Agent for treatment of cancer, and pharmaceutical composition containing same

Abstract

The present invention addresses the problem of providing 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. The present invention solves the problem 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 being used for cancer therapy in which a subject administered with the agent is irradiated with radiation.

Description

Agent for cancer treatment and pharmaceutical composition containing same

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

[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 are activated by that external energy, have been attracting attention.

[0003] Boron neutron capture therapy is a method of treating cancer with boron-10 ( 10 After delivering a boron drug containing B) to cancer cells, neutron irradiation was performed 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 causes 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, 10 The probability of neutron capture by B nuclei is much higher than that of the elements that make up living organisms, and nitrogen ( 14 It is about 2,000 times larger than the neutrons absorbed by the 10 Emitted from the B nucleus 4 The range of He nuclei (alpha rays) is about 9 μm. 7 The range of the Li nucleus is about 4 μm, which is shorter than the diameter of a typical cell.10 By introducing a compound containing B atoms into target cancer cells and irradiating them with neutrons, it is possible to selectively kill only the cancer cells while sparing normal cells such as immune cells that exist in the surrounding area. As of June 2020, BNCT has been covered by health insurance for cancer treatment of "unresectable locally advanced or locally recurrent head and neck cancer."

[0004] In BNCT, the main factors are the energy of the neutron beam and the amount of radiation introduced into the target cells. 10 The therapeutic effect is determined by the amount of B atoms, so there must be a large number of B atoms in the target cells. 10 The development of boron drugs capable of introducing B atoms is crucial. 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 alpha rays and 7 Because the amount of Li nuclei generated can be reduced, it is expected that cancer cells can be killed with fewer side effects.

[0005] Various boron agents 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 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 concentrations in tumors to be treated, L-BPA is administered at a high concentration of 500 mg / kg. In addition, L-BPA binds one atom of phenylalanine to one molecule of the amino acid phenylalanine. 10B is a modified compound, and is thought to be taken up by target cells via LAT-1 (L-type amino acid transporter 1), an amino acid transporter 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 L-BPA to various cancer tissues. 10 There is a strong demand for the development of new boron agents that can accumulate B atoms.

[0007] Boron clusters such as carborane contain many boron atoms in one molecule, which allows them to have a high level of activity against target cells. 10 Boron clusters have attracted attention as boron drugs that can dramatically increase the amount of boron atoms introduced. However, boron clusters are difficult to internalize and selectively accumulate in cancer tissues. Therefore, various delivery systems have been developed to improve the cellular uptake and accumulation of boron clusters in cancer tissues. Among these, nanoparticle delivery systems are expected to be a delivery method that can efficiently increase the concentration of boron drugs in cancer tissues by utilizing 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 delivery systems for nanoparticle-type boron clusters, 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 best of 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 of cancer treatment in which a photosensitizer capable of producing reactive oxygen species when irradiated with light of a specific wavelength is delivered to the target cancer and then irradiated with light of the same wavelength, thereby generating reactive oxygen species specifically targeted to the cancer. Among reactive oxygen species, singlet oxygen, which is considered to be particularly cytotoxic, has a short lifespan and a diffusion distance of approximately 10-50 nm in vivo. Therefore, it is expected that introducing a photosensitizer into target cancer cells and exciting the photosensitizer will selectively kill only the cancer cells. Photodynamic therapy has been put to practical use, and examples of photosensitizers for photodynamic therapy include talaporfin sodium (trade name: Laserphyrin (registered trademark), Meiji Seika Pharma Co., Ltd.) commercially available.

[0009] Boron neutron capture therapy uses boron-10 ( 10 B) resulting from nuclear fission reactions 4 He nucleus (alpha ray) 7 While photodynamic therapy destroys cancer tissue with Li nuclei, photodynamic therapy destroys cancer tissue with reactive oxygen species. Therefore, combining the two therapies is expected to provide complementary and synergistic cancer treatment effects. Attempts at combining boron neutron capture therapy and photodynamic therapy have been described, for example, in Patent Document 4, Non-Patent Document 6, and Non-Patent Document 7. However, to the inventors' knowledge, these attempts to combine boron neutron capture therapy and photodynamic therapy all involve neutron irradiation to achieve the therapeutic effect of BNCT and near-infrared light to achieve the therapeutic effect of PDT, thus requiring the irradiation of two types of external energy. Near-infrared light, which is used to achieve the therapeutic effect of PDT, is known to have relatively high tissue penetration, but its penetration depth is still limited to approximately 2 cm. When targeting deep-seated cancers, it is difficult to deliver the excitation light, near-infrared light, to the vicinity of the target cancer, making it difficult to achieve both the therapeutic effects of BNCT and PDT.

[0010] JP 2008-074817 A International Publication No. WO2014 / 030715 JP 2015-044920 A JP 2013-227233 A

[0011] Stevoronine intravenous infusion bag 9000mg / 300mL, pharmaceutical interview form, revised May 2020 (2nd edition) P. Wongthai, et al., Cancer Sci. 2015, 106(3), 279-286.H. Yanagie, et al., British Journal of Cancer 1991, 63, 522-526.K. Shelly, et al. al,. Proc. Natl. Acad. Sci. USA 1992, 89, 9039-9043.P. Mi, et al., J. Control. Release 2017, 254, 1-9.R. Hiramatsu, et al., Journal of Pharmaceutical Sciences 2015, 104, 962-970.E. Friso, et al., Photochem. Photobiol. Sci., 2006, 5, 39-50.V. Lioret, et al., J. Med. Chem. 2020, 63, 9446-9456.N. Liu, et al., Theranostics 2022, 12, 17, 7404-7418.

[0012] The present invention has been made in view of the problems of the conventional art as described above, and an object of the present invention is to provide an agent for cancer treatment, particularly an agent that can treat cancer by combining boron neutron capture therapy and photodynamic therapy.

[0013] In the course of intensive research efforts to solve the above problems, the present inventors carried out an intensive research project to evaluate the in vivo kinetics of a carrier for accumulating a boron compound and / or a photosensitizer at a cancer site. The present inventors loaded indocyanine green, a photosensitizer excitable by near-infrared light, onto the carrier and evaluated its in vivo kinetics. When the tumor site of a mouse administered with the carrier loaded with indocyanine green was irradiated with neutrons in an experimental nuclear reactor, the inventors 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 because it primarily absorbs light with wavelengths between 600 nm and 800 nm, it is typically excited by near-infrared light with a wavelength of approximately 800 nm, which is its maximum absorption wavelength. The discovery that indocyanine green functions as a radiosensitizer and exerts an antitumor effect when administered to a subject and irradiated with radiation, more specifically, reactor-type neutron beams, rather than near-infrared excitation light, was unprecedented and completely unexpected, even to the present inventors.

[0015] The present invention is based primarily on the above findings, and in one aspect, solves the above problems by providing an agent for treating cancer, the agent comprising a cancer-accumulating carrier and a photosensitizer covalently and / or non-covalently bound to the carrier, and the agent is used to treat cancer by irradiating a subject to which the agent has been administered with radiation.

[0016] In a preferred embodiment, the photosensitizer may be a photosensitizer having an absorption maximum wavelength in the wavelength range of 600 to 900 nm, more preferably in the wavelength range of 700 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 a formula (II):

[0017] (In the above formula (II), R １ and R ４ are each independently an optionally substituted alkyl group; R２ and R ３ are H or groups which combine with each other to form a cyclic structure; X is H or 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 a compound represented by the following formula (III):

[0019] (In the above formula (III), 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 ring structure; R ５ and R ６ are H or groups which combine 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] The agent according to one aspect of the present invention has the advantage that, compared to when a photosensitizer is excited by irradiation with near-infrared light, it can be used to treat cancers located deeper in the body. Furthermore, since the agent can be excited by the same external energy as that used in boron neutron capture therapy, i.e., by irradiation with neutron-containing radiation or a neutron beam, when the agent further contains a boron-containing compound, it has the advantage that the therapeutic effects of both boron neutron capture therapy and photodynamic therapy can be obtained by irradiation with a single 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 believed that Cherenkov radiation is involved in the mechanism by which the above-mentioned photosensitizers exert their therapeutic effects on cancer upon irradiation. Cherenkov radiation is a phenomenon in which light is emitted when charged particles, such as alpha and beta particles, move through a medium such as water or air at a speed faster than the speed of light. The light emitted in this case is called Cherenkov light, and is typically white to bluish-white light with a wavelength of approximately 250 to 600 nm. There are many possible causes for Cherenkov light produced by radiation exposure, and it is thought that the combined effects of these factors produce Cherenkov light. For example, in nuclear reactors, which are sources of radiation, beta particles are emitted during the beta decay of xenon-133, a fission product of uranium-235. When these beta particles are irradiated onto a living body along with neutrons, Cherenkov light can be emitted as they pass through the body, including water molecules (Figure 21). It is also possible that gamma rays are generated when irradiated neutrons react with hydrogen contained in the living body, and that these gamma rays are then Compton scattered, producing electrons that may then generate Cherenkov light.

[0022] Photodynamic therapy using Cherenkov light or Cherenkov radiation has been reported at the research level as Cherenkov-induced PDT (CR-PDT) (e.g., Non-Patent Documents 8 and 9). However, all of the existing research on CR-PDT has focused on the fact that Cherenkov light has a shorter wavelength than near-infrared light, which excites photosensitizers. The research attempts to indirectly excite the photosensitizer by conjugating a donor molecule that absorbs Cherenkov light with the photosensitizer and utilizing the FRET (Förster resonance energy transfer) reaction between the donor molecule and the photosensitizer. To the best of the inventors' knowledge, there have been no reports of indocyanine green being activated in vivo by irradiation with neutron beams from a nuclear reactor, without utilizing the FRET reaction, and thus achieving a photodynamic therapy-like therapeutic effect. This finding was completely unexpected. The Cerenkov light generated in vivo may directly excite the photosensitizer, or may be shifted to longer wavelengths by FRET via endogenous fluorescent dyes such as porphyrins (e.g., heme), which excite the photosensitizer (Figure 21).

[0023] In other words, in one aspect, the present invention also provides use of the above-described photosensitizer as a radiosensitizer. A radiosensitizer is a drug that is administered to a patient who is a target of radiotherapy and can enhance the therapeutic effect of the irradiated radiation. As will be described later, 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 tissue when administered to a cancer-bearing organism, as long as it can accumulate a photosensitizer and / or a boron-containing compound (described later) in the cancer tissue. However, one preferred embodiment is a microparticle containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block. The inventors have focused particularly on microparticles formed from amphiphilic polymers having polysarcosine as the hydrophilic block and polylactic acid as the hydrophobic block, and have conducted extensive research. Polylactic acid is known to be highly biocompatible and have excellent biodegradability, making it a polymeric material that can be advantageously incorporated into pharmaceuticals for use in living organisms, particularly the human body. Meanwhile, polysarcosine is a polymer of sarcosine (N-methylglycine), and is known to exhibit excellent solubility in water, excellent biodegradability, non-immunogenicity, and low nonspecific protein adsorption. Therefore, microparticles formed from amphiphilic polymers containing polysarcosine as the hydrophilic block are expected to exhibit high blood retention 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, and can therefore be particularly suitably used as the above-mentioned carrier exhibiting cancer accumulation.

[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 in that by irradiating a subject to which the agent has been administered with radiation containing neutrons, it is possible to simultaneously obtain the therapeutic effects of both boron neutron capture therapy and photodynamic therapy. The boron-containing compound that can be contained in the agent according to one aspect of the present invention may essentially be any compound, and there is no particular limitation on the type. However, from the viewpoint of efficiently increasing the boron concentration in target cells, a boron cluster is preferred, and a hydrophobic boron cluster such as a carborane or a derivative thereof is more preferred. When microparticles containing an amphiphilic polymer having a hydrophilic block and a hydrophobic block are used as a carrier exhibiting cancer accumulation, the hydrophobic boron cluster can be efficiently supported on the hydrophobic core of the microparticles.

[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):

[0027] (In the above formula (I), ● is C, each ○ is independently BH or B-halogen, and each R is independently H or a substituent having an optionally substituted saturated or unsaturated alkyl group.) The present inventors have found that, compared with m-carborane or p-carborane, 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, particularly on microparticles containing an amphiphilic polymer containing polylactic acid as the hydrophobic block.

[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 formula (I) above, wherein the alkyl group in the substituent R has three or more carbon atoms. The inventors have discovered that a boron-containing compound represented by formula (I) above, having two alkyl groups with three or more carbon atoms at the ortho positions, acts as a molecular glue that binds polylactic acid chains together, thereby dramatically improving the stability in an aqueous medium of microparticles formed from amphiphilic polymers containing polylactic acid as a hydrophobic block (Figure 20). As shown in the experimental examples described below, microparticles containing a boron-containing compound represented by formula (I) above, having two alkyl groups with three or more carbon atoms at the ortho positions, significantly increase the boron concentration in cancer cells and exhibit superior in vivo therapeutic effects compared to L-BPA, a boron-containing compound traditionally used in boron neutron capture therapy.

[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, the radiation irradiation device being 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 reiterate, to the inventors' knowledge, there is no known finding 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-described 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, for example, protons, electrons, and / or neutrons. As described above, the agent or the like administered to the subject may contain a boron-containing compound. When the agent or the like 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 suitable for cancer treatment using a combination of boron neutron capture therapy and photodynamic therapy.

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

[0033] The AB type polysarcosine-poly(L-lactic acid) block polymer (PSar 106 -block-PLLA 32) and the structures of o-carborane, m-carborane, and p-carborane. (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. (a) Outline of the experimental procedure for the cellular uptake test. (b) Intracellular boron concentration in mouse breast cancer cells 4T1 exposed to o-carborane-loaded PSAR-PLLA nanoparticles or L-BPA (boron concentration: 2 mM) for a specified time. The release behavior of o-carborane from o-carborane-loaded PSAR-PLLA nanoparticles in PBS. 1 shows the changes in particle size and polydispersity when (a) PSAR-PLLA nanoparticles not carrying carborane, (b) PSAR-PLLA nanoparticles carrying o-carborane, and (c) PSAR-PLLA nanoparticles carrying m-carborane are dialyzed in PBS for 24 hours.

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[0082] [0083 (d) Transmission electron micrograph of PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane. (a) Release behavior of o-carborane derivatives from PSAR-PLLA nanoparticles loaded with 1,2-dimethyl-o-carborane and PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane in PBS.This figure shows the changes in particle size and polydispersity when (a) PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane and (b) PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were dialyzed in PBS for 24 hours. This figure shows the intracellular boron concentration in 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, which were contacted with (a) PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane or PSAR-PLLA nanoparticles carrying o-carborane. (a) This figure shows an outline of the experimental procedure for the cytotoxicity test. (b) Cell viability of mouse breast cancer 4T1 cells in contact 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. (A) In vivo near-infrared fluorescence imaging of a xenograft mouse administered with ICG-carrying PSAR-PLLA nanoparticles. (B) Time-dependent changes in the intensity of ICG-derived fluorescence in a xenograft mouse administered with ICG-carrying PSAR-PLLA nanoparticles. (C) Ex vivo near-infrared fluorescence imaging of a xenograft mouse administered with ICG-carrying PSAR-PLLA nanoparticles. (A) This figure shows the results of ICP-AES measurement of boron content in various tissues of Xenograft mice administered with PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane. (B) This figure shows the T / N ratio and T / B ratio in Xenograft mice administered with PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane. (A) This figure shows 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 specified period of time. The dose of the boron compound was 2 mM in boron equivalent.(B) Photographs showing typical cell morphology of AsPC-1 cells treated with PSAR-PLLA nanoparticles, PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane, or L-BPA after 40 minutes of neutron irradiation. In the figure, "**" indicates p < 0.01, and "*" indicates p < 0.05. AsPC-1 / CMV-Luc tumor-bearing mice were treated with PSAR-PLLA nanoparticles, PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane, or L-BPA, and 24 hours later were irradiated with neutrons for 40 minutes. (A) Mouse weight changes and (B) tumor size were monitored for 21 days after neutron irradiation. In the figure, "**" indicates p < 0.01, and "*" indicates p < 0.05. (A) This figure shows the cell viability of AsPC-1 cells administered with ICG-loaded PSAR-PLLA nanoparticles after neutron irradiation for a specified period of time. (B) This figure shows a typical photograph of AsPC-1 cells administered with ICG-loaded PSAR-PLLA nanoparticles after neutron irradiation for a specified period of time. In the figure, "**" indicates p < 0.01, and "*" indicates p < 0.05. AsPC-1 / CMV-Luc tumor-bearing mice were administered with ICG-loaded PSAR-PLLA nanoparticles and irradiated with neutrons 24 hours later for 40 minutes. After neutron irradiation, (A) changes in mouse weight and (B) tumor size were monitored for 21 days. In the figure, "**" indicates p < 0.01, and "*" indicates p < 0.05. (a) This figure shows an outline of the experimental procedure for the in vivo kinetics study. (b) shows the results of in vivo near-infrared fluorescence imaging of a Xenograft mouse administered with PSAR-PLLA nanoparticles carrying ICG and 1,2-dihexyl-o-carborane. (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.1 is a conceptual diagram illustrating the interaction between polylactic acid and a boron-containing compound in PSAR-PLLA nanoparticles according to one embodiment of the present invention, and FIG. 2 is a conceptual diagram illustrating the mechanism by which the agent according to one aspect of the present invention provides a BNCT effect and a mechanism by which the agent provides a PDT effect.

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

[0035] <Photosensitizer> A photosensitizer is a compound 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, a photosensitizer can react with oxygen (O2) in the ground triplet state to efficiently generate singlet oxygen. Singlet oxygen is a highly reactive chemical species that damages cells that constitute lesions such as tumors, leading to cell death. As shown in the experimental examples below, the photosensitizer described below is excited by radiation exposure and functions as a radiosensitizer that can enhance the effectiveness of radiation therapy.

[0036] The photosensitizer that can be used in the present invention may basically be any type, and various photosensitizers applicable to photodynamic therapy can be used, such as, but not limited to, cyanine-based photosensitizers, porphyrin-based photosensitizers, phthalocyanine-based photosensitizers, naphthoquinone-based photosensitizers, diimmonium-based photosensitizers, and azo-based photosensitizers. Specific examples of such photosensitizers include, but are not limited to, zinc phthalocyanine (ZnPc), zinc naphthalocyanine, 5,10,15,20-tetrakis(pentafluorophenyl)chlorin derivatives (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 can be one that has an absorption maximum wavelength within the wavelength range of 600 to 900 nm, more preferably within the 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 wavelengths of 600 to 900 nm, can be excited by irradiation with radiation, including neutron beams, and can exhibit significant antitumor effects.

[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]

[0040] In the above formula (II), R １ and R ４ are each independently an optionally substituted alkyl group. R2 and R3 are H or groups which combine with each other to form a cyclic structure. X is H or 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), the cyclic structure that can be formed by R2 and R3 may be substituted. In addition, 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]

[0043] In the above formula (III), R5 and R6 are both 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 same applies to R7 and R8; they are both 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 the above formula (II), so explanations are omitted.

[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]

[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. Meanwhile, 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-covalently and / or covalently 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 usable 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), i.e., a conjugate of indocyanine green and polylactic acid. In formula (XIII), n is an integer, preferably 5 to 50, more preferably 10 to 50, and even more preferably 15 to 50.

[0048]

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

[0050] <Boron-Containing Compound> Meanwhile, the boron-containing compound that can be contained in the agent according to one aspect of the present invention refers to a compound containing a boron atom in its molecular structure. The boron-containing compound may basically be any compound, and there is no particular limitation on the number of boron atoms contained in one molecule of the boron-containing compound. For example, it may contain one boron atom, such as the aforementioned L-BPA, or it may contain two 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, a boron cluster containing a large number of boron atoms in one molecule is preferable.

[0051] The term "boron cluster" refers to a boron-containing compound having a polyhedral structure formed by the aggregation of multiple boron atoms. Examples of boron clusters include 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 suitable boron clusters include, but are not limited to, clusters of boron hydrides such as boron hydrides (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 the skeletal atoms that constitute the clusters. Examples of boron clusters containing boron atoms and carbon atoms include carboranes. Examples of carboranes include closo-carborane ([CB 11 H 12 ] - ), closo-carborane (C2B 10 H 12 ) and nido-carborane ([C2B9H 11 ] - ), but are not limited to these. In addition, dodecaborate ([B 12 H 11 SH] 2- , BSH), Dodecaborate ([B 12 H 11 OH] 2- ), Dodecaborate with an amino group ([B 12 H 11 NH3] - ) 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]

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

[0055] Carborane, a boron cluster containing a boron atom and a carbon atom, or a derivative thereof is 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, an o-carborane derivative having the structure represented by the above formula (I) can be particularly suitably used because it can be efficiently and stably supported by microparticles formed from an amphiphilic polymer containing polylactic acid as a hydrophobic block, as compared with p-carborane derivatives and m-carborane derivatives, as shown in the experimental examples described below.

[0056] In a preferred embodiment, in formula (I), each R independently represents a substituent having an optionally substituted saturated or unsaturated alkyl group, and the alkyl group may have three or more carbon atoms. When the substituent R in formula (I) has an alkyl group having three or more carbon atoms, when the boron-containing compound is incorporated into microparticles formed from an amphiphilic polymer containing polylactic acid as a hydrophobic block, the alkyl group interacts with the polylactic acid chains that form the hydrophobic core of the microparticles, particularly the polylactic acid chains that form an α-helical structure, acting as a molecular glue to bind the two polylactic acid chains together, thereby dramatically improving the stability of the microparticles in aqueous media (Figure 20). From the perspective of improving the stability of the microparticles, the alkyl group preferably has three or more carbon atoms, more preferably four or more, even more preferably five or more, and even more preferably six or more. On the other hand, although there is no particular upper limit on the number of carbon atoms in the alkyl group, 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 may form aggregates by itself, which may make it difficult for the boron-containing compound to be supported on microparticles. From this perspective, the number of carbon atoms in 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 in the alkyl group is preferably 3 or more, more preferably 3 to 11, even more preferably 4 to 10, even more preferably 4 to 9, even more preferably 5 to 8, even more preferably 5 to 7, and particularly preferably 6. As shown in the experimental examples described below, when the number of carbon atoms in 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 significant stabilization 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, with an optionally substituted saturated or unsaturated alkyl group being preferred, an optionally substituted saturated alkyl group being more preferred, and an unsubstituted saturated alkyl group being more preferred. The substituent having an alkyl group may be either linear or branched, with a linear structure being preferred.

[0058] Further specific examples of the substituent having an alkyl group having 3 or more carbon atoms include groups represented by the general formula (-(CH2)CH3), such as an n-propyl group (-(CH2)CH3), an n-butyl group (-(CH2)CH3), an n-pentyl group (-(CH2)CH3), an n-hexyl group (-(CH2)CH3), an n-heptyl group (-(CH2)CH3), and an n-octyl group (-(CH2)CH3). n alkyl groups represented by the general formula (-O(CH2)CH3) (where n is 2 or more), n-propoxy groups (-O(CH2)2CH3)), n-butoxy groups (-O(CH2)3CH3)), n-pentoxy groups (-O(CH2)4CH3)), and n-hexyloxy groups (-O(CH2)5CH3) n alkoxy groups represented by the general formula (-S(CH2)CH3) (where n is 2 or more), n-propylthio group (-S(CH2)2CH3)), n-butylthio group (-S(CH2)3CH3)), n-pentylthio group (-S(CH2)4CH3)), n-hexylthio group (-S(CH2)5CH3)), etc. nExamples of the alkylthio group include, but are not limited to, alkylthio groups represented by the formula (CH3) (where n is 2 or more). When the substituent having an alkyl group with 3 or more carbon atoms is an alkyl group, alkoxy group, or 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, even more preferably 3 or more and 8 or less, even more preferably 4 or more and 7 or less, still more preferably 4 or more and 6 or less, and particularly preferably 5. A specific example of the boron-containing compound represented by formula (I) above and having such an alkyl group-containing substituent as R is 1,2-dihexyl-o-carborane, which will be described in the experimental examples below.

[0059] In this specification, the term "optionally substituted" includes both cases where the substitutable hydrogen atoms constituting the group are not substituted and cases where they are substituted. "The case where the substitutable hydrogen atoms are not substituted" means that all of the substitutable hydrogen atoms are hydrogen atoms. On the other hand, "the case where the substitutable hydrogen atoms are substituted" 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 are no particular limitations on the type of atom that serves as the substituent, but from the viewpoint of binding to the α-helix structure formed by polylactic acid, it is preferable that the atom be relatively 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 derivatives represented by the formula (I) by any suitable 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, followed by reaction with an appropriate alkyl halide. Furthermore, 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 synthesizing an o-carborane derivative having hydroxyl groups (—OH) at the 1st and 2nd positions (1,2-(OH)2-1,2-closo-CB2B10 H 10 ; CAS number 868382-62-1) or an o-carborane derivative with thiol groups at the 1st and 2nd positions (1,2-(SH)2-1,2-closo-C2B 10 H 10 o-Carborane derivatives can be obtained by reacting o-carborane derivatives (CAS No. 23810-63-1) with an appropriate alkyl halide in the presence of a strong base such as KOH to alkoxylate or alkylthiolate them. However, the synthesis method for o-carborane derivatives is not limited to the above, and those skilled in the art can use appropriate methods (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 It is preferable that the material contains 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 B isotope ratio 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, chemical exchange distillation is known.

[0062] <Carrier> Meanwhile, the agent according to one aspect of the present invention includes, in addition to the boron-containing compound and / or photosensitizer described above, a carrier for delivering the boron-containing compound and photosensitizer to tumors, i.e., a cancer-accumulating carrier. A cancer-accumulating carrier refers to a carrier that accumulates in cancer tissues or cancer cells when administered to a living body with cancer, such as a patient suffering from cancer, and increases the amount of the boron-containing compound and / or photosensitizer accumulated in cancer tissues or cancer cells compared to when the agent does not contain a cancer-accumulating carrier. Such a carrier may essentially be any type, but may be one or more of, for example, an antibody, a fragment antibody, a peptide, an amino acid, a nucleic acid, an aptamer, a vitamin, a sugar (including monosaccharides, oligosaccharides, and polysaccharides), a sugar chain, and a microparticle. The boron-containing compound and / or photosensitizer can be covalently and / or non-covalently bound to and supported by such a carrier.

[0063] In a preferred embodiment, the carrier is preferably a microparticle. The microparticle may have an average particle diameter of 20 to 500 nm as measured by dynamic light scattering. From the viewpoint of enhancing accumulation in cancer tissues or enhancing accumulation and penetration into cancer tissues, the average particle diameter (diameter) as measured by dynamic light scattering is preferably 20 to 150 nm, more preferably 20 to 140 nm, even more preferably 20 to 130 nm, and even more preferably 20 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 cancer-accumulating carrier.

[0064] On the other hand, there is no particular limitation on the type of microparticles that can be used in the present invention, and they 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 refers to a polymer chain that is hydrophobic as a whole block. Here, the hydrophobicity of the hydrophobic block refers to low solubility in water, and more preferably low solubility in water to the extent that an amphiphilic polymer comprising a hydrophilic block and a hydrophobic block can self-assemble or aggregate in an aqueous medium to form micellar or vesicle-like microparticles. Those skilled in the art can confirm the formation of micellar or vesicle-like microparticles, 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" refers to a polymer formed by polymerization of lactic acid. Because polylactic acid has excellent biocompatibility and biodegradability, amphiphilic polymers containing polylactic acid as a component can be advantageously used as pharmaceutical materials. Lactic acid, the monomer of polylactic acid, exists as L-lactic acid and its optical isomer, D-lactic acid. Polylactic acid obtained primarily by polymerizing L-lactic acid is called poly(L-lactic acid) (PLLA), polylactic acid obtained primarily by polymerizing D-lactic acid is called poly(D-lactic acid) (PDLA), and polylactic acid obtained by polymerizing L-lactic acid and D-lactic acid is called poly(DL-lactic acid) (PDLLA).

[0067] According to the findings of the present inventors, the α-helix structure formed by polylactic acid chains has a hydrophobic portion on its outer surface, which strongly interacts with the boron-containing compound described below, i.e., o-carborane having two alkyl groups at the ortho positions, thereby stabilizing the microparticles (Figure 20). Therefore, in a preferred embodiment, the "polylactic acid" usable in the present invention is preferably one capable of forming an α-helix higher-order structure. Such polylactic acid may be, for example, poly(L-lactic acid) or poly(D-lactic acid), with poly(L-lactic acid) being particularly preferred. Note that the poly(L-lactic acid) or poly(D-lactic acid) usable in one embodiment of the present invention does not necessarily have to have 100% optical purity or 0% isomer content, as long as it is capable of forming an α-helix higher-order structure. For example, the isomer content may be poly(L-lactic acid) or poly(D-lactic acid) 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] "Containing polylactic acid as the hydrophobic block" includes both cases where the entire hydrophobic block is polylactic acid and cases where only a portion of the hydrophobic block is polylactic acid. That is, the hydrophobic block that can be used in the present invention may contain monomer units other than lactic acid units to the extent that polylactic acid can form a higher-order structure. There are no particular restrictions on 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% of the total monomer units (including lactic acid units) contained in the hydrophobic block.

[0069] The type of monomer unit other than the lactic acid unit that can be contained in the hydrophobic block is not particularly limited, 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 hydroxy acids other than lactic acid include glycolic acid, 2-hydroxyisobutyric acid, and 3-hydroxyisobutyric acid.

[0070] There are no particular limitations on the number of lactic acid units constituting the polylactic acid that can be contained in the hydrophobic block, and this can be appropriately adjusted so that the hydrophobic block exhibits the above-mentioned hydrophobicity, but it can be, for example, 5 to 200, preferably 10 to 100, more preferably 15 to 80, even more preferably 20 to 60, and still more preferably 25 to 45. In this specification, a numerical range indicated by "to" means a numerical range that includes 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 a hydrophobic block containing polylactic acid, and more preferably means that the hydrophilicity is such that an amphiphilic polymer containing a hydrophilic block and a hydrophobic block can self-assemble or aggregate 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 that the polymer 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 can 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 can be 10 to 300, preferably 20 to 250, more preferably 30 to 200, even more preferably 40 to 150, and still more preferably 50 to 100.

[0074] The hydrophilic block may be linear or branched. When the hydrophilic block has branches, 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 still 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. In a preferred embodiment, however, the amphiphilic polymer has a hydrophilic block containing polysarcosine. Polysarcosine refers to a polymer formed by polymerization of sarcosine (also known as "N-methylglycine"). Polysarcosine is highly biocompatible, exhibits low nonspecific protein adsorption, and is low immunogenic, allowing it to avoid the accelerated blood clearance (ABC) phenomenon (K. Son et al., J. Controlled Rel. 2020, 322(10), 209-216). Therefore, it is particularly suitable for use as the hydrophilic block of the amphiphilic polymer that can be contained in the 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, a block copolymer formed by bonding polysarcosine and polylactic acid. Polysarcosine-polylactic acid is known to self-assemble in an aqueous medium to form microparticles with diameters of approximately 20 nm to 200 nm (M. Akira et al., Chemistry Letters, 2007, 36(10), 1220-1221). Furthermore, microparticles formed by polysarcosine-polylactic acid have high blood retention and minimal liver accumulation. For these reasons, a block copolymer of polysarcosine and polylactic acid is particularly suitable as an amphiphilic polymer to be contained in the microparticles of the present invention. Polysarcosine may be linear, branched, or contain both. Examples of polysarcosine-polylactic acid containing linear polysarcosine include AB-type polysarcosine-polylactic acid block polymers. AB-type polysarcosine-polylactic acid block polymers and methods for synthesizing them are disclosed, for example, in International Publication WO 2009 / 148121. On the other hand, polysarcosine-polylactic acid containing branched polysarcosine includes, for example, A3B type polysarcosine-polylactic acid block polymer, which is a block polymer 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 repeating sarcosine units constituting the hydrophilic block, polysarcosine, is not particularly limited as long as the hydrophilic block as a whole exhibits the above-described 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 still more preferably 50 to 100. When the polysarcosine is a branched polysarcosine, the number of repeating sarcosine units in the entire hydrophilic block may be, for example, 10 to 300, preferably 20 to 250, more preferably 30 to 200, even more preferably 40 to 150, and still 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, and may be, for example, 10 to 100, preferably 15 to 80, more preferably 15 to 60, even more preferably 20 to 50, and still 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 microparticles by self-assembly of the amphiphilic polymer, it is preferable that the number of branches be equal to or less than the number of branches of the hydrophilic block.

[0078] 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 with the boron-containing compound and / or the photosensitizer in an aqueous medium. The boron-containing compound and / or the photosensitizer may be covalently or non-covalently bound to the amphiphilic polymer. There are no particular limitations on the method for preparing such microparticles. For example, as described below, they can be prepared by a preparation method including the steps of mixing an amphiphilic polymer containing polylactic acid as the hydrophobic block with the boron-containing compound and / or the photosensitizer to obtain a mixture, and contacting the mixture with an aqueous medium.

[0079] Furthermore, there are no particular limitations on the form of microparticles containing amphiphilic polymers having hydrophilic and hydrophobic blocks, but they can typically be polymeric micelle-like microparticles. Polymeric micelle-like microparticles are typically core-shell microparticles containing amphiphilic polymers, with a shell composed of a hydrophilic block and a core composed of a hydrophobic block. Alternatively, the microparticles may be hollow vesicle-like microparticles. For example, those skilled in the art can appropriately prepare vesicle-like hollow microparticles formed from amphiphilic polymers having polylactic acid as the hydrophobic block (H. Yin et al., Macromolecules 2009, 42(19), 7456-7464; H. Qi et al., Nature Communications 9, Article number: 3005 (2018)).

[0080] Furthermore, when using polymeric micelle-type microparticles containing amphiphilic polymers, the diameter of the microparticles can be controlled appropriately by adjusting the molecular weight of the amphiphilic polymer constituting the microparticles, the ratio of hydrophilic blocks to hydrophobic blocks, etc., which is extremely advantageous. For example, when preparing microparticles using an amphiphilic polymer containing polysarcosine as the hydrophilic block and polylactic acid as the hydrophobic block, using an A3B type polysarcosine-polylactic acid block polymer can prepare nanoparticles with smaller diameters than 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. A targeting molecule is a chemical substance that can enhance the accumulation of microparticles containing the target molecule in target tissues or the binding of microparticles to target cells compared to microparticles that do not contain the targeting molecule. Typically, the targeting molecule is a molecule that has the property of selectively binding to substances such as proteins that are specifically and highly expressed in target cells. There are no particular limitations 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. Note that, since targeting molecules are molecules that selectively bind to specific substances present in target cells, they are preferably present on the surface of the microparticles.

[0082] Those skilled in the art can prepare such microparticles having targeting molecules on their surface by any suitable method. For example, they may be prepared by using an amphiphilic polymer in which a targeting molecule is bound to the end of the hydrophilic block contained in the amphiphilic polymer. On the other hand, microparticles carrying targeting molecules may be prepared by preparing microparticles using a conjugate of polylactic acid and a targeting molecule in which the targeting molecule is bound to the end of polylactic acid in addition to the amphiphilic polymer and a boron-containing compound and / or a photosensitizer.

[0083] A specific example of a targeting molecule that can be used is a small molecule antibody variant (scFv) against mesothelin (MSLN), which is known to be highly expressed in various types of cancer, such as mesothelioma, pancreatic adenocarcinoma, ovarian cancer, and lung adenocarcinoma (H. Yakushiji et al., Cancer Science. 2019, 110(9), 2722-2733). In a preferred embodiment, a cell-penetrating peptide may also be used. A cell-penetrating peptide is a peptide that has the ability to penetrate the cell membrane of cells, particularly human cells, and microparticles containing the cell-penetrating peptide can be efficiently introduced into cells. There are no particular limitations on the type of cell-penetrating peptide that can be included in the agent of the present invention, and examples include, but are not limited to, TAT peptide, penetratin, polyarginine (R9), transportan, and Pep-1. 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. Furthermore, in addition to the amphiphilic polymer and the boron-containing compound and / or photosensitizer, microparticles carrying cell-penetrating peptides can also be prepared by preparing microparticles using a conjugate of polylactic acid and a cell-penetrating peptide in which the 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] Although there is no particular limitation on the amount of boron supported in the agent according to one aspect of the present invention, a larger amount of boron is preferred as long as the agent according to one aspect of the present invention can exist stably. 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 limitation on the ratio of the amphiphilic polymer to the boron-containing compound, but for example, the molar ratio (A / B) of boron contained in (A) the boron-containing compound to (B) the amphiphilic polymer may 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 of the present invention contains the agent or radiosensitizer described above. There are no particular limitations on the disease to be treated, but the composition may be particularly suitable for treating cancer, more preferably for treating 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 ameliorating cancer symptoms, and delaying cancer progression. Furthermore, subjects to which the pharmaceutical composition may be administered include humans and non-human animals.

[0086] There are no particular limitations on the type of cancer for which the pharmaceutical composition of the present invention can be used, and examples 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. Note that boron neutron capture therapy is known to be effective in treating malignant melanoma and head and neck cancers, such as highly malignant brain tumors in which cancer cells and normal cells are mixed, and therefore 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 are no particular limitations on the method of administration of the pharmaceutical composition of the present invention, and an appropriate administration method may be selected depending on the subject of application, including, but not limited to, oral administration, sublingual administration, intravenous administration, intraarterial administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, local administration including intratumoral administration, and infusion administration.

[0088] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be an appropriate dosage form depending on the application and administration method. For example, the pharmaceutical composition may be in the form of an injection, tablet, powder, granule, liquid, syrup, capsule, etc., but is not limited to these. It may also be in the form of a preparation to be prepared just before use. Needless to say, the pharmaceutical composition of the present invention may further contain pharmaceutically 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] Excipients that may be included in the pharmaceutical composition of the present invention may include, but are not limited to, lactose, mannitol, crystalline cellulose, cornstarch, etc. Binders may include, but are not limited to, crystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc. Disintegrants may include, but are not limited to, hydroxypropyl cellulose, carboxymethyl cellulose, cornstarch, croscarmellose, etc. Lubricants may include, but are not limited to, magnesium stearate, calcium stearate, talc, carnauba wax, etc. Coating agents may include, but are not limited to, hydroxypropyl methylcellulose, polyethylene glycol, talc, titanium oxide, iron oxide, etc. Solvents may include, but are not limited to, water, ethanol, propylene glycol, glycerin, vegetable oil, etc. Buffers may include, but are not limited to, phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer, amino acid, etc. Preservatives may include, but are not limited to, quaternary ammonium salts such as benzalkonium chloride, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, benzyl alcohol, sorbic acid and its salts, thimerosal, parabens, cetylpyridinium chloride, catechin, etc. Chelating agents may include, but are not limited to, sodium edetate and citric acid. Antioxidants may include, but are not limited to, sodium bisulfite, sodium sulfite, sodium metabisulfite, ascorbic acid, ascorbic acid glycoside, etc. Isotonicity agents may include, but are not limited to, glucose and table salt. Stabilizers may include, but are not limited to, sodium edetate, citric acid, ascorbic acid, sodium chloride, polyols, polysorbates, etc. Furthermore, the pharmaceutical composition of the present invention may contain one or more active ingredients that are incorporated into other pharmaceutical compositions, such as anticancer agents, nucleic acids such as siRNA and mRNA, etc.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 cancer treatment by neutron irradiation but also for cancer treatment by RNA interference (RNAi).

[0090] When cancer is treated using the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention, the subject to which the agent, radiosensitizer, or pharmaceutical composition according to one aspect of the present invention 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-described 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 administered subject is preferably radiation capable of generating Cerenkov light in the living body. Such radiation may be, for example, radiation containing protons, electrons, and / or neutrons. Irradiation with such radiation excites the photosensitizer, thereby achieving the therapeutic effect of photodynamic therapy.

[0092] On the other hand, in the above-mentioned treatment 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 cancer by utilizing the nuclear reaction between the boron-containing compound and neutrons, thereby achieving the therapeutic effect of boron neutron capture therapy.

[0093] In the above-described therapeutic method according to one aspect of the present invention, when the agent or pharmaceutical composition administered to a subject contains both a boron-containing compound and a photosensitizer, the subject to which the agent or pharmaceutical composition has been administered is irradiated with radiation containing at least neutrons, thereby exciting the photosensitizer to damage the cancer and utilizing the nuclear reaction of the boron-containing compound with neutrons to damage the cancer. This 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 with neutrons and the therapeutic effect of photodynamic therapy using the photosensitizer, can be extremely advantageously used as a method of cancer treatment using radiation. The irradiated radiation need only 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, Cerenkov light can be generated more efficiently to excite the photosensitizer.

[0094] There are no particular limitations 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 radiation irradiations may be one or multiple. A medical professional may select an appropriate number of irradiations depending on the location and condition of the tumor disease to be treated, or taking into consideration the age, sex, and treatment period of the subject. There are also no particular limitations on the radiation irradiation time per session.

[0095] Radiation is preferably administered 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 can be administered 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 administration, and may be administered during radiation administration.

[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 may be performed 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, a radiosensitizer, or a pharmaceutical composition containing them described above has been administered. Such a radiation irradiation device may be an accelerator-type radiation irradiation device.

[0098] <Method for Producing Microparticles> A method for producing microparticles according to one aspect of the present invention includes the steps of mixing an amphiphilic polymer with a boron-containing compound and / or a photosensitizer to obtain a mixture, and contacting the mixture with an aqueous medium. The amphiphilic polymer has the property of forming microparticles by self-assembly when contacted 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 microparticles formed by the amphiphilic polymer by contacting the mixture of the amphiphilic polymer with the boron-containing compound and the photosensitizer with an aqueous solvent. The boron-containing compound and the photosensitizer described above can be used as the hydrophobic boron-containing compound and the photosensitizer.

[0099] Examples of methods for producing microparticles that include the above steps include the film method and the injection method (International Publication No. WO2012 / 176885). Here, as shown in the experimental examples described below, the film method is a method in which a film-like mixture obtained by drying a solution containing an amphiphilic polymer and a boron-containing compound in an organic solvent is contacted with an aqueous medium to obtain a dispersion in which microparticles are dispersed in the aqueous medium. More specifically, the film method can include the following steps: (a1) preparing a mixture 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 a photosensitizer; and (a3) ​​adding an aqueous medium to the film. The above steps (a1), (a2), and (a3) ​​are usually performed in this order.

[0100] The organic solvent used in step (a1) above is not particularly limited as long as it can dissolve the amphiphilic polymer, the boron-containing compound, and / or the photosensitizer; for example, chloroform, dimethylformamide, dimethyl sulfoxide, etc., are suitable. The method for removing the organic solvent in step (a2) above is not particularly limited; a person skilled in the art can employ 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 step (a3) ​​above is not particularly limited as long as it is water or a solvent containing water as the main component; for example, distilled water for injection, physiological saline, buffer solutions (e.g., PBS), etc., are suitable.

[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 of dispersing a solution containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent in an aqueous medium to obtain a dispersion in which fine particles are dispersed in the aqueous medium, and more specifically, it can be a method including: (b1) a step of preparing a mixed solution containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent; (b2) a step of dispersing the mixed solution in an aqueous medium; and (b3) a step of removing the organic solvent. The above steps (b1), (b2), and (b3) are usually performed 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, dimethyl sulfoxide, etc. can be suitably used.

[0104] Meanwhile, the above-mentioned step (b2) involves 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 containing microparticles containing the amphiphilic polymer and the boron-containing compound and / or the photosensitizer. The aqueous medium used in this step is not particularly limited as long as it is water or a solvent primarily composed of water. Suitable examples include distilled water for injection, physiological saline, and buffer solutions (e.g., PBS). The method for dispersing the mixture containing an amphiphilic polymer and a boron-containing compound and / or a photosensitizer in an organic solvent in the aqueous medium is not particularly limited, and can be carried out using, for example, a stirrer. The method for removing the organic solvent from the aqueous medium in the above-mentioned step (b3) is not particularly limited, and those skilled in the art can employ any suitable method. For example, the organic solvent may be removed under reduced pressure.

[0105] In a preferred embodiment, the above-described production method may further include an appropriate purification step before or after step (b3). Examples of purification methods that may be used include, but are not limited to, gel filtration chromatography, filtration, and ultracentrifugation. Furthermore, these purification methods may be combined as necessary.

[0106] In addition, the boron-containing compound represented by the 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 formula (I) in which the substituent R has 3 or more carbon atoms is expected to stabilize microparticles formed not only of amphiphilic polymers but also of 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 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 a similar perspective, in one aspect, the present invention also provides a stabilizer for polylactic acid-containing microparticles, the active ingredient of which is a boron-containing compound represented by the above formula (I), wherein the substituent R has 3 or more carbon atoms. As explained above, the boron-containing compound represented by the above formula (I), wherein the substituent R has 3 or more carbon atoms, is expected to stabilize microparticles formed from polymers containing polylactic acid. Therefore, the boron-containing compound represented by the above formula (I), wherein the substituent R has 3 or more carbon atoms, can be suitably used as a stabilizer for polylactic acid-containing microparticles. Note that a stabilizer for polylactic acid-containing microparticles 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. Those skilled in the art can evaluate the stability or disintegration of microparticles in an aqueous medium, for example, by observing the change in particle size of the microparticles over time using dynamic light scattering.

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

[0109] The present invention will be explained in more detail below by way of 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> Unless otherwise specified, the following reagents were used in the experiments described below: 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 buffer solution (DPBS), crystal violet, chloroform, hydrogen peroxide (H2O2), perchloric acid (HClO4), and nitric acid (HNO3) were 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 as a fructose complex (BPA-F) prepared by the method described by 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.) Briefly, the procedure is as follows: A solution containing 90 mg / mL of L-BPA (Interpharma Praha, As(IPP), Prague, Czech Republic) and 75 mg / mL of fructose was gently stirred, and 5.0 M NaOH was added to adjust the pH to 9.5-10.0, followed by stirring 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 use in the experiments described below.

[0112] <Experiment 1. Carborane-loaded polysarcosine-poly(L-lactic acid) nanoparticles (PSAR-PLLA nanoparticles)> Nanoparticles loaded with carborane as a boron-containing compound were prepared. The amphiphilic polymer used to prepare the nanoparticles was a 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, an AB-type polysarcosine-poly(L-lactic acid) block polymer (PSAR 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 is sometimes referred to as "PSAR-PLLA polymer," and nanoparticles prepared using this block polymer are sometimes referred to as "polysarcosine-polylactic acid nanoparticles" (PSAR-PLLA nanoparticles). Three isomers of carborane were used: o-carborane, m-carborane, and p-carborane (Katchem, Ltd., Prague, Czech Republic). The PSar used in this experiment 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. Separately, 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 PSAR-PLLA polymer solution and 100 μL of the carborane solution were added to a test tube, mixed, and evaporated to remove the organic solvent, yielding a film containing PSAR-PLLA polymer and carborane. 2.0 mL of phosphate-buffered saline (PBS) was added to the test tube containing the film, and the mixture was sonicated for 20 minutes using a bath sonicator. 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 0.22 μm syringe filter before use 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 (PDI) of the PSAR-PLLA nanoparticles were measured by dynamic light scattering using a Zetasizer Nano ZSP (Malvern, Malvern, United Kingdom). Boron Loading and Boron Loading Ratio. The boron loading of the PSAR-PLLA nanoparticles was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using an ICP atomic emission spectrometer (Shimadzu Corporation). Based on the boron loading measured by ICP-AES, the boron loading ratio (DLC (%)) of the PSAR-PLLA nanoparticles was calculated using 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]

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

[0117]

[0118] As shown in Table 1, the boron loading (DLC (%)) of PSAR-PLLA nanoparticles loaded with o-carborane, m-carborane, or p-carborane was 13.0%, 3.49%, and 1.80%, respectively, confirming that all carboranes were loaded onto PSAR-PLLA nanoparticles. In particular, the boron loading of o-carborane, one of the three carborane isomers, was 13.0%, significantly higher than that of m-carborane or p-carborane. The boron loading of the three carborane isomers increased in the order o-carborane > m-carborane > p-carborane, which coincided with the order of 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]

[0121] As shown in Table 2, the PSAR-PLLA nanoparticles loaded with o-carborane, m-carborane, or p-carborane all had particle sizes ranging from 60 nm to 72 nm and PDIs of approximately 0.20, comparable to those of PSAR-PLLA nanoparticles without carborane. Among these, the PSAR-PLLA nanoparticles loaded with o-carborane had the smallest polydispersity index and a highly monodisperse particle size distribution. Figure 2 shows histograms of particle size distributions obtained by dynamic light scattering for the PSAR-PLLA nanoparticles loaded with o-carborane and those without carborane. As shown in Figure 2, the PSAR-PLLA nanoparticles loaded with o-carborane are highly monodisperse, similar to the PSAR-PLLA nanoparticles without carborane. Furthermore, when the o-carborane-loaded PSAR-PLLA nanoparticles were examined using a transmission electron microscope, 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 agent for BNCT, was used as a comparison.

[0123] An outline of the experimental procedure is shown in Figure 3(a). In detail, mouse breast cancer cells 4T1 (ATCC, Rockville, Maryland, USA) were cultured at 0.5 × 10 5A cell suspension containing o-carborane-loaded PSAR-PLLA nanoparticles or L-BPA in cell culture medium at a concentration of 10 ... The number of boron atoms per cell was calculated based on the obtained boron content. 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 experiments for each sample.

[0124] As shown in Figure 3(b), the number of boron atoms per cell in 4T1 cells exposed to o-carborane-loaded PSAR-PLLA nanoparticles was greater than that in 4T1 cells exposed to L-BPA at all incubation times. The difference became more pronounced with shorter incubation times, and especially after 2 hours of incubation, the number of boron atoms per cell in 4T1 cells exposed to o-carborane-loaded PSAR-PLLA nanoparticles 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 with o-carborane-loaded PSAR-PLLA nanoparticles. This result indicates that o-carborane-loaded PSAR-PLLA nanoparticles can more efficiently increase the boron concentration in cancer cells compared to L-BPA, suggesting that o-carborane-loaded PSAR-PLLA nanoparticles 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 examine the stability of PSAR-PLLA nanoparticles carrying o-carborane under physiological conditions, the release of o-carborane from the PSAR-PLLA nanoparticles was evaluated over time when the PSAR-PLLA nanoparticles were incubated in PBS.

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

[0127] The results are shown in Figure 4. As shown in Figure 4, when o-carborane-loaded PSAR-PLLA nanoparticles were incubated in physiological saline, o-carborane was released relatively rapidly 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 rapidly under physiological conditions. Meanwhile, the release of o-carborane from PSAR-PLLA nanoparticles was more pronounced in PBS at 37°C than in PBS at 4°C.

[0128] To further investigate the stability of o-carborane-loaded PSAR-PLLA nanoparticles under physiological conditions, we incubated o-carborane-loaded PSAR-PLLA nanoparticles in saline and then evaluated their particle size and polydispersity using dynamic light scattering. Specifically, after 24 hours of dialysis, the aqueous solution remaining in the dialysis tubing was appropriately diluted and subjected to dynamic light scattering measurements. For comparative analysis, we also performed dialysis using PSAR-PLLA nanoparticles without carborane and PSAR-PLLA nanoparticles loaded with m-carborane instead of o-carborane using the same procedure. After 24 hours of dialysis, the aqueous solution remaining in the dialysis tubing was appropriately diluted and subjected to dynamic light scattering measurements.

[0129] The results are shown in Figure 5. As shown in Figure 5(b), after 24 hours of dialysis at 37°C, the size and polydispersity of the o-carborane-loaded PSAR-PLLA nanoparticles increased significantly. This indicates that at 37°C, the body temperature, the o-carborane-loaded PSAR-PLLA nanoparticles gradually became unstable and their structure gradually collapsed. Meanwhile, the size and polydispersity of the PSAR-PLLA nanoparticles without o-carborane are shown in Figure 5(a). As shown in Figure 5(a), increases in size and polydispersity were also observed for the PSAR-PLLA nanoparticles without o-carborane after 24 hours of dialysis at 37°C.

[0130] As described above, PSAR-PLLA nanoparticles carrying o-carborane, like PSAR-PLLA nanoparticles, gradually destabilized at 37°C. The release of o-carborane from the PSAR-PLLA nanoparticles shown in Figure 4 is likely due to the leakage of hydrophobic compounds physically encapsulated within the PSAR-PLLA nanoparticles, as well as the destabilization of the carrier PSAR-PLLA nanoparticles themselves. The polylactic acid in PSAR-PLLA nanoparticles contains carbonyl groups (-C(=O)-), which are known to undergo nucleophilic reactions with boron atoms (B) located near the two ortho carbon atoms (C) 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-lactic acid) may interfere with the α-helical interactions of poly(L-lactic acid) that constitute the hydrophobic core of PSAR-PLLA nanoparticles, destabilizing the PSAR-PLLA nanoparticles and leading 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] Furthermore, for PSAR-PLLA nanoparticles loaded with m-carborane instead of o-carborane, a more significant increase in particle size and polydispersity was observed after 24 hours of dialysis at 37°C (Figure 5(c)). As shown in Table 1, the m-carborane-loaded PSAR-PLLA nanoparticles loaded with less carborane than the o-carborane-loaded PSAR-PLLA nanoparticles. Nevertheless, the 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 loaded with o-carborane derivatives. Experiment 1 demonstrated that, of the three carborane isomers (o-carborane, m-carborane, and p-carborane), o-carborane was most efficiently loaded onto PSAR-PLLA nanoparticles and may also be advantageous in terms of the stability of the PSAR-PLLA nanoparticles. Therefore, to further enhance the stability of PSAR-PLLA nanoparticles and enable better intracellular boron delivery, we focused on o-carborane and its derivatives. We prepared PSAR-PLLA nanoparticles loaded with o-carborane derivatives in which alkyl groups with specific carbon chain lengths were introduced to the two ortho-carbon atoms of o-carborane, and evaluated their physical properties.

[0133] As in Experiment 1, the amphiphilic polymer used to form nanoparticles was a block copolymer of polysarcosine and polylactic acid (PSar 106 -block-PLLA 32 ) (weight-average molecular weight: 10,001 Da; manufactured by Kobe Natural Products Chemical Co., Ltd.) was used. The o-carborane derivatives used were 1,2-dimethyl-o-carborane, 1,2-dihexyl-o-carborane, and 1,2-didodecyl-o-carborane (Katchem, Ltd., Prague, Czech Republic). 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 PSAR-PLLA nanoparticles were prepared using the same procedure 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 resulting PSAR-PLLA nanoparticles were measured by dynamic light scattering using the same procedure as in Experiment 1. The amount of boron loaded was also measured by ICP-AES, and the boron loading rate (DLC (%)) was calculated based on the obtained amount of boron loaded. The results are shown in Table 3.

[0135]

[0136] As shown in Table 3, the boron loading (DLC (%)) of PSAR-PLLA nanoparticles loaded with 1,2-dimethyl-o-carborane, 1,2-dihexyl-o-carborane, and 1,2-didodecyl-o-carborane was 1.30%, 2.86%, and 0.61%, respectively, confirming that all carborane derivatives were loaded onto PSAR-PLLA nanoparticles. The boron loading (DLC (%)) of 1,2-dihexyl-o-carborane, which has a longer alkyl chain length (i.e., C6), was approximately twice as high as that of 1,2-dimethyl-o-carborane, which has a C1 alkyl chain. However, the boron loading rate did not necessarily increase with the carbon number in the alkyl chain. The boron loading rate of PSAR-PLLA nanoparticles bearing 1,2-didodecyl-o-carborane, which has an even higher carbon number (C12), was only about one-quarter to one-fifth of that of PSAR-PLLA nanoparticles bearing 1,2-dihexyl-o-carborane, which has an alkyl chain of C6. Considering that o-carborane derivatives, like o-carborane, are presumed to be physically encapsulated in PSAR-PLLA nanoparticles primarily through hydrophobic interactions with the polylactic acid that constitutes the hydrophobic core of PSAR-PLLA nanoparticles, the finding that PSAR-PLLA nanoparticles bearing 1,2-dihexyl-o-carborane had the highest boron loading rate was quite unexpected. While 1,2-dihexyl-o-carborane can strongly interact with polylactic acid due to its 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 the 1,2-didodecyl-o-carboranes, which is thought to be the reason why efficient interaction with PSAR-PLLA nanoparticles does not occur.

[0137] The particle size and polydispersity of PSAR-PLLA nanoparticles carrying three types of o-carborane derivatives, as measured by dynamic light scattering, are shown in Table 4, and the particle size distribution histograms are shown in Figures 7(a) to 7(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]

[0139] As shown in Table 4 and Figures 7(a)-(c), the PSAR-PLLA nanoparticles loaded with 1,2-dimethyl-o-carborane had a particle size of 63.7 nm and a PDI of 0.23, comparable to those of PSAR-PLLA nanoparticles without carborane. This result is similar to the results for PSAR-PLLA nanoparticles loaded with the three carborane isomers shown in Experiment 1-2. Meanwhile, the particle sizes of the nanoparticles loaded with 1,2-dihexyl-o-carborane or 1,2-didodecyl-o-carborane ranged from 93.2 nm to 104 nm, larger than those of PSAR-PLLA nanoparticles without carborane, demonstrating differences between the sizes of the three carborane isomers and the PSAR-PLLA nanoparticles loaded with 1,2-dimethyl-o-carborane. Furthermore, when 1,2-dihexyl-o-carborane and 1,2-didodecyl-o-carborane were compared, it was found that the polydispersity index was as low as 0.15 when 1,2-dihexyl-o-carborane was used, and it was found that PSAR-PLLA nanoparticles with an extremely monodisperse particle size distribution were formed. Therefore, when the particle morphology of PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane was observed using a transmission electron microscope, the formation of monodisperse nanoparticles with particle sizes comparable to the particle size distribution obtained by dynamic light scattering was confirmed.

[0140] Experiment 2-2. Evaluation of the Stability of o-Carborane-Loaded PSAR-PLLA Nanoparticles Next, the stability of PSAR-PLLA nanoparticles loaded with 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 loaded with 1,2-dimethyl-o-carborane or 1,2-dihexyl-o-carborane were used instead of o-carborane-loaded PSAR-PLLA nanoparticles.

[0141] The results of measuring the release of o-carborane derivatives are shown in Figure 8. As shown in Figure 8(a), the release of 1,2-dimethyl-o-carborane was observed over time during dialysis from PSAR-PLLA nanoparticles loaded with 1,2-dimethyl-o-carborane. The release of 1,2-dimethyl-o-carborane was more pronounced at 37°C than at 4°C, and after 24 hours of dialysis at 37°C, as much as 60% of the total amount of 1,2-dimethyl-o-carborane was released. This result is similar to that obtained with PSAR-PLLA nanoparticles loaded with o-carborane. In contrast, as shown in Figure 8(b), surprisingly, almost no release of 1,2-dihexyl-o-carborane was observed from PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane during 24 hours of dialysis, both at 4°C and 37°C.

[0142] Figure 9 shows the change in particle size of PSAR-PLLA nanoparticles before and after 24 hours of dialysis in PBS. As shown in Figure 9(a), PSAR-PLLA nanoparticles loaded with 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. However, no significant changes in particle size or polydispersity were observed after 24 hours of dialysis at 4°C. These results indicate that PSAR-PLLA nanoparticles loaded with 1,2-dimethyl-o-carborane are significantly destabilized at 37°C. In contrast, as shown in Figure 9(b), PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane showed little change in particle size or polydispersity even after 24 hours of dialysis at 37°C. These results indicate that PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane have excellent stability even at 37°C in PBS, a temperature and salt concentration similar to the biological environment. Because the polylactic acid that 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 surprising finding that the stability of PSAR-PLLA nanoparticles in aqueous media can be dramatically improved by supporting 1,2-dihexyl-o-carborane is extremely important for the application of PSAR-PLLA nanoparticles as pharmaceuticals that require storage and use in aqueous media.

[0143] <Experiment 2-3. Investigation of the production conditions for PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane> In order to investigate the production conditions for PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane were prepared by varying the ratio of 1,2-dihexyl-o-carborane to PSAR-PLLA polymer.

[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 was dissolved in chloroform. 106 -block-PLLA 32 100 μL of a 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 A film 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 film, which was then dried to form another film. 2.0 mL of PBS was added to the film 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, Buckinghamshire, United Kingdom). The eluate containing the nanoparticles was passed through a 0.22 μm pore size syringe filter followed by a 0.10 μm pore size syringe filter (Merck Millipore, Dublin, Ireland) before use in the experiments described below. The particle size and polydispersity of the PSAR-PLLA nanoparticles were measured by dynamic light scattering using the same procedure as in Experiment 1. The boron loading was also measured by ICP-AES, and the boron loading rate (DLC (%)) was calculated based on the obtained boron loading. The results are shown in Table 5.

[0145]

[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 resulting 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 resulting PSAR-PLLA nanoparticles increased to 83.7 nm, 91.8 nm, and 111 nm. This result indicates that the particle size of 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 cellular 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 be more effective in increasing intracellular boron concentrations than L-BPA, a boron drug conventionally used in BNCT.

[0149] The experimental procedure is outlined in Figure 3(a). Specifically, mouse breast cancer cells 4T1, mouse colon cancer cells CT26, human pancreatic adenocarcinoma cells AsPC-1, or 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 cells / mL, or 1.0 x 10 6 A cell suspension containing 1,2-dihexyl-o-carborane in cell culture medium at a concentration of 10 μmol / mL was seeded into each well of a 6-well microplate and incubated for 24 hours at 37°C in 5% CO2 according to standard procedures. Next, PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane (prepared by the same procedure as in Experiment 2-3) at a ratio of 10 μmol of 1,2-dihexyl-o-carborane to 1.0 μmol of PSAR-PLLA (Ratio 3 in Table 5) or PSAR-PLLA nanoparticles loaded with o-carborane (prepared by the same procedure 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 the cells were incubated for 2 hours. After incubation, the cells were washed three times with PBS according to standard procedures and then harvested by trypsinization. The resulting solution was centrifuged at 1,800 rpm for 5 minutes, and the supernatant was removed to obtain a pellet. The resulting pellet was treated with 60% nitric acid (1 mL) overnight at room temperature. The solution was then filtered through a 0.45 μm syringe filter, and purified water was added to bring the total volume to 10 mL. The resulting solution was subjected to ICP-AES to measure the boron content. The results are shown in Figure 10. Note that Figure 10 shows the mean ± standard error (SEM) of values ​​obtained from three independent tests for each sample.

[0150] As shown in Figure 10, in all cell types, including human-derived cells and mouse-derived cells, ranging from breast cancer to colon cancer, pancreatic adenocarcinoma, and gastric cancer, cells contacted with PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane had a significantly higher number of boron atoms per cell than cells contacted with PSAR-PLLA nanoparticles carrying o-carborane. The minimum number of boron atoms per cell required to obtain a therapeutic effect from BNCT is 1 x 10 9 pieces / cell (up to 20 ng boron / 10 6 As shown in Figure 10, when PSAR-PLLA nanoparticles carrying o-carborane were used, the number of cells increased to 1 × 10 when treated with boron at a concentration of 0.5 mM or 2 mM. 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 boron at a concentration of 0.5 mM or 2 mM, the number of boron atoms per cell was 1 × 10 9 This was far more than the number of cells 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. The intracellular boron concentration of various cancer cells was increased to the 1×10 level required for the therapeutic effect of BNCT. 9 PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane, which can be increased to a level exceeding 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 Figure 11(a). Mouse breast cancer cells 4T1 were placed in a 96-well microplate at a cell density of 5 × 10 cells per well. 3 Cells were seeded at a density of 1000 cells / cell and incubated for 24 hours in a 5% CO2, 37°C environment according to standard procedures. Next, PSAR-PLLA nanoparticles carrying o-carborane or m-carborane, prepared as in Experiment 1-1, or PSAR-PLLA nanoparticles carrying 1,2-dimethyl-o-carborane or 1,2-dihexyl-o-carborane, prepared as in Experiment 2-1, were added to the cell culture medium to achieve boron concentrations of 5, 25, 50, 100, or 250 ppm, and the cells were incubated for 24 hours. Cytotoxicity tests were performed using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the kit's instructions. Optical density (OD) measurements at 450 nm were performed using a microplate reader (Sunrise). TM The assay was performed using a 100% ELISA kit (Tecan Trading AG, Mannedorf, Switzerland). Cell viability was calculated as a percentage of the cell viability of untreated cells (control), which was set at 100%. The 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 Figure 11(b), PSAR-PLLA nanoparticles loaded with o-carborane, m-carborane, 1,2-dimethyl-o-carborane, or 1,2-dihexyl-o-carborane did not exhibit significant cytotoxicity in the boron concentration range of 5 ppm to 250 ppm. The maximum concentration tested in this experiment, 250 ppm, corresponds to 25 mM in molar concentration. Considering this result together with the results of Experiments 2-3, the concentration range of 1 x 10, which is suitable for BNCT, is within the range of concentrations that do not exhibit significant cytotoxicity. 9These results suggest that PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane derivatives, which can achieve boron concentrations exceeding 1 / cell, are highly useful as low-toxicity and effective boron agents for BNCT.

[0155] <Experiment 3. Evaluation of the pharmacokinetics of PSAR-PLLA nanoparticles> To evaluate the in vivo kinetics of PSAR-PLLA nanoparticles, ICG-loaded PSAR-PLLA nanoparticles were prepared and administered to xenograft model mice, followed by near-infrared fluorescence imaging.

[0156] Experiment 3.1 Preparation of ICG-loaded PSAR-PLLA nanoparticles ICG-loaded PSAR-PLLA nanoparticles were prepared by the following procedure. First, PSAR dissolved in chloroform was 106 -block-PLLA 32 (1 μmol / mL; 1 mL) and a predetermined amount of ICG-PLLA 34 (1 mol%) were mixed in a test tube, and the solvent was removed by evaporation to obtain PSar 106 -block-PLLA 32 and ICG-PLLA 34 A film containing ICG-PLLA was prepared. 1.9 mL of DPBS was added to the test tube containing the obtained film 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 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 here, but the ICG-PLLA 34 Regardless of the amount of , 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 inoculating 2 × 10 AsPC-1 / CMV-Luc cells into the mouse model 2 weeks before the experiment. 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 6-week-old female nude mice (BALB / c nu / nu) (purchased from Charles River).

[0158] Experiment 3.3 Near-Infrared Fluorescence Imaging: 100 μL of a solution containing ICG-loaded PSAR-PLLA nanoparticles prepared as described above was administered intravenously via the tail vein to the xenograft model mice. In vivo near-infrared fluorescence imaging was performed 1, 3, 6, 12, 24, 48, and 72 hours after intravenous injection of the nanoparticle solution. After 72 hours of in vivo near-infrared imaging, the mice were euthanized, and blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestine, muscle, and tumor were collected and weighed. The fluorescence intensity in each organ was also measured by ex vivo near-infrared fluorescence imaging. In these 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, emission wavelength 840 nm).

[0159] Figures 12(A) and 12(B) show the in vivo kinetics of PSAR-PLLA nanoparticles evaluated by in vivo near-infrared biofluorescence imaging. As shown in Figures 12(A) and 12(B), strong ICG-derived fluorescence was observed at the tumor site in the right thigh of mice injected with ICG-loaded PSAR-PLLA nanoparticles via the tail vein. The intensity of ICG-derived fluorescence at the tumor site peaked 12 hours after administration of the 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 accumulate at the tumor site. The gradual increase in the ratio of the fluorescence intensity at the tumor site to that in normal tissues suggests that PSAR-PLLA nanoparticles accumulate at the tumor site due to the EPR effect.

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

[0161] <Experiment 4. Evaluation of in vivo kinetics of boron> Next, to evaluate the in vivo kinetics 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 loaded with a boron-containing compound PSAR-PLLA nanoparticles loaded with o-carborane or 1,2-dihexyl-o-carborane were prepared using the same procedure 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 use in the experiments described below.

[0163] <Experiment 4.2: Preparation of Xenograft Model> The xenograft model was prepared using the same procedure as in Experiment 3.2. Two weeks before the experiment, 2 × 10 AsPC-1 / CMV-Luc cells were added to the xenograft model. 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 6-week-old female nude mice (BALB / c nu / nu) (purchased from Charles River).

[0164] Experiment 4.3: Quantification of Boron Accumulation by ICP-AES. 100 μL of a solution containing PSAR-PLLA nanoparticles loaded with boron-containing compounds, prepared as described above, was administered intravenously via the tail vein to the xenograft model mice described above at a boron dose of 5 mg of B equivalent / kg. 24 hours after intravenous injection of the nanoparticle solution, the mice were euthanized, and blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestine, muscle, and tumor were collected and weighed. The collected organs were digested by immersion in a 1:1 mixture of HClO4 and H2O2 at 90°C for 2 hours. The digested sample was diluted with 10 mL of purified water and passed through a 0.5 μm syringe filter before being subjected to ICP-AES analysis.

[0165] The results of ICP-AES measurements of boron accumulation in each organ are shown in Figure 13(A), and the ratios of the boron concentration at the tumor site to that in normal tissue (muscle) (T / N) and the ratios of the boron concentration at the tumor site to that in blood (T / B) are shown in Figure 13(B). As shown in Figure 13(A), the amount of boron accumulated at the tumor site was 16 μg / g for the PSAR-PLLA nanoparticles loaded with o-carborane and 18 μg / g for the PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane, with the PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane showing a slightly higher result. Furthermore, as shown in Figure 13(B), the ratio of boron concentration in the tumor site to that in normal tissue (muscle) (T / N) was 3.3 / 1 for the PSAR-PLLA nanoparticles loaded with o-carborane and 3.6 / 1 for the PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane. Although the ratio was slightly higher for the PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane, the T / N ratio for all PSAR-PLLA nanoparticles was 3 or greater. In BNCT, a T / N ratio of 3 or greater is considered ideal (Tsurubuchi, T. et al., Cells 2020, 9(5), 1-20). The results that PSAR-PLLA nanoparticles loaded with o-carborane or 1,2-dihexyl-o-carborane can increase the T / N ratio at the tumor site to 3 or more suggest 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. BNCT Therapeutic Effect> Next, the in vitro and in vivo BNCT therapeutic effect of PSAR-PLLA nanoparticles carrying o-carborane or 1,2-dihexyl-o-carborane as a boron-containing compound was examined.

[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 Cells were seeded into 6-well microplates at a seeding density of 1000 cells / well and incubated for 24 hours at 37°C in 5% CO2 according to standard procedures. Next, o-carborane-loaded PSAR-PLLA nanoparticles, 1,2-dihexyl-o-carborane-loaded PSAR-PLLA nanoparticles, or L-BPA, prepared as described in Experiment 4.1, were added to cell culture medium to achieve a boron concentration of 2 mM and incubated for 2 hours. As controls, DPBS or blank PSAR-PLLA nanoparticles were added instead of the boron-loaded PSAR-PLLA nanoparticles and L-BPA, and the incubation was repeated for 2 hours in the same manner as for the cells treated with boron-loaded PSAR-PLLA nanoparticles and BPA. After incubation, cells were collected into test tubes and subjected to neutron irradiation (thermal power 1 MW; thermal neutron flux 1.3 × 10 9 neutrons / cm -2 / s -1 ) were irradiated for 0, 10, 20, and 40 minutes. The absorbed doses of neutrons at each irradiation time were 0 Gy (0 minutes), 0.142 Gy (10 minutes), 0.369 Gy (20 minutes), and 0.555 Gy (40 minutes), respectively. Neutron irradiation was performed at the Kyoto University Research Reactor (KUR). After neutron irradiation, neutron-irradiated AsPC-1 / CMV Luc (or AsPC-1) cells were seeded onto 60-mm culture dishes and cultured for two weeks for a colony formation assay. The cultured cells were stained in a 20% methanol solution containing 0.5% crystal violet (CV). Stained colonies were counted using an aCOLyte 3 automated colony counter (Synoptics Ltd.). The results are shown in Figure 14. The results shown in Figure 14 represent the mean ± standard error of the mean (SEM) of values ​​obtained from four independent experiments for each sample.

[0168] As shown in Figure 14, cell proliferation was inhibited in all groups treated with 1,2-dihexyl-o-carborane-loaded PSAR-PLLA nanoparticles, o-carborane-loaded PSAR-PLLA nanoparticles, and L-BPA, and the cell proliferation inhibitory effect tended to increase with increasing neutron irradiation time. In particular, the group treated with 1,2-dihexyl-o-carborane-loaded PSAR-PLLA nanoparticles exhibited a significantly greater proliferation inhibitory effect than the groups treated with o-carborane-loaded PSAR-PLLA nanoparticles or L-BPA.

[0169] <Experiment 5.2 In vivo test> The in vivo BNCT therapeutic effects of PSAR-PLLA nanoparticles loaded with o-carborane and PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane were investigated using the following procedure. Xenograft models, i.e., tumor-bearing mice (n=6) inoculated with AsPC-1 / CMV Luc cells, were prepared using the same procedure as in Experiment 3.2. 10 days after the inoculation of the cancer cells, o-carborane-loaded PSAR-PLLA nanoparticles, 1,2-dihexyl-o-carborane-loaded PSAR-PLLA nanoparticles, or L-BPA, prepared using the same procedure as in Experiment 4.1, were administered at 5 mg / mL. 10 Boron was administered via the tail vein at a dose of 100 B / kg. 24 hours after administration, the rats were irradiated with neutrons. The neutron irradiation was carried out at the 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 neutron irradiation, mice were evaluated for weight and tumor size twice weekly for 21 days. A hot control group was administered without boron compound-loaded PSAR-PLLA nanoparticles and neutron irradiation, and a cold control group was administered without boron compound-loaded PSAR-PLLA nanoparticles and neutron irradiation. The results are shown in Figure 15. Figure 15(A) shows the change in mouse weight, and Figure 15(B) shows the change in tumor size. The results shown in Figure 15 represent the mean ± standard error (SEM) of values ​​obtained from six independent mice for each sample.

[0170] As shown in Figure 15(A), the changes in body weight after neutron irradiation in all neutron-irradiated groups of mice were comparable to those in the cold control group. However, the changes in tumor size over the same period were suppressed in the neutron-irradiated hot control group, the group administered o-carborane-loaded PSAR-PLLA nanoparticles and neutron-irradiated, and the group administered 1,2-dihexyl-o-carborane-loaded PSAR-PLLA nanoparticles and neutron-irradiated, compared with the cold control group. In particular, the group administered PSAR-PLLA nanoparticles carrying o-carborane and neutron irradiation, and the group administered PSAR-PLLA nanoparticles carrying 1,2-dihexyl-o-carborane and neutron irradiation, showed significantly superior tumor suppression effects compared to the hot control group irradiated with neutrons without PSAR-PLLA nanoparticles. These results indicate that administration of PSAR-PLLA nanoparticles carrying boron-containing compounds and neutron irradiation can provide excellent cancer therapeutic effects.

[0171] <Experiment 6. PDT Therapeutic Effect> Next, 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 were examined.

[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 Cells were seeded into a 6-well microplate at a seeding density of 1000 cells / well and incubated for 24 hours at 37°C in 5% CO2 according to standard procedures. Next, ICG-loaded PSAR-PLLA nanoparticles, prepared according to the procedure described in Experiment 3.1, were added to cell culture medium and incubated for 2 hours. The measured ICG concentration in the cell culture medium was 1.6 μM, and the calculated PSAR-PLLA polymer concentration was 1 mM. As controls, DPBS or empty 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 treated with ICG-loaded PSAR-PLLA nanoparticles. After incubation, the cells were collected into test tubes and subjected to neutron irradiation (thermal power 1 MW; thermal neutron flux 1.3 × 10 9 neutrons / cm -2 / s -1 ) were irradiated for 0, 10, 20, and 40 minutes. The absorbed doses of neutrons at each irradiation time were 0 Gy (0 minutes), 0.142 Gy (10 minutes), 0.369 Gy (20 minutes), and 0.555 Gy (40 minutes), respectively. Neutron irradiation was performed at the Kyoto University Research Reactor (KUR). After neutron irradiation, neutron-irradiated AsPC-1 / CMV Luc (or AsPC-1) cells were seeded onto 60-mm culture dishes and cultured for two weeks for a colony formation assay. The cultured cells were stained in a 20% methanol solution containing 0.5% crystal violet (CV). Stained colonies were counted using an aCOLyte 3 automated colony counter (Synoptics Ltd.). The results are shown in Figure 16. The results shown in Figure 16 represent the mean ± standard error of the mean (SEM) of values ​​obtained from four independent experiments for each sample.

[0173] As shown in Figure 16, the number of viable cells in the group treated with PSAR-PLLA nanoparticles was equivalent to that in 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, cancer cell proliferation was significantly suppressed compared to the control group. Furthermore, the cell proliferation inhibitory effect increased as the neutron irradiation time increased. These results indicate that neutron irradiation of cells administered with ICG-loaded PSAR-PLLA nanoparticles can exert a cell proliferation inhibitory effect.

[0174] <Experiment 6.2 In vivo study> The in vivo therapeutic effect of ICG-loaded PSAR-PLLA nanoparticles was investigated using the following procedure. The xenograft model prepared using the above procedure, i.e., tumor-bearing mice (n=6) inoculated 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 using the procedure described in Experiment 3.1 at a measured ICG dose of 8.6 mg / kg (calculated PSAR-PLLA dose of 200 mg / kg). 24 hours after administration, the mice were irradiated with neutrons. The neutron irradiation was performed using the Kyoto University Research Reactor (KUR) under the following irradiation conditions: 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 neutron irradiation, mice were evaluated for weight and tumor size twice weekly for 21 days. As controls, a hot control group was administered without ICG-loaded PSAR-PLLA nanoparticles and neutron irradiation, and a cold control group was administered without ICG-loaded PSAR-PLLA nanoparticles and neutron irradiation. The results are shown in Figure 17. Figure 17(A) shows the change in mouse weight, and Figure 17(B) shows the change in tumor size. The results shown in Figure 17 represent the mean ± standard error (SEM) of values ​​obtained from six independent mice for each sample.

[0175] As shown in Figure 17(A), the weight change after neutron irradiation in both groups of mice was comparable to that in the cold control group. Meanwhile, the change in tumor size over the same period was suppressed in the neutron-irradiated hot control group and the group administered ICG-loaded PSAR-PLLA nanoparticles and neutron-irradiated compared to the cold control group. Surprisingly, the group administered ICG-loaded PSAR-PLLA nanoparticles and neutron-irradiated showed a significantly superior tumor-suppressing 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 produce a therapeutic effect on cancer. The above results, which show that administering PSAR-PLLA nanoparticles loaded with indocyanine green as a photosensitizer and irradiating them with neutron beams can produce an excellent therapeutic effect on cancer, are unexpected and represent a highly surprising finding.

[0177] Experiment 7. Preparation of PSAR-PLLA nanoparticles loaded with boron-containing compounds and photosensitizers, and evaluation of their in vivo dynamics

[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. First, PSAR was dissolved in chloroform. 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. 2.0 mL of PBS was then added 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 a syringe filter with a pore size of 0.10 μm before being 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 Figure 18(a). 6 Xenograft models were created by subcutaneously inoculating 100 μL of the resulting suspension into the right thigh of 6-week-old female nude mice (BALB / c nu / nu) (purchased from Charles River). Two weeks after inoculation, 100 μL of a solution containing boron-containing compounds and photosensitizer-loaded PSAR-PLLA nanoparticles prepared as described above was administered intravenously via the tail vein. In vivo near-infrared fluorescence imaging was performed 1, 3, 6, 12, 24, 48, and 72 hours after intravenous injection of the nanoparticle solution. After 72 hours of in vivo near-infrared fluorescence imaging, the mice were euthanized, and blood, heart, lungs, liver, spleen, pancreas, kidneys, stomach, intestine, muscle, and tumor were collected and weighed. Fluorescence intensity in each organ was also 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, emission wavelength 840 nm). The collected organs were treated with HNO3 according to standard methods and then subjected to ICP-AES to measure the boron concentration in each organ.

[0180] Figure 18(b) shows the in vivo kinetics of PSAR-PLLA nanoparticles evaluated by in vivo near-infrared biofluorescence imaging. As shown in Figure 18(b), strong ICG-derived fluorescence was observed at the tumor site in the right thigh of a mouse that received tail vein injection of PSAR-PLLA nanoparticles loaded with 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 the 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 loaded with 1,2-dihexyl-o-carborane and ICG accumulated at the tumor site. The gradual increase in the ratio of the fluorescence intensity at the tumor site to that in normal tissues suggests that PSAR-PLLA nanoparticles accumulate at the tumor site due to the EPR effect.

[0181] Meanwhile, the ICG-derived fluorescence intensity in each organ collected 72 hours after intravenous injection of PSAR-PLLA nanoparticles was measured by ex vivo near-infrared fluorescence imaging. The results are shown in Figure 19(a). As shown in Figure 19(a), the highest 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 loaded with 1,2-dihexyl-o-carborane and ICG accumulated most significantly at the tumor site, which is consistent with the in vivo near-infrared fluorescence imaging results shown in Figure 18(b).

[0182] Furthermore, the boron accumulation in each organ was measured by ICP-AES (Figure 19(b)). As shown in Figure 19(b), the boron concentration in the tumor site was more than three times higher than that in normal tissue (muscle). In BNCT, the ratio of boron concentration in the tumor site (T) to that in normal tissue (muscle) (T / N ratio) is considered ideal to be 3 or greater (Tsurubuchi, T. et al., Cells 2020, 9(5), 1-20). The results that PSAR-PLLA nanoparticles loaded with 1,2-dihexyl-o-carborane and ICG can increase the T / N ratio to 3 or greater in the tumor site suggest that these nanoparticles are highly useful for cancer treatment using BNCT.

[0183] In the above experiments, nanoparticles formed from AB-type polysarcosine-polylactic acid block polymers were used to support carborane or o-carborane derivatives. However, nanoparticles formed from so-called A3B-type polysarcosine-polylactic acid block polymers, in which three polysarcosine chains are linked 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 smaller particle sizes and are expected to reduce the ABC phenomenon compared to nanoparticles formed from AB-type polysarcosine-polylactic acid block polymers. On the other hand, in the above experiments, nanoparticles carrying o-carborane or o-carborane derivatives are believed to have demonstrated excellent accumulation in tumor tissues based on the EPR effect. However, to further enhance the accumulation of nanoparticles in tumor tissues and, if necessary, to increase the amount of nanoparticles taken up into cells, it goes without saying that nanoparticles can be modified with ligands that specifically bind to target cells and enable intracellular drug delivery. While there are no particular limitations on such ligands, for example, a small molecule antibody variant (scFv) against mesothelin (MSLN), which is known to be highly expressed in various 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.

[0184] The agent according to one aspect of the present invention, or a pharmaceutical composition containing the same, can achieve a higher intracellular boron concentration than the boron-containing compounds conventionally used in boron neutron capture therapy, and is therefore expected to improve the therapeutic effect of boron neutron capture therapy. On the other hand, when the agent according to one aspect of the present invention contains a photosensitizer, excellent photodynamic therapeutic effects can be achieved by irradiating with radiation instead of near-infrared light. The agent according to one aspect of the present invention, which can achieve the photodynamic therapeutic effect of the photosensitizer in addition to boron neutron capture therapy, and a pharmaceutical composition containing the same, are expected to further improve the therapeutic effect of cancer. As such, 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 said carrier, said agent being used to treat cancer by irradiating a subject to which said agent has been administered with radiation.

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

3. The photosensitizer has the following formula (II): (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 has the following formula (III): (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 any one of claims 1 to 4, 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, the hydrophobic block comprising polylactic acid.

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

8. The agent according to any one of claims 1 to 7, further comprising a boron-containing compound, and wherein the radiation comprises at least neutrons.

9. The boron-containing compound is represented by the following formula (I): 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 containing 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): (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): (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. Use of the radiosensitizer according to claim 13 or 14 for treating cancer.

16. A radiation irradiation device having a radiation source for irradiating radiation, the radiation irradiation device being used to treat cancer by irradiating a subject to which the agent described in any one of claims 1 to 11, the pharmaceutical composition described in claim 12, and / or the radiosensitizer described in claim 13 or 14 has been administered.

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