Radiation sensitizer

Abstract

To provide a radiation sensitizer containing fullerene in which the radiation sensitizing effect is high.SOLUTION: A radiation sensitizer contains fullerene modified with polyether.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to radiosensitizers. [Background technology]

[0002] In radiation therapy, a preparation called a radiosensitizer may be administered to the patient prior to radiation irradiation. A radiosensitizer is a preparation that exerts a radiation sensitizing effect by exposing the subject to radiation, such as cells, and enhances the cell killing ability of radiation exposure. By using a radiosensitizer in radiation therapy, the cancer cell killing effect of radiation therapy can be enhanced, which is expected to increase the therapeutic effect of radiation therapy and reduce side effects by reducing the number of radiation exposures required.

[0003] Regarding radiosensitizers, for example, Non-Patent Document 1 describes C 60 Nano-C, a nano-sized cluster of fullerene 60 About nano-C 60 It has been reported that cell membrane damage and increased apoptosis were observed in cells exposed to gamma rays after exposure to C. 60 Hydrate of fullerene molecules (C 60 It is described that UHFM) has an antioxidant effect, prevents damage to DNA and proteins caused by active oxygen generated by radiation exposure, and has a protective effect against damage caused by oxidative stress due to radiation exposure in mice. In addition, Non-Patent Document 3 describes a C 60 Fullerene (C 60 (OH) 24 ) has been disclosed to enhance the activity of antioxidant enzymes in irradiated erythroleukemia-derived cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Jin Ni et al., "Cytotoxicand radiosensitizing effects of nano-C60 on tumor cells in vitro.", J. Nanopart. Res. (2008) 10:643-651. [Non-Patent Document 2] Sergey V. Gudkov et al., "Unmodified hydrated С60 fullerene molecules exhibit antioxidant properties, prevent damage to DNA and proteins induced by reactiveoxygen species and protect mice against injuries caused by radiation-induced oxidative stress.", Nanomedicine: Nanotechnology, Biology, and Medicine 15(2019) 37-46. [Non-Patent Document 3] Bogdanovic V. et al., "FullerenolC60(OH)24 effects on antioxidative enzymes activity in irradiatedhuman erythroleukemia cell line.", J. Radiat. Res. 2008;49:321-7. Summary of the Invention [Problem to be solved by the invention]

[0005] When fullerene is to be used as an active ingredient in a radiosensitizer, the method described in Non-Patent Document 1 requires the use of C 60 Nano-C, a nano-sized cluster prepared by dissolving fullerene in a mixed solvent of water and tetrahydrofuran 60 However, the radiation sensitization effect is low. For example, nano-C 60Even when cells contacted with the fullerene were irradiated with gamma rays at a dose of 10 gray, the cell survival rate did not decrease to 1% or less. On the other hand, when an attempt was made to increase the effective concentration by increasing the water solubility of fullerene to enhance the sensitizing effect, as described in Non-Patent Documents 2 and 3, there was a problem that if fullerene is hydrated or modified with a hydroxyl group, it would actually exert a protective effect against radiation.

[0006] An object of the present disclosure is to provide a radiosensitizer that contains fullerene and has a high radiation sensitizing effect. [Means for solving the problem]

[0007] The present inventors have found that a radiosensitizer containing a fullerene modified with a polyether has a high radiosensitizing ability, and have also found that contacting a subject to be irradiated with a fullerene modified with a polyether can increase the sensitivity of the subject to radiation.

[0008] The present invention includes the following aspects. [1] A radiosensitizer containing a polyether-modified fullerene. [2] The radiosensitizer according to [1], wherein the polyether is a polyalkylene oxide. [3] The radiosensitizer according to [2], wherein the alkylene oxide group in the polyalkylene oxide has 2 or 3 carbon atoms. [4] A method for increasing the sensitivity of an object to radiation by contacting the object with a fullerene modified with a polyether. [5] The method according to [4], wherein the polyether is a polyalkylene oxide. [6] The method according to [5], wherein the alkylene oxide group in the polyalkylene oxide has 2 or 3 carbon atoms. Effect of the Invention

[0009] According to the present disclosure, a radiosensitizer having high radiosensitizing ability is provided. According to the present disclosure, a method for increasing the sensitivity to radiation in a subject is provided. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a graph showing the concentration dependence of cell viability evaluated by a colony formation method for C60 fullerene modified with polyether (A) in Test Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes an embodiment of the present invention, but the present invention is not limited to the following embodiment.

[0012] A first embodiment of the present invention is a radiosensitizer that contains a polyether-modified fullerene.

[0013] In the present disclosure, a radiosensitizer refers to an agent that enhances the sensitivity to radiation of a target to which the sensitizer is applied. In one embodiment, the radiosensitizer may be a preparation that has the effect of enhancing the sensitivity to radiation of a cell contacted with the sensitizer. It is known that irradiation of cells reduces the viability of cells through DNA fragmentation, etc., but when cells are irradiated in the presence of a radiosensitizer, the killing ability of the cells due to such irradiation can be enhanced.

[0014] The radiation may be electromagnetic radiation (electromagnetic waves) or particle radiation (particle beam). Examples of electromagnetic radiation (electromagnetic waves) include X-rays (X-rays) and gamma rays (γ-rays). Examples of particle radiation (particle beam) include alpha rays, beta rays, and neutron rays. In one embodiment, the radiation may be electromagnetic radiation, preferably X-rays or gamma rays. The wavelength of the radiation (electromagnetic radiation) is 1.0×10 -15 m or more or 1.0×10 -13 m or more, and may be 1.0×10 -8m or less or 1.0×10 -9 m or less.

[0015] The radiosensitizer contains a fullerene. The carbon number of the fullerene may be 60 or more, and may be 90 or less, 84 or less, 76 or less, or 70 or less. In one embodiment, the fullerene is selected from the group consisting of C 60 Fullerene, C 70 Fullerene, C 72 Fullerene, C 76 Fullerene, C 82 Fullerene and C 84 fullerenes, preferably C 60 Fullerene and C 70 fullerene, more preferably C 60 It may be a fullerene.

[0016] The fullerene according to one embodiment of the present disclosure may have 10 or less, 7 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less substituents on a carbon atom, or may have 1 or more substituents; however, from the viewpoint of enhancing radiosensitizing ability, it is preferable that the number of substituents is small, and it is more preferable that the fullerene has no substituents.

[0017] The fullerene is modified with a polyether. The radiosensitizer has high radiosensitizing ability because the fullerene contained therein is modified with a polyether. The polyether may be physically adsorbed to the fullerene.

[0018] The polyether may be a linear or cyclic polyether. When the polyether is a linear polyether, the linear polyether may be branched or linear.

[0019] The polyether is preferably a polyalkylene oxide. The number of carbon atoms in the alkylene oxide group in the polyalkylene oxide may be 1 or more or 2 or more and 4 or less or 3 or less, and is preferably 2 or 3.

[0020] Examples of such polyalkylene oxides include homopolymers (so-called homopolymers) such as polymethylene oxide, polyethylene oxide, polypropylene oxide, and polybutylene oxide, and copolymers thereof. The copolymer may be a random copolymer or a block copolymer. Among these, block copolymers are preferred, and more specifically, copolymers having a polyethylene oxide block and a polypropylene oxide block, polyethylene oxide, and polypropylene oxide are preferred. As polyalkylene oxides, from the viewpoint of improving radiation sensitization ability by being more excellent in dispersibility in an aqueous dispersion medium, copolymers having a polyethylene oxide block and a polypropylene oxide block, and polyethylene oxide are preferred. As polyalkylene oxides, from the viewpoint of biocompatibility, polyethylene oxide is preferred, and from the viewpoint of higher hydrophobicity, higher affinity with fullerene, and further improved dispersibility, copolymers consisting of ethylene oxide and propylene oxide, and particularly block copolymers thereof, are preferred.

[0021] The copolymer having a polyethylene oxide block and a polypropylene oxide block may have a plurality of polyethylene oxide blocks or a plurality of polypropylene oxide blocks. Examples of the copolymer having a polyethylene oxide block and a polypropylene oxide block include a polyethylene oxide-polypropylene oxide-polyethylene oxide block copolymer and a polypropylene oxide-polyethylene oxide-polypropylene oxide block copolymer.

[0022] The polyethylene oxide-polypropylene oxide-polyethylene oxide block copolymer may be, for example, a compound represented by the following formula (1-1): The polypropylene oxide-polyethylene oxide-polypropylene oxide block copolymer may be, for example, a compound represented by the following formula (1-2): [ka]

[0023] In formula (1-1), a1, a2, b1 and b2 each independently represent an integer of 1 or more. In formula (1-2), c1, d1 and d2 each independently represent an integer of 1 or more. In formulas (1-1) and (1-2), "*" represents a hydrogen atom or a hydrocarbon group (e.g., an alkyl group, preferably an alkyl group having 1 to 4 carbon atoms) when the compound represented by formula (1-1) or (1-2) is linear, and represents that the bonds to which it is attached are bonded to each other to form a ring when the compound represented by formula (1-1) or (1-2) is cyclic.

[0024] In formula (1-1), a1+a2 may be, for example, 4 or more, 8 or more, 12 or more, 16 or more, or 20 or more. In addition, in formula (1-1), a1+a2 may be, for example, 500 or less, 300 or less, 200 or less, 100 or less, or 80 or less. In formula (1-1), b1+b2 may be, for example, 4 or more, 8 or more, 12 or more, 16 or more, or 20 or more. In addition, in formula (1-1), b1+b2 may be, for example, 400 or less, 200 or less, 100 or less, 80 or less, or 50 or less. In formula (1-1), (a1+a2) / (b1+b2) may be, for example, 0.02 or more, 0.1 or more, 0.2 or more, 0.5 or more, or 0.8 or more. In addition, in formula (1-1), (a1+a2) / (b1+b2) may be, for example, 50 or less, 10 or less, 5 or less, 2 or less, or 1.2 or less.

[0025] In formula (1-2), c1 may be, for example, 4 or more, 8 or more, 12 or more, 16 or more, or 20 or more. In addition, in formula (1-2), c1 may be, for example, 500 or less, 300 or less, 200 or less, 100 or less, or 80 or less. In formula (1-2), d1+d2 may be, for example, 4 or more, 8 or more, 12 or more, 16 or more, or 20 or more. In addition, in formula (1-2), d1+d2 may be, for example, 400 or less, 200 or less, 100 or less, 80 or less, or 50 or less. In formula (1-2), c1 / (d1+d2) may be, for example, 0.02 or more, 0.1 or more, 0.2 or more, 0.5 or more, or 0.8 or more. In formula (1-2), c1 / (d1+d2) may be, for example, 50 or less, 10 or less, 5 or less, 2 or less, or 1.2 or less.

[0026] The polyether may have a molecular weight distribution. The number average molecular weight Mn of the polyether may be, for example, 500 or more, 1000 or more, 1500 or more, 2000 or more, 5000 or more, or 8000 or more. The number average molecular weight Mn of the polyether may be, for example, 100000 or less, 50000 or less, 20000 or less, or 15000 or less.

[0027] The molecular weight distribution of the polyether (ratio of weight average molecular weight to number average molecular weight, Mw / Mn) is not particularly limited, and may be, for example, 5 or less, 3 or less, 2 or less, or 1.8 or less. The lower limit of the molecular weight distribution is 1, and the molecular weight distribution of the polyether according to the present disclosure may be 1 or more.

[0028] In the radiosensitizer, the mass ratio of polyether to fullerene (polyether / fullerene) is not particularly limited, and may be appropriately changed depending on the type, size, specific surface area, etc. of the fullerene and polyether.

[0029] In the radiosensitizer, the mass ratio of polyether to fullerene (polyether / fullerene) may be, for example, 1000 or less, 500 or less, 100 or less, or 30 or less. In the radiosensitizer, the mass ratio of polyether to fullerene (polyether / fullerene) may be, for example, 0.01 or more, 0.1 or more, 1 or more, or 3 or more.

[0030] In addition to the polyether-modified fullerene, the radiosensitizer may contain other components such as a dispersion medium, a buffer, and a pH adjuster. Examples of the buffer include sodium phosphate buffer, TRIS buffer, and HEPES buffer. Examples of the pH adjuster include hydrochloric acid, citric acid, sodium carbonate, sodium bicarbonate, and sodium hydroxide.

[0031] The dispersion medium may be any dispersion medium capable of dispersing fullerene modified with polyether. From the viewpoint of more suitably exerting the radiation sensitizing ability, the dispersion medium is preferably a polar solvent, and more preferably an aqueous dispersion medium (a dispersion medium containing water). Examples of polar solvents include water, alcohols (e.g., monools such as methanol, ethanol, and propanol, and polyols such as ethylene glycol, propylene glycol, and glycerin), ethers (e.g., tetrahydrofuran, dioxane, ethylene glycol monomethyl ether, and propylene glycol monomethyl ether), ketones (e.g., acetone, methyl ethyl ketone), and esters (ethyl acetate, ethylene glycol monomethyl ether acetate, and the like). The polar solvent may be a mixed solvent of these. The aqueous dispersion medium may further contain another dispersion medium other than water. Examples of the other dispersion medium include the polar solvents other than water described above.

[0032] The radiosensitizer may be, for example, a liquid formulation. When the radiosensitizer is a liquid formulation, for example, the radiosensitizer contains a fullerene modified with polyether and a dispersion medium.

[0033] When the radiosensitizer is a liquid formulation, the concentration of the polyether-modified fullerene in the liquid formulation is not particularly limited, and the fullerene concentration may be, for example, 0.001 g / L or more, 0.01 g / L or more, 0.1 g / L or more, or 0.2 g / L or more, and may be 50 g / L or less, 20 g / L or less, 10 g / L or less, or 8 g / L or less.

[0034] The radiosensitizer can be produced, for example, by a production method including a mixing step of mixing a fullerene and a polyether in a dispersion medium.

[0035] In the mixing step, the amount of polyether added to the dispersion medium may be, for example, 0.1 parts by mass or more, 1 part by mass or more, 10 parts by mass or more, or 100 parts by mass or more, relative to 100 parts by mass of fullerene. In addition, in the mixing step, the amount of polyether added to the dispersion medium may be, for example, 10,000 parts by mass or less, 5,000 parts by mass or less, or 2,000 parts by mass or less, relative to 100 parts by mass of fullerene.

[0036] In the mixing step, a mixed solution containing fullerene is obtained. The above production method may further include a concentration step of concentrating the mixed solution to obtain a concentrate, and an extraction step of adding a dispersion medium to the concentrate obtained in the concentration step, mixing the mixture, and then recovering a supernatant component by centrifugation. In this case, a radiosensitizer containing fullerene is obtained by the extraction step.

[0037] The subject to which the radiosensitizer is administered is not particularly limited, and may be a human or a non-human animal. The non-human animal may be, for example, a mammal such as a dog, a cat, a hamster, a mouse, or a rat. These subjects may be, for example, subjects suffering from solid cancer. The subject to which the radiosensitizer is administered may also be, for example, a cultured cell.

[0038] The radiosensitizer may be administered so that the maximum local concentration and the concentration at the site irradiated with radiation in the administered subject are 5 μM or less, 4 μM or less, 3.5 μM or less, or 3 μM or less in terms of fullerene concentration. When the maximum local concentration and the concentration at the site irradiated with radiation in the administered subject are below the above upper limit, cytotoxicity in cells present in sites other than the site irradiated with radiation (affected site) can be suppressed.

[0039] The radiosensitizer may be administered so that the concentration of the fullerene at the site of radiation exposure in the subject to which it is administered is 1 μM or more, 2 μM or more, 2.5 μM or more, or 3 μM or more. When the concentration of the radiosensitizer at the site of radiation exposure in the subject to which it is administered is equal to or more than the above lower limit, the radiosensitizer can more suitably sensitize radiation and can highly efficiently kill cells irradiated with radiation.

[0040] The radiosensitizer may be for example one for sensitizing radiation of 3 Gray or more, 4 Gray or more, 5 Gray or more, or 6 Gray or more, or may be one for sensitizing radiation of 100 Gray or less, 50 Gray or less, 20 Gray or less, 10 Gray or less, 9 Gray or less, 8 Gray or less, 7 Gray or less, or 6 Gray or less. These radiation doses may be, for example, the dose at the site where radiation is sensitized (the site where cell death is induced by radiation, the affected area) as a result of attenuation in the process of radiation irradiated to the subject passing through the living body.

[0041] The radiosensitizer has excellent radiosensitizing ability. For example, the radiosensitizer may have excellent radiosensitizing ability when compared to the cell survival rate when irradiated with the addition of the radiosensitizer in a radiation irradiation test on cultured cells (e.g., SCC-VII cells) without the addition of the radiosensitizer. Also, for example, the radiosensitizer may have excellent radiosensitizing ability when compared to the cell survival rate when irradiated with the addition of the radiosensitizer in a radiation irradiation test on cultured cells (e.g., SCC-VII cells) without the addition of the radiosensitizer, the cell survival rate when irradiated with the addition of the radiosensitizer is 80% or less, 60% or less, 50% or less, 40% or less, or 30% or less. For example, a radiosensitizer having excellent radiosensitizing ability may mean that in a radiation irradiation test using cultured cells (e.g., SCC-VII cells), when the cultured cells are added with a concentration of the radiosensitizer that reduces cell viability by less than 10% in the absence of radiation, the cell viability is less than 50% or less than 30% when irradiated with 4 Gray radiation, or less than 20% or less than 10% when irradiated with 6 Gray radiation. In these cases, the cell viability may be evaluated, for example, 3, 5, 10, or 30 days after radiation irradiation.

[0042] The second embodiment of the present invention is a method for increasing the sensitivity of an object to radiation by contacting the object to be irradiated with a polyether-modified fullerene. The polyether-modified fullerene is contacted with the object by adding the polyether-modified fullerene to the object. The object, radiation, and polyether-modified fullerene in the method according to the second embodiment can be those described in the radiosensitizer according to the first embodiment.

[0043] The time for which the polyether-modified fullerene is contacted with the target is not particularly limited, and may be, for example, 1 minute or more, 3 minutes or more, 6 minutes or more, 10 minutes or more, or 30 minutes or more, and may be 168 hours or less, 24 hours or less, 6 hours or less, or 2 hours or less.

[0044] The concentration of the polyether-modified fullerene contacted with the target is not particularly limited. The concentration of the polyether-modified fullerene contacted with the target's radiation-irradiated portion (e.g., cell population) may be, for example, 1 μM or more, 2 μM or more, 2.5 μM or more, or 3 μM or more, and may be 5 μM or less, 4 μM or less, 3.5 μM or less, or 3 μM or less, and may be 3 μM as an example. When the fullerene concentration is within the above range, the sensitivity to radiation can be highly efficiently increased while suppressing cytotoxicity in the absence of radiation irradiation. EXAMPLES

[0045] The present disclosure will be described more specifically below using examples, but the present disclosure is not limited to the following examples.

[0046] [Preparation Example 1: Preparation of polyether-modified fullerene] 2 mg of fullerene (C 60 , Sigma-Aldrich; 483036-1G), 20 mg of polyethylene oxide-polypropylene oxide-polyethylene oxide block copolymer (Pluronic L64, total number of ethylene oxide units: about 40, total number of propylene oxide units: about 45, Sigma-Aldrich; 435449-250ML, hereinafter also referred to as "polyether (A)"), and 2 mL of water were placed in a vial, stirred with a vortex mixer, and then irradiated with ultrasound for 30 minutes. Next, centrifugation was performed at 11 kG for 15 minutes, and the supernatant (about 80% of the sample) was collected. This supernatant was used as a measurement sample in the following test examples.

[0047] [Test Example 1: Evaluation of the effect of administration of polyether-modified fullerene on cell viability using the colony formation method] SCC-VII cells, a cell line derived from mouse squamous cell carcinoma, were seeded on a 60 mm diameter flat dish and incubated in 5 mL of αMEM medium containing 10% by volume of inactivated FBS at 37°C and 5% CO2 for 12 hours. 12 hours after seeding, the medium was removed and the cells were incubated with C modified with polyether (A). 60 2.5 mL of αMEM containing 10% by volume of inactivated FBS containing fullerene at a fullerene concentration of 1 μM, 3 μM, 5 μM, or 10 μM was added, and the mixture was incubated at 37° C. and 5% CO2 for 5 days. 60 Incubation was also carried out in the same manner for a group to which the same amount of polyether (A) was added instead of fullerene, and a group to which only the medium was added. After 5 days of incubation, the ratio of the number of colonies formed per number of cells seeded in the group to which only the medium was added was 1.0 to 1.0. 60 The cell viability was evaluated using the ratio of the number of colonies formed per number of cells seeded in the group to which fullerene was added or the group to which polyether (A) was added as an index. 60 Group with fullerene added at a fullerene concentration of 1 μM, group with polyether (A) modified C 60 The number of cells seeded in the groups containing 1 μM or 3 μM fullerene and the same amount of polyether (A) and the group containing only medium was 2 × 10 1 The number of cells seeded in the other groups was 4 × 10 1 The number was calculated as 1 / mL.

[0048] The results are shown in Figure 1. The results in Figure 1 are expressed as the average value of n=3, and the error bars indicate the standard deviation. According to Figure 1, the C modified with polyether (A) 60 It was revealed that fullerene does not cause a significant decrease in cell viability at a concentration of at least 3 μM or less. Based on this result, the effect of polyether (A)-modified C 60 The concentration of fullerene added was determined to be 3 μM.

[0049] [Test Example 2: Evaluation test of the radiosensitizing ability of polyether-modified fullerene by colony formation method] SCC-VII cells, a cell line derived from mouse squamous cell carcinoma, were seeded on a 60 mm diameter flat dish and incubated in 5 mL of αMEM medium containing 10% by volume of inactivated FBS at 37°C and 5% CO2 for 12 hours. 12 hours after seeding, the medium was removed and the cells were incubated with C modified with polyether (A). 60 2.5 mL of αMEM containing 10% by volume of inactivated FBS containing fullerene at a fullerene concentration of 1 μM, 3 μM, 5 μM, or 10 μM was added, and incubated for 1 hour under conditions of 37°C and 5% CO2. 60 The same incubation was carried out for the group to which the same amount of polyether (A) was added instead of fullerene, and the group to which only the medium was added. After 1 hour of incubation, the cells were irradiated with X-rays at 2, 4 or 6 grays using X-RAD225iR manufactured by PrecisionX-Ray Inc. In addition, a control group was also prepared that was not irradiated with X-rays (0 gray). These cells were further incubated for 5 days under conditions of 37°C and 5% CO2. After 5 days of incubation, the ratio of the number of colonies formed in the group to which only the medium was added per number of cells seeded, which was modified with polyether (A), to the number of colonies formed in the group to which only the medium was added, was 1.0 g / mL. 60 The cell survival rate was evaluated using the ratio of the number of colonies formed per number of cells seeded in the group to which fullerene or polyether (A) was added as an index. The number of cells seeded in the non-irradiated group and the group irradiated with 2 gray was 2 × 10 1 The number of cells seeded in the group irradiated with 4 Gray was 4 × 10 1 The number of cells seeded in the group irradiated with 6 Gray was 1 × 10 2 The number was calculated as 1 / mL.

[0050] The results are shown in Table 1. The results in Table 1 show the average value ± standard deviation for n=3. According to Table 1, the C modified with polyether (A) 60 In the group to which fullerene was added, a significant decrease in cell viability was observed depending on the radiation dose, compared to the group to which only medium was added and the group to which polyether (A) was added, and in particular, in the group irradiated with 6 gray, the cell viability decreased to less than 10%. From the above, it was revealed that the radiosensitizer containing polyether-modified fullerene has high radiosensitizing ability. [Table 1]

Claims

1. A radiosensitizer comprising a polyether-modified fullerene.

2. 2. The radiosensitizer of claim 1, wherein the polyether is a polyalkylene oxide.

3. 3. The radiosensitizer according to claim 2, wherein the alkylene oxide group in the polyalkylene oxide has 2 or 3 carbon atoms.

4. A method for increasing the sensitivity of an object to radiation by contacting the object with a fullerene modified with a polyether.

5. The method of claim 4 , wherein the polyether is a polyalkylene oxide.

6. The method according to claim 5 , wherein the alkylene oxide group in the polyalkylene oxide has 2 or 3 carbon atoms.

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