WO2007061036A1 - Contrast medium using fullerene derivative - Google Patents

Contrast medium using fullerene derivative Download PDF

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Publication number
WO2007061036A1
WO2007061036A1 PCT/JP2006/323406 JP2006323406W WO2007061036A1 WO 2007061036 A1 WO2007061036 A1 WO 2007061036A1 JP 2006323406 W JP2006323406 W JP 2006323406W WO 2007061036 A1 WO2007061036 A1 WO 2007061036A1
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fullerene
contrast agent
fullerene derivative
molecule
agent according
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PCT/JP2006/323406
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French (fr)
Japanese (ja)
Inventor
Masatoshi Yamada
Akira Masuda
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Nippon Kayaku Kabushiki Kaisha
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Priority to JP2007546496A priority Critical patent/JPWO2007061036A1/en
Publication of WO2007061036A1 publication Critical patent/WO2007061036A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C01B32/152—Fullerenes
    • C01B32/156—After-treatment

Definitions

  • the present invention relates to a contrast agent using a fullerene derivative or an aggregate thereof.
  • X-ray diagnostic methods including X-ray CT, ultrasonic diagnostic methods, and magnetic resonance imaging (MRI) diagnostic methods, which are important techniques for disease diagnosis and treatment.
  • MRI magnetic resonance imaging
  • Gadolinium (III) diethylenetriaminepentaacetic acid (Gd—DTP ⁇ ) is a relatively low molecular weight (molecular weight 538) contrast agent used in clinical practice.
  • the half-life in blood of Gd-DTPA is 20
  • the Gd-DTPA biological period (measurable in vivo) in humans is approximately 90 minutes, so use Gd-DT PA for MRI angiography in organs other than the brain. Has its limits.
  • Gd-DTPA is a small molecule that easily migrates from capillaries to tissues, resulting in a rapid decrease in the signal ratio of vascular Z tissue, making it difficult to accurately detect abnormalities and diseases.
  • Gd-DTPA is antigenic and therefore suitable for repeated administration to the same patient.
  • Contrast agents include natural polymers (eg, proteins and polysaccharides) having many metal chelate-forming sites such as DTPA, and synthetic polymers, which are higher than Gd-DTPA.
  • DTPA metal chelate-forming sites
  • synthetic polymers which are higher than Gd-DTPA.
  • Gd-DTPA metal chelate-forming sites
  • an agent that binds DTPA to serum albumin is immediately recognized by macrophages and is immediately recognized by albumin receptors on vascular endothelial cells. Therefore, it is difficult to extend the blood half-life significantly.
  • there is a risk of immunogenicity and toxicity and there is a limit to use for MRI.
  • contrast agents can be used as therapeutic agents (Patent Documents 1 and 2).
  • Power growth agents such as cell growth inhibitors are used, and should be applied to patients other than those suffering from cancer. There are many problems.
  • Fullerenes, porphyrin derivatives and the like are known to generate active oxygen such as singlet oxygen and superoxide-on when irradiated with visible light or the like.
  • Fullerene is a force physically stable that is a general term for C (carbon) clusters, and carbon containing pure carbon materials such as c and c and metals (or metal oxides) depending on the number of n. cluster
  • Non-patent Document 1 Fullerenes are insoluble in water and are difficult to administer to living bodies. For this reason, polyethylene glycol, polybulal alcohol, dextran, pullulan, starch are used to provide water solubility to enable in vivo administration.
  • Non-patent Documents 2 and 3 water-soluble polymers such as derivatives of these polymers to fullerenes.
  • fullerene derivatives to which water-soluble polymers are bound are considered.
  • the use as a contrast agent is known.
  • Patent Document 1 JP-T-8-501097
  • Patent Document 2 Special Publication 2005—No. 519861
  • Patent Document 3 Japanese Patent Laid-Open No. 9-235235
  • Patent Document 4 International Publication No. 2005Z095494 Pamphlet
  • Patent Document 1 Chemistry, 50 (6) (1995)
  • Non-Patent Document 2 BIO INDUSTRY, Vol. 14, No. 7, 30- 37 (1997)
  • Non-Patent Document 3 Matsumura et al., Cancer and Chemotherapy, Vol. 14, No. 3, 821-829 (1987) Disclosure of the Invention
  • the present invention relates to the following inventions (1) to (21):
  • a contrast agent comprising a fullerene derivative linked via a molecule
  • the contrast agent according to any one of 5);
  • Chelate-forming molecules are diethylenetriaminepentaacetic acid, ethylene bis (oxy —Ethylene-trimethyl) tetraacetic acid, ethylenediaminetetraacetic acid, 1, 4, 7, 10-tetraaza cyclododecane 1, 4, 7, 10-tetraacetic acid, 1, 4, 8, 11-tetraazacyclotetradeca
  • the water-soluble polymer is a polyethylene glycol having an inert group at one end and a reactive group at the other end or a reactive group at both ends, and a chelate-forming molecular force coordinated with a metal ion Gd
  • a contrast agent comprising an aggregate of the fullerene derivative according to any one of (1) to (12) above;
  • the contrast agent according to (13) above comprising an aggregate having an aggregate size of 20 to 400 nm.
  • the contrast agent of the present invention is suitable as a contrast agent for X-rays or MRI, and has an excellent effect that it accumulates in various tumors for a long time, has a long measurement time in vivo, and has low immunogenicity.
  • image contrast such as MRI can be clarified, and active oxygen can be generated by physical stimulation, preferably light irradiation or ultrasonic irradiation. Therefore, it can also be used as a photodynamic therapeutic agent, an ultrasonic mechanical therapeutic agent, etc. for cancer.
  • the contrast agent of the present invention comprises a molecule in which a water-soluble high molecule and a chelate-forming molecule coordinated with a metal ion or a magnetic substance are bound to a fullerene which may have a functional group in the molecule. It contains a fullerene derivative bonded through a linking molecule as necessary.
  • the linking molecule is not particularly limited, and is preferably an amino acid or an aminoalkylene urethane! /.
  • Amino acids mean amino acids and peptides with amide bonds. Particularly preferably, one basic amino acid such as lysine, S-2-aminoethylcysteine and the like can be mentioned.
  • Aminoalkyleneurethane means a linking molecule having an aminoethyleneurethane structure, an aminotrimethyleneurethane structure, etc., and those having an amino C16 alkylurethane structure such as aminoethyleneurethane are preferred.
  • the fullerene in the fullerene derivative used in the contrast agent of the present invention is not particularly limited. Those that generate active oxygen are preferred. For example, pure carbon material C fullerene with 60 carbon atoms, pure carbon material C fullerene with 70 carbon atoms, pure carbon material
  • Nanotube fullerenes various higher-order fullerenes, metal-encapsulated fullerenes, and the like.
  • the metal-encapsulated fullerene includes, for example, Mn, Fe, Co, Gd, Eu, Tb, Er, etc., in which 1 to 3 of these metals are contained.
  • the fullerene derivative used in the contrast agent of the present invention has a functional group in the molecule!
  • the functional group of fullerene that may be used include a carboxy group, an amino group, a hydroxyl group, a cyan group, and a thiol group.
  • the number of bonds is preferably 1-5, more preferably 1-2.
  • Particularly preferred is one carboxy group, and such fullerene is commercially available from a reagent company such as Science Laboratories, Inc., and may be used. Thus, it may be synthesized by the method described in the document “Tetrahedron Letters, Vol. 36, No. 38, 6843 (1995)”!
  • the water-soluble polymer in the fullerene derivative used in the contrast agent of the present invention is not particularly limited, and those capable of reacting with a linking molecule are preferred.
  • Nonionic water-soluble polymers such as polybulurpyrrolidone, dextran, pullulan, starch, hydroxyethyl starch, hydroxypropyl starch; modified products thereof; copolymers or complexes of these two or three components; hyaluron Examples include acid; chitosan; chitin derivatives, etc., and preferably 1 to 5 fullerene derivative molecules are contained.
  • polyethylene glycols that are nonionic water-soluble polymers having a molecular weight of 1,000-1,000,000 are preferred, and polyethylene glycols of 4,000,000-50,000 are particularly preferred.
  • these water-soluble polymers commercially available ones may be used, or they may be prepared and used by a method described in known literature or a method referring to known literature.
  • polyethylene glycols having an inert group at one end and a reactive group at the other end or a reactive group at both ends are particularly preferred. Examples of the inert group include a C1 C6 alkyl group, a benzyl group, and other commonly used protecting groups, and a C1-C6 alkyl group is preferable.
  • Examples of the C1-C6 alkyl group include a methyl group, an ethyl group, an n propyl group, an isopropyl group, an n butyl group, a sec butyl group, a tert-butyl group, an n pentyl group, and an n-hexyl group.
  • a methyl group is preferred because of its availability.
  • the reactive group is not particularly limited as long as it has reactivity with other molecules constituting the fullerene derivative, and examples thereof include a carboxy group, an amino group, a hydroxyl group, a cyano group, and a thiol group.
  • An amino group is more preferred, which is preferably a group having dehydration condensation reactivity such as a group, an amino group, or a hydroxyl group.
  • the bonding position of the reactive group is not particularly limited and is preferably located at the end of the water-soluble polymer.
  • a reactive group may be bonded through an alkylene group or the like. When using a water-soluble polymer that does not have a reactive group, it is necessary to introduce a reactive group.
  • Polyethylene glycol having a methyl group at one end and a trimethyleneamino group at the other end is particularly preferable as the polyethylene glycol.
  • the chelate-forming molecule in the fullerene derivative used in the contrast agent of the present invention is not particularly limited, and various chelate molecules known in the literature can be used.
  • the number of bonds in the fullerene derivative is 1 to: LO is preferred, especially 1 to 3 is preferred.
  • Examples of the chelate-forming molecule include diethylenetriaminepentaacetic acid, ethylenebis (oxyethylene-triethyl) tetraacetic acid, ethylenediaminetetraacetic acid, 1, 4, 7, 10-tetraazacyclododecane 1 , 4, 7, 10-tetraacetic acid, 1, 4, 8, 11-tetraazacyclotetradecane 1, 4, 8, 11-tetraacetic acid or derivatives thereof, and diethylenetriaminepentaacetic acid is preferred. .
  • the metal ion coordinated to the chelate-forming molecule in the fullerene derivative used in the contrast agent of the present invention is not particularly limited as long as it has a contrasting action.
  • atomic numbers 21-29, 42, 44, 56-71, Metal element force of 76, 82, 83 Cations and radioactive ions of the selected element can be mentioned, for example Gd, Fe, Mn, Bi, Pb or Ba cations or release
  • the emissive ions 51 Cr, 57 Co, 9G Y, 99m Tc or 111 In are preferred, especially Gd 3+ or Mn 2+ for MRI.
  • examples of the magnetic substance include magnetite, hematite, ferrite, and the like.
  • examples of the molecule to which the magnetic substance is bonded include molecules such as a polymer of alkoxysilane.
  • the fullerene derivative used in the contrast agent of the present invention preferably has water solubility that allows administration to a living body. Further, the fullerene derivative used in the present invention forms an aggregate of a certain size in an aqueous solvent, and this aggregate can also be used as a contrast agent. This aggregate is also included in the present invention. .
  • the aqueous solvent include water, water-acetonitrile and the like.
  • the particle size as an aggregate is preferably about 20 to 400 nm when measured by the light scattering method in consideration of the ease of migration and accumulation into tissues such as cancer and the migration to normal cells. About 30 to 200 nm is more preferable.
  • the fullerene derivative in the contrast agent of the present invention has various components, that is, fullerene which may have a functional group in the molecule, a water-soluble polymer, and chelate formation in which a metal ion is coordinated.
  • This is a compound obtained by combining a molecule, a molecule to which a magnetic substance is bound, or a linking molecule as necessary in combination, and the compound is also included in the present invention, and includes, for example, the above formulas (1) to (3) ) (M is preferably a metal ion, particularly Gd 3+ or Mn 2+ ).
  • polyclonal antibodies or monoclonal antibodies against tumor antigens such as enzymes, homologs of enzyme substrates, lectins, sugar chains recognized by lectins, adhesion molecules, folic acid, sialic acid, etc.
  • a fullerene derivative in which an antibody or an antibody fragment is bound to enhance tumor directivity and a contrast agent containing the fullerene derivative are also included in the present invention.
  • the method for producing a fullerene derivative contained in the contrast agent of the present invention includes, for example, fullerenes having a functional group in the molecule via a linking molecule, as shown in the following examples,
  • a method of further coordinating a metal ion for detection can be mentioned.
  • the fullerene derivative to be obtained is the bonding order, bonding mode, etc. of these constituent parts.
  • the structure can be changed as appropriate.
  • a linking molecule binds to fullerene, and a formed molecule in which a water-soluble polymer and a metal ion are coordinated or a molecule to which a magnetic substance is bonded may bind to the bonded linking molecule.
  • the binding molecule may be bound to the water-soluble polymer, and the linking molecule may be bound to the forming molecule in which the metal ion is coordinated or the molecule to which the magnetic substance is bound.
  • Examples of the reaction include known reactions that generate chemical bonds such as condensation reactions, addition reactions, and substitution reactions. Dehydration condensation reactions and substitution reactions that generate amide bonds and ester bonds are preferred. In particular, a dehydration condensation reaction that generates a carboxy group and an amino group amide bond or a substitution reaction that generates an acid anhydride and an amino group amide bond is preferred. Examples of the dehydration condensation reaction include ordinary peptide condensation reactions.
  • Examples of the dehydration condensation agent include dicyclohexylcarbodiimide and diisopropylcarbodiimide; carbodiimide such as 1-dimethylaminopropyl-3-ethylcarbodiimide, benzotriazole-1 — Phosphorus salts such as yltris (dimethylamino) phospho-hexafluorophosphate; diphenylphosphoryl azide and the like, and diisopropylcarbodiimide is preferred.
  • the amount of the dehydrating condensing agent used is 0.5 to: LO molar equivalent, preferably 1 to 2 molar equivalent with respect to the carboxy group. The reaction is carried out in the presence or absence of an additive.
  • the additive examples include N-hydroxysuccinimide, 1-hydroxybenzotriazole, 4-nitrophenol, and pentafluorophenol, and preferably 1-hydroxybase. Nzotriazole. When an additive is used, the amount used is about 0.5 to 10 molar equivalents, preferably about 1 to 2 molar equivalents relative to the carboxy group.
  • a fullerene having a functional group in its molecule is bonded to polyethylene glycol substituted with an amino group, and then chelated.
  • a method of coordinating a metal ion for detection after binding the forming molecule may be used.
  • the organic solvent used in these reactions is not particularly limited as long as the reaction proceeds.
  • aromatic hydrocarbons such as benzene, toluene, xylene, etc .
  • Halogenated hydrocarbons such as Zen
  • ethers such as jetyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether
  • -tolyls such as acetonitrile and propio-tolyl
  • dimethylformamide and dimethylacetamide Amides such as hexamethylphosphoric triamide; ureas such as N, N dimethylimidazolidinone; mixed solvents of these solvents, and the like.
  • Dimethylformamide is preferred for the reaction of the linking molecule and the water-soluble polymer or the reaction of the linking molecule and the chelate-forming molecule, and the reaction of the linking molecule and fullerene is preferred for bromobenzene.
  • the reaction temperature is 20 to 100 ° C, preferably 0 to 50 ° C, more preferably room temperature to 37 ° C, and the reaction time is 1 to 84 hours, preferably 24 to 72 hours. This reaction is preferably carried out in the dark.
  • the obtained reaction product can be isolated and purified by applying a separation means known per se, for example, concentration under reduced pressure, solvent extraction, crystallization, chromatography, dialysis, lyophilization and the like.
  • the contrast agent of the present invention containing the above-described fullerene derivative can be used as an aqueous solution of a fullerene derivative or a solution of a hydrous solvent in the same manner as a normal contrast agent.
  • it is preferably used as a contrast agent for MRI.
  • active oxygen is generated.
  • active oxygen is generated by light generated by sonoluminescence induced by ultrasonic irradiation, and can be applied to the treatment of cancer.
  • the active oxygen includes singlet oxygen 0), superoxide-on (O-), hydrogen peroxide (H 2 O), hydroxyradi
  • an ultraviolet region (220 to 380 nm) to a visible light region (380 to 780 nm), preferably 300 to 600 nm can be used.
  • the ultrasonic waves to be irradiated those having a frequency of about 100 KHz to 20 MHz, particularly about 0.5 to 3 MHz can be preferably used.
  • Irradiation is preferably performed at an output of about 0.1 to: L0 WattZcm 2 , particularly about 1 to 5 WattZcm 2 .
  • the irradiation time is a force that varies depending on the frequency used and the irradiation output, and is about 5 to 300 seconds, preferably about 30 to 120 seconds.In the case of pulse irradiation, the duty cycle is about 1 to 100%, preferably about 10%. is there.
  • the contrast agent of the present invention can be used for photodynamic therapy and ultrasonic mechanical therapy simultaneously with the diagnosis of cancer. Allows for medical treatment.
  • the contrast agent of the present invention can be in any dosage form such as an injection, a dispersant, a fluid agent, and a solid powder.
  • the contrast agent of the present invention is combined with various additives such as buffers, physiological saline, preservatives, distilled water for injection and the like that are generally used for injections. be able to.
  • the contrast agent of the present invention can be administered intravenously, intraarterially, intramuscularly, subcutaneously, intradermally, and the like.
  • the contrast agent of the present invention containing a fullerene derivative consisting of a water-soluble polymer or fullerene is accumulated in cancer tissue and inflamed tissue when administered to a living body, and at a higher concentration than in normal tissue. It stays in cancer tissue and inflammatory tissue for a long time.
  • the normal agent is more rapidly excreted in the present invention than in cancerous tissue or inflamed tissue.
  • the concentration of the contrast agent is significantly higher than that in normal tissues, and this contrast agent is specifically distributed at a high concentration in cancer tissues and inflamed tissues. Therefore, it can be used as a contrast agent for imaging a tumor site having a long measurement time in a living body that accumulates in various tumors for a long time.
  • water-soluble high molecules such as polyethylene glycols
  • the use of water-soluble high molecules such as polyethylene glycols as a constituent part of the fullerene derivative is also expected to reduce immunogenicity.
  • M dimethylform of polyethylene glycol (Me O— PEG— 0 (CH) NH, molecular weight 5000, manufactured by NOF Corporation) with 50 mM end group methyl group and other end trimethyleneamino group
  • the precipitate separated by filtration was dissolved in distilled water and passed through an anion exchange resin column (DEAE Toyopar 650M, OH type) and a cation exchange resin column (SP-Toyopearl 650M, H + type).
  • the eluate was lyophilized and ⁇ -Z— ⁇ Boc— L Lysine NH— (CH 2) O— PEG— OMe (Z— Lvs (Boc) — NH— (C
  • Diisopropyl ether was added to the reaction solution to obtain a precipitate.
  • Dissolve the precipitate in 50% aqueous acetonitrile pass through an anion exchange resin column, freeze-dry the eluate, and add ⁇ -fullerene with PEG bound to the carboxy group of fullerene via lysine as a linking molecule.
  • N dimethylformamide solution 1.5 mL of N-a-one (tert-butoxycarbo) N ⁇ N ( ⁇ - (2-Ci- ⁇ )-L-lysine, manufactured by Kokusan Kagaku) N, N dimethylforma 10 mL of the imide solution was added, and 1.5-fold molar amount of 1-hydroxybenzotriazole and N, N′-diisopropylcarpositimide were added, and the mixture was stirred at room temperature for 24 hours under light-shielding conditions.
  • Diisopropyl ether was added to the reaction solution to obtain a precipitate.
  • the precipitate separated by filtration is dissolved in distilled water, passed through an anion exchange and cation exchange resin columns, and the eluate is freeze-dried.
  • PEG is bound to the carboxy group of lysine a- Boc- ⁇ — (2— CI— ⁇ ) — L-lysine— NH— (CH) O— PEG— OMe (Boc— Lys (Cl— Z) — NH— (CH)
  • a 20-fold molar amount of DTP A anhydride was added to 5 mL of a tilformamide solution, and after reacting at room temperature for 72 hours, diisopropyl ether was added to obtain a precipitate.
  • the precipitate is redissolved in 10 mL of 50% acetonitrile solution containing 0.1 M sodium hydroxide and treated at room temperature for 24 hours to hydrolyze unreacted acid anhydride, followed by dialysis and lyophilization.
  • the ⁇ -amino group of lysine as the linking molecule is bound to the carboxy group of the fullerene, and PEG and DTPA is linked via a gin, 0 Ding of 0.22 8? Hachiji-PEG is obtained
  • the reaction solution is extracted with an equal volume of distilled water, passed through a cation exchange resin column, and the eluate is freeze-dried.
  • the PEG1 molecule has a hydroxyl group at the end.
  • the amino group of the trimethyleneamino group at one end Thus, 0.46 g of fullerene was obtained.
  • the solution was dissolved to a volume of 8 mL, and sodium chloride manganese was added to this solution to 8 mM and stirred at room temperature for 24 hours.
  • the reaction solution was gel-filtered with PD-10 to remove excess manganese ions, and the fragment represented by formula (2) in which manganese ions were coordinated to DTP AC -PEG.
  • the solution was dissolved to a volume of 8 mL, and sodium chloride manganese was added to this solution to 8 mM and stirred at room temperature for 24 hours.
  • the reaction solution was gel-filtered with PD-10 to remove excess manganese, and the flag represented by formula (3) in which manganese ions were coordinated to C-PEG DTPA.
  • the coordination number of manganese ions was measured for the fullerene derivatives of the present invention obtained in Examples 1 to 3. Using each of the compound solutions obtained in Examples 1 to 3, measurement was performed with an atomic absorption photometer AA-6800 (Shimadzu Corporation) using a 50 mM MES solution of manganese salt manganese as a standard substance. Set. The measurement results are shown in Table 1.
  • the fullerene derivatives of the present invention obtained in Examples 1 to 3 were measured for particle size by a light scattering method.
  • Each compound solution obtained in Examples 1 to 3 was diluted with a 50 mM MES solution to a final concentration of 1 mgZmL. This solution was measured with a light scattering measurement device DLS-7000 (Otsuka Electronics Co., Ltd.), and the measurement result is shown in FIG.
  • the fullerene derivatives of the present invention obtained in Examples 1 to 3 were evaluated for MRI contrast ability.
  • Each compound solution obtained in Examples 1 to 3 was diluted with a 50 mM MES solution to a final concentration of 500 M, M and 5 M.
  • a 50 mM MES solution was diluted with a 50 mM MES solution to a final concentration of 500 M, M and 5 M.
  • Using this solution as a negative standard for a 50 mM MES solution and a positive standard for a 500 / z M salt / manganese MES solution Measured with a gas resonance apparatus BioSpec (Bruker Biospin). The measurement results are shown in Table 2.
  • the solution was diluted with a 50 mM MES solution to a final concentration of 100 M, and 200 ⁇ L of this solution was dispensed into 48-well plates. This was mixed with 160 ⁇ L of 50 mM MES solution and 40 ⁇ L of viable cell count reagent SF, and using a high-power LED light irradiator Blue Phase (Ivoclar Vivadent), a probe with a diameter of 8 mm, Irradiated with 490nm blue visible light at 1. lWattZcm 2 for 4 minutes. After irradiation, the absorbance of the solution at 450 nm was measured with a spectrophotometer DU-650 (Beckman). The solution irradiated with no compound added was used as a control, and Figure 2 shows the amount of O-generated per minute.
  • a test similar to Test Example 4 was carried out by ultrasonic irradiation instead of light irradiation.
  • 2 mL of each compound solution obtained in Examples 1 to 3 diluted with 50 mM MES solution to a final concentration of 100 M, 1.6 mL of 50 mM MES solution and 400 / zL viable cell count
  • a total of 4 mL of the solution mixed with the reagent SF was dropped onto a 1 MHz ultrasonic probe (Ginsen) with a vinyl tape wrapped around the irradiated surface with a diameter of 32 mm.
  • the ultrasonic probe uses the signal generator WF1966 (ENF circuit design block) and the high-speed bipolar power supply HSA4101 (ENF circuit design block) to output a 1 MHz sine wave at the output of 3 WattZcm 2 in the burst mode (Mark50000 cycle, Space50000 cycle). Cycle) and ultrasonic irradiation was performed for 3 minutes. After irradiation, the absorbance of the solution at 45011111 was measured with a spectrophotometer 1; ⁇ -1200 (Shimadzu Corporation). Figure 3 shows the amount of O-generated per minute.
  • the fullerene derivative of the present invention can also be applied to ultrasonic mechanical therapy.
  • the fullerene derivative of the present invention obtained in Example 2 was evaluated for in vivo MRI imaging ability using tumor-bearing mice.
  • Tumor-bearing mice were excised and subsectioned from tumor colons that had been subcultured by BALBZc-nn (female, Charles Japan, Liver Co., Ltd.) under the influence of mouse colon cancer Colon26 cells (provided by the Cancer Chemotherapy Center, Cancer Society).
  • BALBZc-nn female, Charles Japan, Liver Co., Ltd.
  • mouse colon cancer Colon26 cells provided by the Cancer Chemotherapy Center, Cancer Society.
  • mice was transplanted subcutaneously into the right thigh of CDF1 mice (female, Nippon Charles' Riva Co., Ltd.).
  • Mice that formed a tumor with a diameter of about 5 to L Omm subcutaneously after transplantation were used as cancer-bearing mice in the experiments.
  • the contrast effect is a T1-weighted image using the same device and is shown in Figs. From FIG. 5 to FIG. 8 showing the measurement results before administration, 1 hour after administration, 6 hours after administration and 12 hours after administration of the fullerene derivative of the present invention, the fullerene derivative of the present invention is more than 12 hours after administration.
  • the contrast intensity around the tumor site shown in the tomographic image was enhanced, and no change was seen in the normal tissue on the opposite side, indicating that it is a cancer-specific contrast agent.
  • the contrast agent containing the fullerene derivative of the present invention is suitable as a contrast agent for MRI, accumulates specifically in cancer for a long time, has a long measurement time in vivo, and has a low immunogenicity. Therefore, the MRI image contrast can be clarified, and active oxygen can be generated by physical stimulation such as light irradiation and ultrasonic irradiation.
  • FIG. 1 shows the results of measuring the particle size of fullerene derivatives of Examples 1 to 3 by a light scattering method.
  • FIG. 2 shows the results of measuring the amount of active oxygen generated by light irradiation of the fullerene derivatives of Examples 1 to 3.
  • FIG. 3 shows the results of measuring the amount of active oxygen generated by ultrasonic irradiation of the fullerene derivatives of Examples 1 to 3.
  • FIG. 4 shows a T2-weighted image of the tumor site of a tumor-bearing mouse measured with a biological nuclear magnetic resonance apparatus BioSpec (Bruker Biospin).
  • FIG. 5 shows a T1-weighted image of a tumor site of a tumor-bearing mouse measured with a biological nuclear magnetic resonance apparatus BioSpec (Bruker Biospin).
  • FIG. 6 shows a T1-weighted image of the tumor site 1 hour after administration of the fullerene derivative of Example 2.
  • FIG. 7 shows a T1-weighted image of the tumor site 6 hours after administration of the fullerene derivative of Example 2.
  • FIG. 8 shows a Tl-weighted image of the tumor site 12 hours after administration of the fullerene derivative of Example 2.

Abstract

Disclosed is a contrast medium comprising a fullerene derivative. The fullerene derivative has a water-soluble polymer and a chelating molecule having a metal ion coordinated therein or a molecule bound to a magnetic material both attached to a fullerene optionally via a linker molecule, wherein the fullerene may have a functional group in the molecule. The contrast medium is suitable as a contrast medium for MRI and has such excellent properties that it can be accumulated for a long period in a cancer-specific manner, shows a prolonged determination time in a living body, and has a low immunogenicity.

Description

明 細 書  Specification
フラーレン誘導体を用いた造影剤  Contrast media using fullerene derivatives
技術分野  Technical field
[0001] 本発明は、フラーレン誘導体またはその凝集体を用いる造影剤に関する。  [0001] The present invention relates to a contrast agent using a fullerene derivative or an aggregate thereof.
背景技術  Background art
[0002] 患者の身体における異常を的確に検出することは、疾病の診断と適切な治療のた めの必要条件である。そのために、例えば、 X線 CT法を含む X線診断法、超音波診 断法や磁気共鳴画像 (MRI)診断法があり、これらは疾病の診断と治療にとって重要 な手法である。中でも MRIは組織非侵襲性であり、放射線による術者及び患者の被 曝もない特徴を有する。  [0002] Accurate detection of abnormalities in a patient's body is a necessary condition for diagnosis and appropriate treatment of diseases. To that end, there are, for example, X-ray diagnostic methods including X-ray CT, ultrasonic diagnostic methods, and magnetic resonance imaging (MRI) diagnostic methods, which are important techniques for disease diagnosis and treatment. Among these, MRI is non-tissue invasive and has the characteristics that neither the operator nor the patient is exposed to radiation.
MRIにおいては、正常と異常の区別、例えば、正常組織と腫瘍組織の区別は縦緩 和時間 Tl、或いは横緩和時間 Τ2の差に基づいて理論的には可能である。これらの 差は、示差シグナル強度を生じ、 MRIのコントラストとして現れる。し力しながら、病的 あるいは異常な組織でも正常組織と同程度のシグナル強度を持つことが多ぐ実際 上、造影剤を用いなければ正常組織と区別することは困難である。  In MRI, the distinction between normal and abnormal, for example, normal tissue and tumor tissue, is theoretically possible based on the difference in longitudinal relaxation time Tl or lateral relaxation time Τ2. These differences give rise to differential signal intensity and appear as MRI contrast. However, pathological or abnormal tissues often have a signal intensity comparable to that of normal tissues. In practice, it is difficult to distinguish them from normal tissues without using a contrast medium.
[0003] ガドリニウム (III)ジエチレントリァミンペンタ酢酸 (Gd— DTP Α)は臨床に用いられ ている比較的低分子（分子量 538)の造影剤である力 Gd—DTPAの血中の半減 期は 20分以下であり、ヒトにおける Gd— DTPAの生物学的期間（生体中で測定可 能な時間）は約 90分であるため、脳以外の器官において MRI血管造影に Gd—DT PAを使用することには限界がある。更に、 Gd— DTPAは低分子であるため容易に 毛細血管から組織に移行し、結果として血管 Z組織のシグナル比の急速な減少を招 き、異常及び疾病の正確な検出を困難にする。また、 Gd— DTPAは抗原性があるた め同一患者に対する反復投与に向 、て 、な 、。  [0003] Gadolinium (III) diethylenetriaminepentaacetic acid (Gd—DTP Α) is a relatively low molecular weight (molecular weight 538) contrast agent used in clinical practice. The half-life in blood of Gd-DTPA is 20 The Gd-DTPA biological period (measurable in vivo) in humans is approximately 90 minutes, so use Gd-DT PA for MRI angiography in organs other than the brain. Has its limits. In addition, Gd-DTPA is a small molecule that easily migrates from capillaries to tissues, resulting in a rapid decrease in the signal ratio of vascular Z tissue, making it difficult to accurately detect abnormalities and diseases. Gd-DTPA is antigenic and therefore suitable for repeated administration to the same patient.
[0004] 造影剤には、 DTPAのような金属キレート形成部位を多数に有する天然高分子 (例 えば、たんぱく質及び多糖類等)や合成高分子もあり、これらは Gd— DTPAと比較し て高い緩和性を持つ。し力しながら、例えば、 DTPAを血清アルブミンに結合した造 影剤はマクロファージによって認識されやすぐ血管内皮細胞上にアルブミン受容体 があるため、血中半減期を大幅に延長することは難しい。更に、免疫原性や毒性の 恐れもあり、 MRIへの使用には限度がある。 [0004] Contrast agents include natural polymers (eg, proteins and polysaccharides) having many metal chelate-forming sites such as DTPA, and synthetic polymers, which are higher than Gd-DTPA. Has relaxation. However, for example, an agent that binds DTPA to serum albumin is immediately recognized by macrophages and is immediately recognized by albumin receptors on vascular endothelial cells. Therefore, it is difficult to extend the blood half-life significantly. In addition, there is a risk of immunogenicity and toxicity, and there is a limit to use for MRI.
[0005] また、血中の半減期及び免疫原性に関する問題を克服するために、例えば、種々 の天然高分子または合成高分子により修飾されたィ匕合物も使われている。デキストラ ン、合成ポリアミノ酸、またはポリエチレングリコールがよく用いられる力 特に、ポリエ チレングリコール（PEG)またはそのモノメチルエーテル（MPEG)が使用されることが 冬、、。 PEGまたは MPEGを用いた医用画像診断用の造影剤が知られて 、る。  [0005] In order to overcome the problems relating to the half-life and immunogenicity in blood, for example, compounds modified with various natural polymers or synthetic polymers have been used. The power that dextran, synthetic polyamino acids, or polyethylene glycol are often used in particular, winter, when polyethylene glycol (PEG) or its monomethyl ether (MPEG) is used. Contrast agents for medical image diagnosis using PEG or MPEG are known.
[0006] 造影剤の中には治療剤として使用できる物もある（特許文献 1、 2)力 治療剤として 細胞増殖抑制剤等が用いられており、癌の罹患者以外を対象として施用することに は問題が多い。  [0006] Some contrast agents can be used as therapeutic agents (Patent Documents 1 and 2). Power growth agents such as cell growth inhibitors are used, and should be applied to patients other than those suffering from cancer. There are many problems.
[0007] フラーレン、ポルフィリン誘導体等は、可視光等を照射することにより一重項酸素や スーパーォキシドア-オン等の活性酸素を発生することが知られている。  [0007] Fullerenes, porphyrin derivatives and the like are known to generate active oxygen such as singlet oxygen and superoxide-on when irradiated with visible light or the like.
[0008] フラーレンは、 C (炭素）クラスターの総称である力 物理的に安定で、 nの数に応じ て c 、c 等の純炭素物質や金属 (若しくは金属酸ィ匕物)を内包した炭素クラスター [0008] Fullerene is a force physically stable that is a general term for C (carbon) clusters, and carbon containing pure carbon materials such as c and c and metals (or metal oxides) depending on the number of n. cluster
60 70 60 70
等の化合物がある（非特許文献 1)。フラーレンは水不溶性であるため生体内への投 与が困難である。このため生体内への投与を可能とするべく水溶性を付与するため に、ポリエチレングリコール、ポリビュルアルコール、デキストラン、プルラン、デンプン (Non-patent Document 1). Fullerenes are insoluble in water and are difficult to administer to living bodies. For this reason, polyethylene glycol, polybulal alcohol, dextran, pullulan, starch are used to provide water solubility to enable in vivo administration.
、及びこれらの高分子の誘導体等の水溶性高分子をフラーレンと結合させることが検 討されている (非特許文献 2、特許文献 3)が、水溶性高分子を結合したフラーレン誘 導体につ 、て造影剤としての用途は知られて ヽな 、。 In addition, it has been studied to bind water-soluble polymers such as derivatives of these polymers to fullerenes (Non-patent Documents 2 and 3). However, fullerene derivatives to which water-soluble polymers are bound are considered. The use as a contrast agent is known.
また、フラーレンに 1分子の高分子を結合させた分子は凝集体を形成しやすいため 、正常組織に比べて癌組織に移行しやすぐまた、がん組織に長く滞留する傾向が あるという EPR効果（Enhanced Permeation and Retention Effect:非特許 文献 3)が期待される (特許文献 4)。  In addition, since a molecule in which one molecule of polymer is bound to fullerene tends to form an aggregate, the EPR effect is that it tends to stay in the cancer tissue as soon as it migrates to the cancer tissue compared to the normal tissue. (Enhanced Permeation and Retention Effect: Non-Patent Document 3) is expected (Patent Document 4).
[0009] 特許文献 1 :特表平 8— 501097号公報 [0009] Patent Document 1: JP-T-8-501097
特許文献 2：特表 2005— 519861号公報  Patent Document 2: Special Publication 2005—No. 519861
特許文献 3：特開平 9 - 235235号公報  Patent Document 3: Japanese Patent Laid-Open No. 9-235235
特許文献 4:国際公開第 2005Z095494号パンフレット 非特許文献 1 :化学， 50 (6) (1995) Patent Document 4: International Publication No. 2005Z095494 Pamphlet Non-Patent Document 1: Chemistry, 50 (6) (1995)
非特許文献 2 : BIO INDUSTRY, Vol. 14, No. 7, 30- 37 (1997)  Non-Patent Document 2: BIO INDUSTRY, Vol. 14, No. 7, 30- 37 (1997)
非特許文献 3 :松村ら、癌と化学療法、 Vol. 14, No. 3, 821 -829 (1987) 発明の開示  Non-Patent Document 3: Matsumura et al., Cancer and Chemotherapy, Vol. 14, No. 3, 821-829 (1987) Disclosure of the Invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0010] 臨床現場では各種の腫瘍に長時間蓄積するような生物学的期間が長ぐ免疫原性 が低 、造影剤が求められて 、る。 [0010] In the clinical field, there is a need for a contrast agent that has a long biological period of accumulation in various tumors and a low immunogenicity.
課題を解決するための手段  Means for solving the problem
[0011] 上記課題を解決するため、本発明者等は鋭意検討した結果、本発明を完成するに 至った。 [0011] In order to solve the above problems, the present inventors have intensively studied, and as a result, the present invention has been completed.
即ち、本発明は、以下の（1)から（21)の発明に関する：  That is, the present invention relates to the following inventions (1) to (21):
(1) 分子中に官能基を有していてもよいフラーレンに、水溶性高分子と、金属ィォ ンの配位したキレート形成分子、または磁性体の結合した分子を、必要に応じて連結 分子を介して結合させたフラーレン誘導体を含有する造影剤；  (1) Connect a water-soluble polymer and a chelate-forming molecule coordinated with a metal ion or a molecule with a magnetic substance to fullerene, which may have a functional group in the molecule, as necessary. A contrast agent comprising a fullerene derivative linked via a molecule;
(2) 連結分子がアミノ酸類またはアミノアルキレンウレタン類であるフラーレン誘導 体を含有する上記（1)記載の造影剤；  (2) The contrast agent according to (1) above, which contains a fullerene derivative in which the linking molecule is an amino acid or an aminoalkylene urethane;
(3) アミノ酸類が塩基性アミノ酸であるフラーレン誘導体を含有する上記（2)記載 の造影剤；  (3) The contrast agent according to (2), wherein the amino acid contains a fullerene derivative, which is a basic amino acid;
(4) フラーレンの官能基の数が 0〜5個であるフラーレン誘導体を含有する上記（ 1)から（3)の 、ずれか一項に記載の造影剤；  (4) The contrast agent according to any one of (1) to (3) above, which contains a fullerene derivative in which the number of functional groups of fullerene is 0 to 5;
(5) フラーレンの官能基の数が 1個で、官能基がカルボキシ基であるフラーレン誘 導体を含有する上記 (4)記載の造影剤；  (5) The contrast agent according to (4) above, which contains a fullerene derivative in which the number of functional groups of fullerene is one and the functional group is a carboxy group;
(6) フラーレンが C フラーレンであるフラーレン誘導体を含有する上記（1)から（  (6) From the above (1), which contains a fullerene derivative in which fullerene is C fullerene
60  60
5)の 、ずれか一項に記載の造影剤；  The contrast agent according to any one of 5);
(7) 水 ' 14高 チカ 000〜1, 000, 000のポジエチレングジ 一ノレ であるフラーレン誘導体を含有する上記（1)から（6)の ヽずれか一項に記載の造影 剤；  (7) Contrast agent according to any one of (1) to (6) above, which contains a fullerene derivative that is a water '14 high chica 000 to 1,000,000 positive ethylene guji monore;
(8) キレート形成分子がジエチレントリァミンペンタ酢酸、エチレン ビス（ォキシ —エチレン-トリ口)テトラ酢酸、エチレンジアミンテトラ酢酸、 1, 4, 7, 10—テトラァザ シクロドデカン一 1, 4, 7, 10—テトラ酢酸、 1, 4, 8, 11—テトラァザシクロテトラデカ ンー 1, 4, 8, 11ーテトラ酢酸またはその誘導体であるフラーレン誘導体を含有する 上記（1)から（7)の 、ずれか一項に記載の造影剤； (8) Chelate-forming molecules are diethylenetriaminepentaacetic acid, ethylene bis (oxy —Ethylene-trimethyl) tetraacetic acid, ethylenediaminetetraacetic acid, 1, 4, 7, 10-tetraaza cyclododecane 1, 4, 7, 10-tetraacetic acid, 1, 4, 8, 11-tetraazacyclotetradeca The contrast agent according to any one of (1) to (7) above, comprising a fullerene derivative which is N-1, 4, 8, 11-tetraacetic acid or a derivative thereof;
(9) キレート形成分子に配位した金属イオンが原子番号 21— 29、 42、 44、 56— 71、 76、 82、 83の金属元素力も選択される元素の陽イオンまたは放射性のイオンで あるフラーレン誘導体を含有する上記（1)から（8)の ヽずれか一項に記載の造影剤； (9) Fullerenes in which the metal ion coordinated to the chelate-forming molecule is a cation or radioactive ion of an element with a metal element force of atomic number 21—29, 42, 44, 56—71, 76, 82, 83 also selected The contrast agent according to any one of (1) to (8) above, which contains a derivative;
(10) 磁性体がフェライトであるフラーレン誘導体を含有する上記（1)力も (7)の ヽ ずれか一項に記載の造影剤； (10) The contrast agent according to any one of (7) and (7), wherein the magnetic substance contains a fullerene derivative that is ferrite.
(11) 水溶性高分子が、片末端に不活性基を有し他端に反応性基または両端に 反応性基を有するポリエチレングリコール類であり、金属イオンの配位したキレート形 成分子力 Gd3+または Mn2が配位したエチレントリァミンペンタ酢酸であるフラーレ ン誘導体を含有する上記（1)力 (9)の 、ずれか一項に記載の造影剤； (11) The water-soluble polymer is a polyethylene glycol having an inert group at one end and a reactive group at the other end or a reactive group at both ends, and a chelate-forming molecular force coordinated with a metal ion Gd The contrast agent according to any one of the above (1) force (9), which contains a fullerene derivative that is ethylenetriaminepentaacetic acid coordinated with 3+ or Mn 2 ;
(12) 物理的刺激により活性酸素を発生するフラーレン誘導体を含有する上記（1 )から（11)の 、ずれか一項に記載の造影剤；  (12) The contrast agent according to any one of (1) to (11) above, which contains a fullerene derivative that generates active oxygen by physical stimulation;
(13) 上記（ 1)から（ 12)の 、ずれか一項に記載のフラーレン誘導体の凝集体を 含有する造影剤；  (13) A contrast agent comprising an aggregate of the fullerene derivative according to any one of (1) to (12) above;
(14) 凝集体の大きさが 20〜400nmである凝集体を含有する上記（13)の造影 剤。  (14) The contrast agent according to (13) above, comprising an aggregate having an aggregate size of 20 to 400 nm.
(15) 造影剤が MRI用である上記（1)から（14)の 、ずれか一項に記載の造影剤  (15) The contrast agent according to any one of (1) to (14), wherein the contrast agent is for MRI.
(16) 造影部位が腫瘍部位である上記（1)から（15)の 、ずれか一項に記載の造 影剤； (16) The agent according to any one of (1) to (15) above, wherein the contrast-enhanced site is a tumor site;
(17) 上記（ 1)から（ 12)の 、ずれか一項に記載のフラーレン誘導体；  (17) The fullerene derivative according to any one of (1) to (12) above;
(18) 下記式（1)で表される上記（17)に記載のフラーレン誘導体；  (18) The fullerene derivative according to the above (17) represented by the following formula (1);
[化 1] [Chemical 1]
Figure imgf000007_0001
Figure imgf000007_0001
M  M
[式中、 Mは金属イオンを示す] [Wherein M represents a metal ion]
(19) 下記式（2)で表される上記（17)に記載のフラーレン誘導体; [化 2]  (19) The fullerene derivative according to the above (17) represented by the following formula (2);
Figure imgf000007_0002
Figure imgf000007_0002
M  M
[式中、 Mは金属イオンを示す] [Wherein M represents a metal ion]
(20) 下記式（3)で表される上記（17)に記載のフラーレン誘導体; [化 3] (20) The fullerene derivative according to the above (17) represented by the following formula (3);
Figure imgf000008_0001
Figure imgf000008_0001
M  M
[式中、 Mは金属イオンを示す]、及び [Wherein M represents a metal ion], and
[0013] (21) 上記（17)から（20)のいずれか一項に記載のフラーレン誘導体の凝集体。 [0013] (21) An aggregate of the fullerene derivative according to any one of (17) to (20) above.
発明の効果  The invention's effect
[0014] 本発明の造影剤は、 X線や MRIの造影剤として適し、各種の腫瘍に長時間蓄積し 、生体内での測定時間が長ぐ免疫原性が低いという優れた効果を有するもので、特 に、 MRI等の画像コントラストを明瞭にすることができ、更に、物理刺激、好ましくは、 光照射または超音波照射等により活性酸素を発生させることができる。そのために、 癌の光線力学的治療剤、超音波力学的治療剤等としても用いることができる。  [0014] The contrast agent of the present invention is suitable as a contrast agent for X-rays or MRI, and has an excellent effect that it accumulates in various tumors for a long time, has a long measurement time in vivo, and has low immunogenicity. In particular, image contrast such as MRI can be clarified, and active oxygen can be generated by physical stimulation, preferably light irradiation or ultrasonic irradiation. Therefore, it can also be used as a photodynamic therapeutic agent, an ultrasonic mechanical therapeutic agent, etc. for cancer.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0015] 本発明の造影剤は、分子中に官能基を有していてもよいフラーレンに、水溶性高 分子と、金属イオンの配位したキレート形成分子、または磁性体の結合した分子を、 必要に応じて連結分子を介して結合させたフラーレン誘導体を含有する。  [0015] The contrast agent of the present invention comprises a molecule in which a water-soluble high molecule and a chelate-forming molecule coordinated with a metal ion or a magnetic substance are bound to a fullerene which may have a functional group in the molecule. It contains a fullerene derivative bonded through a linking molecule as necessary.
[0016] 連結分子を用いる場合、該連結分子は特に限定されるものではなぐアミノ酸類ま たはアミノアルキレンウレタン類が好まし！/、。  [0016] When a linking molecule is used, the linking molecule is not particularly limited, and is preferably an amino acid or an aminoalkylene urethane! /.
アミノ酸類とは、アミノ酸やそれがアミド結合したペプチドを意味する。特に好ましく は 1個の塩基性アミノ酸、例えば、リジン、 S— 2—アミノエチルシスティン等が挙げら れる。  Amino acids mean amino acids and peptides with amide bonds. Particularly preferably, one basic amino acid such as lysine, S-2-aminoethylcysteine and the like can be mentioned.
アミノアルキレンウレタン類とはアミノエチレンウレタン構造、アミノトリメチレンウレタ ン構造等を有する連結分子を意味し、アミノエチレンウレタンなどのアミノ C1 6アル キルウレタン構造を有するものが好まし 、。  Aminoalkyleneurethane means a linking molecule having an aminoethyleneurethane structure, an aminotrimethyleneurethane structure, etc., and those having an amino C16 alkylurethane structure such as aminoethyleneurethane are preferred.
[0017] 本発明の造影剤に用いるフラーレン誘導体におけるフラーレンは特に限定されるも のではなぐ活性酸素を発生するものが好ましい。例えば、炭素原子数が 60の純炭 素物質 C フラーレン、炭素原子数が 70の純炭素物質 C フラーレン、純炭素物質 The fullerene in the fullerene derivative used in the contrast agent of the present invention is not particularly limited. Those that generate active oxygen are preferred. For example, pure carbon material C fullerene with 60 carbon atoms, pure carbon material C fullerene with 70 carbon atoms, pure carbon material
60 70  60 70
であるナノチューブフラーレン、各種高次フラーレン、金属内包フラーレン等が挙げら れる。該金属内包フラーレンとしては、内包される金属が、例えば、 Mn、 Fe、 Co、 G d、 Eu、 Tb、 Er等でこれらが 1〜3個含まれるものが挙げられる。  Nanotube fullerenes, various higher-order fullerenes, metal-encapsulated fullerenes, and the like. The metal-encapsulated fullerene includes, for example, Mn, Fe, Co, Gd, Eu, Tb, Er, etc., in which 1 to 3 of these metals are contained.
これらのフラーレンとしては市販されているものでもよぐ例えば、本荘ケミカル (株） 、三菱商事 (株)、東京化成工業 (株)等力も入手することができる (商品名： C フラー  These fullerenes may be commercially available. For example, Honjo Chemical Co., Ltd., Mitsubishi Corporation, Tokyo Chemical Industry Co., Ltd., etc. are also available (trade name: C Fuller
60 レン、 C フラーレン、マノレチウォーノレナノチューブ、シングノレウォーノレナノチューブ 60 Len, C Fullerene, Mano-Wolwano nanotube, Singo-Wo wallo nanotube
70 70
等)でもよい。中でも、供給及び取り扱いの容易さの点力も C フラーレンが特に好ま  Etc.). Above all, C fullerene is especially preferred for its ease of supply and handling.
60  60
しい。  That's right.
[0018] 本発明の造影剤に用いるフラーレン誘導体における分子中に官能基を有して！/、て もよいフラーレンの官能基としては、例えば、カルボキシ基、アミノ基、水酸基、シァノ 基、チオール基等が挙げられる。その結合数は 1〜5個が好ましぐ 1〜2個がより好 ましい。特に好ましくは 1個のカルボキシ基であり、このようなフラーレンは、例えば、（ 株)サイエンスラボラトリーズ等の試薬会社から市販されており、それを用いてもよぐ また、以後の参考例 1に示すように、文献「テトラへドロンレターズ， Vol. 36, No. 38 , 6843 (1995)」記載の方法にて合成して用いてもよ!、。  [0018] The fullerene derivative used in the contrast agent of the present invention has a functional group in the molecule! Examples of the functional group of fullerene that may be used include a carboxy group, an amino group, a hydroxyl group, a cyan group, and a thiol group. The number of bonds is preferably 1-5, more preferably 1-2. Particularly preferred is one carboxy group, and such fullerene is commercially available from a reagent company such as Science Laboratories, Inc., and may be used. Thus, it may be synthesized by the method described in the document “Tetrahedron Letters, Vol. 36, No. 38, 6843 (1995)”!
[0019] 本発明の造影剤に用いるフラーレン誘導体における水溶性高分子とは特に限定さ れるものではなぐ連結分子と反応できるものが好ましぐ例えば、ポリエチレングリコ ール、ポリプロピレングリコール、ポリビュルアルコール、ポリビュルピロリドン、デキス トラン、プルラン、デンプン、ヒドロキシェチルデンプン、ヒドロキシプロピルデンプン等 の非イオン性水溶性高分子；これらの修飾物；これらの 2成分若しくは 3成分の共重 合体または複合体；ヒアルロン酸；キトサン；キチン誘導体等が挙げられ、フラーレン 誘導体分子中に 1〜5個含有するのが好ましい。中でも、分子量が 1, 000-1, 000 , 000の非イオン性水溶性高分子であるポリエチレングリコール類が好ましぐ 4, 00 0-50, 000のポリエチレングリコール類が特に好ましい。これら水溶性高分子として は市販されているものを使用しても、公知文献に記載の方法または公知文献を参考 にした方法にて調製して使用してもよい。 [0020] 更に、ポリエチレングリコール類として片末端に不活性基を有し他端に反応性基ま たは両端に反応性基を有するものが殊に好ましい。不活性基としては、例えば、 C1 C6アルキル基、ベンジル基、その他通常保護基として使用されるものが挙げられ 、 C1— C6アルキル基が好ましい。 C1— C6アルキル基としては、例えば、メチル基、 ェチル基、 n プロピル基、イソプロピル基、 n ブチル基、 sec ブチル基、 tert— ブチル基、 n ペンチル基、 n—へキシル基等が挙げられ、入手の容易性からメチル 基が好ましい。反応性基とは、フラーレン誘導体を構成する他の分子との反応性があ れば特に限定されず、例えば、カルボキシ基、アミノ基、水酸基、シァノ基、チオール 基等が挙げられ、中でも、カルボキシ基、アミノ基、水酸基等の脱水縮合反応性を有 する基が好ましぐアミノ基がより好ましい。反応性基の結合位置は特に限定されず、 水溶性高分子の末端に位置するのが好ましい。また、反応性基がアルキレン基等を 介して結合して ヽてもよ ヽ。反応性基を有しな 、水溶性高分子を用いる場合には、 反応性基を導入する必要がある。 [0019] The water-soluble polymer in the fullerene derivative used in the contrast agent of the present invention is not particularly limited, and those capable of reacting with a linking molecule are preferred. For example, polyethylene glycol, polypropylene glycol, polybulal alcohol, Nonionic water-soluble polymers such as polybulurpyrrolidone, dextran, pullulan, starch, hydroxyethyl starch, hydroxypropyl starch; modified products thereof; copolymers or complexes of these two or three components; hyaluron Examples include acid; chitosan; chitin derivatives, etc., and preferably 1 to 5 fullerene derivative molecules are contained. Of these, polyethylene glycols that are nonionic water-soluble polymers having a molecular weight of 1,000-1,000,000 are preferred, and polyethylene glycols of 4,000,000-50,000 are particularly preferred. As these water-soluble polymers, commercially available ones may be used, or they may be prepared and used by a method described in known literature or a method referring to known literature. [0020] Furthermore, polyethylene glycols having an inert group at one end and a reactive group at the other end or a reactive group at both ends are particularly preferred. Examples of the inert group include a C1 C6 alkyl group, a benzyl group, and other commonly used protecting groups, and a C1-C6 alkyl group is preferable. Examples of the C1-C6 alkyl group include a methyl group, an ethyl group, an n propyl group, an isopropyl group, an n butyl group, a sec butyl group, a tert-butyl group, an n pentyl group, and an n-hexyl group. A methyl group is preferred because of its availability. The reactive group is not particularly limited as long as it has reactivity with other molecules constituting the fullerene derivative, and examples thereof include a carboxy group, an amino group, a hydroxyl group, a cyano group, and a thiol group. An amino group is more preferred, which is preferably a group having dehydration condensation reactivity such as a group, an amino group, or a hydroxyl group. The bonding position of the reactive group is not particularly limited and is preferably located at the end of the water-soluble polymer. In addition, a reactive group may be bonded through an alkylene group or the like. When using a water-soluble polymer that does not have a reactive group, it is necessary to introduce a reactive group.
ポリエチレングリコール類としては片末端メチル基で他端トリメチレンァミノ基のポリ エチレングリコールが特に好ましい。  Polyethylene glycol having a methyl group at one end and a trimethyleneamino group at the other end is particularly preferable as the polyethylene glycol.
[0021] 本発明の造影剤に用いるフラーレン誘導体におけるキレート形成分子としては特に 限定されるものではなぐ文献公知の各種キレート分子を使用することができる。フラ 一レン誘導体中におけるその結合数は 1〜： LO個が好ましぐ特に 1〜3個が好ましい [0021] The chelate-forming molecule in the fullerene derivative used in the contrast agent of the present invention is not particularly limited, and various chelate molecules known in the literature can be used. The number of bonds in the fullerene derivative is 1 to: LO is preferred, especially 1 to 3 is preferred.
[0022] 該キレート形成分子としては、例えば、ジエチレントリァミンペンタ酢酸、エチレン ビス（ォキシ一エチレン-トリ口）テトラ酢酸、エチレンジアミンテトラ酢酸、 1, 4, 7, 10 —テトラァザシクロドデカン一 1, 4, 7, 10—テトラ酢酸、あるいは 1, 4, 8, 11—テトラ ァザシクロテトラデカン 1, 4, 8, 11ーテトラ酢酸またはそれらの誘導体等が挙げら れ、ジエチレントリァミンペンタ酢酸が好ましい。 [0022] Examples of the chelate-forming molecule include diethylenetriaminepentaacetic acid, ethylenebis (oxyethylene-triethyl) tetraacetic acid, ethylenediaminetetraacetic acid, 1, 4, 7, 10-tetraazacyclododecane 1 , 4, 7, 10-tetraacetic acid, 1, 4, 8, 11-tetraazacyclotetradecane 1, 4, 8, 11-tetraacetic acid or derivatives thereof, and diethylenetriaminepentaacetic acid is preferred. .
[0023] 本発明の造影剤に用いるフラーレン誘導体におけるキレート形成分子に配位する 金属イオンとしては造影作用を有すれば特に限定されないが、原子番号 21— 29、 4 2、 44、 56— 71、 76、 82、 83の金属元素力 選択される元素の陽イオンや放射性 のイオンが挙げられ、例えば、 Gd、 Fe、 Mn、 Bi、 Pb若しくは Baの陽イオンまたは放 射性のイオンである51 Cr、 57Co、 9GY、 99mTc若しくは111 Inのイオンが好ましぐ特に M RI用として Gd3+または Mn2+が好まし 、。 [0023] The metal ion coordinated to the chelate-forming molecule in the fullerene derivative used in the contrast agent of the present invention is not particularly limited as long as it has a contrasting action. However, atomic numbers 21-29, 42, 44, 56-71, Metal element force of 76, 82, 83 Cations and radioactive ions of the selected element can be mentioned, for example Gd, Fe, Mn, Bi, Pb or Ba cations or release The emissive ions 51 Cr, 57 Co, 9G Y, 99m Tc or 111 In are preferred, especially Gd 3+ or Mn 2+ for MRI.
[0024] 本発明の造影剤のフラーレン誘導体にお!、て、磁性体の結合した分子を用いる場 合に、磁性体としては、例えば、マグネタイト、へマタイト、フェライト等が挙げられ、こ れらの磁性体が結合した分子としては、例えば、アルコキシシランの重合体等の分子 が挙げられる。 In the case of using a molecule to which a magnetic substance is bound in the fullerene derivative of the contrast agent of the present invention, examples of the magnetic substance include magnetite, hematite, ferrite, and the like. Examples of the molecule to which the magnetic substance is bonded include molecules such as a polymer of alkoxysilane.
[0025] 本発明の造影剤に用いるフラーレン誘導体は生体への投与が可能な程度の水溶 性を有しているのが好ましい。また、本発明で用いるフラーレン誘導体は水系溶媒中 では、一定の大きさの凝集体を形成し、この凝集体も造影剤として使用することが可 能であり、この凝集体も本発明に含まれる。水系溶媒としては、例えば、水、水ーァセ トニトリル等が挙げられる。凝集体としての粒径は、癌等の組織への移行と集積のし やすさ及び正常細胞への移行を考慮して、光散乱法による測定にぉ 、て 20〜400n m程度が好ましぐ 30〜200nm程度がより好ましい。  [0025] The fullerene derivative used in the contrast agent of the present invention preferably has water solubility that allows administration to a living body. Further, the fullerene derivative used in the present invention forms an aggregate of a certain size in an aqueous solvent, and this aggregate can also be used as a contrast agent. This aggregate is also included in the present invention. . Examples of the aqueous solvent include water, water-acetonitrile and the like. The particle size as an aggregate is preferably about 20 to 400 nm when measured by the light scattering method in consideration of the ease of migration and accumulation into tissues such as cancer and the migration to normal cells. About 30 to 200 nm is more preferable.
本発明の造影剤におけるフラーレン誘導体は、上記記載のとおり、各種構成部分、 すなわち、分子中に官能基を有していてもよいフラーレン、水溶性高分子、金属ィォ ンの配位したキレート形成分子、または磁性体の結合した分子、必要に応じて連結 分子を適宜組み合わせて結合して得られる化合物であり、該化合物も本発明に含ま れ、例えば、上記の式（1)〜式（3)で表される化合物（Mは金属イオン、特に Gd3+ま たは Mn2+が好まし 、）が挙げられる。 As described above, the fullerene derivative in the contrast agent of the present invention has various components, that is, fullerene which may have a functional group in the molecule, a water-soluble polymer, and chelate formation in which a metal ion is coordinated. This is a compound obtained by combining a molecule, a molecule to which a magnetic substance is bound, or a linking molecule as necessary in combination, and the compound is also included in the present invention, and includes, for example, the above formulas (1) to (3) ) (M is preferably a metal ion, particularly Gd 3+ or Mn 2+ ).
[0026] また、上記記載の各構成部分の他に、酵素、酵素基質のホモログ、レクチン、レクチ ンに認識される糖鎖、接着分子、葉酸、シアル酸等の腫瘍抗原に対するポリクローナ ル抗体若しくはモノクローナル抗体または抗体の断片を結合させ、腫瘍指向性を高 めたフラーレン誘導体及びそれを含有する造影剤も本発明に含まれる。  [0026] In addition to the constituent parts described above, polyclonal antibodies or monoclonal antibodies against tumor antigens such as enzymes, homologs of enzyme substrates, lectins, sugar chains recognized by lectins, adhesion molecules, folic acid, sialic acid, etc. A fullerene derivative in which an antibody or an antibody fragment is bound to enhance tumor directivity and a contrast agent containing the fullerene derivative are also included in the present invention.
[0027] 本発明の造影剤に含有されるフラーレン誘導体の製造方法は、例えば、後記の実 施例に示すように、連結分子を介して、分子中に官能基を有してもよいフラーレン、 水溶性高分子、キレート形成分子または磁性体の結合した分子を結合し、キレート形 成分子を用いた場合には、更に検出用金属イオンを配位させる方法が挙げられる。 この際、これらの構成部分の結合順序、結合態様等は得ようとするフラーレン誘導体 の構造により適宜変更することが可能である。例えば、フラーレンに連結分子が結合 し、この結合した連結分子に、水溶性高分子及び金属イオンが配位した形成分子、 または磁性体の結合した分子が結合してもよぐあるいは、フラーレンに水溶性高分 子が結合し、その水溶性高分子に連結分子が結合し、その連結分子に金属イオンが 配位した形成分子、または磁性体の結合した分子が結合してもよ ヽ。 [0027] The method for producing a fullerene derivative contained in the contrast agent of the present invention includes, for example, fullerenes having a functional group in the molecule via a linking molecule, as shown in the following examples, In the case where a water-soluble polymer, a chelate-forming molecule or a molecule to which a magnetic substance is bound is bound and a chelate-forming molecule is used, a method of further coordinating a metal ion for detection can be mentioned. In this case, the fullerene derivative to be obtained is the bonding order, bonding mode, etc. of these constituent parts. The structure can be changed as appropriate. For example, a linking molecule binds to fullerene, and a formed molecule in which a water-soluble polymer and a metal ion are coordinated or a molecule to which a magnetic substance is bonded may bind to the bonded linking molecule. The binding molecule may be bound to the water-soluble polymer, and the linking molecule may be bound to the forming molecule in which the metal ion is coordinated or the molecule to which the magnetic substance is bound.
[0028] 反応は、例えば、縮合反応、付加反応、置換反応等の化学結合を生成させる公知 の反応が挙げられ、アミド結合やエステル結合を生成させるような脱水縮合反応や置 換反応が好ましぐ特にカルボキシ基とアミノ基力 アミド結合を生成させる脱水縮合 反応あるいは酸無水物とアミノ基力 アミド結合を生成させる置換反応が好ま 、。 該脱水縮合反応としては通常のペプチド縮合反応が挙げられ、この際の脱水縮合剤 としては、ジシクロへキシルカルボジイミド、ジイソプロピルカルボジイミド； 1ージメチル ァミノプロピル— 3—ェチルカルボジイミド等のカルボジイミド、ベンゾトリァゾール— 1 —ィル一トリス（ジメチルァミノ）ホスホ-ゥムへキサフルォロホスフェート等のホスホ- ゥム塩；ジフエ-ルホスホリルアジド等が挙げられ、ジイソプロピルカルボジイミドが好 ましい。脱水縮合剤の使用量は、カルボキシ基に対して 0. 5〜： LOモル当量であり、 好ましくは 1〜2モル当量である。反応は、添加剤存在下または非存在下で行われ、 添加剤としては、 N—ヒドロキシスクシンイミド、 1—ヒドロキシベンゾトリァゾール、 4— ニトロフエノール、ペンタフルオロフェノール等が挙げられ、好ましくは 1ーヒドロキシべ ンゾトリアゾールである。添加剤を使用する場合、その使用量はカルボキシ基に対し て 0. 5〜10モル当量程度、好ましくは 1〜2モル当量程度である。  [0028] Examples of the reaction include known reactions that generate chemical bonds such as condensation reactions, addition reactions, and substitution reactions. Dehydration condensation reactions and substitution reactions that generate amide bonds and ester bonds are preferred. In particular, a dehydration condensation reaction that generates a carboxy group and an amino group amide bond or a substitution reaction that generates an acid anhydride and an amino group amide bond is preferred. Examples of the dehydration condensation reaction include ordinary peptide condensation reactions. Examples of the dehydration condensation agent include dicyclohexylcarbodiimide and diisopropylcarbodiimide; carbodiimide such as 1-dimethylaminopropyl-3-ethylcarbodiimide, benzotriazole-1 — Phosphorus salts such as yltris (dimethylamino) phospho-hexafluorophosphate; diphenylphosphoryl azide and the like, and diisopropylcarbodiimide is preferred. The amount of the dehydrating condensing agent used is 0.5 to: LO molar equivalent, preferably 1 to 2 molar equivalent with respect to the carboxy group. The reaction is carried out in the presence or absence of an additive. Examples of the additive include N-hydroxysuccinimide, 1-hydroxybenzotriazole, 4-nitrophenol, and pentafluorophenol, and preferably 1-hydroxybase. Nzotriazole. When an additive is used, the amount used is about 0.5 to 10 molar equivalents, preferably about 1 to 2 molar equivalents relative to the carboxy group.
酸無水物とアミノ基力 アミド結合を生成させる置換反応は通常のァミノ基の酸無水 物によるァシル化条件を適用すればよい。  For the substitution reaction for forming an amide bond with an acid anhydride and an amino group, the usual conditions for the acylation of an amino group with an acid anhydride may be applied.
[0029] また、特開 2002— 241307号公報に記載の方法に準じて、分子中に官能基を持 たな 、フラーレンとァミノ基が置換したポリエチレングリコールとを結合し、次 、でキレ ート形成分子を結合後、検出用金属イオンを配位させる方法でもよい。  [0029] Further, in accordance with the method described in JP-A-2002-241307, a fullerene having a functional group in its molecule is bonded to polyethylene glycol substituted with an amino group, and then chelated. A method of coordinating a metal ion for detection after binding the forming molecule may be used.
[0030] これらの反応で使用される有機溶媒としては、反応が進行する限り特に限定されず 、例えば、ベンゼン、トルエン、キシレン等の芳香族炭化水素類;塩化メチレン、クロ口 ホノレム、 1, 2—ジクロロエタン、クロ口ベンゼン-ブロモベンゼン- 1, 2—ジクロロベン ゼン等のハロゲン化炭化水素類；ジェチルエーテル、ジイソプロピルエーテル、テトラ ヒドロフラン、ジォキサン、ジメトキシェタン、ジエチレングリコールジメチルエーテル等 のエーテル類；ァセトニトリル、プロピオ-トリル等の-トリル類；ジメチルホルムアミド、 ジメチルァセトアミド、へキサメチルリン酸トリアミド等のアミド類; N, N ジメチルイミダ ゾリジノン等のウレァ類;これら溶媒の混合溶媒等が挙げられる。 [0030] The organic solvent used in these reactions is not particularly limited as long as the reaction proceeds. For example, aromatic hydrocarbons such as benzene, toluene, xylene, etc .; methylene chloride, black mouth honrem, 1, 2 —Dichloroethane, black-ended benzene-bromobenzene-1, 2-dichlorobenzene Halogenated hydrocarbons such as Zen; ethers such as jetyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; -tolyls such as acetonitrile and propio-tolyl; dimethylformamide and dimethylacetamide Amides such as hexamethylphosphoric triamide; ureas such as N, N dimethylimidazolidinone; mixed solvents of these solvents, and the like.
連結分子と水溶性高分子の反応または連結分子とキレート形成分子等の反応はジ メチルホルムアミドが好ましぐ連結分子とフラーレンの反応はブロモベンゼンが好ま しい。  Dimethylformamide is preferred for the reaction of the linking molecule and the water-soluble polymer or the reaction of the linking molecule and the chelate-forming molecule, and the reaction of the linking molecule and fullerene is preferred for bromobenzene.
[0031] 反応温度は、 20〜100°C、好ましくは 0〜50°C、より好ましくは室温〜 37°Cであ り、反応時間は 1〜84時間、好ましくは 24〜72時間である。本反応は遮光下で行う のが好ましい。得られた反応生成物は、自体公知の分離手段、例えば、減圧濃縮、 溶媒抽出、結晶化、クロマトグラフィー、透析、凍結乾燥等を適用して単離、精製する ことができる。  [0031] The reaction temperature is 20 to 100 ° C, preferably 0 to 50 ° C, more preferably room temperature to 37 ° C, and the reaction time is 1 to 84 hours, preferably 24 to 72 hours. This reaction is preferably carried out in the dark. The obtained reaction product can be isolated and purified by applying a separation means known per se, for example, concentration under reduced pressure, solvent extraction, crystallization, chromatography, dialysis, lyophilization and the like.
[0032] 上記したフラーレン誘導体を含有する本発明の造影剤は、フラーレン誘導体の水 溶液や含水溶媒の溶液として、通常の造影剤と同様に使用可能である。特に、 MRI 用造影剤としての使用が好ましい。この造影剤に光を照射すると活性酸素が発生し、 また、超音波照射により惹起されたソノルミネッセンスによって発生する光によっても 活性酸素が発生し、それによる癌の治療にも適用できる。該活性酸素には、一重項 酸素 0 )、スーパーォキシドア-オン (O―)、過酸化水素（H O )、ヒドロキシラジ  [0032] The contrast agent of the present invention containing the above-described fullerene derivative can be used as an aqueous solution of a fullerene derivative or a solution of a hydrous solvent in the same manner as a normal contrast agent. In particular, it is preferably used as a contrast agent for MRI. When this contrast agent is irradiated with light, active oxygen is generated. Also, active oxygen is generated by light generated by sonoluminescence induced by ultrasonic irradiation, and can be applied to the treatment of cancer. The active oxygen includes singlet oxygen 0), superoxide-on (O-), hydrogen peroxide (H 2 O), hydroxyradi
2 2 2 2 カル（·ΟΗ)等が含まれる。  2 2 2 2 Includes kar (· ΟΗ).
[0033] 照射する光の波長としては、紫外線領域（220〜380nm)〜可視光領域（380〜7 80nm)、好ましくは 300〜600nmを使用することができる。 [0033] As the wavelength of light to be irradiated, an ultraviolet region (220 to 380 nm) to a visible light region (380 to 780 nm), preferably 300 to 600 nm can be used.
照射する超音波としては、周波数約 100KHz〜20MHz、特に約 0. 5〜3MHzの ものを好ましく使用することができる。照射は、約 0. 1〜： L0WattZcm2、中でも約 1 〜5WattZcm2の出力で行うのが好ましい。照射時間は、用いる周波数、照射出力 によっても異なる力 約 5〜300秒、好ましくは約 30〜 120秒であり、パルス照射の場 合、その Dutycycleは約 1〜100%、好ましくは約 10%である。 As the ultrasonic waves to be irradiated, those having a frequency of about 100 KHz to 20 MHz, particularly about 0.5 to 3 MHz can be preferably used. Irradiation is preferably performed at an output of about 0.1 to: L0 WattZcm 2 , particularly about 1 to 5 WattZcm 2 . The irradiation time is a force that varies depending on the frequency used and the irradiation output, and is about 5 to 300 seconds, preferably about 30 to 120 seconds.In the case of pulse irradiation, the duty cycle is about 1 to 100%, preferably about 10%. is there.
これより本発明の造影剤は癌の診断と同時に光線力学的治療や超音波力学的治 療を可能とする。 Thus, the contrast agent of the present invention can be used for photodynamic therapy and ultrasonic mechanical therapy simultaneously with the diagnosis of cancer. Allows for medical treatment.
[0034] 本発明の造影剤は、注射剤、分散剤、流動性剤、固形粉末剤等のあらゆる剤型と することができる。例えば、注射剤とする場合には本発明の造影剤を、注射剤に一般 に用いられる緩衝剤、生理食塩水、保存剤、注射用蒸留水等の各種添加剤を配合 して注射剤とすることができる。本発明の造影剤は静脈内、動脈内、筋肉内、皮下、 皮内等に投与することができる。  [0034] The contrast agent of the present invention can be in any dosage form such as an injection, a dispersant, a fluid agent, and a solid powder. For example, in the case of an injection, the contrast agent of the present invention is combined with various additives such as buffers, physiological saline, preservatives, distilled water for injection and the like that are generally used for injections. be able to. The contrast agent of the present invention can be administered intravenously, intraarterially, intramuscularly, subcutaneously, intradermally, and the like.
[0035] 癌組織や炎症組織には、正常組織と比較して高分子物質が移行しやすぐ且つ、 蓄積しやすい。従って、水溶性高分子やフラーレンからなるフラーレン誘導体を含有 する本発明の造影剤は、生体に投与されると正常組織に比べて癌組織や炎症組織 に集積され、正常組織におけるよりも高濃度で長く癌組織や炎症組織に滞留する。 一方、正常組織においては癌組織や炎症組織におけるよりも速やかに本発明の造 影剤が***されるため、造影剤を生体に投与後、ある程度の時間をおけば、癌組織 や炎症組織における本造影剤の濃度は正常組織における濃度よりも有意に高いも のとなり、本造影剤が癌組織や炎症組織に特異的に高濃度で分布することになる。 従って、特に各種の腫瘍に長時間蓄積するような生体内で測定時間が長い腫瘍部 位を造影する造影剤とすることができる。  [0035] In a cancer tissue or an inflamed tissue, a polymer substance is easily transferred and easily accumulated compared to a normal tissue. Therefore, the contrast agent of the present invention containing a fullerene derivative consisting of a water-soluble polymer or fullerene is accumulated in cancer tissue and inflamed tissue when administered to a living body, and at a higher concentration than in normal tissue. It stays in cancer tissue and inflammatory tissue for a long time. On the other hand, the normal agent is more rapidly excreted in the present invention than in cancerous tissue or inflamed tissue. The concentration of the contrast agent is significantly higher than that in normal tissues, and this contrast agent is specifically distributed at a high concentration in cancer tissues and inflamed tissues. Therefore, it can be used as a contrast agent for imaging a tumor site having a long measurement time in a living body that accumulates in various tumors for a long time.
また、フラーレン誘導体の構成部分としてポリエチレングリコール類等の水溶性高 分子の使用により免疫原性の低下も予測される。  In addition, the use of water-soluble high molecules such as polyethylene glycols as a constituent part of the fullerene derivative is also expected to reduce immunogenicity.
[0036] 以下に、実施例、参考例及び試験例を示し本発明を更に詳細に説明するが、本発 明の範囲はこれらに限定されるものではない。  [0036] Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples and Test Examples, but the scope of the present invention is not limited thereto.
参考例 1  Reference example 1
( 1. 2 メタノ「601フラーレン） -61 -カルボン酸の合成  (1.2 Synthesis of methano-601 fullerene) -61-carboxylic acid
テトラへド、ロンレターズ， Vol. 36, No. 38, 6843 (1995)【こ記載の方法【こより得ら れる（1, 2—メタノ [60]フラーレン） 61—力ルボン酸 tert ブチルエステル（540m g, 0. 65mmol)をトルエン（380mL)に溶解し、 4 トルエンスルホン酸 1水和物（22 2mg, 1. 17mmol)を加えて 10時間加熱還流した。析出した褐色沈殿物をろ取し、 トルエン、蒸留水及びエタノールにて順次洗净後、減圧下乾燥して（1, 2—メタノ [60 Tetrahedo, Ron Letters, Vol. 36, No. 38, 6843 (1995) [Method described here [obtained from (1, 2-methano [60] fullerene) 61-force rubonic acid tert butyl ester (540 mg) , 0.65 mmol) was dissolved in toluene (380 mL), 4 toluenesulfonic acid monohydrate (22 2 mg, 1.17 mmol) was added, and the mixture was heated to reflux for 10 hours. The precipitated brown precipitate is collected by filtration, washed successively with toluene, distilled water and ethanol, and dried under reduced pressure (1,2-methano [60
]フラーレン） 61—カルボン酸（338mg,収率 67%)を褐色結晶として得た。 FAB -MS (positive mode) : m/z 779 (M+H) + ; Fullerene) 61-carboxylic acid (338 mg, yield 67%) was obtained as brown crystals. FAB -MS (positive mode): m / z 779 (M + H) + ;
'H-NMR CCDCl /DMSO -d (1 : 1) , ppm) : 5. 15 (1H, s)  'H-NMR CCDCl / DMSO -d (1: 1), ppm): 5. 15 (1H, s)
3 6  3 6
[0037] 参考例 2  [0037] Reference Example 2
C DTPA— PEG の合成  C DTPA— PEG synthesis
—60 5000 ~ U-L^i —60 5000 ~ U - L ^ i
50mMの片末端メチル基で他端トリメチレンァミノ基のポリエチレングリコール (Me O— PEG— 0 (CH ) NH ,分子量 5000, 日本油脂製）の Ν, Ν ジメチルホルム  M, dimethylform of polyethylene glycol (Me O— PEG— 0 (CH) NH, molecular weight 5000, manufactured by NOF Corporation) with 50 mM end group methyl group and other end trimethyleneamino group
2 3 2  2 3 2
アミド溶液 20mLに 1. 5倍モル量の N— a - (ベンジルォキシカルボ-ル） N— ε — (tert ブトキシカルボ-ル）— L リジン（α— Ζ— ε — Boc— L リジン、国産化 学製）の N, N ジメチルホルムアミド溶液 10mLをカ卩え、 1. 5倍モル量の 1ーヒドロキ シベンゾトリアゾール及び N, N'—ジイソプロピルカルボジイミドを添カ卩し、室温、 24 時間、遮光条件下で攪拌した。反応液にジイソプロピルエーテルを添加し沈殿物を 得た。ろ別した沈殿物を蒸留水に溶解後、陰イオン交換榭脂カラム (DEAE トヨパ ール 650M, OH型）及び陽イオン交換榭脂カラム（SP—トヨパール 650M, H+型） に通塔した。溶出液を凍結乾燥し、リジンのカルボキシ基に PEGの結合した α—Z— ε Boc— L リジン NH—（CH ) O— PEG— OMe (Z— Lvs (Boc)— NH—（C  Amide solution in 20 mL 1.5-fold molar amount of N— a-(benzyloxycarbol) N— ε — (tert-butoxycarbole) — L lysine (α— Ζ— ε — Boc— L lysine, domestic production Add 10 mL of N, N dimethylformamide solution (chemical), and add 1.5-fold molar amount of 1-hydroxybenzotriazole and N, N'-diisopropylcarbodiimide at room temperature for 24 hours. Stir below. Diisopropyl ether was added to the reaction solution to obtain a precipitate. The precipitate separated by filtration was dissolved in distilled water and passed through an anion exchange resin column (DEAE Toyopar 650M, OH type) and a cation exchange resin column (SP-Toyopearl 650M, H + type). The eluate was lyophilized and α-Z— ε Boc— L Lysine NH— (CH 2) O— PEG— OMe (Z— Lvs (Boc) — NH— (C
2 3  twenty three
H ) O— PEG— OMe)を得た。  H) O-PEG-OMe) was obtained.
2 3  twenty three
[0038] 2gの Z— Lys (Boc)— NH— (CH ) O— PEG— OMeを 30mLの N, N ジメチル  [0038] 2 g of Z— Lys (Boc) — NH— (CH 2) 2 O— PEG— OMe 30 mL N, N dimethyl
2 3  twenty three
ホルムアミドに溶解し、 0. 4gの 5%パラジウム炭素（N. E. ChemCat製、 55%含水） を加え、水素雰囲気下、 40°C、 2時間攪拌することにより、接触加水素分解を行った 。反応液に活性炭を添加し、ハイフロースーパーセル（国産化学製)を用いてろ過後 、ジイソプロピルエーテルを添加し沈殿物を得た。沈殿物を 5%塩ィ匕ナトリウム水溶液 に溶解後、 HP— 20カラム（三菱ィ匕学製）に通塔した。 5%塩ィ匕ナトリウム水溶液及び 蒸留水で洗浄後、 50%及び 80%ァセトニトリル水溶液で溶出した溶出液を凍結乾 燥し、リジンのベンジルォキシカルボ-ル基が脱離し、カルボキシ基に PEGが結合し た ε — Boc— L リジン— NH— (CH ) O— PEG— OMe (Lvs (Boc)—NH— (C  After dissolving in formamide, 0.4 g of 5% palladium carbon (manufactured by NE ChemCat, containing 55% water) was added, and the mixture was stirred at 40 ° C. for 2 hours in a hydrogen atmosphere to perform catalytic hydrogenolysis. Activated carbon was added to the reaction solution, and after filtration using a high flow supercell (manufactured by Kokusan Kagaku), diisopropyl ether was added to obtain a precipitate. The precipitate was dissolved in a 5% aqueous sodium chloride solution and then passed through an HP-20 column (Mitsubishi Chemical). After washing with 5% sodium chloride aqueous solution and distilled water, the eluate eluted with 50% and 80% aqueous acetonitrile is lyophilized, the benzyloxycarbol group of lysine is eliminated, and PEG is added to the carboxy group. Bonded ε — Boc— L Lysine— NH— (CH) O— PEG— OMe (Lvs (Boc) —NH— (C
2 3  twenty three
H ) O— PEG— OMe)を 1. 42g得た。  1.42 g of H) O-PEG-OMe) was obtained.
2 3  twenty three
[0039] 0. 4gの Lys (Boc)— NH— (CH ) O— PEG— OMeのブロモベンゼン溶液 20m  [0039] 0.4 g Lys (Boc) — NH— (CH 2) 2 O— PEG — OMe in bromobenzene solution 20 m
2 3  twenty three
Lと 1. 6倍モル量の（1, 2—メタノ [60]フラーレン） 61—力ルボン酸を含むブロモ ベンゼン溶液 125mLを混和し、 1. 6倍モル量の 1ーヒドロキシベンゾトリアゾール及 び 3. 3倍モル量の N, N，—ジイソプロピルカルボジイミドを添カ卩し、室温、 72時間、 遮光条件下で攪拌した。反応液を等量の蒸留水にて抽出後、凍結乾燥した。この全 量を 10mLのジクロロメタンに溶解し、 3mLのトリフルォロ酢酸を添カ卩し、 4°C、 18時 間で Boc基の脱保護を行った。反応液にジイソプロピルエーテルを添加し沈殿物を 得た。沈殿物を 50%ァセトニトリル水溶液に溶解し、陰イオン交換榭脂カラム通塔後 、溶出液を凍結乾燥し、フラーレンのカルボキシ基に、連結分子としてリジンを介して PEGが結合した α—フラーレン一 L リジン一 NH— (CH ) O— PEG— OMe (C L and 1.6 times the molar amount of (1,2-methano [60] fullerene) 61-Bromo with strong rubonic acid Mix 125 mL of benzene solution, add 1.6-fold molar amount of 1-hydroxybenzotriazole and 3.3-fold molar amount of N, N, -diisopropylcarbodiimide, and keep at room temperature for 72 hours under light-shielded conditions. Stir. The reaction solution was extracted with an equal volume of distilled water and then lyophilized. The total amount was dissolved in 10 mL of dichloromethane, and 3 mL of trifluoroacetic acid was added thereto, and the Boc group was deprotected at 4 ° C for 18 hours. Diisopropyl ether was added to the reaction solution to obtain a precipitate. Dissolve the precipitate in 50% aqueous acetonitrile, pass through an anion exchange resin column, freeze-dry the eluate, and add α-fullerene with PEG bound to the carboxy group of fullerene via lysine as a linking molecule. Lysine I NH— (CH) O— PEG— OMe (C
2 3 60 2 3 60
> CHCO -Lys -NH - (CH ) O— PEG— OMe) 0. 33gを得た。 > CHCO -Lys -NH-(CH) O-PEG-OMe) 0.33 g was obtained.
2 3  twenty three
[0040] 5. 6mMの C > CHCO— Lys— NH— (CH ) O— PEG— OMeの N, N ジメ  [0040] 5. 6 mM C> CHCO— Lys— NH— (CH) O— PEG— OMe N, N
60 2 3  60 2 3
チルホルムアミド溶液 5mLに 20倍モル量のジエチレントリアミン— N, N, Ν ' , Ν", Ν" ペンタ酢酸無水物（DTPA無水物、和光純薬)を添加し、室温、 72時間反応後 、ジイソプロピルエーテルを添カ卩し沈殿物を得た。沈殿物は 10mLの 0. 1M水酸ィ匕 ナトリウムを含む 50%ァセトニトリル溶液に再溶解し、室温にて、 24時間処理すること により未反応の酸無水物を加水分解した後、透析、凍結乾燥を順次行い、フラーレ ンのカルボキシ基に、連結分子としてのリジンの ーァミノ基が結合し、そのリジンを 介して PEG及び DTPAが結合した、 0. 22gの C DTPA— PEG が得られた。  Diethyltriamine-N, N, Ν ', Ν ", Ν" pentaacetic anhydride (DTPA anhydride, Wako Pure Chemical Industries, Ltd.) was added to 5 mL of tilformamide solution and diisopropyl ether was reacted at room temperature for 72 hours. Was added to obtain a precipitate. The precipitate was redissolved in 10 mL of 50% acetonitrile solution containing 0.1 M sodium hydroxide and treated at room temperature for 24 hours to hydrolyze the unreacted acid anhydride, followed by dialysis and lyophilization. Thus, 0.22 g of C DTPA-PEG was obtained, in which the lysine amino group as a linking molecule was bound to the carboxy group of fullerene, and PEG and DTPA were bound via the lysine.
60 5000  60 5000
[0041] 参考例 3  [0041] Reference Example 3
DTP AC - PEG. の合成  Synthesis of DTP AC-PEG.
60 5000 ~  60 5000 ~
50mMの片末端メチル基で他端トリメチレンァミノ基の PEG (分子量 5000, 日本油 脂製）の N, N ジメチルホルムアミド溶液 20mLに 1. 5倍モル量の N— a一（tert— ブトキシカルボ-ル） N ε 一（2—クロ口べンジルォキシカルボ-ル） L リジン（ a - Boc - ε — (2— CI— Ζ)— L リジン、国産化学製）の N, N ジメチルホルムァ ミド溶液 10mLをカ卩え、 1. 5倍モル量の 1ーヒドロキシベンゾトリアゾール及び N, N ' —ジイソプロピルカルポジイミドを添加し、室温、 24時間、遮光条件下で攪拌した。反 応液にジイソプロピルエーテルを添加し沈殿物を得た。ろ別した沈殿物を蒸留水に 溶解し、陰イオン交換榭脂カラム及び陽イオン交換榭脂カラムに通塔後、溶出液を 凍結乾燥し、リジンのカルボキシ基に PEGが結合した a— Boc— ε — (2— CI— Ζ) — L リジン— NH— (CH ) O— PEG— OMe (Boc— Lys (Cl— Z)— NH— (CH ) 50 mL of one end methyl group and the other end trimethyleneamino group of PEG (molecular weight 5000, manufactured by NOF Corporation) in N, N dimethylformamide solution 1.5 mL of N-a-one (tert-butoxycarbo) N ε N (ε- (2-Ci- 口)-L-lysine, manufactured by Kokusan Kagaku) N, N dimethylforma 10 mL of the imide solution was added, and 1.5-fold molar amount of 1-hydroxybenzotriazole and N, N′-diisopropylcarpositimide were added, and the mixture was stirred at room temperature for 24 hours under light-shielding conditions. Diisopropyl ether was added to the reaction solution to obtain a precipitate. The precipitate separated by filtration is dissolved in distilled water, passed through an anion exchange and cation exchange resin columns, and the eluate is freeze-dried. PEG is bound to the carboxy group of lysine a- Boc- ε — (2— CI— Ζ) — L-lysine— NH— (CH) O— PEG— OMe (Boc— Lys (Cl— Z) — NH— (CH)
2 3 2 2 3 2
O - PEG - OMe)を得た。 O-PEG-OMe) was obtained.
3  Three
[0042] 2gの Boc— Lys (Cl— Z)— NH— (CH ) O— PEG— OMeを 30mLの N, N ジ  [0042] 2 g of Boc—Lys (Cl—Z) —NH— (CH 2) O—PEG—OMe in 30 mL of N, N
2 3  twenty three
メチルホルムアミドに溶解し、 0. 34gの 5%パラジウム炭素をカ卩え、水素雰囲気下 40 °C、 2時間攪拌することにより接触加水素分解を行った。反応液に活性炭を添加し、 ハイフロースーパーセルを用いてろ過後、ジイソプロピルエーテルを添カ卩し沈殿物を 得た。沈殿物を 5%塩化ナトリウム水溶液に溶解後、 HP— 20カラムに通塔した。 5% 塩ィ匕ナトリウム水溶液及び蒸留水で洗浄後、 50%及び 80%ァセトニトリル水溶液で 溶出した溶出液を凍結乾燥し、リジンのベンジルォキシカルボニル基が脱離し、カル ボキシ基に PEGが結合した a— Boc— L リジン— NH— (CH ) O— PEG— OMe  It was dissolved in methylformamide, 0.34 g of 5% palladium carbon was added, and catalytic hydrogenolysis was carried out by stirring at 40 ° C. for 2 hours in a hydrogen atmosphere. Activated carbon was added to the reaction solution, and after filtration using a high flow supercell, diisopropyl ether was added to obtain a precipitate. The precipitate was dissolved in a 5% aqueous sodium chloride solution and then passed through an HP-20 column. After washing with 5% sodium chloride aqueous solution and distilled water, the eluate eluted with 50% and 80% aqueous acetonitrile was lyophilized, the benzyloxycarbonyl group of lysine was eliminated, and PEG was bound to the carboxyl group. a— Boc— L Lysine— NH— (CH) O— PEG— OMe
2 3  twenty three
(Boc-Lys-NH- (CH ) O— PEG— OMe)を 1. 41g得た。  1.41 g of (Boc-Lys-NH- (CH) O—PEG—OMe) was obtained.
2 3  twenty three
[0043] 0. 4gの Boc— Lys— NH— (CH ) O— PEG— OMeのブロモベンゼン溶液 20m  [0043] 0. 4 g of Boc— Lys— NH— (CH 2) 2 O— PEG— OMe in bromobenzene solution 20 m
2 3  twenty three
Lと 1. 6倍モル量の（1, 2—メタノ [60]フラーレン） 61—力ルボン酸を含むブロモ ベンゼン溶液 125mLを混和し、 1. 6倍モル量の 1ーヒドロキシベンゾトリアゾール及 び 3. 3倍モル量の N, N，—ジイソプロピルカルボジイミドを添カ卩し、室温、 72時間、 遮光条件下で攪拌した。反応液を等量の蒸留水にて抽出後、凍結乾燥した。この全 量を 10mLのジクロロメタンに溶解し、 3mLのトリフルォロ酢酸を添カ卩し、 4°C、 18時 間で Boc基の脱保護を行った。反応液にジイソプロピルエーテルを添加し沈殿物を 得た。沈殿物を 50%ァセトニトリル水溶液に溶解し、陰イオン交換榭脂カラム通塔後 、溶出液を凍結乾燥し、フラーレンのカルボキシ基に、連結分子としてリジンを介して PEGが結合した ε —フラーレン一 L リジン一 NH— (CH ) O— PEG— OMe (Lys  Mix L and 125 mL of a bromobenzene solution containing 1.6-fold molar amount of (1,2-methano [60] fullerene) 61-strength rubonic acid, and add 1.6-fold molar amount of 1-hydroxybenzotriazole and 3 A 3-fold molar amount of N, N, -diisopropylcarbodiimide was added, and the mixture was stirred at room temperature for 72 hours under light-shielding conditions. The reaction solution was extracted with an equal volume of distilled water and then lyophilized. The total amount was dissolved in 10 mL of dichloromethane, and 3 mL of trifluoroacetic acid was added thereto, and the Boc group was deprotected at 4 ° C for 18 hours. Diisopropyl ether was added to the reaction solution to obtain a precipitate. Dissolve the precipitate in 50% aqueous solution of acetonitrile and pass through an anion exchange resin column, freeze-dry the eluate, and PEG is bound to the carboxy group of fullerene via lysine as a linking molecule. Lysine I NH— (CH) O— PEG— OMe (Lys
2 3  twenty three
(COCH< C ) -NH- (CH ) O-PEG-OMe) 0. 33gを得た。  0.33 g of (COCH <C) -NH- (CH3) O-PEG-OMe) was obtained.
60 2 3  60 2 3
[0044] 5. 6mMの Lys (COCHく C ) -NH- (CH ) O— PEG— OMeの N, N ジメ  [0044] 5. 6 mM Lys (COCH C) -NH- (CH) O— PEG— OMe N, N
60 2 3  60 2 3
チルホルムアミド溶液 5mLに 20倍モル量の DTP A無水物を添カ卩し、室温、 72時間 反応後、ジイソプロピルエーテルを添カ卩し沈殿物を得た。沈殿物は 10mLの 0. 1M 水酸ィ匕ナトリウムを含む 50%ァセトニトリル溶液に再溶解し、室温にて、 24時間処理 することにより未反応の酸無水物を加水分解した後、透析、凍結乾燥を順次行い、フ ラーレンのカルボキシ基に、連結分子としてのリジンの ε —ァミノ基が結合し、そのリ ジンを介して PEG及び DTPAが結合した、 0. 228の0丁？八じ —PEG が得られ A 20-fold molar amount of DTP A anhydride was added to 5 mL of a tilformamide solution, and after reacting at room temperature for 72 hours, diisopropyl ether was added to obtain a precipitate. The precipitate is redissolved in 10 mL of 50% acetonitrile solution containing 0.1 M sodium hydroxide and treated at room temperature for 24 hours to hydrolyze unreacted acid anhydride, followed by dialysis and lyophilization. The ε-amino group of lysine as the linking molecule is bound to the carboxy group of the fullerene, and PEG and DTPA is linked via a gin, 0 Ding of 0.22 8? Hachiji-PEG is obtained
60 5000 た。  60 5000.
[0045] 参考例 4  [0045] Reference Example 4
C PEG DTP Aの^^  C PEG DTP A ^^
—60 5000 U-L^i —60 5000 U - L ^ i
4mMの片末端トリメチレンァミノ基の PEG (分子量 5000, 日本油脂製）のブロモベ ンゼン溶液 20mLと 1. 5倍モル量の（1, 2—メタノ [60]フラーレン） 61—力ルボン 酸を含むブロモベンゼン溶液 lOOmLを混和し、 1. 5倍モル量の 1ーヒドロキシベン ゾトリアゾール及び 3倍モル量の N, N'—ジイソプロピルカルボジイミドを添カ卩し、室 温、 72時間、遮光条件下で攪拌した。反応液を等量の蒸留水にて抽出し、陽イオン 交換榭脂カラムに通塔後、溶出液を凍結乾燥し、末端に水酸基を有する PEG1分子 力 その片末端トリメチレンァミノ基のアミノ基を介して結合した、 0. 46gのフラーレン を得た。  Contains 20 mL of bromobenzene solution of PEG (molecular weight 5000, manufactured by NOF Corporation) with 4 mM single-end trimethyleneamino group and 1.5-fold molar amount of (1,2-methano [60] fullerene) 61-strength rubonic acid Bromobenzene solution lOOmL was mixed, and 1.5-fold molar amount of 1-hydroxybenzotriazole and 3-fold molar amount of N, N'-diisopropylcarbodiimide were added, and the mixture was stirred at room temperature for 72 hours under light-shielded conditions. . The reaction solution is extracted with an equal volume of distilled water, passed through a cation exchange resin column, and the eluate is freeze-dried. The PEG1 molecule has a hydroxyl group at the end. The amino group of the trimethyleneamino group at one end Thus, 0.46 g of fullerene was obtained.
9. 3mMの得られた該フラーレンのジメチルスルホキシド溶液 4mLに 10倍モル量 の N, N，一カルボ-ルジイミダゾールを添カ卩し、室温、 2時間反応後、 100倍モル量 のエチレンジァミンのジメチルスルホキシド溶液 2mLに攪拌しながら少量ずつ滴下し 、室温、 24時間反応した。反応液を PD— 10 (アマシャムノィォサイエンス製)でゲル ろ過後、凍結乾燥し、末端にアミノ基を有するウレタン結合を持つ PEG1分子が、そ の片末端トリメチレンァミノ基のアミノ基を介して結合した、 0. 18gのフラーレンを得た  9. To 4 mL of 3 mM dimethylsulfoxide solution of the fullerene obtained, add 10-fold molar amount of N, N, monocarbodiimidazole, react for 2 hours at room temperature, and then add 100-fold molar amount of ethylenediamine dimethyl. The solution was added dropwise to 2 mL of the sulfoxide solution while stirring, and reacted at room temperature for 24 hours. The reaction solution was gel-filtered with PD-10 (Amersham Neuroscience), freeze-dried, and the PEG1 molecule having a urethane bond with an amino group at the end was transferred via the amino group of one end of the trimethyleneamino group. 0.18 g fullerene was obtained.
[0046] 1. 3mMの得られた該フラーレンの N, N—ジメチルホルムアミド溶液 20mLに 20 倍モル量の DTP A無水物を添カ卩し、室温、 72時間反応後、ジイソプロピルエーテル を添加し沈殿物を得た。沈殿物は 10mLの 0. 1M水酸ィ匕ナトリウムを含む 50%ァセ トニトリル水溶液に再溶解し、室温にて 24時間処理することにより未反応の酸無水物 を加水分解した後、透析、凍結乾燥を順次行い、末端にアミノ基を有するウレタン結 合を持つ PEG1分子力 その片末端トリメチレンァミノ基のアミノ基を介してフラーレン に結合し、更にそのウレタン結合のァミノ基に DPTAが結合した、 C —PEG DT [0046] 1. Add 20 times molar amount of DTP A anhydride to 20 mL of N, N-dimethylformamide solution of 3 mM of the fullerene obtained, react at room temperature for 72 hours, add diisopropyl ether and precipitate. I got a thing. The precipitate is redissolved in 10 mL of 50% aqueous acetonitrile containing 0.1 M sodium hydroxide and treated at room temperature for 24 hours to hydrolyze the unreacted acid anhydride, followed by dialysis and freezing. PEG1 molecular force with a urethane bond having an amino group at the terminal after sequential drying. Bonded to fullerene via the amino group of its one-end trimethyleneamino group, and DPTA bound to the amino group of the urethane bond. , C —PEG DT
60 5000 60 5000
PAが得られた。 PA was obtained.
[0047] 実施例 1 C DTPA (Mn) -PEG の調製 [0047] Example 1 Preparation of C DTPA (Mn) -PEG
~ 60 5000  ~ 60 5000
参考例 2の化合物 C DTPA-PEG を 0. 5Mの 2 モルホリノエタンスルホン酸  Reference Example 2 Compound C DTPA-PEG 0.5M 2 morpholinoethanesulfonic acid
60 5000  60 5000
緩衝液 (MES, pH 7. 0、同仁ィ匕学）に最終濃度 lOmgZmLになるように溶解し、 この溶液に塩ィ匕マンガンを 8mMになるように添加して、室温で 24時間攪拌した。反 応液を PD— 10でゲルろ過することで、過剰のマンガンイオンを除去し、 C DTPA  It was dissolved in a buffer solution (MES, pH 7.0, Tongren Chemical Co., Ltd.) to a final concentration of lOmgZmL, and manganese chloride was added to this solution to 8 mM and stirred at room temperature for 24 hours. Excess manganese ions are removed by gel filtration of the reaction solution with PD-10, and C DTPA
60 60
-PEG にマンガンイオンが配位した式（1)で表されるフラーレン誘導体（C DT-Fullerene derivative represented by formula (1) in which manganese ion is coordinated to PEG (C DT
5000 605000 60
PA (Mn) PEG )の溶液 (最終濃度 7. lmg/mL)を得た。 A solution of PA (Mn) PEG was obtained (final concentration 7. lmg / mL).
5000  5000
[0048] 実施例 2  [0048] Example 2
DTPA(Mn) C —PEG の調製  Preparation of DTPA (Mn) C —PEG
60 5000  60 5000
参考例 3の化合物 DTPAC — PEG を 0. 5Mの MES溶液に最終濃度 10mg  Compound DTPAC in Reference Example 3 — PEG in 0.5M MES solution, final concentration 10mg
60 5000  60 5000
/mLになるように溶解し、この溶液に塩ィ匕マンガンを 8mMになるように添カロし、室 温で 24時間攪拌した。反応液を PD— 10でゲルろ過することで、過剰のマンガンィォ ンを除去し、 DTP AC -PEG にマンガンイオンが配位した式（2)で表されるフラ  The solution was dissolved to a volume of 8 mL, and sodium chloride manganese was added to this solution to 8 mM and stirred at room temperature for 24 hours. The reaction solution was gel-filtered with PD-10 to remove excess manganese ions, and the fragment represented by formula (2) in which manganese ions were coordinated to DTP AC -PEG.
60 5000  60 5000
一レン誘導体 (DTPA(Mn) C — PEG )の溶液 (最終濃度 7.  A solution of one-lene derivative (DTPA (Mn) C — PEG) (final concentration 7.
60 5000 lmgZmL)を得 た。  60 5000 lmgZmL) was obtained.
[0049] 実施例 3  [0049] Example 3
~ C 60 -PEG 5000 DTPA (Mn)の言 M ~ C 60 -PEG 5000 DTPA (Mn) word M
参考例 4の化合物 C PEG DTP Aを 0. 5Mの MES溶液に最終濃度 10mg  Compound of Reference Example 4 C PEG DTP A in 0.5 M MES solution, final concentration 10 mg
60 5000  60 5000
/mLになるように溶解し、この溶液に塩ィ匕マンガンを 8mMになるように添カロし、室 温で 24時間攪拌した。反応液を PD— 10でゲルろ過することで、過剰のマンガンィォ ンを除去し、 C -PEG DTPAにマンガンイオンが配位した式（3)で表されるフラ  The solution was dissolved to a volume of 8 mL, and sodium chloride manganese was added to this solution to 8 mM and stirred at room temperature for 24 hours. The reaction solution was gel-filtered with PD-10 to remove excess manganese, and the flag represented by formula (3) in which manganese ions were coordinated to C-PEG DTPA.
60 5000  60 5000
一レン誘導体 (C PEG DTPA (Mn) )の溶液 (最終濃度 7. lmgZmL)を得  Obtained a solution (final concentration 7. lmgZmL) of a mono-ene derivative (C PEG DTPA (Mn))
60 5000  60 5000
た。  It was.
[0050] 試験例 1  [0050] Test Example 1
フラーレン誘導体におけるマンガンイオン西 R位数の測定  Measurement of west R-order of manganese ions in fullerene derivatives
実施例 1〜3で得られた本発明のフラーレン誘導体についてマンガンイオンの配位 数を測定した。実施例 1〜3で得られた各化合物溶液を用いて、塩ィ匕マンガンの 50 mMの MES溶液を標準物質とし原子吸光光度計 AA— 6800 (島津製作所）にて測 定した。その測定結果を表 1に示した。 The coordination number of manganese ions was measured for the fullerene derivatives of the present invention obtained in Examples 1 to 3. Using each of the compound solutions obtained in Examples 1 to 3, measurement was performed with an atomic absorption photometer AA-6800 (Shimadzu Corporation) using a 50 mM MES solution of manganese salt manganese as a standard substance. Set. The measurement results are shown in Table 1.
[0051] [表 1] 表 1 ：実施例 1〜 3の各化合物のマンガンイオン [0051] [Table 1] Table 1: Manganese ions of the compounds of Examples 1 to 3
配位数の原子吸光光度計による定量  Determination of coordination number by atomic absorption photometry
Figure imgf000020_0001
Figure imgf000020_0001
[0052] 測定結果より、本発明のフラーレン誘導体はすべて 1分子に 1個のマンガンイオン を含むことが示された。 [0052] From the measurement results, it was shown that all the fullerene derivatives of the present invention contained one manganese ion per molecule.
[0053] 試験例 2 [0053] Test Example 2
フラーレン誘導体の凝奪体の確認  Confirmation of depleted bodies of fullerene derivatives
実施例 1〜3で得られた本発明のフラーレン誘導体について光散乱法による粒径 測定を行った。実施例 1〜3で得られた各化合物溶液を最終濃度 lmgZmLになる ように 50mMの MES溶液で希釈した。この溶液を光散乱測定装置 DLS - 7000 (大 塚電子株式会社)で測定し、その測定結果を図 1に示した。  The fullerene derivatives of the present invention obtained in Examples 1 to 3 were measured for particle size by a light scattering method. Each compound solution obtained in Examples 1 to 3 was diluted with a 50 mM MES solution to a final concentration of 1 mgZmL. This solution was measured with a light scattering measurement device DLS-7000 (Otsuka Electronics Co., Ltd.), and the measurement result is shown in FIG.
測定結果より、本発明のフラーレン誘導体はすべて約 lOOnmを中心粒子径とする 凝集体であることが示された。これらの粒子径は、高分子物質が正常組織に比べて がん組織に移行しやすぐまた、がん組織に長く滞留する傾向があるという EPR効果 (Enhanced Permeation and Retention effect) すのに適し 7こ大ささであ り、本発明の造影剤は EPR効果を有するといえる。  From the measurement results, it was shown that all the fullerene derivatives of the present invention are aggregates having a central particle diameter of about lOOnm. These particle sizes are suitable for the EPR effect (Enhanced Permeation and Retention Effect), in which polymer substances tend to migrate to cancer tissues more quickly than normal tissues and tend to stay longer in cancer tissues. It can be said that the contrast medium of the present invention has an EPR effect.
[0054] 試験例 3 [0054] Test Example 3
フラーレン誘導体の MRI诰影能の評価  Evaluation of MRI shadowing ability of fullerene derivatives
実施例 1〜3で得られた本発明のフラーレン誘導体について MRI造影能を評価し た。実施例 1〜3で得られた各化合物溶液を最終濃度 500 M、 M及び 5 M になるように 50mMの MES溶液で希釈した。この溶液を 50mMの MES溶液を陰性 標準、 500 /z M塩ィ匕マンガンの MES溶液を陽性標準とし、 4. 7テスラの生体用核磁 気共鳴装置 BioSpec (ブルカー'バイオスピン社)で測定した。その測定結果を表 2 に示した。 The fullerene derivatives of the present invention obtained in Examples 1 to 3 were evaluated for MRI contrast ability. Each compound solution obtained in Examples 1 to 3 was diluted with a 50 mM MES solution to a final concentration of 500 M, M and 5 M. Using this solution as a negative standard for a 50 mM MES solution and a positive standard for a 500 / z M salt / manganese MES solution, Measured with a gas resonance apparatus BioSpec (Bruker Biospin). The measurement results are shown in Table 2.
[0055] [表 2] 表 2 ：実施例 1〜 3の化合物の MR I造影能の結果 [0055] [Table 2] Table 2: Results of MR I imaging ability of the compounds of Examples 1 to 3
Figure imgf000021_0001
Figure imgf000021_0001
[0056] 測定結果より、本発明のフラーレン誘導体またはその凝集体は同濃度のマンガンィ オンと同程度以上の造影能を有することが示され、これにより本発明のフラーレン誘 導体が MRI用造影剤となることが示された。 [0056] The measurement results show that the fullerene derivative of the present invention or an aggregate thereof has a contrast ability equal to or higher than that of manganese ion at the same concentration, whereby the fullerene derivative of the present invention is combined with a contrast agent for MRI. It was shown to be.
[0057] 試験例 4 [0057] Test Example 4
フラーレン謙導体の光照射による活件酸素の発牛畺の沏  Fullerene Ken Conductor's Light Oxidized Oxidized Boiled Rice Bowl
受田の報告（DOJIN NEWS, No. 96, 1—6 (2000) )を参考に生細胞数測定試 薬 SFを用いて、本発明のフラーレン誘導体について、光照射による活性酸素 (スー パーォキシドア-オン， O―)の発生量を測定した。実施例 1〜3で得られる各化合物  With reference to the report by Toda (DOJIN NEWS, No. 96, 1-6 (2000)), the active cell count measurement reagent (superoxide-on) by photoirradiation of the fullerene derivative of the present invention was measured using SF. , O-) was measured. Each compound obtained in Examples 1 to 3
2  2
溶液を最終濃度 100 Mになるように 50mMの MES溶液で希釈し、該溶液を 48ゥ エルプレートに 200 μ Lずつ分注した。これに 160 μ Lの 50mMの MES溶液と 40 μ Lの生細胞数測定試薬 SFを混合し、高出力 LED光照射器ブルーフェーズ (イボクラ ールビバデント社）を用いて、直径 8mmのプローブで、 430〜490nmの青色可視光 を 1. lWattZcm2の出力で 4分間照射した。照射後、溶液の 450nmの吸光度を分 光光度計 DU— 650 (ベックマン社)で測定した。化合物を添加せずに光照射を行つ た溶液をコントロールとし、 1分間当りの O—発生量を図 2に示した。 The solution was diluted with a 50 mM MES solution to a final concentration of 100 M, and 200 μL of this solution was dispensed into 48-well plates. This was mixed with 160 μL of 50 mM MES solution and 40 μL of viable cell count reagent SF, and using a high-power LED light irradiator Blue Phase (Ivoclar Vivadent), a probe with a diameter of 8 mm, Irradiated with 490nm blue visible light at 1. lWattZcm 2 for 4 minutes. After irradiation, the absorbance of the solution at 450 nm was measured with a spectrophotometer DU-650 (Beckman). The solution irradiated with no compound added was used as a control, and Figure 2 shows the amount of O-generated per minute.
2  2
測定結果より、本発明のフラーレン誘導体またはその凝集体は、その構造に関わら ず、高効率で o—の発生が観察された。このことは本発明のフラーレン誘導体が光照 射による癌の光線力学的治療剤に適用できる事を示している。 From the measurement results, generation of o- was observed with high efficiency in the fullerene derivative of the present invention or its aggregate regardless of its structure. This is because the fullerene derivative of the present invention is illuminated. It is shown that it can be applied to photodynamic therapeutic agents for cancer caused by shooting.
[0058] 試験例 5  [0058] Test Example 5
フラーレン誘導体の超音波照射による活件酸素の発牛.量の測定  Catalysis of active oxygen by ultrasonic irradiation of fullerene derivatives.
試験例 4と同様の試験を光照射に替えて超音波照射で実施した。実施例 1〜3で 得られた各化合物溶液を最終濃度 100 Mになるように 50mMの MES溶液で希釈 した溶液 2mLと、 1. 6mLの 50mMの MES溶液及び 400 /z Lの生細胞数測定試薬 SFを混合した合計 4mLの溶液を、直径 32mmの照射面の周囲にビニールテープを 巻いた 1MHz用超音波プローブ (ギンセン社製）に滴下した。超音波プローブは、信 号発生器 WF1966 (ェヌエフ回路設計ブロック）と高速バイポーラ電源 HSA4101 ( ェヌエフ回路設計ブロック）を用いて、 3WattZcm2の出力で 1MHzの正弦波をバ 一ストモード（Mark50000サイクル、 Space50000サイクル）で振動させ、 3分間超 音波照射を行った。照射後、溶液の45011111の吸光度を分光光度計1；¥—1200 (島 津製作所)で測定した。 1分間当りの O—発生量を図 3に示した。 A test similar to Test Example 4 was carried out by ultrasonic irradiation instead of light irradiation. 2 mL of each compound solution obtained in Examples 1 to 3 diluted with 50 mM MES solution to a final concentration of 100 M, 1.6 mL of 50 mM MES solution and 400 / zL viable cell count A total of 4 mL of the solution mixed with the reagent SF was dropped onto a 1 MHz ultrasonic probe (Ginsen) with a vinyl tape wrapped around the irradiated surface with a diameter of 32 mm. The ultrasonic probe uses the signal generator WF1966 (ENF circuit design block) and the high-speed bipolar power supply HSA4101 (ENF circuit design block) to output a 1 MHz sine wave at the output of 3 WattZcm 2 in the burst mode (Mark50000 cycle, Space50000 cycle). Cycle) and ultrasonic irradiation was performed for 3 minutes. After irradiation, the absorbance of the solution at 45011111 was measured with a spectrophotometer 1; ¥ -1200 (Shimadzu Corporation). Figure 3 shows the amount of O-generated per minute.
2  2
測定結果より、本発明のフラーレン誘導体またはその凝集体は、超音波照射によつ ても O _の発生が観察され、特に実施例 3の C -PEG DTPA (Mn)は高い O _ From the measurement results, the fullerene derivative of the present invention or its aggregates were observed to generate O_ even by ultrasonic irradiation, and in particular, C_PEG DTPA (Mn) in Example 3 was high in O_.
2 60 5000 2 発生を示した。従って、本発明のフラーレン誘導体は超音波力学的治療にも適用で きる。 2 60 5000 2 Occurrence occurred. Therefore, the fullerene derivative of the present invention can also be applied to ultrasonic mechanical therapy.
[0059] 試験例 6 [0059] Test Example 6
フラーレン誘導体の in vivoでの MRI诰影能の評価  Evaluation of MRI imaging ability of fullerene derivatives in vivo
実施例 2で得られた本発明のフラーレン誘導体につ ヽて担癌マウスを用いた in vi voでの MRI造影能を評価した。担癌マウスは、マウス結腸癌 Colon26細胞 (癌研究 会 癌化学療法センターより供与）を BALBZc— nn (雌、 日本チャールズ 'リバ一社 )皮下移植にて継代した腫瘍隗を摘出し、細切片を CDF1マウス (雌、 日本チヤール ズ 'リバ一社)の右大腿部皮下に移植することによって作製した。移植後に皮下に直 径 5〜： L Omm程度の腫瘤を形成したマウスを担癌マウスとして実験に用いた。  The fullerene derivative of the present invention obtained in Example 2 was evaluated for in vivo MRI imaging ability using tumor-bearing mice. Tumor-bearing mice were excised and subsectioned from tumor colons that had been subcultured by BALBZc-nn (female, Charles Japan, Liver Co., Ltd.) under the influence of mouse colon cancer Colon26 cells (provided by the Cancer Chemotherapy Center, Cancer Society). Was transplanted subcutaneously into the right thigh of CDF1 mice (female, Nippon Charles' Riva Co., Ltd.). Mice that formed a tumor with a diameter of about 5 to L Omm subcutaneously after transplantation were used as cancer-bearing mice in the experiments.
1. 8%イソフルラン (フォーレン、ダイナボット社)持続麻酔下で 120回 Z分の呼吸 同調を行った担癌マウスに、実施例 2で得られたィ匕合物溶液を最終濃度 14mMにな るように Centriplus YM— 3 (ミリポア社)を用いて濃縮し、マウス尾静脈内に 300 L投与した。 4. 7テスラの生体用核磁気共鳴装置 BioSpec (ブルカー'バイオスピン 社)で測定した担癌マウスの下肢の T2強調画像 (腫瘍などの病変部位が強調される 画像)を図 4に示す。図 4中の右側の灰色の部分が腫瘍組織である。 1. 8% isoflurane (Foren, Dynabot Co., Ltd.) A cancer-bearing mouse that has been respiratory-synchronized 120 times under continuous anesthesia with Z-respiratory solution obtained in Example 2 to a final concentration of 14 mM. Concentrate with Centriplus YM-3 (Millipore) and add 300 L was administered. 4. A T2-weighted image of the lower limb of a tumor-bearing mouse (image highlighting a lesion such as a tumor) measured with a 7 Tesla nuclear magnetic resonance system BioSpec (Bruker Biospin) is shown in Fig. 4. The gray part on the right side in Fig. 4 is the tumor tissue.
造影効果は同装置を用いた T1強調画像にて、図 5から図 8に示した。本発明のフ ラーレン誘導体の投与前、投与 1時間後、投与 6時間後及び投与 12時間後の測定 結果を示す図 5〜図 8から、本発明のフラーレン誘導体は投与後 12時間以上、 T2強 調画像で示された腫瘍部位周辺の造影強度を増強し、反対側の正常組織では変化 が見られなカゝつたことから、癌特異的造影剤であることを示して 、る。  The contrast effect is a T1-weighted image using the same device and is shown in Figs. From FIG. 5 to FIG. 8 showing the measurement results before administration, 1 hour after administration, 6 hours after administration and 12 hours after administration of the fullerene derivative of the present invention, the fullerene derivative of the present invention is more than 12 hours after administration. The contrast intensity around the tumor site shown in the tomographic image was enhanced, and no change was seen in the normal tissue on the opposite side, indicating that it is a cancer-specific contrast agent.
産業上の利用可能性  Industrial applicability
[0060] 本発明のフラーレン誘導体を含有する造影剤は、 MRIの造影剤として適しており、 癌に特異的に長時間蓄積し、生体内での測定時間が長ぐ免疫原性が低いという優 れた効果を有するので、 MRIの画像コントラストを明瞭にすることができ、更に、光照 射や超音波照射等の物理的刺激により活性酸素を発生させることができる。 [0060] The contrast agent containing the fullerene derivative of the present invention is suitable as a contrast agent for MRI, accumulates specifically in cancer for a long time, has a long measurement time in vivo, and has a low immunogenicity. Therefore, the MRI image contrast can be clarified, and active oxygen can be generated by physical stimulation such as light irradiation and ultrasonic irradiation.
図面の簡単な説明  Brief Description of Drawings
[0061] [図 1]実施例 1〜3のフラーレン誘導体の粒子径を光散乱法により測定した結果を示 す。  [0061] FIG. 1 shows the results of measuring the particle size of fullerene derivatives of Examples 1 to 3 by a light scattering method.
[図 2]実施例 1〜3のフラーレン誘導体の光照射による活性酸素発生量を測定した結 果を示す。  FIG. 2 shows the results of measuring the amount of active oxygen generated by light irradiation of the fullerene derivatives of Examples 1 to 3.
[図 3]実施例 1〜3のフラーレン誘導体の超音波照射による活性酸素発生量を測定し た結果を示す。  FIG. 3 shows the results of measuring the amount of active oxygen generated by ultrasonic irradiation of the fullerene derivatives of Examples 1 to 3.
[図 4]生体用核磁気共鳴装置 BioSpec (ブルカー'バイオスピン社)で測定した担癌 マウスの腫瘍部位の T2強調画像を示す。  FIG. 4 shows a T2-weighted image of the tumor site of a tumor-bearing mouse measured with a biological nuclear magnetic resonance apparatus BioSpec (Bruker Biospin).
[図 5]生体用核磁気共鳴装置 BioSpec (ブルカー'バイオスピン社)で測定した担癌 マウスの腫瘍部位の T1強調画像を示す。  FIG. 5 shows a T1-weighted image of a tumor site of a tumor-bearing mouse measured with a biological nuclear magnetic resonance apparatus BioSpec (Bruker Biospin).
[図 6]実施例 2のフラーレン誘導体の投与後 1時間の腫瘍部位の T1強調画像を示す [図 7]実施例 2のフラーレン誘導体の投与後 6時間の腫瘍部位の T1強調画像を示す [図 8]実施例 2のフラーレン誘導体の投与後 12時間の腫瘍部位の Tl強調画像を示 す。 FIG. 6 shows a T1-weighted image of the tumor site 1 hour after administration of the fullerene derivative of Example 2. FIG. 7 shows a T1-weighted image of the tumor site 6 hours after administration of the fullerene derivative of Example 2. FIG. 8 shows a Tl-weighted image of the tumor site 12 hours after administration of the fullerene derivative of Example 2.

Claims

請求の範囲 The scope of the claims
[I] 分子中に官能基を有していてもよいフラーレンに、水溶性高分子と、金属イオンの 配位したキレート形成分子、または磁性体の結合した分子を、必要に応じて連結分 子を介して結合させたフラーレン誘導体を含有する造影剤。  [I] A molecule in which a water-soluble polymer and a chelate-forming molecule coordinated with a metal ion or a magnetic substance are bonded to a fullerene, which may have a functional group in the molecule, are linked molecules as necessary. Contrast agent containing a fullerene derivative bonded through an intermediate.
[2] 連結分子がアミノ酸類またはアミノアルキレンウレタン類であるフラーレン誘導体を 含有する請求項 1記載の造影剤。  [2] The contrast agent according to claim 1, wherein the linking molecule contains a fullerene derivative which is an amino acid or an aminoalkylene urethane.
[3] アミノ酸類が塩基性アミノ酸であるフラーレン誘導体を含有する請求項 2記載の造 影剤。 [3] The agent according to claim 2, wherein the amino acid contains a fullerene derivative which is a basic amino acid.
[4] フラーレンの官能基の数力^〜 5個であるフラーレン誘導体を含有する請求項 1から [4] A fullerene derivative having a number of functional groups of fullerene ^ 5 is included.
3の 、ずれか一項に記載の造影剤。 3. The contrast agent according to any one of items 1 to 3.
[5] フラーレンの官能基の数が 1個で、官能基がカルボキシ基であるフラーレン誘導体 を含有する請求項 4記載の造影剤。 5. The contrast agent according to claim 4, comprising a fullerene derivative in which the number of functional groups of fullerene is one and the functional group is a carboxy group.
[6] フラーレンが C フラーレンであるフラーレン誘導体を含有する請求項 1から 5のい [6] The fullerene derivative according to any one of claims 1 to 5, wherein the fullerene contains a fullerene derivative which is C fullerene.
60  60
ずれか一項に記載の造影剤。  The contrast agent according to one item.
[7] 水溶性高分子が分子量 1, 000〜1, 000, 000のポリエチレングリコール類である フラーレン誘導体を含有する請求項 1から 6のいずれか一項に記載の造影剤。  7. The contrast agent according to any one of claims 1 to 6, wherein the water-soluble polymer contains a fullerene derivative which is a polyethylene glycol having a molecular weight of 1,000 to 1,000,000.
[8] キレート形成分子がジエチレントリァミンペンタ酢酸、エチレン—ビス（ォキシ—ェチ レン-トリ口）テトラ酢酸、エチレンジアミンテトラ酢酸、 1, 4, 7, 10—テトラァザシクロ ドデカン 1, 4, 7, 10—テトラ酢酸、 1, 4, 8, 11ーテトラァザシクロテトラデカン 1 , 4, 8, 11ーテトラ酢酸またはその誘導体であるフラーレン誘導体を含有する請求項 1から 7の 、ずれか一項に記載の造影剤。  [8] The chelate-forming molecule is diethylenetriaminepentaacetic acid, ethylene-bis (oxy-ethylene-triethyl) tetraacetic acid, ethylenediaminetetraacetic acid, 1, 4, 7, 10-tetraazacyclododecane 1, 4, 7, 10 —Tetraacetic acid, 1, 4, 8, 11-tetraazacyclotetradecane 1, 4, 8, 11-tetraacetic acid or a fullerene derivative which is a derivative thereof, according to any one of claims 1 to 7 Contrast agent.
[9] キレート形成分子に配位した金属イオンが原子番号 21— 29、 42、 44、 56— 71、 7 6、 82、 83の金属元素力 選択される元素の陽イオンまたは放射性のイオンであるフ ラーレン誘導体を含有する請求項 1から 8のいずれか一項に記載の造影剤。  [9] The metal ion coordinated to the chelate-forming molecule is the metal element force of atomic number 21—29, 42, 44, 56—71, 7 6, 82, 83. The cation or radioactive ion of the selected element The contrast agent according to any one of claims 1 to 8, comprising a fullerene derivative.
[10] 磁性体カ^ヱライトであるフラーレン誘導体を含有する請求項 1から 7の 、ずれか一 項に記載の造影剤。  [10] The contrast agent according to any one of [1] to [7], comprising a fullerene derivative that is a magnetic material.
[II] 水溶性高分子が、片末端に不活性基を有し他端に反応性基または両端に反応性 基を有するポリエチレングリコール類であり、金属イオンの配位したキレート形成分子 力 Gd3+または Mn2が配位したエチレントリァミンペンタ酢酸であるフラーレン誘導 体を含有する請求項 1から 9のいずれか一項に記載の造影剤。 [II] The water-soluble polymer is a polyethylene glycol having an inert group at one end and a reactive group at the other end or a reactive group at both ends, and a chelate-forming molecule coordinated with a metal ion 10. The contrast agent according to claim 1, comprising a fullerene derivative which is ethylenetriaminepentaacetic acid coordinated with force Gd 3+ or Mn 2 .
[12] 物理的刺激により活性酸素を発生するフラーレン誘導体を含有する請求項 1から 1[12] The fullerene derivative which generates active oxygen by physical stimulation is contained.
1の 、ずれか一項に記載の造影剤。 1. The contrast agent according to any one of the above.
[13] 請求項 1から 12のいずれか一項に記載のフラーレン誘導体の凝集体を含有する造 影剤。 [13] A projector comprising the aggregate of the fullerene derivative according to any one of claims 1 to 12.
[14] 凝集体の大きさが 20〜400nmである凝集体を含有する請求項 13記載の造影剤。  14. The contrast agent according to claim 13, comprising an aggregate having an aggregate size of 20 to 400 nm.
[15] 造影剤が MRI用である請求項 1から 14のいずれか一項に記載の造影剤。 15. The contrast agent according to any one of claims 1 to 14, wherein the contrast agent is for MRI.
[16] 造影部位が腫瘍部位である請求項 1から 15の 、ずれか一項に記載の造影剤。 16. The contrast agent according to any one of claims 1 to 15, wherein the contrast site is a tumor site.
[17] 請求項 1から 12のいずれか一項に記載のフラーレン誘導体。 [17] The fullerene derivative according to any one of claims 1 to 12.
[18] 下記式（1)で表される請求項 17に記載のフラーレン誘導体。 [18] The fullerene derivative according to claim 17, which is represented by the following formula (1).
[化 1]  [Chemical 1]
Figure imgf000026_0001
Figure imgf000026_0001
[式中、 Mは金属イオンを示す] [Wherein M represents a metal ion]
下記式（2)で表される請求項 17に記載のフラーレン誘導体。  The fullerene derivative according to claim 17, which is represented by the following formula (2).
[化 2] [Chemical 2]
Figure imgf000027_0001
Figure imgf000027_0001
Figure imgf000027_0002
Figure imgf000027_0002
[式中、 Mは金属イオンを示す] [Wherein M represents a metal ion]
[21] 請求項 17から 20のいずれか一項に記載のフラーレン誘導体の凝集体, [21] Aggregates of fullerene derivatives according to any one of claims 17 to 20,
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CN110627835A (en) * 2019-08-16 2019-12-31 河南农业大学 Paramagnetic fullerene-metal nano complex and preparation method and application thereof
CN110627835B (en) * 2019-08-16 2021-07-13 河南农业大学 Paramagnetic fullerene-metal nano complex and preparation method and application thereof

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