WO1998023293A1 - Contrast compound, contrast medium for mri, and method for mri - Google Patents

Contrast compound, contrast medium for mri, and method for mri Download PDF

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Publication number
WO1998023293A1
WO1998023293A1 PCT/JP1997/004343 JP9704343W WO9823293A1 WO 1998023293 A1 WO1998023293 A1 WO 1998023293A1 JP 9704343 W JP9704343 W JP 9704343W WO 9823293 A1 WO9823293 A1 WO 9823293A1
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Prior art keywords
compound
mri
sugar chain
chain polymer
imaging
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PCT/JP1997/004343
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French (fr)
Japanese (ja)
Inventor
Toshihiro Akaike
Masato Mikawa
Atsushi Maruyama
Masaya Takahashi
Tomoaki Miyazawa
Naoto Miwa
Original Assignee
Nihon Schering K.K.
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Priority to AU51356/98A priority Critical patent/AU5135698A/en
Publication of WO1998023293A1 publication Critical patent/WO1998023293A1/en

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    • 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
    • A61K49/128—Linear polymers, e.g. dextran, inulin, PEG comprising multiple complex or complex-forming groups, being either part of the linear polymeric backbone or being pending groups covalently linked to the linear polymeric backbone
    • 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
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the MRI diagnostic method is a new diagnostic method that has recently received a great deal of attention not only from the field of radiation diagnosis but also from the whole medical community.
  • MR I contrast media are superior in concentration resolution in tissues, and have high safety without X-ray exposure, indicating the presence of lesions, normality and disease. It has been pointed out that it is clinically useful for understanding the anatomical and functional images of the target site.
  • a contrast agent that 1) can detect target cells such as tumors at a low concentration (low dose) with high sensitivity specifically, 3) has low toxicity, and 4) can be rapidly excreted from the body. Have been.
  • Contrast agents using monoclonal antibodies, peptides, polysaccharides, ribosomes, etc. have been developed as a means for delivering contrast agents for MRI to target sites and organs.
  • the agent needs to be localized at a relatively high concentration, and there is no practical target-directed contrast agent at present.
  • MR I contrast agents are recognized as foreign substances in vivo, undergo phagocytosis by the reticuloendothelial system (RES), and provide sufficient target fingers. No tropism was obtained.
  • RES reticuloendothelial system
  • MRI contrast-enhancing compounds have specific organs and sites as glycopolymers corresponding to glycoprotein receptors expressed specifically in tumors and organs.
  • target directivity such as tumor tissue directivity and / or specific organ directivity.
  • the present invention is as follows.
  • OMRI contrast-enhancing compound especially a compound obtained by modifying magnetite with a sugar chain high molecule that specifically recognizes a target site.
  • Target site-directed contrast agents for MRI including MRI contrasting compounds, especially compounds obtained by modifying magnetite with high sugar chain molecules that specifically recognize target sites.
  • MRI-enhancing compounds are modified with high-sugar-chain molecules that specifically recognize the target site.
  • target site-directed MRI contrast agents including MRI contrasting compounds, especially compounds obtained by modifying magnetite with high molecular weight sugar chains that specifically recognize target sites MR I imaging method characterized by the following.
  • Imaging of tumor cells or liver by imaging tumor cells or liver with an MRI imaging agent that contains a compound that modifies MRI imaging compounds, especially magnetite with hyaluronic acid Method.
  • MRI imaging compounds especially compounds obtained by modifying magnetite with a sugar chain polymer that specifically recognizes the target site, for the production of a target site-directed MRI contrast agent .
  • Figure 1 HA-SPIO (1a and 2a), P VLA-SPIO (lb and 2b) and CDx-SPIO (1c and 2c) and EL4 cells fluorescently labeled with FITC at 4 ° C. Histogram showing the relationship between cell fluorescence intensity and cell number when incubated for 2 hours (1a, 1b and 1c) or at 37 ° C for 6 hours (2a, 2b and 2c) .
  • the solid line (1) shows the results when incubating in the presence of the fluorescently labeled MRI contrast agent described above, and the dashed line (1) shows the results without using the fluorescently labeled MRI contrast agent as a control. Only went to
  • FIG. 9 is a view showing a change in relative signal intensity of MRI in the liver 24 hours later.
  • the contrast mechanism of the MR I contrast agent is very different from other contrast agents.
  • the high X-ray absorption of the contrast agent itself directly affects the brightness on the image, whereas the contrast agent for MRI is not directly depicted itself,
  • the contrast agent activates the relaxation phenomenon of surrounding protons and indirectly increases the brightness of the image. Contrast is achieved by lowering the volume.
  • Two types of contrast agents for MRI are known: T1-weighted contrast agents and T2-weighted contrast agents.
  • the T1-weighted contrast medium is a positive contrast medium, and the brightness of the area where the contrast medium is present increases and glows white on the image.
  • the T2-weighted contrast medium is the negative contrast medium and the brightness of the area where the contrast medium is present is low on the image. It drops and looks dark.
  • a commonly used MRI contrast-enhancing compound can be used, and preferably, magnetite, which is a T2-weighted contrast agent that shortens the transverse relaxation time (T2) of the proton, is used.
  • magnetite which is a T2-weighted contrast agent that shortens the transverse relaxation time (T2) of the proton.
  • a superparamagnetic iron oxide fine particle (Superparamagnetic Iron Oxide: SPI0) colloided with a dextran derivative is used.
  • the sugar chain polymer used in the present invention is not particularly limited as long as it can specifically recognize a target site, for example, an organ such as a tumor tissue or a liver.
  • Hyaluronan that specifically recognizes the hyaluronan receptor that is highly expressed in cells, spleen, lymph nodes, and tumor tissues. And the like.
  • glucose-binding polystyrene or the like that specifically recognizes a glucose transporter from Tengen is exemplified. More preferably, it has a low flexibility and has a high hydrophilicity, so that it tends to have an extended linear structure.
  • the adsorption of the protein is suppressed with the hydrodynamic properties of the hydrophilic chain, and the phagocytic action of RES can be avoided. That is, nonspecific uptake of RES by phagocytosis can be suppressed, vascular retention can be improved, and uptake into tumors and the like can be increased.
  • Hyaluronic acid is strongly negatively charged because it has a carboxyl group in its glucanoic acid residue.
  • Examples of a method for modifying an MRI contrast-enhancing compound with these sugar chain polymers include a method in which the two are bonded by ionic bonding. Specifically, depending on the sugar chain polymer used For example, when SPI0 is modified with hyaluronic acid, ferric chloride and ferric chloride are added to an aqueous solution of hyaluronic acid to add 60 to 100 ° C, preferably about 80 ° C. The reaction is carried out by adding sodium hydroxide solution and neutralizing the pH. It is more preferable to perform ultrafiltration treatment to further purify and increase the purity.
  • the binding ratio between the c- glycan polymer and the MRI imaging compound varies depending on the type of the glycan polymer and the MRI imaging compound used.
  • the sugar chain polymer is 0.2 to 5 parts by weight for hyaluronic acid, preferably 1 part by weight, and 0.2 for lactose per 1 part by weight of magnetite. To 5 parts by weight, preferably 1 part by weight.
  • hydrophilic polymer In order to more specifically deliver the MRI contrast-enhancing compound to a target organ or tumor tissue, it is preferable to further bind a hydrophilic polymer to the MRI contrast agent of the present invention.
  • the hydrophilic polymer that can be used is not particularly limited as long as it has the above-mentioned characteristics, but specific examples include polyethylene glycol, polyvinyl alcohol, and polyvinylolidone. These hydrophilic polymers can be bound using the same method as the above-mentioned method for binding the sugar chain polymer to the MRI contrast-enhancing compound.
  • the contrast agent for MRI of the present invention is not particularly limited as long as it includes a compound obtained by modifying an MRI contrast-enhancing compound with a sugar chain polymer that specifically recognizes a target site.
  • Specific examples thereof include a compound of the present invention, and a compound of the present invention suspended or dissolved in a solvent such as distilled water for injection, physiological saline, or Ringer's solution.
  • Additives such as physically acceptable carriers and excipients can be included.
  • the contrast agent can be applied to cells, etc. and administered to the living body by intravascular (venous or arterial) administration, oral administration, rectal administration, vaginal administration, lymphatic administration, intraarticular administration, etc. It is preferably administered in the form of a solution, emulsion or suspension.
  • Additives included in the MRI contrast agent of the present invention vary depending on the administration form, administration route, etc. Specifically, in the case of an injection, a buffer, an antibacterial agent, a stabilizer, a dissolution aid Preparations and vehicles are used alone or in combination. For oral administration (specifically, solutions, syrups, emulsions or suspensions, etc.), coloring agents, preservatives, stabilizers, A turbidity agent, an emulsifier, a thickener, a sweetener, a fragrance and the like are used alone or in combination. As the various additives, those usually used in the art are used.
  • the contrast agent of the present invention is directed to a target site, it is necessary to specifically image liver and tumor cells when hyaluronic acid is selected as a desired cell, tissue, or organ, particularly as a sugar chain polymer used for modification. Becomes possible. Furthermore, by binding hyaluronic acid, the function as a buffer component of joints and the like which hyaluronic acid originally has can be imparted to the present contrast agent. That is, when the present contrast agent is directly administered to a joint or the like, it becomes suitable for diagnosis of a joint disease such as rheumatism.
  • the dose of the MRI contrast agent of the present invention is determined according to the conventional MRI contrast agent, and the age, body size and target of the subject to be administered are determined.
  • the amount of the MRI contrast-enhancing compound that can be increased or decreased as appropriate depending on the organ to be used, or the amount of iron (F e) in the case of magnetite is 10 to 100 ⁇ mo 1 F ekg, preferably It is selected in the range of 10 to 20 / zmo 1 Fe / kg.
  • the contrast agent of the present invention can be suitably used as a contrast agent for various animals in addition to humans.
  • the administration form, administration route, dose, etc. depend on the weight and condition of the target animal. Select as appropriate.
  • the compound of the present invention and the contrast medium for MRI can be used for specific detection of a hyaluronic acid receptor (such as CD44) specifically expressed in tumor cells and sinusoidal endothelial cells of the liver. Be expected.
  • a hyaluronic acid receptor such as CD44
  • Hyaluronic acid is highly expressed on liver sinusoidal endothelial cells, spleen, lymph nodes and tumor tissue. It is known that it is specifically recognized by a given hyaluronic acid receptor.
  • 9 g of hyaluronic acid (manufactured by Denki Kagaku Kogyo Co., Ltd., average molecular weight: 590,000) is dissolved in 900 ml of water, and 180 mg of hyaluronidase (manufactured by Sigma) is added, and 50 ° The enzyme was digested with C for 3 hours. Thereafter, the mixture was boiled at 100 ° C. for 10 minutes to deproteinize. The reaction solution was cooled with water and filtered to remove proteins. The filtrate was freeze-dried to obtain hyaluronic acid having an average molecular weight of about 8,000.
  • PVLA Polyvinylvinyl-lactonamide
  • VLA Vinyl benzyl-lactone amide
  • AIBN azobisisobutyronitrile
  • mM a polymerization initiator
  • the reaction solution was dropped into ethanol, and the precipitate was collected.
  • the recovered precipitate was dissolved in water, dialyzed (fraction molecular weight: 3,500), and lyophilized to obtain PVLA.
  • Carboxydextran is non-specifically taken up by Kupffer cells of the liver.
  • a product manufactured by Meito Sangyo Co., Ltd. was used.
  • the magnetite was coated with the various sugar chains obtained in (1) above.
  • a magnetite suspension (6.25 mg Fe / l Oml) was added dropwise to the aqueous solution (50 mg of 4 Oml) of PVL A obtained in ii) of (1) above, and after stirring, Ultrafiltration (fraction molecular weight: 20 kDa) gave PVL A magnetite.
  • CDx 8 g was dissolved in 15 ml of water, and CDx and magnetite were bound in the same manner as in a) above in the case of hyaluronic acid to obtain CDX magnetite.
  • the particle size of SPIO was calculated by observation with a transmission electron microscope.
  • the sugar chain polymer concentration is! ! ! In the case of (8) and (8), it was calculated from the fluorescence intensity of each sugar chain fluorescently labeled with ITC, and in the case of CDX, it was determined by colorimetry at a wavelength of 630 nm (VIS) using anthrone. The electric potential is 10 The pH of the 0-fold diluted solution was adjusted to about 7, and measured under the following conditions.
  • Measuring device Zeta master particle electrophoresis analyzer (Ma 1V ern) Measuring temperature: 25 ° C
  • the magnetic balance was measured using a simple magnetic balance MS B MK1 (Fohnson Matthey) and a standard sample (N105) as a control. Note c The results are shown in Table 1, p H in the table are shown the pH of the preparation of the Magunetai bets that combines sugar chain macromolecule.
  • PVLA-SP 10 25.0 54.3 155.2 7.1 29079 7.0
  • the DLS particle diameter of SPIO0 to which the sugar chain polymer was bound was observed to be about 50 to 50 nm.
  • the potential value showed a strong positive potential (approximately 35 mV) in the case of SPI 0 alone, but by binding to a glycan polymer, for example, in the case of PVL A, a weak positive potential (about 35 mV) was obtained. (7 mV), the hyaluronic acid and CDX changed to a strong negative potential (about 150 mV), suggesting that a neutral or negative potential sugar chain polymer was bound. .
  • the value of the magnetic field shows that each of them has a strong magnetic property of about 20,000 to 30,000 regardless of the binding of the sugar chain polymer, confirming that the MRI contrast property is maintained.
  • cell binding assays and endcytosis assays were performed in vitro on each sugar chain polymer-bound magnetite.
  • EL4 cells a mouse lymphoma-derived cell line
  • EL 4 cells that are known to express the hyaluronic acid receptor on the cell membrane surface were used.
  • EL 4 cells were subcultured at 37 ° C and 5% CO 2 in RPM 1-1640 medium supplemented with 10% fetal calf serum inactivated by heat. .
  • HA hyaluronic acid
  • Example 1 550 mg of hyaluronic acid (HA) prepared in i) of (1) of Example 1 was dissolved in 15 ml of honolemamide.
  • a solution prepared by dissolving 250 mg of FITC (Fluorescein isothiocyanate; manufactured by Dojin), 100 mg of dibutyltin dilaurate and 100 mg of sodium hydrogen carbonate in 15 ml of dimethyl sulfoxide (DMS 0) Add 9 5. Stir and mix with C for 20 minutes. The mixture is dropped into ethanol, and a few drops of a saturated aqueous sodium chloride solution are further dropped to cause precipitation. The obtained precipitate is dissolved in formamide, and the solution is again precipitated in ethanol. This operation is repeated a total of 5 times.
  • FITC Fluorescein isothiocyanate
  • DMS 0 dimethyl sulfoxide
  • the PVL A 3 0111 prepared in (1) ii) of Example 1 was dissolved in 0] ⁇ 50 of 3111]. 30 mg of F ITC and 9 mg of dibutyltin dilaurate were added to the solution.
  • CDX 30 Omg was dissolved in 5 ml of DMSO.
  • a solution prepared by dissolving 30 mg of FITC and 9 mg of dibutyltin dilaurate in 0.5 ml of DMSO is added to the solution, and the mixture is stirred and mixed at 90 ° C for 1 hour.
  • the mixture is dropped into ethanol to cause precipitation.
  • the resulting precipitate is dissolved in DMS0, the solution is again dropped into ethanol, and a few drops of a saturated aqueous sodium chloride solution are dropped to form a precipitate. Repeat this operation three times.
  • the finally obtained precipitate is dried under reduced pressure, redissolved in water, and filtered through a cellulose acetate membrane having a pore size of 0.2 ⁇ m.
  • the filtrate was freeze-dried over a period to obtain FITC-labeled CDX [yield: 21.4 mg (yield: 65%)].
  • the labeled compound obtained under labeling under these conditions contains 0.9 mol of FITC per 100 glucose repeating structural units. Will be. )
  • HA-SP10 specifically recognizes the HA receptor, that is, it is a contrast agent having target site directivity.
  • CBHZH e mice were anesthetized with 40 mg / kg of pentobarbital sodium administered intraperitoneally, a contrast agent for MRI was injected from the tail vein with a butterfly needle, and placed in the center of the magnetic field.
  • HA-SPI0, PVLA-SPI0 and CDX-SPI0 prepared in Example 1 were used as contrast agents for MR I, and the concentrations of Fe contained in each were adjusted to 30 umo 1 / k. .
  • the relative signal intensity (%) [RSI (%)] detected in the liver MRI image is calculated by the following formula.
  • SI (after muscle): Signal intensity in muscle after injection of contrast agent SI (immediately before liver): Signal intensity in liver before injection of contrast agent SI (muscle-front): Signal intensity in muscle before injection of contrast agent
  • Modification of the MR I imaging compound with a sugar chain macromolecule corresponding to a glycoprotein receptor that is specifically expressed in tumors and organs allows the target site-directed MR I imaging compound and MR I A contrast agent is obtained.
  • hyaluronic acid having high hydrophilicity as the sugar chain polymer to be modified, it is expected that phagocytosis of RES will be avoided. Since the target site can be specifically imaged, diagnosis can be performed with a low dose, that is, it is possible to provide a contrast agent for MRI with reduced toxicity.

Abstract

A compound comprising an MRI contrast compound and a sugar chain polymer bonded thereto, especially hyaluronic acid, corresponding to a glycoprotein receptor specifically manifesting in a tumor or organ; an MRI contrast medium containing the compound; and a method for imaging a tumor and/or a liver with the MRI contrast medium. An MRI contrast compound and an MRI contrast medium both having a target site directivity are obtained by modifying an MRI contrast compound with a sugar chain polymer corresponding to a glycoprotein receptor specifically manifesting in a tumor or organ. In particular, selection of hyaluronic acid, which is highly hydrophilic, as the modifying sugar chain polymer is expected to be especially effective in avoiding the phagocytosis by RES. Since target sites can be imaged specifically, the contrast medium enables diagnosis in a small dose, i.e., toxicity can be reduced.

Description

明細書  Specification
造影性化合物、 MR I用造影剤および MR I造影方法  Contrast compound, contrast agent for MR I and MR I contrast method
技術分野  Technical field
MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾してな る化合物、 該化合物を含んでなる標的部位指向性 MR I用造影剤および腫瘍細胞 およびノまたは肝臓の造影方法に関する。  A compound obtained by modifying an MRI imaging compound with a sugar chain polymer that specifically recognizes a target site, a target site-directed contrast agent for MRI comprising the compound, and a tumor cell and / or liver Related to the imaging method.
背景技術  Background art
近年の臨床画像診断の進歩は著しく、 X線 CT (コンビユタ一断層撮影） 、 超 音波画像診断、 MR I (磁気共鳴画像） 診断、 シンチグラフィ一等、 実に様々な 画像診断法が全身のほぼ全ての分野に用いられている。 さらに、 それらの画像診 断法に適した各種造影剤が開発され、 それらの有用性が報告されている。  In recent years, clinical imaging diagnosis has progressed remarkably, and a variety of imaging methods such as X-ray CT (combination tomography), ultrasonic imaging, MR I (magnetic resonance imaging), scintigraphy, etc. Used in the field. In addition, various contrast agents suitable for these diagnostic imaging methods have been developed and their usefulness has been reported.
特に MR I診断法は、 最近放射線診断分野のみならず医学界全体から著しい注 目を集めている新しい診断法である。 MR I用の造影剤は、 他の造影剤と比較し た場合、 組織中の濃度分解能に優れており、 X線被曝等がなく高い安全性が認め られており、 病変の指摘、 正常および病的部位の解剖学的および機能的イメージ の把握に臨床上有用であることが指摘されている。  In particular, the MRI diagnostic method is a new diagnostic method that has recently received a great deal of attention not only from the field of radiation diagnosis but also from the whole medical community. Compared with other contrast media, MR I contrast media are superior in concentration resolution in tissues, and have high safety without X-ray exposure, indicating the presence of lesions, normality and disease. It has been pointed out that it is clinically useful for understanding the anatomical and functional images of the target site.
しかしながら、 その検出能についてはまだ十分とはいいがたく、 一部の疾病や 部位に限定されており、 より高機能な造影剤の開発が期待されている。  However, its detectability is still insufficient, but is limited to some diseases and sites, and the development of more sophisticated contrast agents is expected.
とりわけ、 1 ) 低濃度 （低投与量） で、 2) 腫瘍等の標的細胞を特異的に高感 度で検出し、 3 ) 毒性が低く、 4) 速やかに体内から***されうる造影剤が望ま れている。  In particular, it is desirable to have a contrast agent that 1) can detect target cells such as tumors at a low concentration (low dose) with high sensitivity specifically, 3) has low toxicity, and 4) can be rapidly excreted from the body. Have been.
MR I用造影剤を標的部位および臓器に送達させるための手段として、 モノク ロナール抗体、 ペプチド類、 多糖類およびリボソーム等を用いた造影剤が開発さ れているが、 これら既存の MR I用造影剤は比較的高濃度に局在化する必要があ り実用的な標的指向性造影剤はいまだ存在しないのが現状である。  Contrast agents using monoclonal antibodies, peptides, polysaccharides, ribosomes, etc. have been developed as a means for delivering contrast agents for MRI to target sites and organs. The agent needs to be localized at a relatively high concentration, and there is no practical target-directed contrast agent at present.
また、 従来の MR I用造影剤では、 生体内で異物として認識され、 細網内皮系 (reticuloendothelial system; RE S) による貪食作用を受け、 十分な標的指 向性が得られなかった。 In addition, conventional MR I contrast agents are recognized as foreign substances in vivo, undergo phagocytosis by the reticuloendothelial system (RES), and provide sufficient target fingers. No tropism was obtained.
本発明の目的は、 M R I造影性化合物よりなる、 R E Sによる貪食作用を回避 し、 より実用的な標的部位指向性のある化合物、 該化合物を含む標的部位指向性 M R I用造影剤および腫瘍細胞および Zまたは肝臓の造影方法を提供することに ある。  An object of the present invention is to provide a more practical target site-directed compound comprising an MRI contrast-enhancing compound, which avoids phagocytosis by RES, a target-site-directed MRI contrast agent containing the compound, a tumor cell, and a Z cell. Another object of the present invention is to provide a liver imaging method.
発明の開示  Disclosure of the invention
本発明者らは上記課題に鑑み鋭意研究を重ねた結果、 M R I造影性化合物に、 腫瘍や臓器特異的に発現している糖タンパク質レセプターに対応する糖鎖高分子 として特定の臓器、 部位を特異的に認識する糖鎖高分子、 特にヒアルロン酸を選 択し結合させることにより該 M R I造影性化合物に腫瘍組織指向性およびノまた は特異的臓器指向性といった標的指向性を賦与することに成功し、 本発明を完成 するに到った。  The present inventors have conducted intensive studies in view of the above-mentioned problems, and as a result, MRI contrast-enhancing compounds have specific organs and sites as glycopolymers corresponding to glycoprotein receptors expressed specifically in tumors and organs. By selectively binding and binding to a sugar chain polymer, particularly hyaluronic acid, which has been successfully recognized, we succeeded in imparting the MRI-imaging compound with target directivity such as tumor tissue directivity and / or specific organ directivity. Thus, the present invention has been completed.
即ち、 本発明は以下の通りである。  That is, the present invention is as follows.
OM R I造影性化合物、 就中マグネタイ トを標的部位を特異的に認識する糖鎖高 分子にて修飾してなる化合物。 OMRI contrast-enhancing compound, especially a compound obtained by modifying magnetite with a sugar chain high molecule that specifically recognizes a target site.
2)糖鎖高分子がヒアルロン酸である上記 1)記載の化合物。  2) The compound according to 1) above, wherein the sugar chain polymer is hyaluronic acid.
3) M R I造影性化合物、 就中マグネタイ トを標的部位を特異的に認識する糖鎖高 分子にて修飾してなる化合物を含む標的部位指向性 M R I用造影剤。  3) Target site-directed contrast agents for MRI, including MRI contrasting compounds, especially compounds obtained by modifying magnetite with high sugar chain molecules that specifically recognize target sites.
4)糖鎖高分子がヒアルロン酸である上記 3)記載の標的部位指向性 M R I用造影剤 c 4) The target site-directed MRI contrast agent c according to 3) above, wherein the sugar chain polymer is hyaluronic acid
5)標的部位指向性が肝臓または腫瘍に対してである上記 4)記載の標的部位指向性 M R I用造影剤。 5) The contrast agent for target site-directed MRI according to 4) above, wherein the target site-directivity is for a liver or a tumor.
6) M R I造影性化合物、 就中マグネタイ トを標的部位を特異的に認識する糖鎖高 分子にて修飾することを特徴とする、 M R I造影性化合物を標的部位を特異的に 認識する糖鎖高分子にて修飾してなる化合物の製造方法。  6) MRI-enhancing compounds, particularly, magnets, are modified with high-sugar-chain molecules that specifically recognize the target site. A method for producing a compound modified with a molecule.
7)糖鎖高分子がヒアルロン酸である上記 6)記載の製造方法。  7) The production method according to the above 6), wherein the sugar chain polymer is hyaluronic acid.
8) M R I造影性化合物、 就中マグネタイ トを標的部位を特異的に認識する糖鎖高 分子にて修飾してなる化合物を含む標的部位指向性 M R I用造影剤を用いること を特徴とする MR I造影方法。 8) Use of target site-directed MRI contrast agents, including MRI contrasting compounds, especially compounds obtained by modifying magnetite with high molecular weight sugar chains that specifically recognize target sites MR I imaging method characterized by the following.
9)糖鎖高分子がヒアルロン酸である上記 8)記載の造影方法。  9) The imaging method according to 8) above, wherein the sugar chain polymer is hyaluronic acid.
10) MR I造影性化合物、 就中マグネタイ トをヒアルロン酸にて修飾してなる化 合物を含有する MR I用造影剤にて腫瘍細胞または肝臓を造影することによる腫 瘍細胞または肝臓の造影方法。  10) Imaging of tumor cells or liver by imaging tumor cells or liver with an MRI imaging agent that contains a compound that modifies MRI imaging compounds, especially magnetite with hyaluronic acid Method.
11) MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾して なる化合物、 および当該化合物を MR I造影に使用しうるかまたは使用すべきで あることを記載した書類を含む商業的パッケージ。  11) A compound obtained by modifying an MRI contrast-enhancing compound with a sugar chain polymer that specifically recognizes the target site, and a document stating that the compound can or should be used for MRI imaging Including commercial packages.
12) 標的部位指向性 MR I用造影剤の製造の為の、 MR I造影性化合物、 就中マ グネタイ 卜を標的部位を特異的に認識する糖鎖高分子にて修飾してなる化合物の 使用。  12) Use of MRI imaging compounds, especially compounds obtained by modifying magnetite with a sugar chain polymer that specifically recognizes the target site, for the production of a target site-directed MRI contrast agent .
13) 糖鎖高分子がヒアルロン酸である上記 12) 記載の使用。  13) The use according to 12) above, wherein the sugar chain polymer is hyaluronic acid.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
図 1 それぞれ F I TCで蛍光標識した HA— S P I O (1 aおよび 2 a) 、 P VLA— S P I O (l bおよび 2 b) および CDx— S P I O (1 cおよび 2 c) と E L 4細胞を 4 °Cで 2時間 （ 1 a、 1 bおよび 1 c ) あるいは 3 7 °Cで 6時間 (2 a, 2 bおよび 2 c) インキュベートした場合の細胞の蛍光強度と細胞数の 関係を示したヒス トグラムである。 実線 （一） は上記の蛍光標識した MR I用造 影剤存在下でィンキュベ一 トした場合の結果を、 破線 （一） は対照として蛍光標 識した MR I用造影剤を用いずにィンキュベ一トのみ行ったものである。 Figure 1 HA-SPIO (1a and 2a), P VLA-SPIO (lb and 2b) and CDx-SPIO (1c and 2c) and EL4 cells fluorescently labeled with FITC at 4 ° C. Histogram showing the relationship between cell fluorescence intensity and cell number when incubated for 2 hours (1a, 1b and 1c) or at 37 ° C for 6 hours (2a, 2b and 2c) . The solid line (1) shows the results when incubating in the presence of the fluorescently labeled MRI contrast agent described above, and the dashed line (1) shows the results without using the fluorescently labeled MRI contrast agent as a control. Only went to
図 2 各糖鎖高分子で標識した S P I 0の注入前、 および注入直後、 2 0分後、Figure 2 Before and immediately after injection of SPIO labeled with each sugar chain polymer, 20 minutes later,
2 4時間後の肝臓における MR Iの相対シグナル強度の変化を示した図である。 FIG. 9 is a view showing a change in relative signal intensity of MRI in the liver 24 hours later.
発明の詳細な説明  Detailed description of the invention
MR I用造影剤の造影機序は他の造影剤とは大きく異なっている。 例えば X線 造影剤では、 造影剤自体の X線吸収度が高いことが画像上の輝度に直接影響を与 えているのに対して、 MR I用造影剤では、 それ自体は直接描写されないで、 造 影剤が周囲のプロ トンの緩和現象を賦活して間接的に画像の輝度を上昇させたり 低下させたりすることによりコン トラス トをつけている。 M R I用造影剤として は T 1強調型造影剤および T 2強調型造影剤の 2種類が知られている。 T 1強調 型造影剤は陽性造影剤で、 画像上では造影剤の存在部位の輝度が上昇し白く光る T 2強調型造影剤は陰性造影剤で、 画像上では造影剤の存在部位の輝度が低下し 暗く写る。 The contrast mechanism of the MR I contrast agent is very different from other contrast agents. For example, in the case of an X-ray contrast agent, the high X-ray absorption of the contrast agent itself directly affects the brightness on the image, whereas the contrast agent for MRI is not directly depicted itself, The contrast agent activates the relaxation phenomenon of surrounding protons and indirectly increases the brightness of the image. Contrast is achieved by lowering the volume. Two types of contrast agents for MRI are known: T1-weighted contrast agents and T2-weighted contrast agents. The T1-weighted contrast medium is a positive contrast medium, and the brightness of the area where the contrast medium is present increases and glows white on the image.The T2-weighted contrast medium is the negative contrast medium and the brightness of the area where the contrast medium is present is low on the image. It drops and looks dark.
本発明においては通常用いられている MR I造影性化合物が使用できるが、 好 ましくはプロ トンの横緩和時間 （T 2 ) を短縮する T 2強調型造影剤であるマグ ネタイ 卜が用いられ、 具体的には超常磁性酸化鉄微粒子 （Superparamagnet i c I ron Ox i de : S P I 0 ) をデキストランの誘導体でコロイ ド化したものが用いら れる。  In the present invention, a commonly used MRI contrast-enhancing compound can be used, and preferably, magnetite, which is a T2-weighted contrast agent that shortens the transverse relaxation time (T2) of the proton, is used. Specifically, a superparamagnetic iron oxide fine particle (Superparamagnetic Iron Oxide: SPI0) colloided with a dextran derivative is used.
本発明において用いられる糖鎖高分子としては、 標的部位、 例えば腫瘍組織や 肝臓等の臓器を特異的に認識することができるものであれば特に限定はされない 力^ 具体的には肝類洞内皮細胞、 脾臓、 リンパ節および腫瘍組織に多く発現して いるヒアルロン酸レセプターを特異的に認識するヒアルロン酸ゃ肝実質細胞に多 く発現しているァシァロ糖タンパク質レセプターを特異的に認識するラク ト一ス 等が挙げられる。 さらに他の標的指向能を有する糖鎖高分子としては滕臓のグル コーストランスポーターを特異的に認識するグルコース結合ポリスチレン等が例 示される。 より好ましくは柔軟性が小さく、 しかも親水性が大きいために伸びた 直鎖構造をとりやすぃヒアル口ン酸が好ましい。 この様な親水性の大きな糖鎖高 分子を選択することにより、 その親水性鎖の流動力学的な特性をもってタンパク 質の吸着が抑制され、 R E Sの貪食作用を回避することができる。 即ち、 R E S の貪食作用による非特異的な取り込みを抑制し、 血管滞留性を向上させ、 腫瘍等 への取り込みを高めることができる。  The sugar chain polymer used in the present invention is not particularly limited as long as it can specifically recognize a target site, for example, an organ such as a tumor tissue or a liver. Hyaluronan that specifically recognizes the hyaluronan receptor that is highly expressed in cells, spleen, lymph nodes, and tumor tissues. And the like. Further, as another sugar chain polymer having a targeting ability, glucose-binding polystyrene or the like that specifically recognizes a glucose transporter from Tengen is exemplified. More preferably, it has a low flexibility and has a high hydrophilicity, so that it tends to have an extended linear structure. By selecting such a high-molecular sugar chain having a high hydrophilicity, the adsorption of the protein is suppressed with the hydrodynamic properties of the hydrophilic chain, and the phagocytic action of RES can be avoided. That is, nonspecific uptake of RES by phagocytosis can be suppressed, vascular retention can be improved, and uptake into tumors and the like can be increased.
また、 ヒアルロン酸はそのグルク口ン酸残基にカルボキシル基を有するため強 く負に帯電している。  Hyaluronic acid is strongly negatively charged because it has a carboxyl group in its glucanoic acid residue.
これらの糖鎖高分子で M R I造影性化合物を修飾する方法としては、 イオン結 合により両者を結合させる方法が挙げられる。 具体的には用いる糖鎖高分子によ つて条件が異なるが、 例えばヒアルロン酸で S P I 0を修飾する場合、 ヒアルロ ン酸水溶液に塩化第 2鉄および塩化第 3鉄を添加し 6 0〜 1 0 0 °C、 好ましくは 8 0 °C程度で攪拌、 水酸化ナトリウム溶液を添加した後 p Hを中和することによ り行う。 さらに精製し純度を高める為に限外濾過処理を行うことがより好ましい c 糖鎖高分子と M R I造影性化合物の結合比は使用する糖鎖高分子および M R I 造影性化合物の種類によって異なるが、 例えばマグネタイ 卜を使用する場合、 マ グネタイ ト 1重量部に対して糖鎖高分子は、 ヒアルロン酸であれば 0 . 2〜5重 量部、 好ましくは 1重量部、 ラク トースであれば 0 . 2〜5重量部、 好ましくは 1重量部である。 Examples of a method for modifying an MRI contrast-enhancing compound with these sugar chain polymers include a method in which the two are bonded by ionic bonding. Specifically, depending on the sugar chain polymer used For example, when SPI0 is modified with hyaluronic acid, ferric chloride and ferric chloride are added to an aqueous solution of hyaluronic acid to add 60 to 100 ° C, preferably about 80 ° C. The reaction is carried out by adding sodium hydroxide solution and neutralizing the pH. It is more preferable to perform ultrafiltration treatment to further purify and increase the purity.The binding ratio between the c- glycan polymer and the MRI imaging compound varies depending on the type of the glycan polymer and the MRI imaging compound used. When magnetite is used, the sugar chain polymer is 0.2 to 5 parts by weight for hyaluronic acid, preferably 1 part by weight, and 0.2 for lactose per 1 part by weight of magnetite. To 5 parts by weight, preferably 1 part by weight.
より特異的に標的とする臓器や腫瘍組織に MR I造影性化合物を送達させるた めには、 さらに本発明の M R I用造影剤に親水性高分子を結合させることが好ま しい。 使用できる親水性高分子は上記の特性を有するものであれば特に限定はさ れないが具体的にはポリエチレングリコール、 ポリ ビニルアルコ一ル、 ポリ ビニ ルビロリ ドン等が挙げられる。 これらの親水性高分子は上記の糖鎖高分子と M R I造影性化合物との結合と同様の手法を用いて結合させることができる。  In order to more specifically deliver the MRI contrast-enhancing compound to a target organ or tumor tissue, it is preferable to further bind a hydrophilic polymer to the MRI contrast agent of the present invention. The hydrophilic polymer that can be used is not particularly limited as long as it has the above-mentioned characteristics, but specific examples include polyethylene glycol, polyvinyl alcohol, and polyvinylolidone. These hydrophilic polymers can be bound using the same method as the above-mentioned method for binding the sugar chain polymer to the MRI contrast-enhancing compound.
本発明の M R I用造影剤としては、 M R I造影性化合物を標的部位を特異的に 認識する糖鎖高分子にて修飾してなる化合物を含んでし、れば特に限定されない。 具体的には本発明の化合物、 本発明の化合物を注射用蒸留水、 生理食塩水やリ ン ゲル液等の溶媒に懸濁、 または溶解したもの等が挙げられ、 さらに必要に応じて、 薬理学的に許容されうる担体、 賦形剤等の添加剤を含めることができる。 当該造 影剤は、 細胞などに適用しうる他、 血管 （静脈、 動脈） 内投与、 経口投与、 直腸 内投与、 膣内投与、 リンパ管内投与、 関節内投与等によって生体内に投与するこ とができ、 好ましくは、 水剤、 乳剤または懸濁液等の形態で投与する。 本発明の M R I造影剤に含められる添加剤としては、 その投与形態、 投与経路等によって も異なるが具体的には、 注射剤の場合には、 緩衝剤、 抗菌剤、 安定化剤、 溶解補 助剤や陚形剤等が単独または組み合わせて用いられ、 経口投与剤 （具体的には水 剤、 シロップ剤、 乳剤または懸濁液等） の場合、 着色剤、 保存剤、 安定化剤、 懸 濁化剤、 乳化剤、 粘稠剤、 甘味剤、 芳香剤等が単独または組み合わせて用いられ る。 各種添加剤は通常当分野で用いられるものが使用される。 The contrast agent for MRI of the present invention is not particularly limited as long as it includes a compound obtained by modifying an MRI contrast-enhancing compound with a sugar chain polymer that specifically recognizes a target site. Specific examples thereof include a compound of the present invention, and a compound of the present invention suspended or dissolved in a solvent such as distilled water for injection, physiological saline, or Ringer's solution. Additives such as physically acceptable carriers and excipients can be included. The contrast agent can be applied to cells, etc. and administered to the living body by intravascular (venous or arterial) administration, oral administration, rectal administration, vaginal administration, lymphatic administration, intraarticular administration, etc. It is preferably administered in the form of a solution, emulsion or suspension. Additives included in the MRI contrast agent of the present invention vary depending on the administration form, administration route, etc. Specifically, in the case of an injection, a buffer, an antibacterial agent, a stabilizer, a dissolution aid Preparations and vehicles are used alone or in combination. For oral administration (specifically, solutions, syrups, emulsions or suspensions, etc.), coloring agents, preservatives, stabilizers, A turbidity agent, an emulsifier, a thickener, a sweetener, a fragrance and the like are used alone or in combination. As the various additives, those usually used in the art are used.
本発明の造影剤は標的部位指向性であるので、 所望の細胞や組織、 臓器、 特に 修飾に用いる糖鎖高分子としてヒアルロン酸を選択した場合には肝臓や腫瘍細胞 を特異的に造影することが可能となる。 さらにヒアル口ン酸を結合させることに より、 ヒアルロン酸が本来有する関節等の緩衝剤成分としての機能を本造影剤に 付与することができる。 即ち、 本造影剤は、 関節等に直接投与することによりリ ゥマチ等の関節疾患の診断に好適なものとなる。  Since the contrast agent of the present invention is directed to a target site, it is necessary to specifically image liver and tumor cells when hyaluronic acid is selected as a desired cell, tissue, or organ, particularly as a sugar chain polymer used for modification. Becomes possible. Furthermore, by binding hyaluronic acid, the function as a buffer component of joints and the like which hyaluronic acid originally has can be imparted to the present contrast agent. That is, when the present contrast agent is directly administered to a joint or the like, it becomes suitable for diagnosis of a joint disease such as rheumatism.
本発明の M R I用造影剤の投与量は、 例えば本発明の化合物を投与する場合に は、 従来の M R I用造影剤に準じて決定され、 投与される対象の年齢や身体の大 きさおよび標的とする臓器等によつて適宜増減できるが通常中に含まれる M R I 造影性化合物の量、 マグネタイ トであれば鉄 （F e ) の量として 1 0 ~ 1 0 0〃 m o 1 F e k g、 好ましくは 1 0〜2 0 /z m o 1 F e / k gの範囲で選択され る。  The dose of the MRI contrast agent of the present invention, for example, when the compound of the present invention is administered, is determined according to the conventional MRI contrast agent, and the age, body size and target of the subject to be administered are determined. The amount of the MRI contrast-enhancing compound that can be increased or decreased as appropriate depending on the organ to be used, or the amount of iron (F e) in the case of magnetite is 10 to 100〃mo 1 F ekg, preferably It is selected in the range of 10 to 20 / zmo 1 Fe / kg.
また、 本発明の造影剤はヒ 卜以外にも各種動物用の造影剤としても好適に用い ることができ、 その投与形態、 投与経路、 投与量等は対象となる動物の体重や状 態によって適宜選択する。  In addition, the contrast agent of the present invention can be suitably used as a contrast agent for various animals in addition to humans. The administration form, administration route, dose, etc., depend on the weight and condition of the target animal. Select as appropriate.
さらに、 本発明の化合物ならびに M R I用造影剤は、 腫瘍細胞や肝臓の類洞内 皮細胞に特異的に発現しているヒアルロン酸レセプタ— （C D 4 4等） の特異的 な検出への利用が期待される。  Furthermore, the compound of the present invention and the contrast medium for MRI can be used for specific detection of a hyaluronic acid receptor (such as CD44) specifically expressed in tumor cells and sinusoidal endothelial cells of the liver. Be expected.
実施例 Example
以下に本発明をより詳細に説明するため実施例および実験例を示すが本発明は これらによりなんら限定されるものではない。  Hereinafter, examples and experimental examples will be described in order to explain the present invention in more detail, but the present invention is not limited thereto.
実施例 1 各種糖鎖高分子結合マグネタィ トの調製 Example 1 Preparation of various sugar chain polymer-bound magnetites
( 1 ) 糖鎖高分子の調製  (1) Preparation of sugar chain polymer
i ) ヒアルロン酸  i) Hyaluronic acid
ヒアルロン酸は肝類洞内皮細胞、 脾臓、 リ ンパ節および腫瘍組織に多く発現し ているヒアルロン酸レセプタ一に特異的に認識されることが知られている。 ヒァ ル口ン酸 （電気化学工業社製、 平均分子量 5 9 0, 0 0 0 ) 9 gを 9 0 0 m lの 水に溶解し、 ヒアルロニダーゼ （シグマ社製） 1 8 Omgを加え、 5 0°Cで 3時 間酵素分解した。 その後、 1 0 0°Cで 1 0分間煮沸し脱タンパクさせた。 反応液 を水冷後濾過し、 タンパク質を除去した。 濾液を凍結乾燥し、 平均分子量約 8, 0 0 0のヒアルロン酸を得た。 Hyaluronic acid is highly expressed on liver sinusoidal endothelial cells, spleen, lymph nodes and tumor tissue. It is known that it is specifically recognized by a given hyaluronic acid receptor. 9 g of hyaluronic acid (manufactured by Denki Kagaku Kogyo Co., Ltd., average molecular weight: 590,000) is dissolved in 900 ml of water, and 180 mg of hyaluronidase (manufactured by Sigma) is added, and 50 ° The enzyme was digested with C for 3 hours. Thereafter, the mixture was boiled at 100 ° C. for 10 minutes to deproteinize. The reaction solution was cooled with water and filtered to remove proteins. The filtrate was freeze-dried to obtain hyaluronic acid having an average molecular weight of about 8,000.
ii) P VL A  ii) P VL A
ポリービニノレべンジノレーラク トンアミ ド (poly-vinylbenzyl-lactonamide; P V L A) は非還元型ガラク ト一スを末端に持つラク トースを有するポリスチレン 誘導体の合成高分子であり、 肝実質細胞中のァシァ口糖タンパク質レセプターに 特異的に認識され取り込まれる。  Polyvinylvinyl-lactonamide (PVLA) is a synthetic polymer of non-reduced galactose-terminated lactose-derived polystyrene derivatives. It is specifically recognized and incorporated.
ビニルベンジルーラク トンアミ ド （VLA) 5 mM (2. 3 7 1 5 6 m 1 DMS O) を重合管中に入れ、 重合開始剤としてァゾビスイソプチロニト リル (A I B N) 0. 0 1 mM ( 1. 6 4 2 mg) を加え、 真空融封し 6 0 °Cで 1 2 時間反応させ重合させた。 反応液をエタノール中に滴下し、 沈澱物を回収した。 回収した沈澱物を水に溶解し透析 （分画分子量 3， 5 0 0 ) した後、 凍結乾燥し て P V L Aを得た。  Vinyl benzyl-lactone amide (VLA) 5 mM (2.3715.6 m1 DMS O) is placed in a polymerization tube, and azobisisobutyronitrile (AIBN) 0.01 mM as a polymerization initiator (1.642 mg) was added, vacuum sealed, and reacted at 60 ° C for 12 hours to polymerize. The reaction solution was dropped into ethanol, and the precipitate was collected. The recovered precipitate was dissolved in water, dialyzed (fraction molecular weight: 3,500), and lyophilized to obtain PVLA.
iii ) カルボキシデキス トラン （CDx)  iii) Carboxydextran (CDx)
カルボキシデキストランは肝臓のクッパー細胞より非特異的に取り込まれる。 本実施例においては名糖産業社製のものを用いた。  Carboxydextran is non-specifically taken up by Kupffer cells of the liver. In this example, a product manufactured by Meito Sangyo Co., Ltd. was used.
(2) 糖鎖とマグネタイ 卜との結合  (2) Binding of sugar chains to magnetite
マグネタイ 卜を上記 （ 1 ) で得られた各種糖鎖でコ一ティ ングした。  The magnetite was coated with the various sugar chains obtained in (1) above.
) ヒアルロン酸マグネタイ ト  ) Hyaluronic acid magnetite
上記 （ 1 ) の i ) で得られたヒアルロン酸 2. 1 1 gを 2 5 m 1の水に溶解し， 窒素パージし、 F e C l 2 - 4 H 2 O O. 1 7 9 gおよび l MF e C l 3 1. 8 6 m lを加え、 8 0。Cで 2 0分間攪拌する。 3 N Na OH 4 m lを加え、 8 0 °Cで 1 0分間攪拌し、 I N HC 1で中和し、 2時間還流させ、 ヒアルロン酸マ グネタイ 卜中間体を得た。 得られた中間体を限外濾過 （分画分子量 2 0 kD a) し、 ヒアルロン酸マグネタイ トを得た。 The hyaluronic acid 2. 1 1 g obtained in i) of (1) was dissolved in 2 of 5 m 1 water, purged with nitrogen, F e C l 2 - 4 H 2 O O. 1 7 9 g and l MF e C l 3 1. Add 86 ml and add 80. Stir at C for 20 minutes. Add 4 ml of 3 N NaOH, stir at 80 ° C for 10 minutes, neutralize with IN HC1, reflux for 2 hours, A gnetite intermediate was obtained. The obtained intermediate was subjected to ultrafiltration (fraction molecular weight: 20 kDa) to obtain hyaluronic acid magnetite.
b ) P V L Aマグネタイ ト  b) PVLA magnetite
上記 （ 1 ) の ii) で得られた PVL Aの水溶液 （5 0 mgノ 4 Om l ) にマグ ネタイ ト懸濁液 （6. 2 5 mgF e/ l Om l ) を滴下し、 攪拌後、 限外濾過 (分画分子量 2 0 k D a) して PVL Aマグネタイ トを得た。  A magnetite suspension (6.25 mg Fe / l Oml) was added dropwise to the aqueous solution (50 mg of 4 Oml) of PVL A obtained in ii) of (1) above, and after stirring, Ultrafiltration (fraction molecular weight: 20 kDa) gave PVL A magnetite.
c ) C D Xマグネ夕イ ト  c) C D X Magne evening
CDx 8 gを 1 5m lの水に溶解し、 上記 a ) のヒアル口ン酸の場合と同様に して CD xとマグネタイ トを結合させ CD Xマグネタイ トを得た。  8 g of CDx was dissolved in 15 ml of water, and CDx and magnetite were bound in the same manner as in a) above in the case of hyaluronic acid to obtain CDX magnetite.
実験例 1 各種糖鎖高分子結合マグネタィ トの物理化学的特性 Experimental Example 1 Physicochemical properties of various sugar-chain polymer-bound magnets
実施例 1で調製したヒアルロン酸を結合させた S P I O (HA— S P I O) 、 PVL Aを結合させた S P I 0 (PVLA— S P I O) および CDxを結合させ た S P I O (CDx— S P I O) の物理化学的特性として、 MR I造影能として の鉄の定量、 糖鎖高分子濃度、 動的光散乱 （dynamic light scattering： DL S) 粒子径、 Γ電位および磁気天秤について調べた。 鉄の定量として、 各糖鎖高分子 結合 S P I O溶液の鉄濃度を 1 %ο—フヱナンスロ リン試液による UV 4 2 0 η mの比色定量により算出した。 DL S粒子径は各糖鎖高分子結合 S P I 0溶液の 1 0 0倍希釈溶液について、 次の条件で測定した。  The physicochemical properties of SPIO (HA-SPIO) conjugated with hyaluronic acid, SPI0 (PVLA-SPIO) conjugated with PVL A, and SPIO (CDx-SPIO) conjugated with CDx prepared in Example 1 Quantitative determination of iron as MRI contrast ability, sugar chain polymer concentration, dynamic light scattering (DLS) particle size, zeta potential and magnetic balance were investigated. For iron quantification, the iron concentration of each sugar chain polymer-bound SPIO solution was calculated by colorimetric quantification of UV 420 ηm with a 1% o-pentanothroline reagent solution. The DLS particle diameter was measured on a 100-fold diluted solution of each sugar chain polymer-bound SPI0 solution under the following conditions.
測定条件：  Measurement condition:
測定機器： PC S 4 7 0 0 (Ma l v e r n社製）  Measuring equipment: PC S4700 (Malvern)
測定温度： 2 5 °C  Measurement temperature: 25 ° C
測定角度： 9 0 °  Measuring angle: 90 °
測定波長： 5 1 4 n m  Measurement wavelength: 5 1 4 nm
S P I 0の粒子径は、 透過型電子顕微鏡観察により算出した。  The particle size of SPIO was calculated by observation with a transmission electron microscope.
糖鎖高分子濃度は、 ！！八と 八の場合は I T Cで蛍光標識した各糖鎖の 蛍光強度から算出し、 また CD Xの場合はアンスロンによる波長 6 3 0 nm (V I S) での比色定量より求めた。 電位は各糖鎖高分子結合 S P I 0溶液の 1 0 0倍希釈溶液の pHを約 7に調整して、 次の条件で測定した。 The sugar chain polymer concentration is! ! In the case of (8) and (8), it was calculated from the fluorescence intensity of each sugar chain fluorescently labeled with ITC, and in the case of CDX, it was determined by colorimetry at a wavelength of 630 nm (VIS) using anthrone. The electric potential is 10 The pH of the 0-fold diluted solution was adjusted to about 7, and measured under the following conditions.
測定条件：  Measurement condition:
測定機器： ゼータマスター粒子電気泳動分析器 （Ma 1 V e r n社製） 測定温度： 2 5 °C  Measuring device: Zeta master particle electrophoresis analyzer (Ma 1V ern) Measuring temperature: 25 ° C
測定時間 ： 2 0 s e c  Measurement time: 20 sec
レーザー ： 5 mW H e N eレーザー、 6 3 3 nm  Laser: 5 mW HeNe laser, 63.3 nm
磁気天秤は簡易型磁気天秤 MS B MK 1 (F o h n s o n Ma t t h e y 社製） を用い標準試料 （N 1 0 5) を対照に測定した。 以上の結果を表 1に示す c 尚、 表中の p Hは該糖鎖高分子を結合したマグネタイ トを調製した時の pHを示 している。 The magnetic balance was measured using a simple magnetic balance MS B MK1 (Fohnson Matthey) and a standard sample (N105) as a control. Note c The results are shown in Table 1, p H in the table are shown the pH of the preparation of the Magunetai bets that combines sugar chain macromolecule.
(mg/ml) 粒子サイズ ポテンシャル x (mg / ml) Particle size Potential x
铁 議高好 (nm) (mV) c.g.sxi06 PH铁 Mitaka (nm) (mV) cgsxi0 6 PH
SP I 0 12. 5 5. 1 35. 6 28353 6. 5SP I 0 12.5 5. 1 35.6 28 353 6.5
HA-SP I 0 , 20. 0 60. 0 48. 6 -46. 3 20798 7. 0HA-SP I 0, 20.0 60.0 48.6 -46.3 20798 7.0
PVLA-SP 10 25. 0 54. 3 155. 2 7. 1 29079 7. 0PVLA-SP 10 25.0 54.3 155.2 7.1 29079 7.0
CDX 一 SP I 0 28. 5 30. 3 55. 9 —49. 4 29717 6. 5 χ i (magnetic susceptibility balance、 EMU/g F e) CD X- SPI 0 28.5 30.3 55.9 —49.4 29717 6.5 χ i (magnetic susceptibility balance, EMU / g Fe)
S P I Oの粒子径は約 5 nmであるのに対し、 糖鎖高分子を結合させた S P I 0の D L S粒子径は約 5 0から I 5 0 nmと観察された。 また、 電位の値にお いて S P I 0のみの場合では強い正電位 （約 3 5 mV) を示していたものが、 糖 鎖高分子と結合させることにより、 例えば PVL Aの場合は弱い正電位 （7 mV) に、 ヒアルロン酸や CD Xに到っては強い負電位 （約一 5 0 mV) へと変化した ことは、 中性または負電位の糖鎖高分子が結合したことが推察される。 一方、 磁 気天枰の値から、 糖鎖高分子の結合に関わらずいずれも約 2万から 3万の強い磁 性を有することが示され MR I造影性を保持していることが確認された。 While the particle diameter of SPIO was about 5 nm, the DLS particle diameter of SPIO0 to which the sugar chain polymer was bound was observed to be about 50 to 50 nm. Also, the potential value showed a strong positive potential (approximately 35 mV) in the case of SPI 0 alone, but by binding to a glycan polymer, for example, in the case of PVL A, a weak positive potential (about 35 mV) was obtained. (7 mV), the hyaluronic acid and CDX changed to a strong negative potential (about 150 mV), suggesting that a neutral or negative potential sugar chain polymer was bound. . On the other hand, the value of the magnetic field shows that each of them has a strong magnetic property of about 20,000 to 30,000 regardless of the binding of the sugar chain polymer, confirming that the MRI contrast property is maintained. Was.
実験例 2 各種糖鎖高分子結合マグネタイ 卜の in vitroでの標的指向性 Experimental example 2 Target directivity of various sugar-chain polymer-bound magnetites in vitro
標的指向性の指標として各糖鎖高分子結合マグネタイ 卜における in V roでの 細胞結合ァッセィおよびェンドサイ トーシスァッセィを行った。  As an indicator of target directivity, cell binding assays and endcytosis assays were performed in vitro on each sugar chain polymer-bound magnetite.
( 1 ) 標的細胞の調製  (1) Preparation of target cells
標的細胞としては、 その細胞膜表面にヒアルロン酸レセプターを発現している ことが知られている E L 4細胞 （マウスリンパ腫由来細胞株） を用いた。 E L 4 細胞は熱で非働化したゥシ胎児血清を 1 0 %添加した R PM 1 - 1 6 4 0培地で、 3 7°C、 5 %C O2 条件下で継代培養したものを用いた。 As target cells, EL4 cells (a mouse lymphoma-derived cell line) that are known to express the hyaluronic acid receptor on the cell membrane surface were used. EL 4 cells were subcultured at 37 ° C and 5% CO 2 in RPM 1-1640 medium supplemented with 10% fetal calf serum inactivated by heat. .
( 2 ) 糖鎖高分子結合 S P I 0の蛍光標識  (2) Fluorescent labeling of sugar chain polymer-bound SPIO
i ) 糖鎖高分子の蛍光標識  i) Fluorescent labeling of sugar chain polymer
①ヒアルロン酸の F I TC標識化  ①FITC labeling of hyaluronic acid
実施例 1の （ 1 ) の i ) で調製したヒアルロン酸 （HA) 5 5 0 mgを 1 5 m 1のホノレムアミ ドに溶解した。 該溶液に F I TC (Fluorescein isothiocyanate; D o j i n社製） 2 5 0 mg、 ジブチルチンジラウレート 1 0 0 mgおよび炭酸 水素ナトリウム 1 0 O mgを 1 5 m lのジメチルスルホキシド （DMS 0) に溶 解した溶液を添加し、 9 5。Cで 2 0分間攪拌混合する。 混合液をエタノール中に 滴下、 さらに数滴の飽和塩化ナトリゥム水溶液を滴下することにより沈澱を生じ させる。 得られた沈澱をホルムアミ ドに溶解し、 再び該溶液をエタノール中で沈 澱させ、 この操作を合計 5回橾り返す。 最終的に得られた沈澱を減圧乾燥した後、 水に再溶解し、 2 mの孔サイズのセルロースアセテート膜で濾過してごみ 等の不溶物の除去操作を行った。 濾液をー晚かけて凍結乾燥して、 F I TC標識 した H Aを得た 〔収量 5 3 1 mg (収率 6 6 %) 〕 。 （この条件下で標識すると 得られた檩識化合物は H A 1 0 0繰り返し構造単位あたり 0. 6 mo 1の F I T Cを含有することになる。 ） 550 mg of hyaluronic acid (HA) prepared in i) of (1) of Example 1 was dissolved in 15 ml of honolemamide. A solution prepared by dissolving 250 mg of FITC (Fluorescein isothiocyanate; manufactured by Dojin), 100 mg of dibutyltin dilaurate and 100 mg of sodium hydrogen carbonate in 15 ml of dimethyl sulfoxide (DMS 0) Add 9 5. Stir and mix with C for 20 minutes. The mixture is dropped into ethanol, and a few drops of a saturated aqueous sodium chloride solution are further dropped to cause precipitation. The obtained precipitate is dissolved in formamide, and the solution is again precipitated in ethanol. This operation is repeated a total of 5 times. After drying the finally obtained precipitate under reduced pressure, It was redissolved in water and filtered through a cellulose acetate membrane with a pore size of 2 m to remove insolubles such as dust. The filtrate was freeze-dried under reduced pressure to obtain FITC-labeled HA [yield 531 mg (yield 66%)]. (The labeling compound obtained under labeling under these conditions contains 0.6 mol of FITC per HA 100 repeating structural unit.)
② P VL Aの F I TC標識化  ② F ITC labeling of PVL A
実施例 1の （ 1 ) の ii) で調製した PVL A 3 0 0111 を 3111】 の0]^50に 溶解した。 該溶液に F I TC 3 0 mgおよびジブチルチンジラウレート 9mgを The PVL A 3 0111 prepared in (1) ii) of Example 1 was dissolved in 0] ^ 50 of 3111]. 30 mg of F ITC and 9 mg of dibutyltin dilaurate were added to the solution.
2 m 1の DM S 0に溶解した溶液を添加し、 9 0 °Cで 2時間攪拌混合する。 混合 液をェタノール中に滴下することにより沈澱を生じさせる。 得られた沈澱を DMA solution dissolved in 2 ml of DMSO is added, and the mixture is stirred and mixed at 90 ° C for 2 hours. The mixture is dropped into ethanol to cause precipitation. The resulting precipitate is DM
SOに溶解し、 再び該溶液をエタノール中で沈澱させ、 この操作を合計 5回繰り 返す。 最終的に得られた沈澱を減圧乾燥した後、 水に再溶解し、 0. 2 ^mの孔 サイズのセルロースァセテ一ト膜で濾過する。 濾液をー晚かけて凍結乾燥して、Dissolve in SO, precipitate the solution again in ethanol, and repeat this operation a total of 5 times. The finally obtained precipitate is dried under reduced pressure, redissolved in water, and filtered through a cellulose acetate membrane having a pore size of 0.2 m. The filtrate is freeze-dried over
F I丁じ標識した し八を得た 〔収量 2 4 5 mg (収率 7 4 %) 〕 。 （この条 件下で標識すると得られた標識化合物は PVL A 1 0 0繰り返し構造単位あたりFI-labeled shihachi was obtained [yield 24.5 mg (74% yield)]. (The labeling compound obtained by labeling under these conditions will have a
1. 5 mo 1の F I TCを含有することになる。 ） It will contain 1.5 mo 1 of F ITC. )
③ CD Xの F I TC標識化  ③ F ITC labeling of CD X
C D X 3 0 Omgを 5m lの DM SOに溶解した。 該溶液に F I T C 3 0 m g およびジブチルチンジラウレー ト 9 m gを 0. 5m lの DM S Oに溶解した溶液 を添加し、 9 0 °Cで 1時間攪拌混合する。 混合液をエタノール中に滴下すること により沈澱を生じさせる。 得られた沈澱を DMS 0に溶解し、 再び該溶液をエタ ノール中に滴下、 さらに数滴の飽和塩化ナトリゥム水溶液を滴下することにより 沈澱を生じさせる。 この操作を合計 3回繰り返す。 最終的に得られた沈澱を減圧 乾燥した後、 水に再溶解し、 0. 2〃mの孔サイズのセルロースアセテート膜で 濾過する。 濾液を一晚かけて凍結乾燥して、 F I T C標識した CD Xを得た 〔収 量 2 1 4 mg (収率 6 5 %) 〕 。 （この条件下で標識すると得られた標識化合物 はグルコース 1 0 0繰り返し構造単位あたり 0. 9 mo 1の F I TCを含有する ことになる。 ） CDX 30 Omg was dissolved in 5 ml of DMSO. A solution prepared by dissolving 30 mg of FITC and 9 mg of dibutyltin dilaurate in 0.5 ml of DMSO is added to the solution, and the mixture is stirred and mixed at 90 ° C for 1 hour. The mixture is dropped into ethanol to cause precipitation. The resulting precipitate is dissolved in DMS0, the solution is again dropped into ethanol, and a few drops of a saturated aqueous sodium chloride solution are dropped to form a precipitate. Repeat this operation three times. The finally obtained precipitate is dried under reduced pressure, redissolved in water, and filtered through a cellulose acetate membrane having a pore size of 0.2 μm. The filtrate was freeze-dried over a period to obtain FITC-labeled CDX [yield: 21.4 mg (yield: 65%)]. (The labeled compound obtained under labeling under these conditions contains 0.9 mol of FITC per 100 glucose repeating structural units. Will be. )
ii) 蛍光標識された糖鎖高分子と S P I Oとの結合  ii) Binding of fluorescently labeled sugar chain polymer to SPIO
上記 i ) で得られた F I TC標識した糖鎖高分子の水溶液 （5 0 mg//4 0 m 1 ) に S P I O溶液 (6. 2 S mg F e/ l O m l ) を滴下し攪拌した。 得られ た混合液を限外濾過 （分画分子量 5 k D a ) して濃縮し、 該濃縮液を 0. 2 jum のセルロースァセテ一卜膜で濾過し、 各 F I T C標識された糖鎖高分子結合 S PWas stirred dropwise SPIO solution (6. 2 S mg F e / l O ml) in an aqueous solution of FI TC labeled sugar polymer obtained (5 0 mg / / 4 0 m 1) above i). The resulting mixture was concentrated by ultrafiltration (molecular weight cut off: 5 kDa), and the concentrated solution was filtered through a 0.2 jum cellulose acetate membrane to obtain a FITC-labeled sugar chain. Molecular bond SP
1 0を得た。 10 was obtained.
( 3 ) 結合およびエンドサイ ト一シスアツセィ  (3) Coupling and end-site access
E L 4細胞における各糖鎖高分子結合 S P I 0の 4 °Cでの細胞表面への接着 (結合） 特性および 3 7 °Cでの細胞内への取り込み （エンドサイ ト一シス） 特性 をフローサイ トメ トリー法により検討した。 上記 （ 1 ) で準備した E L 4細胞を 3 5 mm径の培養皿に 1 x 1 0 6 個の細胞数で撒きこんだ。 撒きこみ後 3 Ί。CでFlow cytometry of the adhesion (binding) characteristics of SPI0 to the cell surface at 4 ° C and the uptake into cells (end-site lysis) at 37 ° C at 4 ° C in EL 4 cells It was examined by the method. Yelling plated at 1 x 1 0 6 single cell number in a culture dish of 3 5 mm diameter EL 4 cells prepared in the above (1). 3 後 after spraying. In C
2 4時間培養した後、 まず 4 °Cで 1 0分間細胞をプレイ ンキュベーショ ンした。 続いて 1 0 0 gの F I T C標識した糖鎮高分子結合 S P I 0を各々培養液中に 添加し 4。 で 2時間ィンキュベー卜あるいは 3 7 で 6時間ィンキュベートした c 洗浄後、 細胞表面に結合した F I T C標識された糖鎖高分子結合 S P I 0および 細胞によって取り込まれた F I T C標識された糖鎖高分子結合 S P I 0をフロー サイ トメ一夕一 （Cyto ACE- 150, J a s c o社製） で解析した。 結果を図 1に示 す。 さらにこれらの細胞を共焦点レーザー顕微鏡 （DMR、 ライカ社製） で観察 した。 After culturing for 24 hours, the cells were first pre-incubated at 4 ° C for 10 minutes. Subsequently, 100 g of SPI0 labeled with FITC-labeled glucoside polymer was added to each culture solution4. Incubate for 2 hours at 37 ° C or incubate at 37 ° C for 6 hours, wash, and then wash FITC-labeled glycan-polymer-bound SPI 0 bound to cell surface and FITC-labeled glycan-bound SPI 0 taken up by cells Was analyzed using a flow cytometer (Cyto ACE-150, manufactured by Jasco). The results are shown in Figure 1. Furthermore, these cells were observed with a confocal laser microscope (DMR, manufactured by Leica).
図 1にも示される様に E L 4細胞は HA— S P I 0を添加し、 4 °Cおよび 3 7 °Cで培養した場合のみに蛍光強度の增加が観察された。 一方、 PVL A— S P I 0および C D X— S P I Oを E L 4細胞に添加した場合には 4 °Cおよび 3 7ての いずれの温度で培養した場合にも蛍光強度の增加は認められなかつた。  As shown also in FIG. 1, the increase in the fluorescence intensity was observed only when the EL4 cells were cultured at 4 ° C. and at 37 ° C. after addition of HA—SPI0. On the other hand, when PVL A-SPIO and CDX-SPIO were added to the EL4 cells, no increase in the fluorescence intensity was observed when the cells were cultured at any of 4 ° C and 37 ° C.
また、 共焦点レーザー顕微鏡における E L 4細胞内部への H A— S P 1 0の取 り込みの様子を観察すると、 細胞中心部に強い蛍光強度が見られ、 細胞内部に蛍 光標識した H A— S P I 0が取り込まれていることを確認した。 HA- S P I Oが E L 4細胞の表面に結合しさらにェンドサイ 卜一シスによつ て取り込まれたことから H A— S P I 0の E L 4細胞への指向性が示唆された。 一方 H A以外の糖鎖、 即ち PVL Aおよび CD Xを結合させたものについては有 意な結合もェン ドサイ トーシスも観察されなかった。 これらの結果より HA— S P I 0は H Aレセプタ一を特異的に認識する、 即ち標的部位指向性を有する造影 剤であることが確認された。 Observation of the incorporation of HA-SP10 into EL4 cells using a confocal laser microscope revealed that strong fluorescence intensity was observed in the center of the cell, and that HA-SPI0 was labeled inside the cell. Has been captured. HA-SPIO bound to the surface of EL4 cells and was taken up by end-site lysis, suggesting the directivity of HA-SPI0 to EL4 cells. On the other hand, no significant binding or end-cytosis was observed for sugar chains other than HA, ie, those linked to PVLA and CDX. From these results, it was confirmed that HA-SPI0 specifically recognizes the HA receptor, that is, it is a contrast agent having target site directivity.
実験例 3 各種糖鎖結合マグネタイ トの in vivo での標的指向性 Experimental Example 3 Target directivity of various sugar chain-bound magnetites in vivo
( 1 ) MR I造影  (1) MR I imaging
C BHZH eマウスを 4 0 m g/k gのペントバルビタールナ卜リゥムを腹腔 内投与して麻酔、 バタフライ針で尾静脈から MR I用造影剤を注入し、 磁場の中 央に置いた。 MR I用造影剤には実施例 1で調製した HA— S P I 0、 PVLA — S P I 0および C D X - S P I 0を用い、 それぞれ含まれる F eの濃度が 3 0 umo 1 /k になるように用いた。  CBHZH e mice were anesthetized with 40 mg / kg of pentobarbital sodium administered intraperitoneally, a contrast agent for MRI was injected from the tail vein with a butterfly needle, and placed in the center of the magnetic field. HA-SPI0, PVLA-SPI0 and CDX-SPI0 prepared in Example 1 were used as contrast agents for MR I, and the concentrations of Fe contained in each were adjusted to 30 umo 1 / k. .
4. 7 T a n i m a l i m a g e r (Om e g a C S I — 2， GE - B r u k e r社製） を用いて MR I造影を行った。 MR I画像は T 1および T 2 の合成一 W I CTR/T E = 6 0 0 /2 4. 5 m s ) を用いて得られた。  MRI imaging was performed using 4.7 Tanimalimager (Omega s CSI-2, manufactured by GE-Bruker). MRI images were obtained using a composite of T1 and T2-WICTR / TE = 600 / 4.5ms).
造影剤を注入する前、 注入直後、 注入後 2 0分および 2 4時間経過した肝臓上 で 8枚の 2. 0 mm厚の連続薄切画像を得た。  Eight 2.0 mm-thick serial slice images were obtained on the liver before, immediately after, 20 minutes and 24 hours after the injection of the contrast medium.
( 2 ) MR I画像解析  (2) MR I image analysis
種々の造影剤について注入前、 注入直後、 注入後 2 0分および 2 4時間経過し た肝臓での MR I シグナルの強さの違いを解析した。  The differences in the intensity of the MRI signal in the liver before, immediately after, 20 minutes and 24 hours after injection were analyzed for various contrast agents.
肝臓での MR I画像において検出される相対シグナル強度 （％) [R S I (%) ) は以下の式で算出される。  The relative signal intensity (%) [RSI (%)] detected in the liver MRI image is calculated by the following formula.
R S I (%) = (S I (肝臓一後） ZS I (筋肉一後） 〕  R S I (%) = (S I (after the liver) ZS I (after the muscle)]
/ 〔S I (肝臓一前） ZS I (筋肉一前） 〕 X 1 0 0  / [S I (before liver) ZS I (before muscle)] X 1 0 0
S I (肝臓一後） ：造影剤の注入後の肝臓におけるシグナル強度  S I (after liver): Signal intensity in liver after injection of contrast agent
S I (筋肉一後） ：造影剤の注入後の筋肉におけるシグナル強度 S I (肝臓一前） ：造影剤の注入前の肝臓におけるシグナル強度 S I (筋肉—前） ：造影剤の注入前の筋肉におけるシグナル強度 SI (after muscle): Signal intensity in muscle after injection of contrast agent SI (immediately before liver): Signal intensity in liver before injection of contrast agent SI (muscle-front): Signal intensity in muscle before injection of contrast agent
結果を図 2に示す。  The result is shown in figure 2.
いずれの場合も、 投与直後から急激な信号強度の低下が示された。 これらの結 果より HA— S P 10は PVLA— S P I Oおよび CDx— S P I 0同様十分に 肝臓を造影できることが示された。  In each case, the signal intensity sharply decreased immediately after administration. From these results, it was shown that HA-SP10 was able to image liver as well as PVLA-SPIO and CDx-SPIO.
産業上の利用可能性  Industrial applicability
MR I造影性化合物を腫瘍や臓器特異的に発現している糖夕ンパク質レセプタ 一に対応する糖鎖高分子で修飾することにより、 標的部位指向性のある MR I造 影性化合物および MR I用造影剤が得られる。 特に修飾させる糖鎖高分子として 親水性の大きいヒアルロン酸を選択することにより RE Sの貪食作用の回避が期 待できる。 標的部位を特異的に造影することが可能となったので、 低投与量で診 断が可能、 即ち毒性が低減された MR I用造影剤の提供が可能となる。 本出願は日本で出願された平成 8年特許願第 3 1 8 3 5 0号を基礎としており それらの内容は本明細書に全て包含されるものである。  Modification of the MR I imaging compound with a sugar chain macromolecule corresponding to a glycoprotein receptor that is specifically expressed in tumors and organs allows the target site-directed MR I imaging compound and MR I A contrast agent is obtained. In particular, by selecting hyaluronic acid having high hydrophilicity as the sugar chain polymer to be modified, it is expected that phagocytosis of RES will be avoided. Since the target site can be specifically imaged, diagnosis can be performed with a low dose, that is, it is possible to provide a contrast agent for MRI with reduced toxicity. This application is based on Japanese Patent Application No. 318350/1996 filed in Japan, the contents of which are incorporated in full herein.

Claims

請求の範囲 The scope of the claims
I . MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾して なる化合物。  I. MRI A compound obtained by modifying a contrast-enhancing compound with a sugar chain polymer that specifically recognizes the target site.
2. MR I造影性化合物がマグネタイ トである請求の範囲 1記載の化合物。 2. The compound according to claim 1, wherein the MRI contrast-enhancing compound is magnetite.
3. 糖鎖高分子がヒアルロン酸である請求の範囲 1または 2記載の化合物。3. The compound according to claim 1, wherein the sugar chain polymer is hyaluronic acid.
4. MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾して なる化合物を含む標的部位指向性 MR I用造影剤。 4. A target site-directed contrast agent for MR I containing a compound obtained by modifying a MR I contrast compound with a sugar chain polymer that specifically recognizes the target site.
5. MR I造影性化合物がマグネタイ トである請求の範囲 4記載の標的部位指向 性 MR I用造影剤。  5. The target-site-directed contrast agent for MRI according to claim 4, wherein the MRI contrast-enhancing compound is magnetite.
6. 糖鎖高分子がヒアルロン酸である請求の範囲 4または 5記載の標的部位指向 性 MR I用造影剤。  6. The target site-directed MRI contrast agent according to claim 4, wherein the sugar chain polymer is hyaluronic acid.
7. 標的部位指向性が肝臓または腫瘍に対してである請求の範囲 6記載の標的部 位指向性 MR I用造影剤。  7. The target site-directed MR I contrast agent according to claim 6, wherein the target site-directivity is for a liver or a tumor.
8. MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾する ことを特徴とする、 MR I造影性化合物を標的部位を特異的に認識する糖鎖高分 子にて修飾してなる化合物の製造方法。  8. MRI contrast-enhancing compound is modified with a sugar chain polymer that specifically recognizes the target site. A method for producing a compound obtained by modification.
9. MR I造影性化合物がマグネタイ トである請求の範囲 8記載の製造方法。 9. The production method according to claim 8, wherein the MRI contrast-enhancing compound is magnetite.
1 0. 糖鎖高分子がヒアルロン酸である請求の範囲 8または 9記載の製造方法。10. The production method according to claim 8, wherein the sugar chain polymer is hyaluronic acid.
I I . MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾し てなる化合物を含む標的部位指向性 MR I用造影剤を用いることを特徴とする M R I造影方法。 I I. A MRI contrasting method comprising using a target site-directed MRI contrast agent containing a compound obtained by modifying a MRI imaging compound with a sugar chain polymer that specifically recognizes a target site.
1 2. MR I造影性化合物がマグネタイ トである請求の範囲項 1 1記載の造影方 法。  12. The imaging method according to claim 11, wherein the MRI imaging compound is magnetite.
1 3. 糖鎖高分子がヒアルロン酸である請求の範囲 1 1または 1 2記載の造影方 法。  13. The imaging method according to claim 11, wherein the sugar chain polymer is hyaluronic acid.
1 4. MR I造影性化合物をヒアルロン酸にて修飾してなる化合物を含有する M R I用造影剤にて腫瘍細胞または肝臓を造影することによる腫瘍細胞または肝臓 の造影方法。 1 4. Tumor cells or liver by imaging tumor cells or liver with MRI contrast agent containing a compound obtained by modifying MR I imaging compound with hyaluronic acid Imaging method.
1 5. MR I造影性化合物がマグネタイ 卜である請求の範囲 1 4記載の腫瘍細胞 の造影方法。  15. The method for imaging tumor cells according to claim 14, wherein the MRI imaging compound is magnetite.
1 6. MR I造影性化合物を標的部位を特異的に認識する糖鎖高分子にて修飾し てなる化合物、 および当該化合物を MR I造影に使用しうるかまたは使用すべき であることを記載した書類を含む商業的パッケージ。  1 6. A compound obtained by modifying an MR I-imaging compound with a sugar chain polymer that specifically recognizes a target site, and that the compound can or should be used for MR I-imaging Commercial package containing documents.
1 7. 標的部位指向性 MR I用造影剤の製造の為の、 MR I造影性化合物を標的 部位を特異的に認識する糖鎖高分子にて修飾してなる化合物の使用。  1 7. Target site directivity Use of a compound obtained by modifying an MR I imaging compound with a sugar chain polymer that specifically recognizes the target site for the manufacture of an MR I contrast agent.
1 8. MR I造影性化合物がマグネタイ 卜である請求の範囲 1 7記載の使用。 1 8. Use according to claim 17, wherein the MRI contrast-enhancing compound is magnetite.
1 9. 糖鎖高分子がヒアルロン酸である請求の範囲 1 7または 1 8記載の使用。 19. The use according to claim 17 or 18, wherein the sugar chain polymer is hyaluronic acid.
PCT/JP1997/004343 1996-11-28 1997-11-27 Contrast compound, contrast medium for mri, and method for mri WO1998023293A1 (en)

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US6409990B1 (en) 1999-05-14 2002-06-25 The Regents Of The University Of California Macromolecular carrier for drug and diagnostic agent delivery
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US8906345B2 (en) 2006-09-20 2014-12-09 Isis Innovation Limited Multimeric particles
US9439985B2 (en) 2009-01-30 2016-09-13 Navidea Biopharmaceuticals, Inc. Compositions for radiolabeling diethylenetriaminepentaacetic acid (DTPA)-dextran
JP2010208979A (en) * 2009-03-09 2010-09-24 Tokyo Institute Of Technology Mri contrast medium for imaging tissue
JP2019522004A (en) * 2016-06-29 2019-08-08 ソウル ナショナル ユニバーシティ アールアンドディービー ファウンデーション Hydrated gel-based nanoemulsion for selective labeling of cancer lesions and method for producing the same
US11103600B2 (en) 2016-06-29 2021-08-31 Seoul National University R & Db Foundation Hydrogel-based nanoenulsion for selectively labeling cancer lesion, and preparation method therefor
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