that no precipitate had formed, and the dispersibility in phosphate buffered saline 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 the hydrodynamic diameter, respectively.
The IR measurement was carried out using an IR Prestige-21 (trade name) manufactured by Shimadzu Corporation. FTIR spectra were obtained by collecting 32 scans of spectra at a resolution of 4 cm ⁻¹ in the wavenumber range of 4000 to 500 cm ⁻¹ .
2 shows the FTIR spectra of _{the 10B4C} ^- PG nanoparticles of Example 1 and _{the 10B4C} ^{nanoparticles} of Comparative Example 1. In the FTIR spectrum, the ^10B4C nanoparticles exhibit peaks at ₁₁₃₀ cm ^-1 and 1612 cm- ¹ , which are attributed to icosahedral _B4C , and a peak around ¹⁴¹⁵ cm ^-1 , which is associated with the ^10B -O vibration on the surface of the _10B4C . Compared with _B4C containing ^10B and ^11B (natural abundances of 19% and 81%, respectively), all peaks of ^10B4C are shifted to higher wavenumbers because the atomic weight of B _{in 10B4C} is lighter than that ^of _B4C . This indicates that the nanoparticles used in Example 1 are enriched (enriched) with ^10B . The FTIR spectrum of _{the 10B4C} ^- PG nanoparticles shows typical polyglycerol peaks around 1140 cm ^-1 , 2900 cm ^-1 , and 3500 cm ^-1 , corresponding to C-O-C, C-H, and O-H stretching, respectively, indicating a polyglycerol coating on _{the 10B4C} ^surface .

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

DLS was measured five times per sample in number mode using a Microtrac Nanotrac UPA-UT151 system. The median diameter (d50) of ^10B4C _{nanoparticles} in water was 62.1 nm, while the median diameter of ^10B4C -PG nanoparticles increased to 72.5 nm. From this, the thickness of the polyglycerol layer of ^the _10B4C -PG nanoparticles in Example 1 is considered to be 5.2 _nm .

Preparation of Polyglycerol-Modified Boron Carbide ( ¹⁰ B ₄ C-PG) Nanoparticles in Examples 2 to 4 ^{The 10} B ₄ C-PG nanoparticles in Examples 2 to 4 were prepared using a method similar to that described in Example 1. The results are shown in Table 1 below together with the results of Example 1. Note that 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 With a view to application to in vivo BNCT described below, a pharmacokinetics experiment of the above-mentioned ¹⁰ B ₄ C-PG nanoparticles was carried out as follows.
Animals and Tumor Models: Eight- to 11-week-old female BALB/c mice were housed in the KUR Animal Breeding Facility (Kyoto University, Kumatori Campus) with free access to water and standard food. All animal experiments were conducted in accordance with standards approved by the Kyoto University Ethics Committee. Approximately 1 × ¹⁰ CT26 cells suspended in 100 μL of PBS were implanted subcutaneously into the right leg of BALB/c mice 7 to 9 days before injection of the nanoformulations. Mice were maintained, handled, and housed under pathogen-free conditions in accordance with the guidelines of the Science Council of Japan. All animal experiments were conducted in accordance with the standards of the Experimental Animal Center of Kyoto University.

Pharmacokinetics Experiment After the tumor reached a longitudinal length of 10 mm, a PBS dispersion of ^10B4C -PG nanoparticles (200 _μL ) was injected into tumor-bearing BALB/c mice via the tail vein. After a certain period of time, the mice were euthanized and the boron content in the blood, tumor, and organs (liver, spleen, and kidney) was evaluated. The samples were placed in Teflon tubes, and the ^10B concentration of each sample was measured using a rapid γ-ray microanalysis system to obtain a ppm-order concentration (μg [ ^10B ]/g [biomaterial]) (see Non-Patent Document 7).

Pharmacokinetics of ¹⁰ B ₄ C-PG Nanoparticles in Example 3 Pharmacokinetic experiments of ¹⁰ B ₄ C-PG nanoparticles in Example 3 were performed by injecting a PBS dispersion of ¹⁰ B ₄ C-PG nanoparticles (200 μL) at a dose of 30.9 mg [ ¹⁰ B]/kg [mouse].
4 shows the concentrations of the ^10B4C -PG nanoparticles of Example 3 in the main tissues, tumor, and blood versus time after injection of _{the 10B4C} ^- PG nanoparticles. The _10B concentrations in the spleen, liver, and blood were much higher than those in the kidney and tumor at an early stage. ^{The 10B} concentrations in the spleen and liver remained roughly the same up to 48 hours, but ^{the 10B} concentration in the tumor gradually increased, peaking at 24 hours (36.7 ppm) and remaining above 20 ppm up to 48 hours.
Figure 5 shows the ^10B concentration in the tumor (T), the ^10B concentration in the blood (B), and the ratio of the ^10B concentration in the tumor (T) to the ^10B concentration in the blood (B) (T/B ratio) versus time from injection of ^10B4C -PG nanoparticles. In BNCT for cancer, it is more preferable that the ^10B concentration in the tumor (T) be 20 ppm or higher, _and in Figure 5, this condition was met for a fairly long period of time, from 18 to 48 hours.
Furthermore, since no mice (n=6) died over a period of 48 hours, ^{the 10} B ₄ C-PG nanoparticles of Example 3 did not exhibit acute toxicity.

While a sufficient ^10B concentration level for BNCT was maintained in the tumor for 30 hours (18 to 48 hours), the ^10B concentration in the blood gradually decreased with a half-life of 7.3 hours. Therefore, the T/B ratio increased over time and reached 4.4 at 48 hours, which satisfied the T/B of 3 or more, which is considered preferable for BNCT. Based on the time showing the maximum ^10B concentration in the tumor (36.7 ppm) and the time showing the maximum T/B ratio in Figure ₅ , we decided to irradiate with neutrons 24 and 48 hours after injection of the ^10B4C -PG nanoparticles.

In vivo BNCT of polyglycerol-modified boron carbide ( 10B4C _- ^PG ) nanoparticles
In vivo BNCT of ¹⁰ B ₄ C-PG nanoparticles was performed as follows.
Mice were injected with ^10B4C -PG nanoparticles using a method similar to that described in the pharmacokinetics experiment. 24 or 48 hours after injection of ^the _10B4C -PG nanoparticles, the 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% ^6LiF (96% ^6Li ) to block thermal neutrons. The tumor-bearing thigh was stretched over the hole and irradiated with neutrons at a fluence of 5 × ¹⁰⁹ cm ^-2 s ^-1 for 12 minutes or 6.5 × ¹⁰⁸ cm ^- 2 s ^-1 for 60 minutes using the Kyoto University research reactor (KUR). The day of neutron irradiation was designated as day 0. The tumor volumes of all mice were measured continuously by the same observer using the same caliper throughout the experiment. The tumor volumes were calculated and statistically analyzed according to the following formula 1:

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

Pharmacokinetic Data Analysis Data analysis was performed using Graphpad Prism 9 (Graphpad Software, Inc., San Diego, CA). Blood concentration data were fitted using a one-phase decay model, which considers the concentration curve as the sum of exponential declines. The decay of the nanoparticle blood concentration was approximated by the following equation 3:
Formula 3: C _t = (C ₀ - plateau) x e ^{- kt} + plateau (t is time (h), C _t is concentration (μg [ ¹⁰ B]/g [blood]), and k is the rate constant (h ^-1 )).

In vivo BNCT of ¹⁰ B ₄ C-PG nanoparticles of Example 1
^{The 10} B ₄ C-PG nanoparticles of Example 1 were injected intravenously into each mouse at a dosage of 34.3 mg [ ¹⁰ B]/kg [mouse]. 24 or 48 hours after injection of the ¹⁰ B ₄ C-PG nanoparticles, the mice were irradiated with neutrons at a fluence of 3.6×10 ¹² cm ^-2 . After neutron irradiation, tumor size and body weight were monitored for 29 days.
Simultaneously, the ^10B concentrations in the tumor and blood were measured ⁸ , ₂₄ , and 48 hours after injection of ^10B4C -PG nanoparticles using a method similar to that described in the pharmacokinetics experiment above.

6 and 7 show the in vivo BCNT results for ^{the 10} B ₄ C-PG nanoparticles of Example 1.
6, BCNT24h and BCNT48h represent the relative tumor size versus the number of days from the time point when _{neutron irradiation was performed 24 hours or 48 hours after the injection of the 10B4C} ^- PG nanoparticles of Example 1. The cold control represents a condition in which ^{the 10B4C} -PG nanoparticles of Example 1 were not injected and no neutron irradiation was performed, the hot control represents a _condition in _which the ^10B4C -PG nanoparticles of Example 1 were not injected but were irradiated with neutrons, and the injection control represents a condition in which _the ^10B4C -PG nanoparticles of Example 1 were injected but were not irradiated with neutrons.
The fact that neutron irradiation inhibits tumor growth can be seen by comparing the cold control, injection control, and hot control. It can be seen that BNCT24h and BNCT48h inhibit tumor size more efficiently than the hot control. After 29 days, BNCT24h and BNCT48h inhibited tumor size by 13% and 12%, respectively, compared to the cold control. After 29 days, BNCT24h and BNCT48h inhibited tumor size by 20% and 19%, respectively, compared to the hot control.

7, BCNT24h and BCNT48h show the relative body weights of mice relative to the number of days from the time point when neutron irradiation was performed 24 hours or 48 hours after the injection of ¹⁰ B ₄ C-PG nanoparticles of Example 1. Cold control, hot control, and injection control show the same experimental conditions as those in FIG.
It can also be seen from Figure 7 that neutron irradiation suppresses weight change. As in Figure 6, BNCT 24h and BNCT 48h showed more efficient suppression of tumor growth than the hot control.

8 shows the ^10B concentration in the tumor ( _T ), the 10B concentration in the blood (B), and the ratio of the ^10B concentration in the tumor (T) to the ^10B concentration in the blood (B) (T/B ratio) ⁸ , 24, and 48 hours after injection of the ^10B4C -PG nanoparticles of Example 1 into _mice . The ^10B4C -PG nanoparticles of Example 1 exhibited pharmacokinetics similar to that of the ^10B4C -PG nanoparticles of Example 3 shown in FIG. 5. The ^10B concentration in the tumor (T) remained above 20 ppm from 24 to 48 hours after injection. Furthermore, since _no mice (n=7) died over a 48-hour period, it was concluded that ^{the 10BC} -PG nanoparticles of Example 1 had no acute toxicity.

In vivo BNCT of ¹⁰ B ₄ C-PG nanoparticles of Example 2
^{The 10} B ₄ C-PG nanoparticles of Example 2 were injected intravenously into each mouse at a dose of 27.9 mg [ ¹⁰ B]/kg [mouse]. 24 or 48 hours after injection of the ¹⁰ B ₄ C-PG nanoparticles, the mice were irradiated with neutrons at a fluence of 2.3 × 10 ¹² cm ^-2 . After neutron irradiation, tumor size and body weight were monitored for 20 days.
Simultaneously, the ^10B concentrations in the tumor and blood were measured ⁸ , ₂₄ , and 48 hours after injection of ^10B4C -PG nanoparticles using a method similar to that described in the pharmacokinetics experiment above.

Similar trends to those shown in Figures 6 and 7 were observed in the in vivo BNCT of the ^10B4C -PG nanoparticles of Example 2. Here, too, experiments were conducted under the same conditions as in Figures ₆ and 7, using cold control, injection control, hot control, BNCT24, and BNCT48. The hot control suppressed tumor size more than the cold control and injection control. BNCT24h and BNCT48h further suppressed tumor size than the hot control. After 20 days, BNCT24h and BNCT48h suppressed tumor size by 52% and 39%, respectively, compared to the cold control. After 20 days, BNCT24h and BNCT48h suppressed tumor size by 63% and 48%, respectively, compared to the hot control.

TEM observation of tumors 24 and 48 hours after injection of the ¹⁰ B ₄ C-PG nanoparticles of Example 1 into mice
To further understand the BCNT due to the 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 observed using a transmission electron microscope (TEM).
Figure 9a shows a TEM image of a section of tumor tissue taken 24 hours after injection of the ^10B4C -PG nanoparticles of Example 1, _and Figure 9b shows a TEM image of a section of tumor tissue taken _{48 hours after injection of the 10B4C} ^- PG nanoparticles of Example 1. In both Figures 9a and 9b, numerous particles were observed as black dots, supporting the accumulation of ^10B in the tumor tissue, as confirmed in the pharmacokinetic experiments.

The modified boron carbide particle powder of an embodiment of the present invention comprises boron carbide particles, at least a portion of the surface of at least some of the boron carbide particles being modified with polyglycerol. Therefore, drugs containing the modified boron carbide particle powder of an embodiment of the present invention have advantageous effects such as inherently low toxicity, shorter administration time (superior water solubility or water dispersibility), the ability to accumulate boron at higher concentrations within tumors, and the ability to persist within tumors for a long period of time while being rapidly removed from the blood, making them suitable for use in boron neutron capture therapy.

Claims (6)

A boron carbide particle powder comprising boron carbide particles, at least a portion of the surface of at least some of the boron carbide particles being modified with polyglycerol by a chemical reaction . 2. The modified boron carbide particle powder of claim 1, wherein the median diameter is greater 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 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. The modified boron carbide particle powder according to claim 1, wherein the average particle size of the boron carbide particles is less than 1000 nm. The modified boron carbide particles powder according to any one of claims 1 to 4, which have a spherical shape. A neutron capture therapy drug comprising the modified boron carbide particle powder according to any one of claims 1 to 5.