WO2007061036A1 - Produit de contraste utilisant un dérivé d'un fullerène - Google Patents

Produit de contraste utilisant un dérivé d'un fullerène Download PDF

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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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English (en)
Japanese (ja)
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Masatoshi Yamada
Akira Masuda
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Nippon Kayaku Kabushiki Kaisha
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Priority to JP2007546496A priority Critical patent/JPWO2007061036A1/ja
Publication of WO2007061036A1 publication Critical patent/WO2007061036A1/fr

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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.

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Abstract

L'invention concerne un produit de contraste comprenant un dérivé d'un fullerène. Le dérivé de fullerène comprend un polymère soluble dans l'eau et une molécule chélatante ayant un ion métallique coordiné dans celle-ci ou une molécule liée à une matière magnétique tous deux attachés à un fullerène éventuellement via une molécule de liaison, ledit fullerène pouvant avoir un groupe fonctionnel dans la molécule. Le produit de contraste convient comme produit de contraste pour l'IRM et a d'excellentes propriétés qui font qu'il peut s'accumuler sur une longue durée d'une manière spécifique à un cancer, qu'il présente une très longue durée de mesurabilité dans un corps vivant et qu'il a une faible immunogénicité.
PCT/JP2006/323406 2005-11-25 2006-11-24 Produit de contraste utilisant un dérivé d'un fullerène WO2007061036A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011501761A (ja) * 2007-10-22 2011-01-13 ルナ イノベーションズ インコーポレイテッド 金属フラーレン造影剤
CN102827149A (zh) * 2012-09-06 2012-12-19 中国工程物理研究院核物理与化学研究所 富勒烯单大环多胺衍生物中间体及其制备方法
CN102827150A (zh) * 2012-09-06 2012-12-19 中国工程物理研究院核物理与化学研究所 富勒烯单大环多胺衍生物及其制备方法
CN109568607A (zh) * 2018-12-30 2019-04-05 河南农业大学 一种钆基金属富勒烯水溶性氮宾衍生物及其制备方法与应用
CN110627835A (zh) * 2019-08-16 2019-12-31 河南农业大学 一种顺磁性富勒烯-金属纳米配合物及其制备方法与应用

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WO2005095494A1 (fr) * 2004-03-31 2005-10-13 Nippon Kayaku Kabushiki Kaisha Nouveau fullerène soluble dans l'eau, procédé servant à produire celui-ci et générateur d'oxygène actif contenant le fullerène

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011501761A (ja) * 2007-10-22 2011-01-13 ルナ イノベーションズ インコーポレイテッド 金属フラーレン造影剤
US9233177B2 (en) 2007-10-22 2016-01-12 Luna Innovations Incorporated Metallofullerene contrast agents
CN102827149A (zh) * 2012-09-06 2012-12-19 中国工程物理研究院核物理与化学研究所 富勒烯单大环多胺衍生物中间体及其制备方法
CN102827150A (zh) * 2012-09-06 2012-12-19 中国工程物理研究院核物理与化学研究所 富勒烯单大环多胺衍生物及其制备方法
CN109568607A (zh) * 2018-12-30 2019-04-05 河南农业大学 一种钆基金属富勒烯水溶性氮宾衍生物及其制备方法与应用
CN110627835A (zh) * 2019-08-16 2019-12-31 河南农业大学 一种顺磁性富勒烯-金属纳米配合物及其制备方法与应用
CN110627835B (zh) * 2019-08-16 2021-07-13 河南农业大学 一种顺磁性富勒烯-金属纳米配合物及其制备方法与应用

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