Agent for treatment of cancer, and pharmaceutical composition containing the same

Abstract

To provide an agent for the treatment of cancer, in particular, an agent which can be used in combination with boron neutron capture therapy and photodynamic therapy for the treatment of cancer.SOLUTION: The foregoing problem is solved by providing an agent for the treatment of cancer, the agent containing: a carrier that is accumulating in cancer cells; and a boron-containing compound and / or a photosensitizer covalently bonded and / or non-covalently bonded to the carrier, and the agent being used for cancer therapy in which a subject administered with the agent is irradiated with radiation.SELECTED DRAWING: Figure 21

Description

[Technical field]

[0001] The present invention relates to an agent for cancer treatment, in particular, an agent for treating cancer by a combination of boron neutron capture therapy and photodynamic therapy. Throughout this specification, unless otherwise specified, "cancer" refers to "cancer disease", but may also refer to "cancer cells" or "cancer tissues". [Background technology]

[0002] In recent years, cancer treatment methods such as boron neutron capture therapy (BNCT) and photodynamic therapy (PDT), which combine external energy such as radiation or near-infrared light with drugs that become activated in response to that external energy, have been attracting attention.

[0003] Boron neutron capture therapy is a method to capture boron-10( 10 After delivering a boron drug containing B) to cancer cells, it is irradiated with neutrons to increase the activity of boron in tumor tissue. 4 He nucleus (alpha ray) 7 This is a treatment method that releases Li nuclei to kill cancer cells. 10 B) absorbs neutrons contained in the radiation extracted from nuclear reactors and accelerators, and undergoes nuclear fission reactions ( 10 B(n,α) 7 Li reaction) 4 He nucleus (alpha ray) 7 It has the property of emitting a Li nucleus. Neutrons can be captured by various atomic nuclei other than boron, but the probability of neutron capture by the atoms that make up living organisms is not high. In contrast, the probability of neutron capture, expressed as the capture cross section, 10 The probability of neutron capture by the B nucleus is much higher than that of the elements that make up living organisms. The nitrogen nucleus has the largest capture cross section among them ( 14 It is about 2,000 times larger than the neutrons absorbed by the 10 Emitted from B nuclei 4 The range of the He nucleus (alpha ray) is about 9 μm.7 The range of the Li nucleus is about 4 μm, which is shorter than the diameter of an average cell. 10 By introducing a compound containing B atoms into the target cancer cells and irradiating them with neutrons, it is possible to selectively kill only the cancer cells while sparing the surrounding normal cells such as immune cells in the cancer tissue. As for cancer treatment using BNCT, it has been covered by health insurance since June 2020 for "unresectable locally advanced or locally recurrent head and neck cancer."

[0004] In BNCT, the main factors are the energy of the neutrons and the amount of neutrons introduced into the target cells. 10 The therapeutic effect is determined by the amount of B atoms, so there are many B atoms in the target cells. 10 It is extremely important to develop boron drugs that can introduce B atoms. 10 If the amount of B atoms can be increased compared to the surrounding tissue, the energy of the neutron rays that cause radiation damage can be reduced, and the amount of α-particles and 7 Since the amount of Li nuclei generated can be suppressed, it is expected that cancer cells can be killed with fewer side effects.

[0005] Various boron drugs for BNCT have been developed, but only a few have progressed to clinical research. For example, L-BPA (4-borono-L-phenylalanine ( 10 B)), that 18 F-labeled compound [ 18 F]F-BPA( 18 F-fluoro-borono-L-phenylalanine), boric acid, BSH(sodium mercaptododecaborate( 10 B)) etc. As of July 2023, the only boron drug approved by the Ministry of Health, Labour and Welfare in Japan is L-BPA (Stella Pharma Corporation, Non-Patent Document 1).

[0006] However, L-BPA does not accumulate well in tumors, and currently, in order to obtain sufficient boron concentration in the tumor to be treated, L-BPA is administered at a high concentration of 500 mg / kg. In addition, L-BPA binds one atom of the amino acid phenylalanine to one molecule. 10 B is a modified compound, and is thought to be taken up into target cells via LAT-1 (L-type amino acid transporter 1), an amino acid transporter that is highly expressed in cancer cells (Non-Patent Document 2). However, LAT-1 is not necessarily highly expressed in all cancer cells, and there are some cancer types for which it is difficult to apply L-BPA-based BNCT. Therefore, it is difficult to deliver high concentrations of BPA to various cancer tissues. 10 There is a strong demand for the development of new boron drugs that can accumulate B atoms.

[0007] Boron clusters such as carborane contain many boron atoms in one molecule, which allows them to have a greater ability to bind to target cells. 10Boron clusters have attracted attention as boron drugs that can dramatically increase the amount of B atoms introduced. However, boron clusters are difficult to be taken up into cells and difficult to selectively accumulate in cancer tissues, so various delivery systems have been developed to improve the amount of boron clusters taken up into cells and their accumulation in cancer tissues. Among them, nanoparticle-type delivery systems are expected to be a delivery means that can efficiently increase the concentration of boron drugs in cancer tissues by accumulating and storing them in cancer tissues mainly by the EPR effect (Enhanced Permeability and Retention Effect) (J. Fang et al., Advanced Drug Delivery Reviews 63 (2011) 136-151) or active targeting using targeting molecules. As nanoparticle-type boron cluster delivery systems, liposomes carrying boron clusters (Patent Document 1, Patent Document 2, Non-Patent Document 3, Non-Patent Document 4) and polymeric micelles (Patent Document 3, Non-Patent Document 5) have been proposed, but to the inventors' knowledge, BNCT treatment using a nanoparticle-type boron cluster delivery system has not yet been realized.

[0008] On the other hand, photodynamic therapy is a method in which a photosensitizer that has the property of producing active oxygen when irradiated with light having a specific wavelength is delivered to a target cancer, and the light having the above wavelength is irradiated to generate active oxygen specifically in the cancer, thereby obtaining a therapeutic effect on the cancer. Among active oxygen, singlet oxygen, which is considered to have particularly high cytotoxicity, has a short life span and is said to have a diffusion distance of about 10 to 50 nm in the body. Therefore, it is expected that by introducing a photosensitizer into a target cell, a cancer cell, and exciting the photosensitizer, it is possible to selectively kill only the cancer cells. Photodynamic therapy has been put into practical use, and as a photosensitizer for photodynamic therapy, for example, talaporfin sodium (trade name: Laserphyrin (registered trademark), Meiji Seika Pharma Co., Ltd.) is commercially available.

[0009] Boron neutron capture therapy is a method to 10B) caused by nuclear fission reactions 4 He nucleus (alpha ray) 7 Since photodynamic therapy destroys cancer tissues with active oxygen, whereas Li atomic nuclei destroy cancer tissues, it is expected that a complementary and synergistic cancer treatment effect can be achieved if the two can be combined. Attempts at combined therapy of boron neutron capture therapy and photodynamic therapy are also described in, for example, Patent Document 4, Non-Patent Document 6, and Non-Patent Document 7. However, as far as the present inventors know, these attempts to combine boron neutron capture therapy and photodynamic therapy all aim to obtain the therapeutic effect of BNCT by irradiating neutrons and the therapeutic effect of PDT by irradiating near-infrared light, which requires irradiation with two types of external energy. It is known that near-infrared light irradiated to obtain the therapeutic effect of PDT has relatively high tissue penetration, but its penetration is still about 2 cm. When targeting a deep cancer, it is not easy to obtain both the therapeutic effect of BNCT and the therapeutic effect of PDT, because the near-infrared light, which is the excitation light, cannot be delivered to the vicinity of the target cancer. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] JP 2008-074817 A [Patent Document 2] International Publication No. WO2014 / 030715 [Patent Document 3] JP 2015-044920 A [Patent Document 4] JP 2013-227233 A [Non-patent literature]

[0011] [Non-Patent Document 1] Steboronine Infusion Bag 9000mg / 300mL, Pharmaceutical Interview Form, Revised May 2020 (2nd Edition) [Non-Patent Document 2] P. Wongthai, et al., Cancer Sci. 2015, 106(3), 279-286. [Non-Patent Document 3] H. Yanagie, et al., British Journal of Cancer 1991, 63, 522-526. [Non-Patent Document 4] K. Shelly, et al,. Proc. Natl. Acad. Sci. USA 1992, 89, 9039-9043. [Non-Patent Document 5] P. Mi, et al., J. Control. Release 2017, 254, 1-9. [Non-Patent Document 6] R. Hiramatsu, et al., Journal of Pharmaceutical Sciences 2015, 104, 962-970. [Non-Patent Document 7] E. Friso, et al., Photochem. Photobiol. Sci., 2006, 5, 39-50. [Non-Patent Document 8] V. Lioret, et al., J. Med. Chem. 2020, 63, 9446-9456. [Non-Patent Document 9] N. Liu, et al., Theranostics 2022, 12, 17, 7404-7418. Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention has been made in consideration of the problems of the conventional technology as described above, and an object of the present invention is to provide an agent for cancer treatment, in particular an agent capable of treating cancer by combining boron neutron capture therapy and photodynamic therapy. [Means for solving the problem]

[0013] In the course of intensive research efforts to solve the above problems, the present inventors carried out an evaluation of the in vivo dynamics of a carrier for accumulating a boron compound and / or a photosensitizer in a cancer by carrying indocyanine green, a photosensitizer excitable by near-infrared light, on the carrier and evaluating the in vivo dynamics. In this evaluation, the tumor site of a mouse administered with the carrier carrying indocyanine green was irradiated with neutrons in an experimental nuclear reactor, and it was surprisingly found that an extremely excellent antitumor effect was exhibited, even though no boron-containing compound was administered.

[0014] Indocyanine green is known to function as a photosensitizer applicable to PDT, but since it mainly absorbs light with wavelengths of 600 nm to 800 nm, it is usually excited by near-infrared light with a wavelength of about 800 nm, which is the maximum absorption wavelength. It has never been found before that indocyanine green can function as a radiosensitizer and exert an antitumor effect by irradiating a subject administered with indocyanine green with radiation, more specifically, reactor-type neutron rays, instead of excitation light in the near-infrared region, and this finding was completely unexpected even for the present inventors.

[0015] The present invention is based mainly on the above findings, and in one aspect, An agent for treating cancer, comprising: A cancer-accumulating carrier and a photosensitizer covalently and / or non-covalently bound to the carrier, The above-mentioned problems are solved by providing an agent for treating cancer by irradiating a subject administered with the agent with radiation.

[0016] In a preferred embodiment, the photosensitizer may be a photosensitizer having an absorption maximum wavelength within a wavelength range of 600 nm to 900 nm, more preferably within a wavelength range of 700 nm to 900 nm. Such a photosensitizer may be a cyanine-based photosensitizer having a polymethine skeleton, preferably a cyanine-based photosensitizer having a heptamethine skeleton, and more preferably a cyanine-based photosensitizer having the following formula (II):

[0017] [ka] (In the above formula (II), R 1 and R 4 are each independently an optionally substituted alkyl group; R 2 and R 3 is H or a group that combines with each other to form a cyclic structure; X is H or a halogen; ring B and ring D are each independently a nitrogen-containing fused aromatic heterocycle; A - is an anion, and m is 0 or 1.

[0018] In a preferred embodiment, the photosensitizer is represented by the following formula (III):

[0019] [ka] (In the above formula (III), R 1 and R 4 are each independently an optionally substituted alkyl group; R 2 and R 3 is H or groups which combine with each other to form a ring structure; R 5 and R 6 are H or groups which combine together to form an aryl group; R 7 and R 8 are H or groups which combine together to form an aryl group; X is H or a halogen; A - is an anion, and m is 0 or 1. Such photosensitizers include the above-mentioned indocyanine green.

[0020] According to the agent according to one aspect of the present invention, the photosensitizer is excited by irradiation with radiation to exert a therapeutic effect, and therefore, compared to the case where the photosensitizer is excited by irradiation with near-infrared light, the agent has the advantage of being applicable to cancers located deeper. In addition, the agent can be excited by the same external energy as that irradiated in boron neutron capture therapy, that is, by irradiation with radiation containing neutrons or neutron rays, so that when the agent further contains a boron-containing compound, the agent has the advantage that both the therapeutic effect of boron neutron capture therapy and the therapeutic effect of photodynamic therapy can be obtained by irradiation with one type of external energy. Thus, the agent according to one aspect of the present invention can be used extremely advantageously for cancer treatment using radiation.

[0021] It is speculated that Cherenkov radiation is involved in the mechanism by which the photosensitizer exerts its cancer treatment effect when irradiated with radiation. Cherenkov radiation is a phenomenon in which light is emitted when charged particles such as alpha and beta rays move through a medium such as water or air at a speed faster than the speed of light. The light emitted at this time is called Cherenkov light, and is typically a white to bluish-white light with a wavelength of about 250 nm to 600 nm. There are many possible causes for Cherenkov light to be generated by irradiation with radiation, and it is thought that many causes act in combination to generate Cherenkov light. For example, in an atomic reactor, which is a source of radiation, beta particles are released when xenon-133, a fission product of uranium-235, undergoes beta decay. When these beta particles are irradiated to a living body together with neutron rays, Cherenkov light can be generated as they pass through the living body, including water molecules (Figure 21). It is also possible that the irradiated neutrons react with hydrogen contained in the living body to generate gamma rays, which are then Compton scattered to produce electrons that generate Cherenkov light.

[0022] Photodynamic therapy using Cherenkov light or Cherenkov radiation has been reported at the research level as CR-PDT (Cherenkov-induced PDT) (for example, Non-Patent Documents 8 and 9). However, all of the existing studies known about CR-PDT have focused on the fact that Cherenkov light has a shorter wavelength than near-infrared light that excites photosensitizers, and have attempted to indirectly excite the photosensitizer by binding a donor molecule that absorbs Cherenkov light to the photosensitizer and utilizing the FRET (Forster resonance energy transfer) reaction between the donor molecule and the photosensitizer. As far as the present inventors know, there has been no report of a phenomenon in which indocyanine green is activated in a living body without using a FRET reaction and by irradiating a neutron beam from a nuclear reactor, resulting in a therapeutic effect similar to that of photodynamic therapy, and this was a completely unexpected finding. It is thought that the Cherenkov light generated in the body can directly excite the photosensitizer, or it can be shifted to longer wavelengths of light by FRET via endogenous fluorescent dyes such as porphyrins (e.g., heme) present in the body, and thereby excite the photosensitizer (Figure 21).

[0023] In other words, in one aspect, the present invention also provides a use of the above-described photosensitizer as a radiosensitizer. A radiosensitizer means a drug that is administered to a patient who is a subject of radiotherapy and can enhance the therapeutic effect of the irradiated radiation. As described below, the radiation can be radiation that can generate Cherenkov light in a living body, or radiation that contains protons, electrons, and / or neutrons.

[0024] The cancer-accumulating carrier may be any carrier that accumulates in the cancer when administered to a living body having cancer, and may be any carrier as long as it can accumulate the photosensitizer and / or the boron-containing compound described later in the cancer tissue. In one preferred embodiment, the carrier may be a microparticle containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block. The present inventors have focused on microparticles formed from an amphiphilic polymer having polysarcosine as the hydrophilic block and polylactic acid as the hydrophobic block, and have made extensive research efforts. Polylactic acid is known to have high biocompatibility and excellent biodegradability, and is a polymer material that can be advantageously incorporated into pharmaceuticals applied to living organisms, particularly the human body. On the other hand, polysarcosine is a polymer of sarcosine (N-methylglycine), and is known to have excellent solubility in water, excellent biodegradability, no immunogenicity, and little nonspecific adsorption of proteins. Therefore, microparticles formed from an amphiphilic polymer containing polysarcosine as the hydrophilic block are expected to exhibit high blood retention and accumulate and accumulate in tumors due to the EPR effect. As shown in the experimental examples described below, microparticles containing an amphiphilic polymer having polysarcosine as a hydrophilic block and polylactic acid as a hydrophobic block exhibit particularly excellent tumor accumulation properties and can therefore be particularly suitably used as the above-mentioned carrier exhibiting cancer accumulation properties.

[0025] On the other hand, as described above, the agent according to one aspect of the present invention may further contain a boron-containing compound. When the agent according to one aspect of the present invention further contains a boron-containing compound, there is an advantage that both the therapeutic effect of boron neutron capture therapy and the therapeutic effect of photodynamic therapy can be obtained simultaneously by irradiating a subject to which the agent has been administered with radiation containing neutrons. The boron-containing compound that can be contained in the agent according to one aspect of the present invention may basically be any compound, and there is no particular limitation on the type thereof, but from the viewpoint of efficiently increasing the boron concentration in target cells, it is preferably a boron cluster, and more preferably a hydrophobic boron cluster such as carborane or a derivative thereof. When a microparticle containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block is used as a carrier exhibiting cancer accumulation, the hydrophobic boron cluster can be efficiently supported on the hydrophobic core of the microparticle.

[0026] In a preferred embodiment, the boron-containing compound contained in the agent according to one aspect of the present invention has the following formula (I): [ka]

[0027] (In the above formula (I), ● is C, ◯ is each independently BH or B-halogen, and R is each independently H or a substituent having an optionally substituted saturated or unsaturated alkyl group.) or a derivative thereof. According to the findings of the present inventors, o-carborane or a derivative thereof represented by the above formula (I) can be more efficiently and stably supported on microparticles containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block, in particular, on microparticles containing an amphiphilic polymer containing polylactic acid as the hydrophobic block, compared with m-carborane or p-carborane.

[0028] In a preferred embodiment, the boron-containing compound contained in the agent according to one aspect of the present invention may be a boron-containing compound represented by the above formula (I) in which the alkyl group of the substituent R has 3 or more carbon atoms. According to the findings of the present inventors, the boron-containing compound represented by the above formula (I) and having two alkyl groups with 3 or more carbon atoms at the ortho positions acts as a molecular glue that binds polylactic acid chains together, thereby dramatically increasing the stability in an aqueous medium of microparticles formed from an amphiphilic polymer containing polylactic acid as a hydrophobic block (FIG. 20). As shown in the experimental example described below, the microparticles containing the boron-containing compound represented by the above formula (I) and having two alkyl groups with 3 or more carbon atoms at the ortho positions as a boron-containing compound dramatically increase the boron concentration in cancer cells compared to L-BPA, a boron-containing compound that has been used in boron neutron capture therapy, and can also exhibit excellent therapeutic effects in vivo.

[0029] As described above, the agent according to one aspect of the present invention, which contains a photosensitizer and / or a boron-containing compound, and a composition containing the same can be particularly suitably used for cancer treatment by photodynamic therapy (PDT) or boron neutron capture therapy (BNCT), and for combined therapy of boron neutron capture therapy (BNCT) and photodynamic therapy (PDT).

[0030] In one aspect, the present invention also provides a radiation irradiation device equipped with a radiation source for irradiating radiation, which is used to treat cancer by irradiating radiation to a subject administered with the above-described radiosensitizer, or the agent comprising a cancer-accumulating carrier and a photosensitizer covalently and / or non-covalently bound to the carrier, or a pharmaceutical composition containing these. To repeat, as far as the present inventors know, there is no knowledge at all that an excellent antitumor effect can be obtained by irradiating radiation to a subject administered with a photosensitizer, particularly an indocyanine-based photosensitizer. Therefore, the above-mentioned use provided by the present invention is a new use not previously known for a radiation irradiation device equipped with a radiation source for irradiating radiation.

[0031] In a preferred embodiment, the radiation is preferably radiation capable of generating Cherenkov light in a living body, and is preferably radiation containing protons, electrons, and / or neutrons. As described above, the agent administered to the subject may contain a boron-containing compound. When the agent administered to the subject contains a boron-containing compound, the radiation irradiated to the subject is preferably radiation containing at least neutrons. In this case, the radiation irradiation device can be particularly preferably used for cancer treatment using boron neutron capture therapy and photodynamic therapy in combination. Effect of the Invention

[0032] According to one aspect of the present invention, the agent accumulates a photosensitizer and / or a boron-containing compound in the target tissue and is then irradiated with radiation, thereby exciting the photosensitizer to damage the cancer and / or utilizing a nuclear reaction between the boron-containing compound and neutrons to damage the cancer, thereby achieving excellent cancer treatment effects. [Brief description of the drawings]

[0033] [Figure 1]FIG. 1 shows the structures of an AB-type polysarcosine-poly(L-lactic acid) block polymer (PSar106-block-PLLA32) used in the experimental examples, as well as o-carborane, m-carborane, and p-carborane. [Diagram 2] (a) Particle size distribution and transmission electron micrographs of polysarcosine-poly(L-lactic acid) nanoparticles not carrying carborane (hereinafter, polysarcosine-poly(L-lactic acid) nanoparticles may be abbreviated as "PSAR-PLLA nanoparticles") (b) Particle size distribution and transmission electron micrographs of PSAR-PLLA nanoparticles carrying o-carborane. [Diagram 3] (a) A diagram showing an outline of the experimental procedure for the intracellular uptake test, (b) A diagram showing the intracellular boron concentration in mouse breast cancer cells 4T1 contacted with PSAR-PLLA nanoparticles carrying o-carborane or L-BPA (boron concentration: 2 mM) for a given time. [Figure 4] FIG. 1 shows the release behavior of o-carborane from PSAR-PLLA nanoparticles loaded with o-carborane in PBS. [Diagram 5] FIG. 1 shows the change in particle size and polydispersity upon dialysis in PBS for 24 hours for (a) PSAR-PLLA nanoparticles without carborane, (b) PSAR-PLLA nanoparticles loaded with o-carborane, and (c) PSAR-PLLA nanoparticles loaded with m-carborane. [Figure 6] FIG. 1 shows the structures of three o-carborane derivatives used in the experimental examples, namely, 1,2-dimethyl-o-carborane, 1,2-dihexyl-o-carborane, and 1,2-didodecyl-o-carborane. [Figure 7] FIG. 1 shows particle size distributions of (a) PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane, (b) PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, and (c) PSAR-PLLA nanoparticles carrying 1,2-didodecyl-o-carborane, and (d) a transmission electron micrograph of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane. [Figure 8] FIG. 1 shows the release behavior of o-carborane derivatives from (a) PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane and (b) PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane in PBS. [Figure 9] FIG. 1 shows the change in particle size and polydispersity upon dialysis in PBS for 24 hours for (a) PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane and (b) PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane. [Figure 10] FIG. 1 shows intracellular boron concentrations in (a) mouse breast cancer cells 4T1, (b) mouse colon cancer cells CT26, (c) human pancreatic adenocarcinoma cells AsPC-1, and (d) human gastric cancer cells NC1-N87 contacted with 1,2-dihexyl-o-carborane-loaded PSAR-PLLA nanoparticles or o-carborane-loaded PSAR-PLLA nanoparticles. [Figure 11] (a) Schematic diagram of the experimental procedure for the cytotoxicity test (b) Cell viability of mouse breast cancer cells 4T1 contacted with PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane, PSAR-PLLA nanoparticles carrying o-carborane, or PSAR-PLLA nanoparticles carrying m-carborane. [Figure 12] (A) A diagram showing the results of in vivo near-infrared fluorescence imaging of a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying ICG. (B) A diagram showing the time course of the intensity of fluorescence derived from ICG in a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying ICG. (C) A diagram showing the results of ex vivo near-infrared fluorescence imaging of a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying ICG. [Figure 13](A) The boron content in each tissue of a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane was measured by ICP-AES. (B) The T / N ratio and T / B ratio in a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane. [Figure 14] (A) This is a graph showing the cell viability of AsPC-1 cells administered with PSAR-PLLA nanoparticles, PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane, or L-BPA after neutron irradiation for a given period of time. The boron compound was administered at a dose of 2 mM in terms of boron. (B) This is a photograph showing a typical state of cells after neutron irradiation for 40 minutes for AsPC-1 cells administered with PSAR-PLLA nanoparticles, PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane, or L-BPA. In the graph, "**" indicates p < 0.01, and "*" indicates p < 0.05. [Figure 15] AsPC-1 / CMV-Luc tumor-bearing mice were administered PSAR-PLLA nanoparticles, PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane, or L-BPA, and 24 hours later, they were irradiated with neutrons for 40 minutes. After neutron irradiation, (A) changes in mouse body weight and (B) tumor size were observed for 21 days. In the figures, "**" indicates p < 0.01, and "*" indicates p < 0.05. [Figure 16] (A) A graph showing the cell viability of AsPC-1 cells administered with ICG-loaded PSAR-PLLA nanoparticles after neutron irradiation for a given period of time. (B) A photograph showing a typical cell appearance after administration of ICG-loaded PSAR-PLLA nanoparticles after neutron irradiation for a given period of time. In the figure, "**" indicates p < 0.01, and "*" indicates p < 0.05. [Figure 17]AsPC-1 / CMV-Luc tumor-bearing mice were administered ICG-loaded PSAR-PLLA nanoparticles and 24 hours later were irradiated with neutrons for 40 minutes. After neutron irradiation, (A) changes in mouse body weight and (B) tumor size were observed for 21 days. In the figure, "**" indicates p < 0.01 and "*" indicates p < 0.05. [Figure 18] (a) A diagram showing an outline of the experimental procedure for the in vivo kinetics test, (b) A diagram showing the results of in vivo near-infrared fluorescence imaging of Xenograft mice administered with PSAR-PLLA nanoparticles carrying ICG and 1,2-dihexyl-o-carborane. [Figure 19] (a) shows the results of ex vivo near-infrared fluorescence imaging of a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying ICG and 1,2-dihexyl-o-carborane, (b) shows the results of ICP-AES measurement of boron content in each tissue of a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying ICG and 1,2-dihexyl-o-carborane. [Figure 20] FIG. 2 is a conceptual diagram illustrating the interaction between polylactic acid and a boron-containing compound in PSAR-PLLA nanoparticles in one embodiment of the present invention. [Figure 21] FIG. 1 is a conceptual diagram illustrating the mechanism by which an agent according to one aspect of the present invention provides a BNCT effect and a mechanism by which an agent according to one aspect of the present invention provides a PDT effect. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention will now be described in further detail.

[0035] <Photosensitizer> Photosensitizers refer to compounds that can be excited by light of a specific wavelength, typically near-infrared light, to generate reactive oxygen species (ROS). When excited in the presence of oxygen, photosensitizers convert oxygen (O 2) to efficiently generate singlet oxygen. Singlet oxygen is a highly reactive chemical species that damages cells that compose lesions such as tumors, resulting in cell death. As shown in the experimental examples described below, the photosensitizer described below is excited by irradiation with radiation and functions as a radiosensitizer that can enhance the effects of radiation therapy.

[0036] The photosensitizer that can be used in the present invention can be basically any, and various photosensitizers that can be applied to photodynamic therapy can be used.For example, cyanine-based photosensitizers, porphyrin-based photosensitizers, phthalocyanine-based photosensitizers, naphthoquinone-based photosensitizers, diimmonium-based photosensitizers, or azo-based photosensitizers can be used, but are not limited thereto. Specific examples of such photosensitizers include, but are not limited to, zinc phthalocyanine (ZnPc), zinc naphthalocyanine, 5,10,15,20-tetrakis(pentafluorophenyl)chlorin derivative (TFPC), 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TFPP), protoporphyrin IX (PPIX), indocyanine green (ICG), porfimer (Photofrin (registered trademark)), temoporfin (Foscan (registered trademark)), talaporfin sodium (Laserphyrin (registered trademark)), verteporfin (Visudyne (registered trademark)), and palladium bacteriopheophorbide (TOOKAD (registered trademark)).

[0037] In a preferred embodiment, the photosensitizer that can be used in the present invention may be a photosensitizer having an absorption maximum wavelength within a wavelength range of 600 to 900 nm, more preferably within a wavelength range of 700 to 900 nm. An example of a photosensitizer that exhibits such properties is indocyanine green (ICG). As shown in the experimental examples described below, nanoparticles carrying indocyanine green, a photosensitizer that absorbs near-infrared light with a wavelength of 600 to 900 nm, can be excited by irradiation with radiation including neutron beams and exhibit a significant antitumor effect.

[0038] In a preferred embodiment, the photosensitizer that can be used in the present invention is preferably a cyanine-based photosensitizer having a high absorption coefficient for near-infrared light, and more preferably a cyanine-based photosensitizer having a heptamethine skeleton and represented by the following formula (II):

[0039] [ka]

[0040] In the above formula (II), R 1 and R 4 R is each independently an optionally substituted alkyl group. 2 and R 3 are H or groups which combine with each other to form a cyclic structure. X is H or a halogen. Ring B and ring D are each independently a nitrogen-containing fused aromatic heterocycle. A - is an anion, and m is 0 or 1. In the above formula (II), R 2 and R 3 The ring structure which may be formed by the formula (I) may be substituted. Furthermore, ring B and ring D may be a nitrogen-containing bicyclic heterocycle or a tricyclic aromatic heterocycle.

[0041] Further specific examples of the photosensitizer that can be used in the present invention include indocyanine-based photosensitizers represented by the following formula (III).

[0042] [ka]

[0043] In the above formula (III), R 5 and R 6 are each H or a group which combines with each other to form an aryl group. The aryl group may be an optionally substituted benzene ring. R 7 and R 8The same applies to each of the symbols, which are each H or a group that combines with each other to form an aryl group. The aryl group may be an optionally substituted benzene ring. The other symbols are the same as those in formula (II) above, and therefore will not be described.

[0044] Specific examples of the photosensitizer represented by the above formula (II) or (III) include the following photosensitizers, but the photosensitizers that can be used in the present invention are not limited to these.

[0045] [ka]

[0046] The photosensitizer may be non-covalently or covalently supported on the cancer-accumulating carrier contained in the agent according to one aspect of the present invention. On the other hand, the photosensitizer that can be contained in the agent according to one aspect of the present invention may have one or more functional groups for non-covalent and / or covalent bonding to the carrier. Examples of such functional groups include, but are not limited to, a carboxyl group, an amino group, an isothiocyanate group, a thiol group, a maleimide group, an azide group, an alcohol group, and a halogenated alkyl group.

[0047] In a preferred embodiment, when the cancer-accumulating carrier that can be used in the present invention is an amphipathic polymer having a hydrophilic block and a hydrophobic block, the photosensitizer may be a conjugate of the above-described photosensitizer and an amphipathic polymer having a hydrophilic block and a hydrophobic block, or a conjugate of the above-described photosensitizer and a hydrophobic polymer having a hydrophobic block. Specific examples of such conjugates include a conjugate having the structure of the following formula (XIII), that is, a conjugate of indocyanine green and polylactic acid. In the following formula (XIII), n is an integer, and may be preferably 5 to 50, more preferably 10 to 50, and even more preferably 15 to 50.

[0048] [ka]

[0049] As shown in the experimental examples described later, the conjugate of indocyanine green and polylactic acid can be easily supported on microparticles containing an amphiphilic polymer containing polysarcosine as a hydrophilic block and polylactic acid as a hydrophobic block. The conjugate of the above formula can be synthesized by an appropriate method, and the synthesis method is described, for example, in H. Tsujimoto, et. al. Cancer Sci. 105 (2014) 1626-1630.

[0050] <Boron-containing compounds> On the other hand, the boron-containing compound that can be contained in the agent according to one aspect of the present invention means a compound containing a boron atom in its molecular structure. The boron-containing compound may be basically any compound, and there is no particular limit to the number of boron atoms contained in one molecule of the boron-containing compound. For example, it may contain one boron atom like the above-mentioned L-BPA, or it may contain two or more boron atoms or three or more boron atoms. From the viewpoint of more efficiently increasing the concentration of boron atoms in the therapeutic target, it is preferable that the number of boron atoms contained in one molecule of the boron-containing compound is large, and for example, it is preferable that it is a boron cluster containing a large number of boron atoms in one molecule.

[0051] A "boron cluster" is a boron-containing compound that has a polyhedral structure formed by the assembly of multiple boron atoms. An example of a boron cluster is Decaborane (B 10 H 14 ), Decahydrodecaborate ([B 10 H 10 ] 2- ), Dodecaborate ([B 12 H 12 ] 2- ), nido,nido-Octadecaborane(22)(B 18 H 22Examples of boron clusters include, but are not limited to, clusters of boron hydride compounds such as boron hydride (CAS RN:21107-56-2). For example, boron clusters may contain carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, etc., in addition to boron atoms as skeletal atoms that constitute the boron cluster. Examples of boron clusters containing boron atoms and carbon atoms include carborane. Examples of carborane include closo-carborane ([CB 11 H 12 ] - ), closo-carborane (C 2 B 10 H 12 ) and nido-carborane ([C 2 B 9 H 11 ] - ), but are not limited to these. In addition, dodecaborate ([B 12 H 11 SH] 2- , BSH), dodecaborate with hydroxyl group ([B 12 H 11 OH] 2- ), dodecaborate with amino group ([B 12 H 11 NH 3 ] - ) are also known.

[0052] In a preferred embodiment, the boron-containing compound that may be contained in the agent according to one aspect of the present invention is preferably carborane or a derivative thereof, and more preferably a boron-containing compound represented by the following formula (I), i.e., o-carborane or an o-carborane derivative:

[0053] [ka]

[0054] In the above formula (I), ● is C, ◯ is each independently BH or B-halogen, and R is each independently a substituent having an optionally substituted saturated or unsaturated alkyl group or H. Here, in this specification, "each independently" means that each may be the same or different.

[0055] Carborane or its derivatives, which are boron clusters containing boron atoms and carbon atoms, are highly hydrophobic and can be efficiently encapsulated in the hydrophobic core of a self-assembly formed from an amphiphilic polymer having a hydrophilic block and a hydrophobic block, and therefore can be suitably used in the agent according to one aspect of the present invention. Among them, o-carborane derivatives having the structure represented by the above formula (I) can be more efficiently and stably supported by microparticles formed from an amphiphilic polymer containing polylactic acid as a hydrophobic block, as shown in the experimental examples described later, and therefore can be particularly suitably used.

[0056] In a preferred embodiment, R in the above formula (I) is a substituent having a saturated or unsaturated alkyl group which may be substituted, and the number of carbon atoms of the alkyl group may be 3 or more. When the substituent R in the above formula (I) has an alkyl group having 3 or more carbon atoms, when the boron-containing compound is loaded onto a microparticle formed from an amphiphilic polymer containing polylactic acid as a hydrophobic block, the boron-containing compound interacts with the polylactic acid chain constituting the hydrophobic core of the microparticle, particularly the polylactic acid chain forming an α-helix structure, via the alkyl group, thereby acting as a molecular glue binding two polylactic acid chains together, thereby dramatically improving the stability of the microparticle in an aqueous medium (FIG. 20). From the viewpoint of improving the stability of the microparticle, the number of carbon atoms of the alkyl group is preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, and even more preferably 6 or more. On the other hand, there is no particular upper limit on the number of carbon atoms of the alkyl group, but according to the findings of the present inventors, if the number of carbon atoms constituting the alkyl group is too large, the boron-containing compound will form aggregates by itself, and the boron-containing compound may be difficult to support on the microparticles. From this viewpoint, the number of carbon atoms of the alkyl group is preferably 11 or less, more preferably 10 or less, more preferably 9 or less, even more preferably 8 or less, even more preferably 7 or less, and even more preferably 6 or less. In summary, the number of carbon atoms of the alkyl group is preferably 3 or more, more preferably 3 to 11 or less, even more preferably 4 to 10 or less, even more preferably 4 to 9 or less, even more preferably 5 to 8 or less, even more preferably 5 to 7 or less, and particularly preferably 6. As shown in the experimental examples described later, when the number of carbon atoms of the alkyl group is within the above range, the boron-containing compound represented by the above formula (I) can be particularly efficiently supported on microparticles formed from an amphiphilic polymer containing polylactic acid as a hydrophobic block, and can exhibit a more remarkable stabilizing effect on the microparticles.

[0057] Specific examples of the substituent having an alkyl group include an optionally substituted saturated or unsaturated alkyl group, an optionally substituted saturated or unsaturated alkoxy group, or an optionally substituted saturated or unsaturated alkylthio group, preferably an optionally substituted saturated or unsaturated alkyl group, more preferably an optionally substituted saturated alkyl group, and more preferably an unsubstituted saturated alkyl group. The substituent having an alkyl group may be either linear or branched, but is preferably linear.

[0058] Further specific examples of the substituent having an alkyl group having 3 or more carbon atoms include an n-propyl group (-(CH 2 ) 2 CH 3 ), n-butyl group (-(CH 2 ) 3 CH 3 ), n-pentyl group (-(CH 2 ) 4 CH 3 ), n-hexyl group (-(CH 2 ) 5 CH 3 ), n-heptyl group (-(CH 2 ) 6 CH 3 ), n-octyl group (-(CH 2 ) 7 CH 3 ) and the general formula (-(CH 2 ) n CH 3 )) (where n is 2 or more), n-propoxy group (-O(CH 2 ) 2 CH 3 )), n-butoxy group (-O(CH 2 ) 3 CH 3 )), n-pentoxy group (-O(CH 2 ) 4 CH 3 )), n-hexyloxy group (-O(CH 2 ) 5 CH 3 )) and other general formulas (-O(CH 2 )n CH 3 )) (where n is 2 or more), an n-propylthio group (-S(CH 2 ) 2 CH 3 )), n-butylthio group (-S(CH 2 ) 3 CH 3 )), n-pentylthio group (-S(CH 2 ) 4 CH 3 )), n-hexylthio group (-S(CH 2 ) 5 CH 3 )) and other general formulas (-S(CH 2 ) n CH 3 )) (n is 2 or more), but is not limited thereto. When the substituent having an alkyl group with 3 or more carbon atoms is an alkyl group, an alkoxy group, or an alkylthio group represented by any of the above general formulas, n in the general formula is preferably 2 or more and 10 or less, more preferably 3 or more and 9 or less, more preferably 3 or more and 8 or less, more preferably 4 or more and 7 or less, even more preferably 4 or more and 6 or less, and particularly preferably 5. A specific example of the boron-containing compound represented by the above formula (I) having a substituent having an alkyl group as R as described above is, for example, 1,2-dihexyl-o-carborane shown in the experimental example described later.

[0059] In this specification, "may be substituted" includes both cases where the substitutable hydrogen atoms constituting the group are not substituted and cases where they are substituted. When the substitutable hydrogen atoms are not substituted, it means that all the substitutable hydrogen atoms are hydrogen atoms. On the other hand, when the substitutable hydrogen atoms are substituted, it means that at least one of the substitutable hydrogen atoms is substituted with an atom other than a hydrogen atom or a carbon atom. There is no particular restriction on the type of atom to be a substituent, but from the viewpoint of the binding property to the α-helix structure formed by polylactic acid, it is preferable that the atom is relatively highly hydrophobic. Specific examples of such atoms include, but are not limited to, halogens such as fluorine, chlorine, bromine, iodine, and astatine. There may be multiple substitution sites.

[0060] Those skilled in the art can synthesize the o-carborane derivative represented by the above formula (I) by an appropriate method. For example, an o-carborane derivative in which the substituent represented by R in formula (I) is an optionally substituted saturated or unsaturated alkyl group can be obtained by reacting o-carborane with a strong base such as BuLi or PhLi, and then reacting with an appropriate alkyl halide. In addition, an o-carborane derivative in which the substituent represented by R in formula (I) is an optionally substituted alkoxy or alkylthio group can be obtained by reacting an o-carborane derivative having hydroxyl groups (-OH) at the 1st and 2nd positions (1,2-(OH) 2 -1,2-closo-C 2 B 10 H 10 ;CAS number 868382-62-1) or o-carborane derivatives with thiol groups at the 1st and 2nd positions (1,2-(SH) 2 -1,2-closo-C 2 B 10 H 10;CAS number 23810-63-1) with an appropriate alkyl halide in the presence of a strong base such as KOH to alkoxylate or alkylthiolate the o-carborane derivative. However, the synthesis method of the o-carborane derivative is not limited to the above, and a person skilled in the art can use an appropriate method (TL Heying et al., Inorg. Chem. 1963, 2(6), 1097-1105; MM Fein et al., Inorg. Chem. 1963, 2(6), 1115-1119).

[0061] The boron-containing compound that can be contained in the agent according to one aspect of the present invention contains boron 10( 10 B) is preferably contained. Naturally occurring boron isotopes include boron-10 ( 10 B) and boron-11( 11 There are two known isotopes of boron: boron-10, which accounts for approximately 19.9% ​​of naturally occurring boron, and boron-11, which accounts for approximately 80.1%. Boron neutron capture therapy uses these isotopes of boron: 10 Therefore, from the viewpoint of use in boron neutron capture therapy, the boron-containing compound that can be contained in the microparticles of the present invention is 10 The isotope ratio of B is preferably 19.9% ​​or more, more preferably 50% or more, even more preferably 70% or more, and even more preferably 90% or more. 10 The isotope ratio of B is 10 Boron with increased B content ( 10 This can be increased by using B enriched boron as a raw material. 10 As a method for producing B-enriched boron, for example, a chemical exchange distillation method is known.

[0062] <Carrier> On the other hand, the agent according to one aspect of the present invention contains, in addition to the boron-containing compound and / or photosensitizer described above, a carrier for delivering the boron-containing compound and the photosensitizer to a tumor, i.e., a cancer-accumulating carrier. The cancer-accumulating carrier means a carrier that accumulates in cancer tissues or cancer cells when administered to a living body having cancer, such as a patient suffering from a cancer disease, and means a carrier that increases the amount of accumulation of the boron-containing compound and / or the photosensitizer in cancer tissues or cancer cells compared to when the agent does not contain a cancer-accumulating carrier. Such a carrier may basically be any type, and may be, for example, one or more of antibodies, fragment antibodies, peptides, amino acids, nucleic acids, aptamers, vitamins, sugars (including monosaccharides, oligosaccharides, and polysaccharides), sugar chains, and fine particles. The boron-containing compound and / or the photosensitizer may be covalently and / or non-covalently bound to these carriers and carried thereon.

[0063] In a preferred embodiment, the carrier is preferably a microparticle. The microparticle may be, for example, a microparticle having an average particle diameter of 20 nm to 500 nm measured by dynamic light scattering. From the viewpoint of increasing accumulation in cancer tissues or increasing accumulation and permeability in cancer tissues, the average particle diameter (diameter) measured by dynamic light scattering is preferably 20 nm to 150 nm, more preferably 20 nm to 140 nm, even more preferably 20 nm to 130 nm, and even more preferably 20 nm to 120 nm. It has been reported that microparticles having these average particle diameters accumulate and accumulate in cancer tissues mainly due to the EPR effect (Enhanced Permeability and Retention Effect) (J. Fang et al., Advanced Drug Delivery Reviews 63 (2011) 136-151), and can be suitably used as a carrier that accumulates in cancer tissues.

[0064] On the other hand, there is no particular limitation on the type of microparticles that can be used in the present invention, and the microparticles may be, for example, organic particles such as dendrimers and liposomes, or inorganic particles such as gold nanoparticles and silica nanoparticles. In a preferred embodiment, however, the microparticles are preferably microparticles containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block.

[0065] <Hydrophobic block> The "hydrophobic block" of an amphiphilic polymer means a polymer chain that is hydrophobic as a whole block. Here, the hydrophobicity of the hydrophobic block means that it has low solubility in water, and more preferably, that the amphiphilic polymer containing a hydrophilic block and a hydrophobic block has low solubility in water to such an extent that it can self-assemble or aggregate in an aqueous medium to form micellar or vesicle-like particles. Those skilled in the art can confirm the formation of micellar or vesicle-like particles, for example, by dynamic light scattering or transmission electron microscopy.

[0066] The amphiphilic polymer that can be used in the present invention contains polylactic acid as a hydrophobic block. "Polylactic acid" means a polymer formed by polymerization of lactic acid. Since polylactic acid has excellent biocompatibility and biodegradability, amphiphilic polymers containing polylactic acid as a component can be advantageously used as pharmaceutical materials. Lactic acid, which is a monomer of polylactic acid, exists as L-lactic acid and its optical isomer D-lactic acid. Polylactic acid obtained mainly by polymerizing L-lactic acid is called poly(L-lactic acid) (PLLA: poly(L-lactic acid)), polylactic acid obtained mainly by polymerizing D-lactic acid is called poly(D-lactic acid) (PDLA: poly(D-lactic acid)), and polylactic acid obtained by polymerizing L-lactic acid and D-lactic acid is called poly(DL-lactic acid) (PDLLA: poly(DL-lactic acid)).

[0067] According to the findings of the present inventors, the α-helix structure formed by the polylactic acid chain has a hydrophobic portion on the outer surface and can strongly interact with a boron-containing compound described later, i.e., o-carborane having two alkyl groups at the ortho positions, to stabilize the microparticles (FIG. 20). Therefore, in a preferred embodiment, the "polylactic acid" that can be used in the present invention is preferably one that can form an α-helix-type higher-order structure. Such polylactic acid can be, for example, poly(L-lactic acid) and poly(D-lactic acid), with poly(L-lactic acid) being particularly preferred. Note that the poly(L-lactic acid) or poly(D-lactic acid) that can be used in a preferred embodiment of the present invention does not necessarily have to have an optical purity of 100% or an isomer content of 0%, as long as it can form an α-helix-type higher-order structure. For example, it may be poly(L-lactic acid) or poly(D-lactic acid) having an isomer content of 10% or less, preferably 6% or less, more preferably 5% or less, even more preferably 3% or less, even more preferably 2% or less, and even more preferably 1% or less.

[0068] The hydrophobic block containing polylactic acid includes both the case where the entire hydrophobic block is polylactic acid and the case where a part of the hydrophobic block is polylactic acid. That is, the hydrophobic block that can be used in the present invention may have monomer units other than lactic acid units to the extent that polylactic acid can form a higher-order structure. There is no particular limit to the content of monomer units other than lactic acid units that can be contained in the hydrophobic block, but it is preferably 10% or less, more preferably 8% or less, even more preferably 5% or less, even more preferably 1% or less, and particularly preferably 0% based on the total monomer units (including lactic acid units) contained in the hydrophobic block.

[0069] There is no particular limitation on the type of monomer unit other than the lactic acid unit that can be contained in the hydrophobic block, but a hydroxy acid other than lactic acid can be preferably used. Note that a hydroxy acid is an organic compound having a carboxyl group (-COOH) and a hydroxyl group (-OH) in one molecule, and examples of the hydroxy acid other than lactic acid include glycolic acid, 2-hydroxyisobutyric acid, and 3-hydroxyisobutyric acid.

[0070] The number of lactic acid units constituting the polylactic acid that can be contained in the hydrophobic block is not particularly limited, and can be appropriately adjusted so that the hydrophobic block exhibits the above-mentioned hydrophobicity, and can be, for example, 5 to 200, preferably 10 to 100, more preferably 15 to 80, even more preferably 20 to 60, and even more preferably 25 to 45. In this specification, the numerical range indicated by "to" means a numerical range including the upper and lower limits.

[0071] <Hydrophilic block> On the other hand, the term "hydrophilic block" refers to a polymer chain having hydrophilicity. Here, the hydrophilicity of the hydrophilic block means that the hydrophilicity is relatively higher than that of the hydrophobic block containing polylactic acid, and more preferably means that the amphiphilic polymer containing the hydrophilic block and the hydrophobic block is capable of self-assembling or aggregating in an aqueous medium to form micelle- or vesicle-like particles.

[0072] There are no particular limitations on the specific type of polymer constituting the "hydrophilic block" contained in the amphiphilic polymer that can be used in the present invention, but from the viewpoint of increasing the retention of the microparticles in the blood and, in turn, increasing the accumulation of the microparticles at the tumor site to be treated, it is preferable for the polymer to be a hydrophilic polymer with low protein adsorption. Examples of such hydrophilic polymers include polypeptoids such as polysarcosine and other polymers of N-substituted glycines; polyamino acids or derivatives thereof such as polyglutamic acid, poly(hydroxyethyl-L-glutamine), poly(hydroxyethyl-L-asparagine), PAS polypeptides consisting of repeating units of Pro-Ala-Ser, and XTEN polypeptides consisting of repeating units of Gly-Ala-Pro-Glu-Ser-Thr; polysaccharides such as dextran, hyaluronic acid, and polysialic acid; zwitterionic polymers such as phosphobetaine polymers, sulfobetaine polymers, and carboxybetaine polymers; and uncharged synthetic polymers such as polyethylene glycol, polyglycerol, poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), polyvinylpyrrolidone, and poly(2-hydroxyethyl methacrylate).

[0073] The number of monomer units of the polymer constituting the hydrophilic block is not particularly limited, and may be appropriately adjusted depending on the type of polymer or monomer constituting the hydrophilic block so that the hydrophilic block exhibits the above-mentioned hydrophilicity. For example, the number may be 10 to 300, preferably 20 to 250, more preferably 30 to 200, even more preferably 40 to 150, and even more preferably 50 to 100.

[0074] The hydrophilic block may be linear or branched. When the hydrophilic block has a branch, the number of branches is not particularly limited, but is preferably 2 to 3. When the hydrophilic block is branched, the number of monomer units constituting the hydrophilic block as a whole may be, for example, 10 to 300, preferably 20 to 250, more preferably 30 to 200, even more preferably 40 to 150, and even more preferably 50 to 100.

[0075] <Amphiphilic polymer> The amphiphilic polymer that can be used in the agent according to one aspect of the present invention is not particularly limited as long as it is a polymer containing a hydrophilic block and a hydrophobic block as described above, but in a preferred embodiment, it has a hydrophilic block containing polysarcosine. Polysarcosine means a polymer formed by polymerization of sarcosine (also called "N-methylglycine"). Since polysarcosine has high biocompatibility, low nonspecific protein adsorption, and low immunogenicity and can avoid the ABC (Accelerated Blood Clearance) phenomenon (K. Son et al., J. Controlled Rel. 2020, 322(10), 209-216), it can be particularly preferably used as a hydrophilic block of an amphiphilic polymer that can be contained in an agent according to one aspect of the present invention. An amphiphilic polymer having a hydrophobic block containing polylactic acid and a hydrophilic block containing polysarcosine can be, for example, polysarcosine-polylactic acid, which is a block copolymer formed by binding polysarcosine and polylactic acid. It is known that polysarcosine-polylactic acid self-assembles in an aqueous medium to form microparticles with a diameter of about 20 nm to 200 nm (M. Akira et al., Chemistry Letters, 2007, 36(10), 1220-1221). In addition, the microparticles formed by polysarcosine-polylactic acid have high retention in blood and little accumulation in the liver. For these reasons, a block copolymer of polysarcosine and polylactic acid can be particularly preferably used as an amphiphilic polymer that can be contained in the microparticles of the present invention. Note that polysarcosine may be linear, branched, or may contain both. Polysarcosine-polylactic acid containing linear polysarcosine includes, for example, AB-type polysarcosine-polylactic acid block polymers. AB-type polysarcosine-polylactic acid block polymers and a method for synthesizing the same are disclosed, for example, in International Publication No. WO2009 / 148121. On the other hand, examples of polysarcosine-polylactic acid containing branched polysarcosine include A3B type polysarcosine-polylactic acid block polymers, which are block polymers of three-branched polysarcosine and polylactic acid.A3B type polysarcosine-polylactic acid block polymers and their synthesis methods are disclosed, for example, in International Publication No. WO2012 / 176885, MSH Lim et al., Journal of Pharmaceutical Sciences 2021, 110, 1788-1798, and MSH Lim et al., Life 2021, 11(2), 158. Nanoparticles formed from A3B type polysarcosine-polylactic acid block polymers can further reduce the ABC phenomenon compared to nanoparticles formed from AB type polysarcosine-polylactic acid block polymers (Takenaka et al., Drug Delivery System 33-3, 2018; MSH Lim et al., Life 2021, 11(2), 158).

[0076] In the polysarcosine-polylactic acid block copolymer, the number of repetitions of the sarcosine units constituting the hydrophilic block, polysarcosine, is not particularly limited as long as the hydrophilic block as a whole exhibits the above-mentioned hydrophilicity, but may be, for example, 10 to 300, preferably 20 to 250, more preferably 30 to 200, even more preferably 40 to 150, and even more preferably 50 to 100. When the polysarcosine is a branched polysarcosine, the number of repetitions of the sarcosine units in the hydrophilic block as a whole may be, for example, 10 to 300, preferably 20 to 250, more preferably 30 to 200, even more preferably 40 to 150, and even more preferably 50 to 100. When the polysarcosine has branches, the number of branches is not particularly limited, but is preferably 2 to 3.

[0077] On the other hand, the number of repeating lactic acid units constituting the polylactic acid, which is the hydrophobic block of the polysarcosine-polylactic acid block copolymer, is not particularly limited as long as the hydrophobic block as a whole exhibits the above-mentioned hydrophobicity, but may be, for example, 10 to 100, preferably 15 to 80, more preferably 15 to 60, even more preferably 20 to 50, and even more preferably 25 to 45. When the polylactic acid has a branched structure, the number of repeating lactic acid units in the entire hydrophobic block may be, for example, 10 to 400, preferably 20 to 200. When the hydrophobic block is branched, the number of branches of the hydrophobic block is not particularly limited, but from the viewpoint of obtaining fine particles by self-assembly of an amphiphilic polymer, it is preferable that the number of branches is equal to or less than the number of branches of the hydrophilic block.

[0078] The microparticles containing the amphiphilic polymer having a hydrophilic block and a hydrophobic block and further containing a boron-containing compound and / or a photosensitizer can typically be obtained by self-assembly of the amphiphilic polymer containing a hydrophilic block and a hydrophobic block and the boron-containing compound and / or the photosensitizer in an aqueous medium. The boron-containing compound and / or the photosensitizer may be covalently bonded to the amphiphilic polymer or non-covalently bonded to the amphiphilic polymer. There is no particular limitation on the method for preparing such microparticles, but for example, as described below, they can be prepared by a preparation method including a step of mixing an amphiphilic polymer containing polylactic acid as a hydrophobic block with a boron-containing compound and / or a photosensitizer to obtain a mixture, and a step of contacting the mixture with an aqueous medium.

[0079] In addition, there is no particular limitation on the form of the microparticles containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block, but they may typically be polymeric micelle-like microparticles. The polymeric micelle-like microparticles may typically be core-shell type microparticles that contain an amphiphilic polymer and have a shell composed of a hydrophilic block and a core composed of a hydrophobic block. On the other hand, the form of the microparticles may be vesicle-like hollow microparticles. For example, vesicle-like hollow microparticles formed from an amphiphilic polymer having polylactic acid as a hydrophobic block can be appropriately prepared by a person skilled in the art (H. Yin et al., Macromolecules 2009, 42(19), 7456-7464; H. Qi et al., Nature Communications 9, Article number: 3005 (2018)).

[0080] In addition, when using polymer micelle-type microparticles containing amphiphilic polymers, the diameter of the microparticles can be appropriately controlled by adjusting the molecular weight of the amphiphilic polymer constituting the microparticles, the ratio of hydrophilic blocks and hydrophobic blocks, etc., which is extremely advantageous. For example, when preparing microparticles using an amphiphilic polymer containing polysarcosine as a hydrophilic block and polylactic acid as a hydrophobic block, nanoparticles having a smaller particle size can be prepared by using an A3B type polysarcosine-polylactic acid block polymer compared to nanoparticles formed from an AB type polysarcosine-polylactic acid block polymer (MSH Lim et al., Life 2021, 11(2), 158).

[0081] In a preferred embodiment, the microparticles that can be used in the present invention may contain a targeting molecule. The targeting molecule refers to a chemical substance that can increase the accumulation of the microparticles containing the targeting molecule in the target tissue or the binding to the target cell compared to when the microparticles do not contain the targeting molecule, and is typically a molecule that has the property of selectively binding to a substance such as a protein that is specifically and highly expressed in the target cell. There is no particular limitation on such targeting molecules, and they may be, for example, antibodies, low molecular weight antibody variants, proteins, peptides, peptoids, nucleic acids, vitamins, sugars (including monosaccharides, oligosaccharides, and polysaccharides), sugar chains, etc. In addition, since the targeting molecule is a molecule that selectively binds to a specific substance present in the target cell, it is preferable that it is present on the surface of the microparticle.

[0082] In addition, such microparticles having targeting molecules on the particle surface can be prepared by a person skilled in the art using an appropriate method. For example, the microparticles may be prepared by using an amphipathic polymer in which a targeting molecule is bound to the end of the hydrophilic block contained in the amphipathic polymer described above. On the other hand, in addition to the amphipathic polymer and the boron-containing compound and / or the photosensitizer, a conjugate of polylactic acid in which a targeting molecule is bound to the end of polylactic acid may be used to prepare a microparticle carrying a targeting molecule.

[0083] As a specific example of the targeting molecule, for example, a small molecule antibody variant (scFv) against mesothelin (MSLN), which is known to be highly expressed in many types of cancer such as mesothelioma, pancreatic adenocarcinoma, ovarian cancer, and lung adenocarcinoma (H. Yakushiji et al., Cancer Science. 2019, 110(9), 2722-2733), can be preferably used. In a preferred embodiment, a cell-penetrating peptide may be used. A cell-penetrating peptide is a peptide that has the property of penetrating the cell membrane of a cell, particularly a human cell, and a microparticle containing a cell-penetrating peptide can be efficiently introduced into the cell. There is no particular limitation on the type of cell-penetrating peptide that can be contained in the agent according to the present invention, but examples thereof include, but are not limited to, TAT peptide, penetratin, polyarginine (R9), transportan, Pep-1, and the like. Those skilled in the art can use appropriate cell-penetrating peptides (DM Copolovici et al., ACS Nano 2014, 8(3), 1972-1994; MM Khan et al., J. Controlled Rel. 2021, 330(10), 1220-1228). Thus, microparticles containing cell-penetrating peptides can be obtained, for example, by preparing microparticles using an amphiphilic polymer in which a cell-penetrating peptide is bound to the end of the hydrophilic block contained in the amphiphilic polymer described above. In addition to the amphiphilic polymer and the boron-containing compound and / or photosensitizer, a microparticle carrying a cell-penetrating peptide may be prepared by preparing a microparticle using a conjugate of polylactic acid and a cell-penetrating peptide in which a cell-penetrating peptide is bound to the end of polylactic acid. Methods for preparing microparticles carrying cell-penetrating peptides are described, for example, in MSH Lim et al., Journal of Pharmaceutical Sciences 2021, 110, 1788-1798 and MSH Lim et al., Life 2021, 11(2), 158.

[0084] There is no particular limit to the amount of boron carried in the agent according to one aspect of the present invention, but as long as the agent according to one aspect of the present invention can exist stably, the more the amount of boron carried, the better.When the carrier contained in the agent according to one aspect of the present invention is a microparticle containing an amphiphilic polymer, there is no particular limit to the ratio of the amphiphilic polymer to the boron-containing compound, but for example, the molar ratio (A / B) of the boron contained in the (A) boron-containing compound to the (B) amphiphilic polymer can be 0.1 to 30.0, 0.2 to 15.0, 0.3 to 12.0, 0.5 to 8.0, 0.5 to 5.0, or 0.6 to 3.5.

[0085] <Pharmaceutical Composition> The pharmaceutical composition according to the present invention contains the agent or radiosensitizer as described above. There is no particular limitation on the disease to be treated, but it can be particularly used to treat cancer, more preferably to treat cancer by boron neutron capture therapy and / or photodynamic therapy. In this specification, "treatment" of cancer includes not only curing cancer, but also shrinking cancer, alleviating or improving symptoms of cancer, and delaying the progression of cancer. Subjects to which the pharmaceutical composition can be administered include humans and animals other than humans.

[0086] There is no particular limitation on the type of cancer for which the pharmaceutical composition of the present invention can be used, but examples of such cancers include solid cancers and blood cancers, including brain tumors (meningioma, glioma, pituitary tumor, acoustic neuroma, glioblastoma multiforme, etc.), malignant melanoma, head and neck cancer, lung cancer, liver cancer, thyroid cancer, kidney cancer, prostate cancer, breast cancer, pancreatic cancer, colon cancer, bladder cancer, meningioma, mesothelioma, sarcoma, gastric cancer, ovarian cancer, uterine cancer, cervical cancer, pharyngeal cancer, laryngeal cancer, bone cancer, etc. In addition, since boron neutron capture therapy is known to be effective in treating head and neck cancers such as highly malignant brain tumors in which cancer cells and normal cells are mixed, and malignant melanoma, the pharmaceutical composition of the present invention can be particularly suitably used in the treatment of head and neck cancer and malignant melanoma.

[0087] There is no particular limitation on the method of administration of the pharmaceutical composition according to the present invention, and an appropriate administration method may be selected depending on the subject of application, for example, oral administration, sublingual administration, intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, local administration including intratumoral administration, drip administration, etc., but are not limited thereto.

[0088] The dosage form of the pharmaceutical composition according to the present invention is not particularly limited, and may be an appropriate dosage form depending on the application and administration method. For example, the dosage form may be, but is not limited to, injections, tablets, powders, granules, liquids, syrups, capsules, etc. Also, it may be a preparation prepared at the time of use. Needless to say, the pharmaceutical composition according to the present invention may further contain pharma- ceutical acceptable excipients, binders, disintegrants, lubricants, coating agents, solvents, buffers, preservatives, chelating agents, antioxidants, isotonicity agents, stabilizers, etc., depending on the dosage form and administration method.

[0089] Examples of excipients that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, mannitol, crystalline cellulose, corn starch, etc. Examples of binders include, but are not limited to, crystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc. Examples of disintegrants include, but are not limited to, hydroxypropyl cellulose, carboxymethyl cellulose, corn starch, croscarmellose, etc. Examples of lubricants include, but are not limited to, magnesium stearate, calcium stearate, talc, carnauba wax, etc. Examples of coating agents include, but are not limited to, hydroxypropyl methylcellulose, polyethylene glycol, talc, titanium oxide, iron oxide, etc. Examples of solvents include, but are not limited to, water, ethanol, propylene glycol, glycerin, vegetable oil, etc. Examples of buffers include, but are not limited to, phosphate buffers, borate buffers, citrate buffers, tartaric acid buffers, acetate buffers, amino acids, etc. Examples of preservatives include, but are not limited to, quaternary ammonium salts such as benzalkonium chloride, paraoxybenzoic acid esters such as methyl paraoxybenzoate, benzyl alcohol, sorbic acid and its salts, thimerosal, parabens, cetylpyridinium chloride, and catechin. Examples of chelating agents include, but are not limited to, sodium edetate and citric acid. Examples of antioxidants include, but are not limited to, sodium hydrogen sulfite, sodium sulfite, sodium pyrosulfite, ascorbic acid, and ascorbic acid glycoside. Examples of isotonicity agents include, but are not limited to, glucose and table salt. Examples of stabilizers include, but are not limited to, sodium edetate, citric acid, ascorbic acid, sodium chloride, polyols, and polysorbates. In addition, the pharmaceutical product according to the present invention may contain one or more active ingredients that are incorporated into other pharmaceutical products, such as anticancer agents, and nucleic acids such as siRNA and mRNA.A method for preparing microparticles carrying substances such as nucleic acids is described, for example, in MSH Lim et al., Journal of Pharmaceutical Sciences 2021, 110, 1788-179. Microparticles carrying nucleic acids can be effectively used not only for the treatment of cancer by neutron irradiation, but also for the treatment of cancer by RNA interference (RNAi).

[0090] When the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention is used to treat cancer, the subject to which it has been administered may be irradiated with radiation. That is, in one aspect, the present invention also provides a method for treating cancer, comprising the steps of administering the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention to a subject, and irradiating the subject with radiation.

[0091] In the above-mentioned therapeutic method according to one aspect of the present invention, when the agent or pharmaceutical composition administered to the subject contains a photosensitizer, the radiation irradiated to the subject to which the agent or pharmaceutical composition is administered is preferably radiation capable of generating Cerenkov light in a living body. Such radiation may be, for example, radiation containing protons, electrons, and / or neutrons. By irradiating the subject with such radiation, the photosensitizer is excited, and a therapeutic effect of photodynamic therapy can be obtained.

[0092] On the other hand, in the above-mentioned therapeutic method according to one aspect of the present invention, when the agent or pharmaceutical composition administered to the subject contains a boron-containing compound, it is preferable to use radiation containing at least neutrons. By using radiation containing at least neutrons, it is possible to damage the cancer by utilizing the nuclear reaction between the boron-containing compound and neutrons, and to obtain the therapeutic effect of boron neutron capture therapy.

[0093] In the above-mentioned therapeutic method according to one aspect of the present invention, when the agent or pharmaceutical composition administered to the subject contains both a boron-containing compound and a photosensitizer, the subject to which the agent or pharmaceutical composition is administered is irradiated with radiation containing at least neutrons, which excites the photosensitizer to damage the cancer, and also damages the cancer by utilizing the nuclear reaction of the boron-containing compound and neutrons. The above-mentioned therapeutic method according to one aspect of the present invention, which can achieve a synergistic cancer therapeutic effect due to both the therapeutic effect of boron neutron capture therapy based on the nuclear reaction of the boron-containing compound and neutrons, and the therapeutic effect of photodynamic therapy by the photosensitizer, can be extremely advantageously used as a method for treating cancer using radiation. The irradiated radiation may contain at least neutrons, but may also contain other radioactive particles, such as protons (α rays) or electrons (β rays), or may also contain γ rays. When the radiation contains charged particles such as protons (α rays) and electrons (β rays) or electromagnetic waves such as γ rays, it can generate Cherenkov light more efficiently and excite the photosensitizer.

[0094] There is no particular limitation on the radiation irradiation method or radiation source that can be used in the above-mentioned treatment method according to one aspect of the present invention. The radiation source may be a nuclear reactor or an accelerator such as a cyclotron, a linear accelerator, or an electrostatic accelerator. A combination of multiple radiation sources may also be used. Meanwhile, the number of times of radiation irradiation may be one or multiple times. Depending on the site or condition of the tumor disease to be treated, or taking into consideration the age, sex, treatment period, etc. of the subject, a medical professional may select an appropriate number of times. There is also no particular limitation on the radiation irradiation time per time.

[0095] Radiation irradiation is preferably performed after the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention has reached the site to be treated. For example, radiation irradiation can be performed after a predetermined time has elapsed since administration of the agent, radiosensitizer, or pharmaceutical composition. However, the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention does not necessarily need to be administered prior to radiation irradiation, and may be administered during radiation irradiation.

[0096] The cancer treatment using the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention may be performed as a single treatment or in combination with surgery and / or chemotherapy.

[0097] <Radiation irradiation device> In one aspect, the present invention also provides a radiation irradiation device including a radiation source for irradiating radiation, the radiation irradiation device being used to treat cancer by irradiating a subject to which the agent according to one aspect of the present invention described above, a radiosensitizer, or a pharmaceutical composition containing them has been administered. Such a radiation irradiation device may be an accelerator-type radiation irradiation device.

[0098] <Method of producing fine particles> A method for producing fine particles according to one aspect of the present invention is a method for producing fine particles, comprising a step of mixing an amphipathic polymer with a boron-containing compound and / or a photosensitizer to obtain a mixture, and a step of contacting the mixture with an aqueous medium. The amphipathic polymer has a property of forming fine particles by self-assembly when it comes into contact with an aqueous medium. When the boron-containing compound and the photosensitizer are hydrophobic molecules, the boron-containing compound and the photosensitizer can be encapsulated in the fine particles formed by the amphipathic polymer by contacting a mixture of the amphipathic polymer, the boron-containing compound, and the photosensitizer with an aqueous solvent. As the hydrophobic boron-containing compound and the photosensitizer, the boron-containing compound and the photosensitizer described above can be used.

[0099] Examples of the method for producing fine particles including the above steps include the film method and the injection method (International Publication No. WO2012 / 176885). Here, the film method is a method in which, as shown in the experimental examples described below, a solution containing an amphiphilic polymer and a boron-containing compound in an organic solvent is dried to obtain a film-like mixture, and the resulting mixture is brought into contact with an aqueous medium to obtain a dispersion in which fine particles are dispersed in the aqueous medium. More specifically, (a1) preparing a mixed solution containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent; (a2) removing the organic solvent from the mixture to obtain a film containing the amphiphilic polymer and the boron-containing compound and / or the photosensitizer; and (a3) adding an aqueous medium to the film; The above steps (a1), (a2) and (a3) ​​are usually carried out in this order.

[0100] The organic solvent used in the above step (a1) is not particularly limited as long as it can dissolve the amphiphilic polymer and the boron-containing compound and / or the photosensitizer, and for example, chloroform, dimethylformamide, dimethylsulfoxide, etc. can be preferably used. The method for removing the organic solvent in the above step (a2) is not particularly limited, and a person skilled in the art can adopt an appropriate method depending on the organic solvent used. For example, the solvent may be removed by drying under reduced pressure, or by natural drying. On the other hand, the aqueous medium used in the above step (a3) ​​is not particularly limited as long as it is water or a solvent mainly composed of water, and for example, distilled water for injection, physiological saline, buffer solution (e.g., PBS), etc. can be preferably used.

[0101] In a preferred embodiment, the method for producing microparticles according to one aspect of the present invention may further include, after the step (a3), a step of stirring or ultrasonically treating the mixture of the film and the aqueous medium.

[0102] On the other hand, the injection method is a method in which a solution containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent is dispersed in an aqueous medium to obtain a dispersion in which fine particles are dispersed in the aqueous medium. More specifically, the injection method is (b1) preparing a mixed solution containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent; (b2) dispersing the mixture in an aqueous medium; and (b3) removing the organic solvent; The above steps (b1), (b2) and (b3) are usually carried out in this order.

[0103] The organic solvent used in the above step (b1) is not particularly limited as long as it can dissolve the amphiphilic polymer and the boron-containing compound and / or the photosensitizer. For example, dimethylformamide, dimethylsulfoxide, etc. can be suitably used.

[0104] On the other hand, the above step (b2) is a step of injecting and mixing a mixture containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent into an aqueous medium to obtain a dispersion in which microparticles containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer are dispersed. Here, the aqueous medium used in this step is not particularly limited as long as it is water or a solvent mainly composed of water, and for example, distilled water for injection, physiological saline, buffer solution (e.g., PBS), etc. can be suitably used. In addition, there is no particular limit to the method of dispersing a mixture containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent in an aqueous medium, and for example, it can be performed using a stirrer. In addition, in the above step (b3), there is no particular limit to the method of removing the organic solvent from the aqueous medium containing the organic solvent, and a person skilled in the art can adopt an appropriate method. For example, the organic solvent may be removed under a reduced pressure environment.

[0105] In a preferred embodiment, the above-mentioned production method may further include an appropriate purification step before or after the above-mentioned step (b3). For example, the purification method may be, but is not limited to, gel filtration chromatography, filter treatment, ultracentrifugation, etc. Furthermore, these purification methods may be combined as necessary.

[0106] In addition, the boron-containing compound represented by the above formula (I), in which the substituent R has 3 or more carbon atoms, mainly stabilizes the hydrophobic core composed of polylactic acid, thereby stabilizing the microparticles. Therefore, the boron-containing compound represented by the above formula (I), in which the substituent R has 3 or more carbon atoms, is expected to stabilize microparticles formed not only from amphiphilic polymers but also from a wide range of polymers containing polylactic acid. That is, in a preferred embodiment, the method for producing microparticles according to one aspect of the present invention may be a method for producing microparticles, comprising the steps of: mixing a polymer containing polylactic acid with the boron-containing compound represented by the above formula (I), in which the substituent R has 3 or more carbon atoms to obtain a mixture; and contacting the mixture with an aqueous medium.

[0107] From the same viewpoint, in one aspect, the present invention also provides a stabilizer for microparticles containing polylactic acid, the stabilizer comprising a boron-containing compound represented by the above formula (I) and having 3 or more carbon atoms in the substituent R as an active ingredient. As explained above, the boron-containing compound represented by the above formula (I) and having 3 or more carbon atoms in the substituent R is expected to stabilize microparticles formed from a polymer containing polylactic acid, so the boron-containing compound represented by the above formula (I) and having 3 or more carbon atoms in the substituent R can be suitably used as a stabilizer for microparticles containing polylactic acid. The stabilizer for microparticles containing polylactic acid can be an agent that improves the stability of the microparticles in an aqueous medium, or an agent that delays the disintegration of the microparticles in an aqueous medium, compared to microparticles not containing the stabilizer. The stability or disintegration of the microparticles in an aqueous medium can be evaluated by a person skilled in the art, for example, by observing the change in particle size of the microparticles over time using a dynamic light scattering method.

[0108] The disclosures of all documents cited herein are incorporated by reference in their entirety. Also, when this specification is translated into English, the singular words "a," "an," and "the" are intended to include the plural as well as the singular, unless the context clearly indicates otherwise.

[0109] The present invention will be described in more detail below with reference to experimental examples. However, these experimental examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention in any way.

[0110] <Reagents> In the experiments described below, the following reagents were used unless otherwise specified: AB type block polymer of polysarcosine and poly(L-lactic acid) (PSar 106 -block-PLLA 32 ) (weight average molecular weight: 10,001 Da) was purchased from Kobe Natural Products Chemical Co., Ltd. (Kobe, Japan). o-Carborane, m-carborane, p-carborane, 1,2-dimethyl-o-carborane, 1,2-dihexyl-o-carborane, and 1,2-didodecyl-o-carborane were purchased from Katchem, Ltd. (Prague, Czech Republic). 10 B-BPA was purchased from Interpharma Praha, as (IPP) (Prague, Czech Republic). RPMI-1640 medium, Dulbecco's phosphate buffered solution (DPBS), crystal violet, chloroform, hydrogen peroxide (H 2 O 2 ), perchloric acid (HClO 4 ), and nitric acid (HNO 3) was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich, Co., Ltd. (St. Louis, MO, USA). Corning® Matrigel® Matrix was purchased from Corning (Arizona, USA). Boron standard solution, penicillin-streptomycin mixed solution, and 2.5 g / L-trypsin / 1 mmol / L-EDTA solution were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

[0111] In the following experimental examples, L-BPA was used after preparing a complex with fructose (BPA-F) by the method described in Coderre, et al. (Coderre, JA; Button, TM; Micca, PL; Fisher, CD; Nawrocky, MM; Liu, HB Neutron capture therapy of the 91 rat gliosarcoma using the P-boronophenylalanine-fructose complex, Int. J. Radiat. Oncol. Biol. Phys. 1994 30 (3), 643-652.). The procedure is briefly as follows: A solution containing L-BPA (Interpharma Praha, As(IPP), Prague, Czech Republic) at a concentration of 90 mg / mL and fructose at a concentration of 75 mg / mL was gently stirred, and 5.0 M NaOH was added to adjust the pH to 9.5-10.0, and the solution was stirred for 2-3 minutes. Then, HCl was added to adjust the pH to 7.4, and the solution was passed through a syringe filter with a pore size of 0.22 μm before being used in the experiments described below.

[0112] <Experiment 1. Carborane-loaded polysarcosine-polylactide nanoparticles (PSAR-PLLA nanoparticles)> We prepared nanoparticles carrying carborane as a boron-containing compound. As an amphiphilic polymer for preparing the nanoparticles, we used polysarcosine-poly(L-lactic acid) block polymer (PSAR-PLLA) with polysarcosine as the hydrophilic block and poly(L-lactic acid) as the hydrophobic block. More specifically, we used AB-type polysarcosine-poly(L-lactic acid) block polymer (PSAR-PLLA) with polysarcosine and poly(L-lactic acid). 106 -block-PLLA 32 ) (weight average molecular weight: 10,001 Da) (manufactured by Kobe Natural Products Chemical Co., Ltd.) was used. In the following experimental examples, the above-mentioned AB-type polysarcosine-poly(L-lactic acid) block polymer may be referred to as "PSAR-PLLA polymer", and nanoparticles prepared using the block polymer may be referred to as "polysarcosine-polylactic acid nanoparticles" (PSAR-PLLA nanoparticles). As carborane, three isomers, o-carborane, m-carborane, and p-carborane (Katchem, Ltd., Prague, Czech Republic), were used. The PSar 106 -block-PLLA 32 The structures of o-carborane, m-carborane, and p-carborane are shown in Figure 1.

[0113] <Experiment 1-1. Preparation of PSAR-PLLA nanoparticles> PSAR-PLLA nanoparticles carrying o-carborane, m-carborane, and p-carborane were prepared by the film method. 106 -block-PLLA 32) was dissolved in 1.0 mL of chloroform to obtain a solution containing PSAR-PLLA polymer. Meanwhile, 10 μmol of carborane (o-carborane, m-carborane, or p-carborane) was dissolved in 100 μL of chloroform to obtain a solution containing carborane. 1 mL of the solution containing PSAR-PLLA polymer and 100 μL of the solution containing carborane were added to a test tube, mixed, and the organic solvent was removed by evaporation to obtain a film containing PSAR-PLLA polymer and carborane. 2.0 mL of PBS (phosphate buffered saline) was added to the test tube containing the obtained film, and the film was sonicated for 20 minutes using a bath-type ultrasonicator. After sonication, the eluate containing PSAR-PLLA nanoparticles was collected using a PD-10 column (GE Healthcare). The eluate containing PSAR-PLLA nanoparticles was passed through a syringe filter with a pore size of 0.22 μm before being used in the experiments described below. For comparison, PSAR-PLLA nanoparticles not supporting carborane were prepared in the same manner as above, except that the solution containing carborane was not used.

[0114] <Experiment 1-2. Evaluation of PSAR-PLLA nanoparticles> Particle size distribution and polydispersity The particle size and polydispersity index (PDI) of the obtained PSAR-PLLA nanoparticles were measured by dynamic light scattering using a Zetasizer Nano ZSP (Malvern, Malvern, United Kingdom). Boron loading amount and boron loading rate The amount of boron loaded in the PSAR-PLLA nanoparticles was measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) using an ICP optical emission spectrometer (Shimadzu Corporation). Based on the amount of boron loaded measured by ICP-AES, the boron loading rate (DLC (%)) of the PSAR-PLLA nanoparticles was calculated according to the following formula. In the following formula, "weight of loaded drug" is the amount of boron loaded determined by ICP-AES, and "weight of nanoparticles" is the mass of the PSAR-PLLA polymer used to prepare the PSAR-PLLA nanoparticles.

[0115]

number

[0116] The boron loading amount (Loaded B), boron loading rate (DLC (%)), and ratio of boron to PSAR-PLLA polymer in PSAR-PLLA nanoparticles (B / PSAR-PLLA Ratio) are shown in Table 1 for the PSAR-PLLA nanoparticles loaded with three types of carborane isomers (i.e., o-carborane, m-carborane, and p-carborane).

[0117] [Table 1]

[0118] As shown in Table 1, the boron loading rate (DLC (%)) in PSAR-PLLA nanoparticles carrying o-carborane, m-carborane, or p-carborane was 13.0%, 3.49%, and 1.80%, respectively, and it was confirmed that all carboranes were loaded on the PSAR-PLLA nanoparticles. In particular, the boron loading rate when o-carborane was used among the three carborane isomers was 13.0%, which was significantly higher than the boron loading rate when m-carborane or p-carborane was used. The boron loading rate when the three carborane isomers were used was higher in the order of o-carborane > m-carborane > p-carborane, which was consistent with the order of the hydrophobicity of the carborane isomers (o-carborane > m-carborane > p-carborane). This result suggests that the three carborane isomers are primarily loaded onto the hydrophobic core of PSAR-PLLA nanoparticles via hydrophobic interactions.

[0119] Meanwhile, the particle size and polydispersity of PSAR-PLLA nanoparticles carrying three types of carborane were measured by dynamic light scattering, and the results are shown in Table 2. For comparison, Table 2 also shows the particle size and polydispersity of PSAR-PLLA nanoparticles not carrying carborane.

[0120] [Table 2]

[0121] As shown in Table 2, the PSAR-PLLA nanoparticles carrying o-carborane, m-carborane, or p-carborane all had a particle size of about 60 nm to 72 nm and a PDI of about 0.20, which were nanoparticles having a particle size and PDI equivalent to those of PSAR-PLLA nanoparticles not carrying carborane. Among them, the PSAR-PLLA nanoparticles carrying o-carborane had the smallest polydispersity and an extremely monodisperse particle size distribution. The histograms of particle size distribution obtained by dynamic light scattering for the PSAR-PLLA nanoparticles carrying o-carborane and the PSAR-PLLA nanoparticles not carrying carborane are shown in FIG. 2. As shown in FIG. 2, the PSAR-PLLA nanoparticles carrying o-carborane are extremely monodisperse, similar to the PSAR-PLLA nanoparticles not carrying carborane. Furthermore, when the o-carborane-loaded PSAR-PLLA nanoparticles were examined by transmission electron microscopy, nanoparticles with a particle size distribution corresponding to the results shown by dynamic light scattering were confirmed (Figure 2).

[0122] <Experiment 1-3. Evaluation of intracellular uptake behavior> To investigate the effect of increasing intracellular boron concentration by the PSAR-PLLA nanoparticles carrying o-carborane prepared in Experiment 1-1, the intracellular uptake behavior of the PSAR-PLLA nanoparticles was evaluated. In this experiment, L-BPA, which is conventionally used as a boron drug for BNCT, was used as a comparison.

[0123] An outline of the experimental procedure is shown in Fig. 3(a). In detail, mouse breast cancer cells 4T1 (ATCC, Rockville, Maryland, USA) were cultured at 0.5 × 10 5 A cell suspension containing cells in cell culture medium at a concentration of 1000 cells / mL was seeded in a 6-well microplate at 1 mL per well and incubated in 5% CO 2The cells were incubated for 24 hours at 37°C. Next, the PSAR-PLLA nanoparticles or L-BPA carrying o-carborane prepared in Experiment 1-1 were added to the cell culture medium so that the boron concentration in the cell culture medium was 2 mM, and incubated for 2, 4, or 6 hours. After the predetermined incubation time, the cells were washed three times with PBS according to a conventional method, and then the cells were recovered by trypsin treatment. The recovered solution was centrifuged at 1,800 rpm for 5 minutes, the supernatant was removed, and a pellet was obtained. The pellet was treated with 60% nitric acid (1 mL) overnight at room temperature. Then, the solution was filtered through a syringe filter with a pore size of 0.45 μm, and pure water was added to the filtrate obtained to make the total volume 10 mL. The solution thus obtained was subjected to ICP-AES to measure the boron content in the solution. Based on the obtained boron content, the number of boron atoms per cell was calculated. In the above experiments, RPMI-1640 medium containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin solution was used as the cell culture medium. The results are shown in Figure 3(b). Figure 3(b) shows the mean ± standard error (SEM) of the values ​​obtained from three independent tests for each sample.

[0124] As shown in Figure 3(b), the number of boron atoms per cell in 4T1 cells exposed to PSAR-PLLA nanoparticles carrying o-carborane was greater than that in 4T1 cells exposed to L-BPA at all incubation times. The difference between the two was more pronounced at shorter incubation times, and in particular, after 2 hours of incubation, the number of boron atoms per cell in 4T1 cells exposed to PSAR-PLLA nanoparticles carrying o-carborane was 13.2 × 10 9 The number of boron atoms per cell in 4T1 cells exposed to L-BPA at the same boron concentration for 2 hours (4.6 × 10 9This was approximately three times higher than the number of boron particles per cell obtained using PSAR-PLLA nanoparticles carrying o-carborane. This result indicates that PSAR-PLLA nanoparticles carrying o-carborane can increase the boron concentration in cancer cells more efficiently than the case where L-BPA was used, suggesting that PSAR-PLLA nanoparticles carrying o-carborane may be useful as a boron drug for boron neutron capture therapy.

[0125] <Experiment 1-4. Evaluation of the stability of PSAR-PLLA nanoparticles carrying o-carborane> To investigate the stability of o-carborane-loaded PSAR-PLLA nanoparticles under physiological conditions, the release of o-carborane from the PSAR-PLLA nanoparticles was evaluated over time when the nanoparticles were incubated in PBS.

[0126] The release of o-carborane from the PSAR-PLLA nanoparticles was evaluated by dialysis. That is, 2 mL of a solution containing PSAR-PLLA nanoparticles carrying o-carborane prepared according to the procedure described in Experiment 1-1 was added to a dialysis tube (product name "Cellulose Tube 20 / 32", molecular weight cutoff: 12,000 to 14,0000, sold by Nacalai Tesque, Inc.), the dialysis tube was closed, and the dialysis tube was immersed in 300 mL of PBS (phosphate buffered saline) at 37 ° C. or 4 ° C. After dialysis for 0, 1, 3, 6, 12, and 24 hours, 2 mL was removed from the aqueous solution outside the dialysis tube, and 2 mL of PBS was added instead. The amount of boron atoms contained in the aqueous solution outside the removed dialysis tube was measured by ICP-AES, and the content of o-carborane contained in the aqueous solution outside the dialysis tube was calculated based on the measured amount of boron atoms, which was taken as the amount released from the PSAR-PLLA nanoparticles.

[0127] The results are shown in Figure 4. As shown in Figure 4, when the PSAR-PLLA nanoparticles loaded with o-carborane were incubated in saline, it was found that o-carborane was released relatively quickly from the PSAR-PLLA nanoparticles. This result is consistent with the findings in the art that hydrophobic compounds physically encapsulated in polymeric micelles leak relatively quickly under physiological conditions. Meanwhile, the release of o-carborane from the PSAR-PLLA nanoparticles was more prominent in PBS at 37°C than in PBS at 4°C.

[0128] In order to investigate the stability of PSAR-PLLA nanoparticles carrying o-carborane under physiological conditions in more detail, the particle size and polydispersity of PSAR-PLLA nanoparticles carrying o-carborane after incubation in physiological saline were evaluated by dynamic light scattering. Specifically, in the above experiment, after 24 hours of dialysis, the aqueous solution remaining in the dialysis tube was appropriately diluted and subjected to measurement of particle size and polydispersity by dynamic light scattering. For comparison, dialysis was performed in the same manner as in the above experiment using PSAR-PLLA nanoparticles not carrying carborane and PSAR-PLLA nanoparticles carrying m-carborane instead of o-carborane, and after 24 hours of dialysis, the aqueous solution remaining in the dialysis tube was appropriately diluted and the particle size and polydispersity were measured by dynamic light scattering.

[0129] The results are shown in Figure 5. As shown in Figure 5(b), after 24 hours of dialysis at 37°C, the particle size of the PSAR-PLLA nanoparticles carrying o-carborane increased significantly, as did the polydispersity. This result indicates that at 37°C, which is the body temperature, the PSAR-PLLA nanoparticles carrying o-carborane gradually become unstable and their structure gradually collapses. On the other hand, the results of measuring the particle size and polydispersity of the PSAR-PLLA nanoparticles not carrying o-carborane are shown in Figure 5(a). As shown in Figure 5(a), an increase in particle size and polydispersity was observed for the PSAR-PLLA nanoparticles not carrying o-carborane after 24 hours of dialysis at 37°C.

[0130] As described above, it was shown that the PSAR-PLLA nanoparticles carrying o-carborane gradually become unstable at 37°C, similar to the PSAR-PLLA nanoparticles. It is presumed that the release of o-carborane from the PSAR-PLLA nanoparticles carrying o-carborane shown in Figure 4 is due to the leakage of hydrophobic compounds physically encapsulated in the PSAR-PLLA nanoparticles, as well as the destabilization of the carrier PSAR-PLLA nanoparticles themselves. Here, the polylactic acid contained in the PSAR-PLLA nanoparticles has a carbonyl group (-C(=O)-), and it is known that the carbonyl group undergoes a nucleophilic reaction with the boron atom (B) present near the two carbon atoms (C) at the ortho positions in the o-carborane molecule (Ohta, K, et al. Journal of Synthetic Organic Chemistry, Japan. 2007;65(4):320-33). Such interactions between carborane and poly(L-lactide) may destabilize the PSAR-PLLA nanoparticles by interfering with the α-helical interactions of poly(L-lactide) that constitutes the hydrophobic core of the PSAR-PLLA nanoparticles, and may also lead to partial decomposition and polarization of the o-carborane molecules, which may contribute to the release of o-carborane from the PSAR-PLLA nanoparticles.

[0131] In addition, in the case of PSAR-PLLA nanoparticles carrying m-carborane instead of o-carborane, a more significant increase in particle size and polydispersity was observed after dialysis at 37°C for 24 hours (Figure 5(c)). As shown in Table 1, the PSAR-PLLA nanoparticles carrying m-carborane carry a smaller amount of carborane than the PSAR-PLLA nanoparticles carrying o-carborane. Nevertheless, a more significant increase in particle size and polydispersity was observed, suggesting that m-carborane has a stronger destabilizing effect on PSAR-PLLA nanoparticles than o-carborane.

[0132] <Experiment 2. PSAR-PLLA nanoparticles carrying o-carborane derivatives> Experiment 1 showed that, among the three carborane isomers, o-carborane, m-carborane, and p-carborane, o-carborane was most efficiently loaded onto PSAR-PLLA nanoparticles, and was also advantageous in terms of the stability of the PSAR-PLLA nanoparticles. Therefore, in order to further increase the stability of PSAR-PLLA nanoparticles and enable better intracellular boron delivery, we focused on o-carborane and its derivatives, prepared PSAR-PLLA nanoparticles loaded with o-carborane derivatives in which alkyl groups with a specific carbon chain length were introduced to the two ortho carbons of o-carborane, and evaluated their physical properties.

[0133] The amphiphilic polymer used to form nanoparticles was the same as in Experiment 1, a block copolymer of polysarcosine and polylactide (PSar 106 -block-PLLA 32 ) (weight average molecular weight: 10,001 Da; manufactured by Kobe Natural Products Chemical Co., Ltd.) was used. On the other hand, 1,2-dimethyl-o-carborane, 1,2-dihexyl-o-carborane, and 1,2-didodecyl-o-carborane (Katchem, Ltd., Prague, Czech Republic) were used as o-carborane derivatives. The structures of the carborane derivatives used in this experiment are shown in Figure 6.

[0134] <Experiment 2-1. Preparation of PSAR-PLLA nanoparticles and their characterization> The preparation of PSAR-PLLA nanoparticles was carried out in the same manner as in Experiment 1-1, except that the above-mentioned o-carborane derivative was used instead of carborane. The particle size and polydispersity of the obtained PSAR-PLLA nanoparticles were measured by dynamic light scattering in the same manner as in Experiment 1. The amount of boron supported was measured by ICP-AES, and the boron support rate (DLC (%)) was calculated based on the amount of boron supported. The results are shown in Table 3.

[0135] [Table 3]

[0136] As shown in Table 3, the boron loading rate (DLC (%)) in PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane, 1,2-dihexyl-o-carborane, or 1,2-didodecyl-o-carborane was 1.30%, 2.86%, and 0.61%, respectively, confirming that all carborane derivatives were supported on PSAR-PLLA nanoparticles. The boron loading rate (DLC (%)) was about twice as high for 1,2-dihexyl-o-carborane with an alkyl chain having a larger carbon number, i.e., C6, compared with 1,2-dimethyl-o-carborane with an alkyl chain having C1. However, the boron loading rate does not necessarily increase with the carbon number of the alkyl chain, and the boron loading rate of PSAR-PLLA nanoparticles carrying 1,2-didodecyl-o-carborane, which has an alkyl chain with a carbon number of even greater than 12, was only about 1 / 4 to 1 / 5 of that of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, which has an alkyl chain with a carbon number of 6. Considering that the o-carborane derivative, like o-carborane, is presumed to be physically encapsulated in the PSAR-PLLA nanoparticles mainly through hydrophobic interactions with the polylactic acid that constitutes the hydrophobic core of the PSAR-PLLA nanoparticles, the finding that the boron loading rate of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane was the highest was completely unexpected. It is presumed that while 1,2-dihexyl-o-carborane can strongly interact with polylactic acid due to the C6 alkyl chain, 1,2-didodecyl-o-carborane, which has a larger carbon number and a more hydrophobic C12 alkyl group, significantly forms aggregates due to hydrophobic interactions between 1,2-didodecyl-o-carborane groups, and thus does not efficiently interact with PSAR-PLLA nanoparticles.

[0137] On the other hand, the particle size and polydispersity of PSAR-PLLA nanoparticles carrying three types of o-carborane derivatives, measured by dynamic light scattering, are shown in Table 4, and the histograms of particle size distribution are shown in Figures 7(a) to (c). For comparison, Table 4 also shows the particle size and polydispersity of PSAR-PLLA nanoparticles not carrying carborane. A transmission electron microscope photograph of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane is shown in Figure 7(d).

[0138] [Table 4]

[0139] As shown in Table 4 and Figures 7(a) to (c), the particle size of the PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane was 63.7 nm and the PDI was 0.23, which was equivalent to the particle size and PDI of the PSAR-PLLA nanoparticles not carrying carborane. This result is similar to the measurement results of the PSAR-PLLA nanoparticles carrying the three carborane isomers shown in Experiment 1-2. On the other hand, the particle size of the nanoparticles carrying 1,2-dihexyl-o-carborane or 1,2-didodecyl-o-carborane was 93.2 nm to 104 nm, which was larger than that of the PSAR-PLLA nanoparticles not carrying carborane, and differences were observed between the PSAR-PLLA nanoparticles carrying the three carborane isomers and 1,2-dimethyl-o-carborane. In addition, when 1,2-dihexyl-o-carborane and 1,2-didodecyl-o-carborane were compared, it was found that the polydispersity index was as small as 0.15 when 1,2-dihexyl-o-carborane was used, and that PSAR-PLLA nanoparticles with an extremely monodisperse particle size distribution were formed.The particle morphology of the PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane was observed using a transmission electron microscope, and the formation of monodisperse nanoparticles with a particle size similar to that obtained by dynamic light scattering was confirmed.

[0140] <Experiment 2-2. Evaluation of the stability of PSAR-PLLA nanoparticles carrying o-carborane> Next, the stability of PSAR-PLLA nanoparticles carrying three types of o-carborane derivatives was evaluated. In this experiment, 1,2-dimethyl-o-carborane and 1,2-dihexyl-o-carborane, which had relatively high boron loadings in Experiment 2-1, were used. For stability evaluation, the release of o-carborane derivatives and the change in particle size over time in PBS were evaluated according to the procedure described in Experiment 1-2, except that PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane or PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were used instead of PSAR-PLLA nanoparticles carrying o-carborane.

[0141] The results of measuring the amount of o-carborane derivative released are shown in FIG. 8. As shown in FIG. 8(a), the release of 1,2-dimethyl-o-carborane was observed over time during dialysis in PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane. The release of 1,2-dimethyl-o-carborane was more prominent at 37°C than at 4°C, and it was revealed that 60% of the total amount of 1,2-dimethyl-o-carborane was released after 24 hours of dialysis at 37°C. This result is similar to that obtained for PSAR-PLLA nanoparticles carrying o-carborane. In contrast, as shown in FIG. 8(b), surprisingly, the release of 1,2-dihexyl-o-carborane was hardly observed in PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane during 24 hours of dialysis at both 4°C and 37°C.

[0142] On the other hand, the change in particle size of PSAR-PLLA nanoparticles before and after 24 hours of dialysis in PBS is shown in FIG. 9. As shown in FIG. 9(a), the PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane showed a significant change in particle size and an increase in polydispersity after 24 hours of dialysis at 37°C. On the other hand, no significant change in particle size and polydispersity was observed after 24 hours of dialysis at 4°C. The above results indicate that the PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane are significantly destabilized at 37°C. In contrast, as shown in FIG. 9(b), the PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane showed almost no change in particle size and polydispersity even after 24 hours of dialysis at 37°C. This result indicates that the PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane have excellent stability even at 37°C in PBS, a temperature and salt concentration close to the biological environment. Since polylactic acid, which constitutes the hydrophobic core of PSAR-PLLA nanoparticles, gradually hydrolyzes in aqueous media, it was thought that stabilizing PSAR-PLLA nanoparticles in aqueous media would be extremely difficult. However, the discovery that the stability of PSAR-PLLA nanoparticles in aqueous media can be dramatically improved by supporting 1,2-dihexyl-o-carborane is surprising and is of great importance for the application of PSAR-PLLA nanoparticles as pharmaceuticals that require storage and use in aqueous media.

[0143] <Experiment 2-3. Examination of the production conditions of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane> To investigate the conditions for preparing PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, the ratios of 1,2-dihexyl-o-carborane and PSAR-PLLA polymer were varied to prepare PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane.

[0144] The procedure for preparing PSAR-PLLA nanoparticles is as follows. First, PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were prepared by the following procedure. First, PSAR dissolved in chloroform was 106 -block-PLLA 32 100 μL of the solution containing PSar (0.1, 0.5, or 1.0 μmol) was placed in a test tube, and the solvent was removed by evaporation. 106 -block-PLLA 32 was prepared. 100 μL of a solution containing 1,2-dihexyl-o-carborane (10 μmol) dissolved in DMSO was added to the test tube containing the obtained film, and the solution was dried to form a film again. 2.0 mL of PBS was added to the obtained film and stirred at 1,200 rpm for 1 hour. After stirring, the eluent containing the nanoparticles was collected using a PD-10 column (GE Healthcare, Buckinghamshire, United Kingdom). The eluent containing the nanoparticles was passed through a syringe filter with a pore size of 0.22 μm and then a pore size of 0.10 μm (Merck Millipore, Dublin, Ireland) before being used in the experiment described below. The particle size and polydispersity of the obtained PSAR-PLLA nanoparticles were measured by dynamic light scattering in the same manner as in Experiment 1. The amount of boron supported was also measured by ICP-AES, and the boron support rate (DLC (%)) was calculated based on the amount of boron supported. The results are shown in Table 5.

[0145] [Table 5]

[0146] As shown in Table 5, PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were obtained under all preparation conditions. As the ratio (A / B) of the amount of PSAR-PLLA polymer (B) to the amount of 1,2-dihexyl-o-carborane (A) increased from 10 μmol / 0.1 μmol ("Ratio 1" in Table 5), to 10 μmol / 0.5 μmol ("Ratio 2" in Table 5), to 10 μmol / 1.0 μmol ("Ratio 3" in Table 5), the ratio of boron to PSAR-PLLA polymer in the obtained PSAR-PLLA nanoparticles (B / PSAR-PLLA Ratio) decreased to 3.27, 1.30, and 0.80.

[0147] On the other hand, as the ratio (A / B) of the amount of PSAR-PLLA polymer (B) to the amount of 1,2-dihexyl-o-carborane (A) increased from 10 μmol / 0.1 μmol ("Ratio 1" in Table 5), to 10 μmol / 0.5 μmol ("Ratio 2" in Table 5), to 10 μmol / 1.0 μmol ("Ratio 3" in Table 5), the particle size of the obtained PSAR-PLLA nanoparticles increased to 83.7 nm, 91.8 nm, and 111 nm. This result indicates that the particle size of the PSAR-PLLA nanoparticles can be controlled by adjusting the ratio of the amount of PSAR-PLLA polymer to the amount of 1,2-dihexyl-o-carborane.

[0148] <Experiment 2-4. Cellular uptake test> Next, we evaluated the intracellular uptake behavior of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane. In this experiment, we used PSAR-PLLA nanoparticles carrying o-carborane as a comparison, which was shown in the previous experiment to have a better effect on increasing intracellular boron concentration than L-BPA, which is conventionally used as a boron drug for BNCT.

[0149] The experimental procedure is outlined in Fig. 3(a). In detail, mouse breast cancer cells 4T1, mouse colon cancer cells CT26, human pancreatic adenocarcinoma cells AsPC-1, and human gastric cancer cells NC1-N87 (all purchased from ATCC, Rockville, Maryland, USA) were cultured at 0.5 × 10 6 pieces / mL, 0.5×10 6 pieces / mL, 1.0×10 6 / mL, or 1.0 x 10 6 A cell suspension containing cells in cell culture medium at a concentration of 1000 cells / mL was seeded in a 6-well microplate at 1 mL per well and incubated in 5% CO 2 The mixture was incubated for 24 hours at 37°C. Next, PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, which were prepared in the same manner as in Experiment 2-3 with a mixing ratio of 10 μmol of 1,2-dihexyl-o-carborane and 1.0 μmol of PSAR-PLLA (Ratio 3 in Table 5), or PSAR-PLLA nanoparticles carrying o-carborane, which were prepared in the same manner as in Experiment 1-1, were added to the cell culture medium so that the boron concentration in the cell culture medium was 0.15, 0.5, or 2 mM, and incubated for 2 hours. After incubation, the cells were washed three times with PBS according to a conventional method, and then the cells were recovered by trypsin treatment. The obtained recovery solution was centrifuged at 1,800 rpm for 5 minutes, and the supernatant was removed to obtain a pellet. The obtained pellet was treated with 60% nitric acid (1 mL) at room temperature overnight. The solution was then filtered through a syringe filter with a pore size of 0.45 μm, and pure water was added to make the total volume 10 mL. The solution thus obtained was subjected to ICP-AES to measure the boron content. The results are shown in Figure 10. Note that Figure 10 shows the average value ± standard error (SEM) of the values ​​obtained from three independent tests for each sample.

[0150] As shown in Figure 10, in all cell types, including human and mouse cells, from breast cancer to colon cancer, pancreatic adenocarcinoma, and gastric cancer, the number of boron atoms per cell was significantly greater in cells contacted with PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane than in cells contacted with PSAR-PLLA nanoparticles carrying o-carborane. Here, the minimum number of boron atoms per cell required to obtain a therapeutic effect by BNCT is 1 x 10 9 pcs / cell (~20 ng boron / 10 6 As shown in Figure 10, when PSAR-PLLA nanoparticles carrying o-carborane were used, the boron concentration was either 0.5 mM or 2 mM, resulting in a 1 × 10 9 In contrast, when PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were used, the number of boron atoms per cell was 1×10 even at a boron concentration of 0.15 mM. 9 When treated with 0.5 mM or 2 mM boron, the number of boron atoms per cell was 1 × 10 9 This was far more than the number of pieces per cell.

[0151] These results indicate that the use of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane can further increase the boron concentration in cancer cells compared to the use of PSAR-PLLA nanoparticles carrying o-carborane. In various cancer cells, the intracellular boron concentration was increased to the 1×10 required for the therapeutic effect of BNCT. 9 PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, which can be enhanced to a level of more than 1 / cell, can be advantageously used for cancer treatment by BNCT.

[0152] <Experiment 2-5. Cytotoxicity test> The cytotoxicity of PSAR-PLLA nanoparticles carrying carborane or o-carborane derivatives was evaluated.

[0153] The experimental procedure is outlined in Fig. 11(a). Mouse breast cancer cells 4T1 were cultured in a 96-well microplate at 5 × 10 cells per well. 3 The cells were seeded at 100 cells / cell and incubated in 5% CO 2 The cells were incubated at 37°C for 24 hours. Next, PSAR-PLLA nanoparticles carrying o-carborane or m-carborane prepared in the same manner as in Experiment 1-1, or PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane or 1,2-dihexyl-o-carborane prepared in the same manner as in Experiment 2-1, were added to the cell culture medium so that the boron concentration was 5, 25, 50, 100, or 250 ppm, and incubated for 24 hours. The cytotoxicity test was performed using Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manual attached to the kit. The optical density (OD) at a wavelength of 450 nm was measured using a microplate reader (Sunrise TM The assay was performed using a 30-well platelet counting system (Tecan Trading AG, Mannedorf, Switzerland). Cell viability was calculated as a percentage of untreated cells (control) taken as 100%. Results are shown as the mean ± standard error (SEM) of values ​​obtained from four independent samples. The results are shown in Figure 11(b).

[0154] As shown in FIG. 11(b), PSAR-PLLA nanoparticles carrying o-carborane, m-carborane, 1,2-dimethyl-o-carborane, or 1,2-dihexyl-o-carborane did not show significant cytotoxicity in the concentration range of 5 ppm to 250 ppm in terms of boron concentration. The maximum concentration tested in this experiment, 250 ppm, is equivalent to 25 mM in terms of molar concentration. Considering this result together with the results of Experiments 2-3, it is clear that the concentration range of 1×10 9 It is suggested that PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane derivatives, which can achieve boron concentrations exceeding 1 / cell, are extremely useful as low-toxicity and effective boron drugs for BNCT.

[0155] <Experiment 3. Evaluation of pharmacokinetics of PSAR-PLLA nanoparticles> To evaluate the in vivo dynamics of PSAR-PLLA nanoparticles, we prepared PSAR-PLLA nanoparticles carrying ICG, administered them to xenograft model mice, and performed near-infrared fluorescence imaging.

[0156] <Experiment 3.1 Preparation of ICG-loaded PSAR-PLLA nanoparticles> The ICG-loaded PSAR-PLLA nanoparticles were prepared as follows. First, PSAR was dissolved in chloroform. 106 -block-PLLA 32 (1 μmol / mL; 1 mL) and a given amount of ICG-PLLA 34 (1 mol%) were mixed in a test tube, the solvent was removed by evaporation, and PSar 106 -block-PLLA 32 and ICG-PLLA 34A film containing ICG-PLLA was prepared. 1.9 mL of DPBS was added to the test tube containing the obtained film, and the mixture was stirred at 1,200 rpm for 1 hour. After stirring, the eluate containing the nanoparticles was collected using a PD-10 column (GE Healthcare). The eluate containing the nanoparticles was passed through syringe filters with pore sizes of 0.22 μm and 0.10 μm before being used in the experiments described below. Detailed experimental data is omitted, but the ICG-PLLA 34 Regardless of the amount of dopant added, the particle size and PDI of the resulting nanoparticles were similar.

[0157] <Experiment 3.2 Preparation of Xenograft Model> The xenograft model was prepared by injecting 2 × 10 AsPC-1 / CMV-Luc cells into the mouse 2 weeks before the experiment. 6 The cells were suspended in 100 μL of DPBS containing 40% Corning® Matrigel® Matrix (Corning, Arizona, USA), and 100 μL of the resulting suspension was subcutaneously inoculated into the right thigh of a 6-week-old female nude mouse (BALB / c nu / nu) (purchased from Charles River).

[0158] <Experiment 3.3 Near-infrared fluorescence imaging> 100 μL of the solution containing the PSAR-PLLA nanoparticles carrying ICG prepared by the above procedure was administered to the Xenograft model mice by intravenous injection from the tail vein. After intravenous injection of the solution containing the nanoparticles, in vivo near-infrared fluorescence imaging was performed 1, 3, 6, 12, 24, 48, and 72 hours later. After in vivo near-infrared fluorescence imaging 72 hours later, the mice were euthanized, and the blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestines, muscles, and tumor were collected and the weights of each organ were measured. In addition, the fluorescence intensity in each organ was measured by ex vivo near-infrared fluorescence imaging. In the above experiments, near-infrared fluorescence imaging was performed using an IVIS spectrum system (Xenogen, Hopkinton, Massachusetts) equipped with a filter for ICG (excitation wavelength 745 nm, fluorescence wavelength 840 nm).

[0159] The results of evaluating the in vivo dynamics of PSAR-PLLA nanoparticles by in vivo near-infrared biofluorescence imaging are shown in Figure 12 (A) (B). As shown in Figure 12 (A) (B), strong ICG-derived fluorescence was observed at the tumor site in the right thigh of the mouse that had received tail vein administration of ICG-loaded PSAR-PLLA nanoparticles. The intensity of ICG-derived fluorescence at the tumor site peaked 12 hours after administration of PSAR-PLLA nanoparticles. Meanwhile, the ratio of the fluorescence intensity at the tumor site to that in normal tissues continued to gradually increase over 72 hours after administration. These results indicate that ICG-loaded PSAR-PLLA nanoparticles are accumulated at the tumor site. Since the ratio of the fluorescence intensity at the tumor site to that in normal tissues gradually increases, it is inferred that PSAR-PLLA nanoparticles are accumulated at the tumor site due to the EPR effect.

[0160] On the other hand, the fluorescence intensity derived from ICG in each organ collected 72 hours after intravenous injection of PSAR-PLLA nanoparticles was measured by ex vivo near-infrared fluorescence imaging, and the results are shown in Figure 12(C). As shown in Figure 12(C), the strongest ICG fluorescence intensity was observed in the blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestines, muscle, and tumor. This result indicates that the PSAR-PLLA nanoparticles carrying ICG are most highly accumulated at the tumor site, and is consistent with the results of in vivo near-infrared fluorescence imaging shown in Figures 12(A) and (B).

[0161] <Experiment 4. Evaluation of in vivo dynamics of boron> Next, to evaluate the in vivo dynamics of boron when PSAR-PLLA nanoparticles carrying boron compounds were administered, PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane as boron-containing compounds were prepared and administered to xenograft model mice, and the amount of boron accumulated in each organ was measured by ICP-AES.

[0162] <Experiment 4.1 Preparation of PSAR-PLLA nanoparticles carrying boron-containing compounds> Preparation of PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane was carried out in the same manner as in Experiment 1-1, except that o-carborane or 1,2-dihexyl-o-carborane was used as the boron-containing compound. In this experiment, the eluent containing PSAR-PLLA nanoparticles was passed through a syringe filter with a pore size of 0.22 μm and a syringe filter with a pore size of 0.10 μm before being used in the experiments described below.

[0163] <Experiment 4.2 Preparation of Xenograft Model> The xenograft model was prepared in the same manner as in Experiment 3.2. That is, 2 weeks before the experiment, 2 × 10 AsPC-1 / CMV-Luc cells were added to the xenograft. 6The cells were suspended in 100 μL of DPBS containing 40% Corning® Matrigel® Matrix (Corning, Arizona, USA), and 100 μL of the resulting suspension was subcutaneously inoculated into the right thigh of a 6-week-old female nude mouse (BALB / c nu / nu) (purchased from Charles River).

[0164] <Experiment 4.3 Quantification of boron accumulation by ICP-AES> 100 μL of the solution containing the PSAR-PLLA nanoparticles carrying the boron-containing compound prepared by the above procedure was administered to the Xenograft model mice by intravenous injection via the tail vein so that the dose of boron was 5 mg of B equivalent / kg. After intravenous injection of the solution containing the nanoparticles, the mice were euthanized 24 hours later, and the blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestines, muscles, and tumor were collected, and the weight of each organ was measured. The collected organs were then washed with HClO 4 and H 2 O 2 The samples were digested by immersing them in a 1:1 mixture of these two solutions and treating them at 90° C. for 2 hours. The digested samples were diluted with 10 mL of purified water and passed through a syringe filter with a pore size of 0.5 μm before being subjected to ICP-AES analysis.

[0165] The amount of boron accumulated in each organ was measured by ICP-AES and the ratio (T / N) of the boron concentration at the tumor site to the boron concentration in normal tissue (muscle) and the ratio (T / B) of the boron concentration at the tumor site to the boron concentration in blood are shown in Fig. 13(B). As shown in Fig. 13(A), the amount of boron accumulated at the tumor site was 16 μg / g in the PSAR-PLLA nanoparticles carrying o-carborane and 18 μg / g in the PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, showing a slightly higher result in the PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane. In addition, as shown in Figure 13(B), the ratio (T / N) of the boron concentration at the tumor site to the boron concentration in normal tissue (muscle) was 3.3 / 1 for PSAR-PLLA nanoparticles carrying o-carborane and 3.6 / 1 for PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane. Although the result was slightly higher for PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, the T / N ratio was 3 or higher for all PSAR-PLLA nanoparticles. In BNCT, a T / N ratio of 3 or higher is considered ideal (Tsurubuchi, T. et al., Cells 2020, 9(5), 1-20). The result that the T / N ratio at the tumor site can be increased to 3 or more by using PSAR-PLLA nanoparticles loaded with o-carborane or 1,2-dihexyl-o-carborane suggests that PSAR-PLLA nanoparticles loaded with o-carborane or 1,2-dihexyl-o-carborane are extremely useful for cancer treatment by BNCT.

[0166] <Experiment 5. Effect of BNCT treatment> Next, we investigated the in vitro and in vivo BNCT therapeutic effects of PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane as boron-containing compounds.

[0167] <Experiment 5.1 In vitro test> Human pancreatic adenocarcinoma cells AsPC-1 / CMV Luc (or AsPC-1) (purchased from ATCC, Rockville, Maryland, USA) were cultured at 1.5 × 10 6 The cells were seeded in a 6-well microplate at a seeding density of 100 cells / well and incubated in 5% CO 2 The cells were incubated for 24 hours at 37°C. Next, PSAR-PLLA nanoparticles carrying o-carborane, PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, or L-BPA, which were prepared in the same manner as in Experiment 4.1, were added to the cell culture medium so that the boron concentration was 2 mM, and incubated for 2 hours. As a control, DPBS or blank PSAR-PLLA nanoparticles were added instead of the PSAR-PLLA nanoparticles carrying boron compounds and L-BPA, and incubated for 2 hours in the same manner as for the cells to which the PSAR-PLLA nanoparticles carrying boron compounds and BPA were added. After incubation, the cells were collected in a test tube according to a conventional method and irradiated with neutrons (thermal power 1 MW; thermal neutron flux 1.3×10 9 neutrons / cm -2 / s -1 ) was irradiated for 0, 10, 20, and 40 minutes. The absorbed dose of neutron rays at each irradiation time was 0 Gy (0 min), 0.142 Gy (10 min), 0.369 Gy (20 min), and 0.555 Gy (40 min), respectively. Neutron irradiation was performed at Kyoto University Research Reactor (KUR). After neutron irradiation, neutron-irradiated AsPC-1 / CMV Luc (or AsPC-1) cells were seeded on a 60 mm culture dish and cultured for 2 weeks to perform a colony formation assay. The cultured cells were stained in a 20% methanol solution containing 0.5% crystal violet (CV). The stained colonies were counted using an aCOLyte 3 automatic colony counter (Synoptics Ltd.). The results obtained are shown in FIG. 14. The results shown in FIG. 14 represent the mean ± standard error (SEM) of the values ​​obtained from four independent tests for each sample.

[0168] As shown in Figure 14, cell proliferation was inhibited in all groups treated with PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, PSAR-PLLA nanoparticles carrying o-carborane, and L-BPA, and the cell proliferation inhibitory effect tended to increase with the length of neutron irradiation time. In particular, the group treated with PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane showed a particularly excellent proliferation inhibitory effect compared to the groups treated with PSAR-PLLA nanoparticles carrying o-carborane and L-BPA.

[0169] <Experiment 5.2 In vivo test> The in vivo BNCT therapeutic effects of PSAR-PLLA nanoparticles carrying o-carborane and PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were examined by the following procedure. Xenograft models prepared by the same procedure as in Experiment 3.2, i.e., tumor-bearing mice (n=6) seeded with AsPC-1 / CMV Luc cells, were administered 5 mg / mL of PSAR-PLLA nanoparticles carrying o-carborane, PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, or L-BPA, prepared by the same procedure as in Experiment 4.1, 10 days after the seeding of the cancer cells. 10 Boron was administered at a dose of 1000 B / kg via tail vein injection. 24 hours after administration, the rats were irradiated with neutron beams. The neutron beam irradiation was performed at Kyoto University Research Reactor (KUR) under the following conditions: thermal power 5 MW, thermal neutron flux 3.3×10 9 neutrons / cm -2 / s -1The irradiation time was 40 minutes, and the gamma dose was 1.509 Gy. After the neutron irradiation, the body weight and tumor size of the mice were evaluated twice a week for 21 days. In this experiment, as controls, a hot control group was prepared in which the boron compound-loaded PSAR-PLLA nanoparticles were not administered and the mice were irradiated with neutrons, and a cold control group was prepared in which the boron compound-loaded PSAR-PLLA nanoparticles were not administered and the mice were not irradiated with neutrons. The results are shown in FIG. 15. FIG. 15(A) shows the changes in the body weight of the mice, and FIG. 15(B) shows the changes in the tumor size. The results shown in FIG. 15 are the average values ​​± standard error (SEM) of the values ​​obtained from six independent mice for each sample.

[0170] As shown in Figure 15(A), the change in body weight after neutron irradiation in all groups of mice irradiated with neutrons was equivalent to that in the cold control group not irradiated with neutrons. On the other hand, in terms of the change in tumor size during the same period, the increase in tumor size was suppressed in the hot control group irradiated with neutrons, the group administered with PSAR-PLLA nanoparticles carrying o-carborane and irradiated with neutrons, and the group administered with PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and irradiated with neutrons, compared to the cold control group. In particular, the group administered PSAR-PLLA nanoparticles carrying o-carborane and irradiated with neutron beams and the group administered PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and irradiated with neutron beams showed significantly superior tumor suppression effects compared to the hot control group that was not administered PSAR-PLLA nanoparticles and was irradiated with neutron beams. This result indicates that excellent cancer therapeutic effects can be obtained by administering PSAR-PLLA nanoparticles carrying boron-containing compounds and irradiating with neutron beams.

[0171] <Experiment 6. Effect of PDT treatment> Next, we examined the in vitro and in vivo therapeutic effects of PSAR-PLLA nanoparticles that did not carry a boron-containing compound but carried indocyanine green as a photosensitizer.

[0172] <Experiment 6.1 In vitro test> Human pancreatic adenocarcinoma cells AsPC-1 / CMV Luc (or AsPC-1) (purchased from ATCC, Rockville, Maryland, USA) were cultured at 1.5 × 10 6 The cells were seeded in a 6-well microplate at a seeding density of 100 cells / well and incubated in 5% CO 2 The cells were incubated at 37°C for 24 hours. Next, the ICG-loaded PSAR-PLLA nanoparticles prepared according to the procedure described in Experiment 3.1 were added to the cell culture medium and incubated for 2 hours. The ICG concentration (measured value) in the cell culture medium at this time was 1.6 μM, and the PSAR-PLLA polymer concentration (calculated value) was 1 mM. As a control, DPBS or blank PSAR-PLLA nanoparticles were added instead of the ICG-loaded PSAR-PLLA nanoparticles, and incubated for 2 hours in the same manner as for the cells to which the ICG-loaded PSAR-PLLA nanoparticles were added. After incubation, the cells were collected in a test tube according to a conventional method and irradiated with neutrons (thermal power 1 MW; thermal neutron flux 1.3×10 9 neutrons / cm -2 / s -1) was irradiated for 0, 10, 20, and 40 minutes. The absorbed dose of neutron radiation at each irradiation time was 0 Gy (0 min), 0.142 Gy (10 min), 0.369 Gy (20 min), and 0.555 Gy (40 min), respectively. Neutron radiation was performed at Kyoto University Research Reactor (KUR). After neutron irradiation, the neutron-irradiated AsPC-1 / CMV Luc (or AsPC-1) cells were seeded on a 60 mm culture dish and cultured for 2 weeks to perform a colony formation assay. The cultured cells were stained in a 20% methanol solution containing 0.5% crystal violet (CV). The stained colonies were counted using an aCOLyte 3 automatic colony counter (Synoptics Ltd.). The results obtained are shown in FIG. 16. The results shown in FIG. 16 represent the mean ± standard error (SEM) of the values ​​obtained from four independent tests for each sample.

[0173] As shown in Figure 16, the number of live cells in the group treated with PSAR-PLLA nanoparticles was equivalent to that of the control group, and no cell proliferation inhibitory effect was observed. On the other hand, in the group treated with ICG-loaded PSAR-PLLA nanoparticles, the proliferation of cancer cells was significantly suppressed compared to the control group. In addition, the cell proliferation inhibitory effect increased as the neutron irradiation time was extended. The above results indicate that a cell proliferation inhibitory effect can be exerted by irradiating cells administered with ICG-loaded PSAR-PLLA nanoparticles with neutron beams.

[0174] <Experiment 6.2 In vivo test> The in vivo therapeutic effect of ICG-loaded PSAR-PLLA nanoparticles was examined by the following procedure. The xenograft model prepared by the above procedure, i.e., tumor-bearing mice (n=6) seeded with AsPC-1 / CMV Luc cells, was intravenously administered ICG-loaded PSAR-PLLA nanoparticles (average particle size (diameter): 54.6 nm; PDI: 0.20) prepared by the procedure described in Experiment 3.1 at a dose of 8.6 mg / kg of actual ICG (calculated PSAR-PLLA amount: 200 mg / kg) 10 days after the seeding of the cancer cells. Twenty-four hours after the administration, the mice were irradiated with neutron beams. The neutron beam irradiation was performed at Kyoto University Research Reactor (KUR), and the irradiation conditions were thermal power 5 MW, thermal neutron flux 3.3×10 9 neutrons / cm -2 / s -1 The irradiation time was 40 minutes, and the gamma dose was 1.509 Gy. After the neutron irradiation, the body weight and tumor size of the mice were evaluated twice a week for 21 days. In this experiment, as controls, a hot control group was prepared in which the ICG-loaded PSAR-PLLA nanoparticles were not administered and the mice were irradiated with neutrons, and a cold control group was prepared in which the ICG-loaded PSAR-PLLA nanoparticles were not administered and the mice were not irradiated with neutrons. The results are shown in FIG. 17. FIG. 17(A) shows the changes in the body weight of the mice, and FIG. 17(B) shows the changes in the tumor size. The results shown in FIG. 17 are the average values ​​± standard error (SEM) of the values ​​obtained from six independent mice for each sample.

[0175] As shown in Figure 17(A), the change in body weight after neutron irradiation in all groups of mice irradiated with neutrons was equivalent to that in the cold control group not irradiated with neutrons. On the other hand, when looking at the change in tumor size during the same period, the increase in tumor size was suppressed in the hot control group irradiated with neutrons and the group administered ICG-loaded PSAR-PLLA nanoparticles and irradiated with neutrons compared to the cold control group. Surprisingly, the group administered ICG-loaded PSAR-PLLA nanoparticles and irradiated with neutrons showed a significantly superior tumor suppression effect compared to the hot control group.

[0176] To the best of the inventors' knowledge, it has never been known that introducing indocyanine green into a tumor and irradiating it with neutron beams can provide a therapeutic effect for cancer. The above results, which show that excellent therapeutic effects for cancer can be obtained by administering PSAR-PLLA nanoparticles carrying indocyanine green as a photosensitizer and irradiating them with neutron beams, are unexpected and highly unexpected findings.

[0177] <Experiment 7. Preparation of PSAR-PLLA nanoparticles carrying boron-containing compounds and photosensitizers and their in vivo dynamics evaluation>

[0178] PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane as a boron-containing compound and indocyanine green (ICG) as a photosensitizer were prepared as follows. 106 -block-PLLA 32 (1 μmol / mL; 1 mL) and ICG-PLLA 34 (1 mol%; 1 mL) were mixed in a test tube, and the solvent was removed by evaporation to obtain PSar 106 -block-PLLA 32 and ICG-PLLA 34A film containing the nanoparticles was prepared. 1,2-Dihexyl-o-carborane (10 μmol in 1 mL DMSO) was added to the test tube containing the obtained film, dried, and the solvent was removed. Then, 2.0 mL of PBS was added and stirred at 1,200 rpm for 1 hour. After stirring, the eluate containing the nanoparticles was collected using a PD-10 column (GE Healthcare). The eluate containing the nanoparticles was passed through a syringe filter with a pore size of 0.10 μm and then used in the experiments described below.

[0179] On the other hand, the in vivo kinetics test was carried out according to the procedure shown in FIG. 6 The cells were suspended in PBS at a concentration of 100 μL / mL, and 100 μL of the resulting suspension was subcutaneously inoculated into the right thigh of a 6-week-old female nude mouse (BALB / c nu / nu) (purchased from Charles River) to prepare a xenograft model. Two weeks after inoculation of the 4T1 cells, 100 μL of a solution containing the boron-containing compound and PSAR-PLLA nanoparticles carrying a photosensitizer prepared by the above-mentioned procedure was administered by intravenous injection from the tail vein. After intravenous injection of the solution containing the nanoparticles, in vivo near-infrared fluorescence imaging was performed 1, 3, 6, 12, 24, 48, and 72 hours later. After in vivo near-infrared fluorescence imaging 72 hours later, the mice were euthanized, and the blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestines, muscles, and tumor were collected and weighed. In addition, the fluorescence intensity in each organ was measured by ex vivo near-infrared fluorescence imaging. In the above experiments, near-infrared fluorescence imaging was performed using an IVIS spectrum system (Xenogen, Hopkinton, Massachusetts) equipped with a filter for ICG (excitation wavelength 745 nm, fluorescence wavelength 840 nm). 3 After treatment, the boron concentration in each organ was measured by ICP-AES.

[0180] The results of evaluating the in vivo dynamics of PSAR-PLLA nanoparticles by in vivo near-infrared biofluorescence imaging are shown in Figure 18(b). As shown in Figure 18(b), strong ICG-derived fluorescence was observed at the tumor site in the right thigh of the mouse that had received tail vein administration of PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and ICG. The intensity of ICG-derived fluorescence at the tumor site peaked 12 to 24 hours after administration of PSAR-PLLA nanoparticles. Meanwhile, the ratio of the fluorescence intensity at the tumor site to that in normal tissues continued to gradually increase over 72 hours after administration. These results indicate that PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and ICG are accumulated at the tumor site. Since the ratio of the fluorescence intensity at the tumor site to that in normal tissues gradually increases, it is inferred that PSAR-PLLA nanoparticles are accumulated at the tumor site due to the EPR effect.

[0181] On the other hand, the fluorescence intensity derived from ICG in each organ collected 72 hours after intravenous injection of PSAR-PLLA nanoparticles was measured by ex vivo near-infrared fluorescence imaging, and the results are shown in Figure 19(a). As shown in Figure 19(a), the strongest ICG fluorescence intensity was observed in the blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestines, muscle, and tumor. This result indicates that PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and ICG are most highly accumulated at the tumor site, which is consistent with the results of in vivo near-infrared fluorescence imaging shown in Figure 18(b).

[0182] Furthermore, the results of measuring the amount of boron accumulated in each organ by ICP-AES are shown in Figure 19(b). As shown in Figure 19(b), the boron concentration at the tumor site was more than three times higher than that in normal tissue (muscle). In BNCT, it is considered ideal that the ratio (T / N ratio) between the boron concentration at the tumor site (T) and the boron concentration at the normal tissue (muscle) (T / N ratio) is 3 or more (Tsurubuchi, T. et al., Cells 2020, 9(5), 1-20). The result that the T / N ratio at the tumor site can be increased to 3 or more by PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and ICG suggests that PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and ICG are extremely useful for cancer treatment by BNCT.

[0183] In the above experiments, nanoparticles formed from AB-type polysarcosine-polylactic acid block polymers were used as nanoparticles carrying carborane or o-carborane derivatives. Alternatively, nanoparticles formed from so-called A3B-type polysarcosine-polylactic acid block polymers in which three polysarcosine chains are bound to one polylactic acid chain can also be used (MSH Lim et al., Journal of Pharmaceutical Sciences 2021, 110, 1788-179; MSH Lim et al., Life 2021, 11(2), 158; F. Takenaka et al., Drug Delivery System 33-3, 2018). Nanoparticles formed from A3B-type polysarcosine-polylactic acid block polymers have a smaller particle size than nanoparticles formed from AB-type polysarcosine-polylactic acid block polymers, and are expected to reduce the ABC phenomenon. On the other hand, in the above experiments, nanoparticles carrying o-carborane or o-carborane derivatives are considered to have shown excellent accumulation in tumor tissues based on the EPR effect. However, it goes without saying that in order to further increase the accumulation of nanoparticles in tumor tissues and, if necessary, to increase the amount of nanoparticles taken up into cells, the nanoparticles may be modified with a ligand that specifically binds only to the target cells and enables drug delivery into the cells. There are no particular limitations on such ligands, but for example, a small molecule antibody variant (scFv) against mesothelin (MSLN), which is known to be highly expressed in many types of cancer such as mesothelioma, pancreatic adenocarcinoma, ovarian cancer, and lung adenocarcinoma (H. Yakushiji et al., Cancer Science. 2019, 110(9), 2722-2733), may be suitably used. [Industrial Applicability]

[0184] According to the agent according to one aspect of the present invention, or a pharmaceutical composition containing the same, a higher intracellular boron concentration can be achieved compared to boron-containing compounds that have been used in conventional boron neutron capture therapy, and therefore an improvement in the therapeutic effect of boron neutron capture therapy is expected. On the other hand, when the agent according to one aspect of the present invention contains a photosensitizer, an excellent photodynamic therapeutic effect can be achieved by irradiating with radiation instead of near-infrared light. According to the agent according to one aspect of the present invention, which can achieve a photodynamic therapeutic effect due to a photosensitizer in addition to boron neutron capture therapy, and a pharmaceutical composition containing the same, a further improvement in the therapeutic effect of cancer is expected. Thus, the present invention has great industrial applicability.

Claims

1. An agent for treating cancer, comprising: A cancer-accumulating carrier and a photosensitizer covalently and / or non-covalently bound to the carrier, An agent for treating cancer by irradiating a subject to which the agent has been administered with radiation.

2. The agent according to claim 1 , wherein the photosensitizer has an absorption maximum wavelength within a wavelength range of 600 nm to 900 nm.

3. The photosensitizer has the following formula (II): 【Chemistry 1】 (In the above formula (II), R １ and R ４ are each independently an optionally substituted alkyl group; R ２ and R ３ are H or groups which combine with each other to form a cyclic structure; X is H or a halogen; ring B and ring D are each independently a nitrogen-containing fused aromatic heterocycle; A － The photosensitizer according to claim 1 or 2, wherein:

4. The photosensitizer is represented by the following formula (III): 【Chemistry 2】 (In the above formula (III), R １ and R ４ are each independently an optionally substituted alkyl group; R ２ and R ３ is H or groups which combine with each other to form a ring structure; R ５ and R ６ are H or groups which combine together to form an aryl group; R ７ and R ８ is H or groups which combine together to form an aryl group; X is H or a halogen; A － The photosensitizer according to claim 3, wherein:

5. The agent according to claim 1 , wherein the cancer-accumulating carrier is a microparticle having an average particle diameter of 20 nm to 150 nm as measured by dynamic light scattering.

6. The agent according to claim 5 , wherein the microparticles are microparticles comprising an amphiphilic polymer having a hydrophilic block and a hydrophobic block, and the hydrophobic block comprises polylactic acid.

7. The agent of claim 6 , wherein the hydrophilic block comprises polysarcosine.

8. Further comprising a boron-containing compound, The agent according to claim 1 , wherein the radiation comprises at least neutrons.

9. The boron-containing compound has the following formula (I): 【Chemistry 3】 The agent according to claim 8, characterized in that it is represented by the above formula (I): (in the above formula (I), ● is C; ◯ is each independently BH or B-halogen; and R is each independently H or a substituent having an optionally substituted saturated or unsaturated alkyl group.)

10. The agent according to claim 9, wherein in the above formula (I), the alkyl group of the substituent has 3 or more and 11 or less carbon atoms.

11. The agent according to claim 9 or 10, wherein in the above formula (I), the substituent is any one of an optionally substituted saturated or unsaturated alkyl group, an optionally substituted saturated or unsaturated alkoxy group, or an optionally substituted saturated or unsaturated alkylthio group.

12. A pharmaceutical composition comprising the agent according to any one of claims 1 to 11, for treating cancer by irradiating a subject to which the pharmaceutical composition has been administered.

13. The following formula (II): 【Chemistry 4】 (In the above formula (II), R １ and R ４ are each independently an optionally substituted alkyl group; R ２ and R ３ are H or groups which combine with each other to form a cyclic structure; X is H or a halogen; ring B and ring D are each independently a nitrogen-containing fused aromatic heterocycle; A － is an anion, and m is 0 or 1.

14. The compound has the following formula (III): 【Chemistry 5】 (In the above formula (III), R １ and R ４ are each independently an optionally substituted alkyl group; R ２ and R ３ is H or groups which combine with each other to form a ring structure; R ５ and R ６ are H or groups which combine together to form an aryl group; R ７ and R ８ is H or groups which combine together to form an aryl group; X is H or a halogen; A － The radiosensitizer according to claim 13, which is a compound represented by the following formula:

15. 15. Use of the radiosensitizer according to claim 13 or 14 for treating cancer.

16. A radiation irradiation device including a radiation source for irradiating radiation, A radiation irradiation device for treating cancer by irradiating a subject to which the agent according to any one of claims 1 to 11, the pharmaceutical composition according to claim 12, and / or the radiosensitizer according to claim 13 or 14 has been administered with radiation.

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