WO2008059835A1 - Metal chelate complex, proton relaxation rate enhancing agent and mri contrast agent - Google Patents

Metal chelate complex, proton relaxation rate enhancing agent and mri contrast agent Download PDF

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Publication number
WO2008059835A1
WO2008059835A1 PCT/JP2007/072008 JP2007072008W WO2008059835A1 WO 2008059835 A1 WO2008059835 A1 WO 2008059835A1 JP 2007072008 W JP2007072008 W JP 2007072008W WO 2008059835 A1 WO2008059835 A1 WO 2008059835A1
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Prior art keywords
metal chelate
chelate complex
contrast agent
mri contrast
present
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PCT/JP2007/072008
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French (fr)
Japanese (ja)
Inventor
Tetsuji Yamaoka
Yoichi Tachibana
Hidehiro Iida
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Japan Health Sciences Foundation
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Priority to JP2008544149A priority Critical patent/JPWO2008059835A1/en
Publication of WO2008059835A1 publication Critical patent/WO2008059835A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/05—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves 
    • A61B5/055—Detecting, measuring or recording for diagnosis by means of electric currents or magnetic fields; Measuring using microwaves or radio waves  involving electronic [EMR] or nuclear [NMR] magnetic resonance, e.g. magnetic resonance imaging
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/42—Introducing metal atoms or metal-containing groups

Definitions

  • Metal chelate complex Metal chelate complex, proton relaxation rate enhancer, and MRI contrast agent
  • the present invention relates to a novel compound that can be used for diagnostic imaging, and changes over time in a certain area such as a cell or an organ by staying at a certain place without going back and forth between cell membranes.
  • This relates to a proton relaxation rate enhancer or MRI contrast agent that can be verified.
  • the present invention relates to an intracellular stay type MRI contrast agent for staying in a cell and tracking the cell in a regenerative treatment process or the like.
  • image diagnosis plays a major role, but there is an image diagnosis method that can measure the internal form and function of a living body non-destructively and non-invasively and show the result as an image. It has become important.
  • Such diagnostic imaging methods include X-ray diagnostic methods, radionuclide diagnostic methods (Positron C
  • T PET, single photon (SPECT), nuclear magnetic resonance diagnosis (MRI), ultrasound US, etc.
  • MRI is highly versatile because it has the advantages of superior soft tissue resolution, imaging in any direction, and the need for high-power measurement like PET.
  • MRI is a method that utilizes the nuclear magnetic resonance (NMR) phenomenon, and is a method that uses an MR signal obtained from hydrogen atoms (protons) in water molecules present in a living body.
  • the energy level force of protons present in the measurement object The time (relaxation time) or speed (relaxation rate) until it returns to the original energy level after it is once raised by electromagnetic waves irradiated from the MRI apparatus Due to the difference or the difference in proton density in the test object, it appears as contrast on the image.
  • An MRI contrast agent may be used for this MRI.
  • the MRI contrast agent is used for obtaining a clearer image when the contrast is small using a substance having an action of shortening the proton relaxation time in MRI or the like.
  • MRI contrast agents are mainly metals such as gadolinium. And chelate compounds (metal chelates).
  • this metal chelate Since this metal chelate has the action of shortening the relaxation time of protons, the contrast between the region where the contrast agent using the metal chelate stays and the region where it does not stay is more emphasized in the test object. Is done.
  • Omniscan registered trademark, Gd-DTPA-BMA, manufactured by Daiichi Pharmaceutical
  • Prohans registered trademark, Gd (HP—D03A), manufactured by Eisai
  • Magnescope registered trademark, Gd— Low molecular weight compounds such as DOTA, manufactured by Eiken or Tanabe Seiyaku
  • Magnevist registered trademark, Gd-DTPA, manufactured by Schering
  • Non-Patent Documents in which the blood half-life of the above-described low-molecular contrast agent is extended (Non-Patent Documents; see! -3 etc.), Metal-encapsulated micelles targeting cancer, dendrimer-type contrast agents that can bind a large amount of metal chelate to enhance contrast have been developed.
  • Non-Patent Document 1 describes Gd-DTPA and Gd-DOTA bound to polyglutamic acid (PG).
  • Non-Patent Documents 2 and 3 describe a product in which Gd-DTPA-cysteine complex, Gd-D03A, or the like is bound to polyethylene glycol (PEG).
  • organ transplantation has problems such as a shortage of donors, rejection after transplantation, and side effects caused by an immune suppressant to suppress rejection.
  • the above-mentioned contrast agent composed of a low molecular weight compound is small, it can penetrate cell membranes and the like. Even after being administered to a living body, it is free to move and it is not always possible to track the state of the tissue in the target area.
  • polymers having a relatively large molecular weight such as a polymer-bonded contrast agent and a dendrimer type contrast agent have been developed, but these also have an insufficient effect of preventing interaction with the cell membrane. Also, there is a possibility that deviation from the tracking target may occur due to membrane permeation.
  • all of the polymer-bound MRI contrast agents described in non-patent literature are biodegraded after one imaging.
  • Non-patent literature l Bioconjugate Chem .; (Article); 2004; 15 (6); 1408-1415
  • Non-Patent Document 2 Bioconjugate Chem .; (Article); 2004; 15 (6); 1424-1430
  • Non-Patent Document 3 Journal of Alloys and Compounds 249 (1997) 185-190
  • Non-Patent Document 4 J. Pharm. Pharmacol., 1995, 47, 479-486.
  • the present inventors have found that the contrast agent power using polybulualcohol is stable in the cell for a long period of time, and is extremely low in cytotoxicity even though long-term imaging is possible.
  • the invention has reached the invention, and the purpose of the invention is to stay in a certain place without going in and out of the cell membrane, and to stay in a certain area without being limited to one-time imaging.
  • the ability to observe the tissue state of the tissue, especially the regeneration process by cell transplantation over time, and is extremely low in cytotoxicity, metal chelate complex, proton relaxation rate enhancer, MRI contrast agent, or intracellular residence To provide type MRI contrast media.
  • a metal chelate complex comprising one or more metal chelates and polybulal alcohol as constituent components.
  • the metal chelate complex according to the first invention characterized in that the metal chelate is bound to polybulal alcohol via a linker.
  • the chelating agent is one or more chelating agents selected from the group consisting of DTPA, DTP A anhydride, DOTA, D03A, HP—D03A, DTPA BMA.
  • the metal chelate complex according to any one of the three inventions.
  • a proton relaxation rate enhancer comprising the metal chelate complex according to any one of the first to sixth inventions.
  • An MRI contrast agent comprising the metal chelate complex according to any one of the first to sixth inventions.
  • An intracellular stay-type MRI contrast agent comprising the metal chelate complex according to any one of the first to sixth inventions.
  • the invention's effect [0026]
  • the compound of the present invention can be used for diagnostic imaging. By staying in a certain area without going back and forth between cell membranes, the state of the organization in a certain area such as a cell or an organ. It can be used as a proton relaxation rate enhancer, more specifically an MRI contrast agent, etc. that can track the presence or absence of disease and the degree of progression over time.
  • the metal chelate complex of the present invention contains one or more metal chelates and polybulal alcohol (hereinafter sometimes referred to as “PVA”) as constituent components.
  • PVA polybulal alcohol
  • metal ions used for the metal chelate include various magnetic metals.
  • the six lanthanoid elements such as gadolinium ion, cerium ion, samarium ion, upium ion, dysprosium ion, thulium ion, and yttrium ion, chromium ion, molybdenum ion, tungsten ion, etc.
  • Element ions Group 7 element ions such as manganese, Iron ion, Ruthenium ion, Group 8 element ion such as osmium ion, Cobalt ion, Group 9 element ion such as rhodium, Nickel ion, Palladium ion, etc. 10 Ions of group elements, ions of group 11 elements such as copper ions, ions of group 12 elements such as zinc ions and cadmium ions.
  • gadmium (Gd) ions are preferably used from the viewpoint that the complex having the largest number of coordination with the chelating agent atom has the highest stability. This is because the metal chelate complex of the present invention using gadolinium ions is highly effective as an agent having the highest proton relaxation rate enhancement effect.
  • chelating agent used for metal chelates include the well-known DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid butyl methacrylate), DTP A anhydride, DOTA (l, 4, 7, 10 Tetraazacyclododecane 1, 4, 7, 10 Tetraacetate), D03A (1, 4, 7, 10 Tetraazacyclododecane 1, 4, 7 Triacetate), HP—D03A (2 Hydroxypro Pinole 1, 4, 7, 10 tetraazacyclododecane-1, 4, 7 triacetate), etc., but DTPA is particularly preferred because of its long track record of clinical use. Then DOTA is preferred.
  • DOTA having high stability is preferable because long-term cell tracking is required.
  • the metal chelate used in the metal chelate complex of the present invention includes Omniscan (registered trademark, Gd—DTPA—BMA, manufactured by Daiichi Pharmaceutical), Prohans (registered trademark, Gd (HP—D03A), manufactured by Eisai), Magnescope (Registered trademark, Gd—DOTA, manufactured by Eiken or Tanabe Seiyaku), Magnevist (registered trademark, Gd—DTPA, manufactured by Schering), etc. Use force S. You can also.
  • the metal ion and the quenching agent can be produced by, for example, mixing in an aqueous solution at room temperature, normal pressure, pH 6 to 6.5 for 24 hours.
  • the preferred pH range depends on the type of chelating agent. Generally, it is preferable to set the pH value as described above in view of the fact that chelation is promoted.
  • the PVA used in the metal chelate complex of the present invention is a known polymer having butyl alcohol as its structural unit (monomer), and is a highly hydrophilic synthetic resin.
  • the structural component, butyalcohol is unstable in molecular structure and unsuitable as a raw material for polymer production. Therefore, usually, acetic acid bull is first polymerized into polyacetic acid bull, and then the acetate ester residue is obtained. It is produced by converting a group into a hydroxyl group by hydrolysis.
  • PVA used in the metal chelate complex of the present invention may be a straight chain or a part thereof. Have a branched structure, or have a crosslinked structure or a cyclic structure.
  • the property is not particularly limited as long as it does not affect the property of the present invention as a proton relaxation rate enhancer or an MRI contrast agent, but the following are preferable.
  • the weight-average molecular weight is a certain area where the blood half-life is difficult to traverse the cell membrane, or if it is likely to stay in the cell, it is difficult to accumulate more than 2000 in the body! / 100,000 is preferable in terms of the point. More preferably, it is 20,000-50,000, More preferably, it is 30,000-40,000.
  • the saponification degree is preferably 85-99.9%.
  • the glass transition temperature is preferably 10 to 80 ° C.
  • the glass transition temperature is measured by DSC.
  • the solution viscosity is preferably !! ⁇ lOOOOOcps force S.
  • PVA used in the metal chelate complex of the present invention has other high molecular weights within a range that does not adversely affect the properties of the present invention as a proton relaxation rate enhancer or an MRI contrast agent.
  • These block units may be used as block copolymers or other copolymers, and may be blended with other high molecules.
  • the content ratio of the bull alcohol unit (the raw material component is bull acetate) is not particularly limited, but it has a certain half-life in blood, hydrophilicity, and a property that makes it difficult to access the cell membrane. In order to hold it reliably, it is about 70% or more, preferably 80% or more, particularly preferably 90% or more as a butyl alcohol unit among the monomer units constituting the polymer.
  • the chelate complex of the present invention is easy to control the rate of introduction of metal chelate into PVA
  • a linker is included as a constituent component.
  • linker in order to increase the introduction rate of the metal chelate, it is preferable to use a linker.
  • the term “linker” as used in the present invention is a substance capable of binding PVA and metal chelate by binding to PVA and metal chelate at both ends.
  • Examples of the structural component of the linker include polyamines such as diamine and triamine, polyhydric alcohols such as dialcohol, oxycarboxylic acid, amino acid, diisocyanate and the like.
  • One end of diamine forms an amide bond with the carboxyl group of the metal chelate, and the other end of diamine forms a urethane bond with the OH group of PVA using a condensing agent such as imidazole such as carboxyimidazole (CDI).
  • diamine is preferred because it can be performed under mild reaction conditions without the need for special equipment and the like!
  • diamines examples include hydrocarbon compounds such as ethylenediamine, propanediamine, butylenediamine, hexamethylenediamine, diaminodecane and the like.
  • the number of carbon atoms of the linker is not particularly limited, but! ⁇ 10 forces Create a suitable space between the metal chelate to be linked and the PVA, so that a lot of metal chelate can be bound, and water solubility should not be maintained.
  • increasing the rate of introduction of metal chelates is preferred from the standpoint of using biomaterials.
  • a biomaterial 6 or less is preferable, and 2 to 5 is more preferable, and the most preferable is a linker having 3 carbon atoms such as propanediamin, and in particular, 1,3-propanediol. Ammine is preferred.
  • the metal chelate complex of the present invention can be produced by a known method, for example, by directly bonding the chelate COOH group to the OH group of PVA using an esterifying agent or the like and then introducing a metal ion.
  • the ability to produce COOH groups of metal chelates prepared in advance may be combined with the OH groups of PVA.
  • a scheme showing an example of the production process of the metal chelate complex of the present invention is shown in FIG.
  • the metal chelate complex of the present invention contains a linker as a constituent component, for example, after a chelating agent is bonded to PV A via the linker, the metal chelating agent contains a metal It is possible to obtain power S by introducing a method such as ION.
  • the metal ion may be used as a metal chelate by a chelating agent in advance and then used for binding to PVA.
  • the binding of PVA to the linker can be performed as follows, for example, when diamine is used as the linker.
  • PVA is dissolved in DMSO (dimethyl sulfoxide).
  • CDI Carboxidiimidazole
  • the obtained reaction solution is dialyzed in water and the desired product is obtained by lyophilization.
  • Binding of the chelating agent to the linker can be performed by a conventional method.
  • diamine when used, it can be performed as follows.
  • N-hydroxysuccinimide is added to the chelating agent to form an activated ester, which is reacted with amine.
  • the obtained reaction solution is dialyzed in water and freeze-dried to obtain the desired product.
  • the introduction of the metal ion into the chelating agent can be performed by a force S that can be performed by a conventional method, for example, as follows.
  • a chelating agent and metal ions are stirred in water at an optimum pH.
  • the obtained reaction solution is dialyzed in water and the desired product is obtained by lyophilization.
  • the metal chelate prepared in advance can be bound to the linker by a conventional method. For example, it can be applied as follows.
  • the linker contains diamine as a constituent component
  • the dehydration condensing agent and PVA side chain alcohol are reacted to form an active ester intermediate, and diamine is added.
  • the other amine group is amide-bonded to the carboxylate group of the chelating agent.
  • a metal ion is introduced into the chelating agent.
  • the ratio of the linker introduced into the PVA correlates with the amount of linker added. Therefore, the introduction ratio can be adjusted by changing the addition amount.
  • the percentage of units in which the linker is introduced to all the units when technically considering bull alcohol as one unit and introducing it by the above method can range from several percent to 90%. From the viewpoint of exhibiting a sufficient proton relaxation rate enhancing effect and maintaining water solubility, 3% to 40% is preferable, and more preferably about 5% to 30%.
  • the introduction ratio of the chelating agent to the linker is almost 100% if a sufficient amount of the chelating agent is added during the introduction operation.
  • the introduction ratio of the metal ion to the chelating agent is a force S that can be adjusted according to various experimental conditions, and the PVA-linker single chelating agent complex before introduction of the metal ion at room temperature, When mixing metal ions under normal pressure, ⁇ 6 to ⁇ 6 ⁇ 5, approximately 50
  • % To 100% of the chelate can be introduced.
  • the introduction ratio when introducing a chelating agent or metal chelate directly into PVA without using a linker can be adjusted by various experimental conditions and by the amount of chelating agent or metal chelate added. It is.
  • the ratio force S of the unit in which the metal chelate is introduced to the entire unit is the same as in the case of using the linker S, 1.5 to 40%.
  • the force S is preferable, and more preferably 2.5 to 30%.
  • the metal chelate complex of the present invention has the property of hardly passing through the cell membrane.
  • the degree of saponification of PVA is high, it has the property that the adsorption to the cell membrane is small, and there is little advantage of approaching the cell membrane itself! RU
  • the metal chelate complex of the present invention can be used as a later-described proton relaxation rate enhancer, an MRI contrast agent, or the like.
  • it is particularly useful as an intracellular MRI contrast agent because it is difficult to pass through the cell membrane.
  • the proton relaxation rate enhancer of the present invention is characterized by containing the metal chelate complex of ⁇ 1> above.
  • the concentration of the metal chelate complex of the above ⁇ 1> in the proton relaxation rate enhancer of the present invention is preferably from 0.5 to 10% by weight, and more preferably from 0.5 to 5% by weight. This is because when the amount is 1% by weight or more, an effect of enhancing the proton relaxation rate is obtained, and when the amount is 10% by weight or less, the viscosity is suppressed and it is suitable for an injection solution or the like.
  • the proton relaxation rate enhancer of the present invention is a component generally used in injections such as mannitol, in addition to solvents such as water, alcohol, and physiological saline, Alternatively, components generally used in proton relaxation rate enhancers and MRI contrast agents can be further included.
  • an excipient for example, an excipient , Lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, moisturizers, adsorbents, fillers, extenders, moisturizers , And can be formulated by known methods using additives such as preservatives.
  • excipients include organic excipients and inorganic excipients.
  • the proton relaxation rate enhancer of the present invention is a cell treatment for treating transplanted cells even when the form of the solution is preferable, such as injection, catheter solution, oral solution, cell treatment solution, tissue treatment solution, etc. Liquid, tissue treatment liquid is preferred! [0063] (Production method)
  • the proton relaxation rate enhancer of the present invention can be obtained by a method such as mixing the metal chelate complex as the main component and each of the other components such as the second component at room temperature and normal pressure. Touch with S.
  • the proton relaxation rate enhancer of the present invention has the property that the metal chelate complex as the main component is difficult to pass through the cell membrane, and the property that it hardly approaches the cell membrane itself.
  • the proton relaxation rate enhancer of the present invention is an MRI contrast agent described later, in particular, an intracellular residence type.
  • the MRI contrast agent of the present invention comprises the metal chelate complex of ⁇ 1> above.
  • the concentration of the metal chelate complex of the above ⁇ 1> in the MRI contrast agent of the present invention is preferably from 0.5 to 10% by weight, and more preferably from 0.5 to 5% by weight. This is because when the amount is 1% by weight or more, the effect of enhancing the proton relaxation rate is obtained, and when the amount is 10% by weight or less, the viscosity is suppressed, which is suitable for an injection solution or the like.
  • the MRI contrast agent of the present invention includes components generally used for injections such as mannitol in addition to the metal chelate complex of ⁇ 1> above, solvents such as water, alcohol, and physiological saline, or MRI contrast agents In addition, generally used components can be further included. [0070] In addition, other components can be contained within a range that does not adversely affect the properties of the MRI contrast agent of the present invention.
  • an excipient for example, an excipient, a lubricant, Binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, humectants, adsorbents, fillers, extenders, moisturizers, preservatives It can be formulated by a known method using additives such as.
  • excipients include organic excipients and inorganic excipients.
  • the MRI contrast medium of the present invention is a cell treatment solution or tissue for treating transplanted cells, even though the form of the solution is preferable, such as an injection, a catheter solution, an oral solution, a cell treatment solution, a tissue treatment solution, etc.
  • a treatment liquid is preferred.
  • the MRI contrast agent of the present invention can be obtained by a method such as mixing the metal chelate complex as the main component and each of the other components such as the second component at room temperature and normal pressure.
  • the MRI contrast agent of the present invention has the property that the metal chelate complex as the main component is difficult to pass through the cell membrane, and the property of approaching the cell membrane itself is small.
  • the MRI contrast agent of the present invention was introduced into cells, the presence of the MRI contrast agent was observed over almost the entire cell group even after cell division (FIGS. 15 and 16).
  • the MRI contrast agent of the present invention is particularly suitable as an intracellular stay type MRI contrast agent described later.
  • Intracellular MRI contrast agent of the present invention Intracellular MRI contrast agent of the present invention
  • the intracellular MRI contrast agent of the present invention comprises the metal chelate complex of ⁇ 1> above.
  • the concentration of the metal chelate complex of ⁇ 1> above in the intracellular residence type MRI contrast agent of the present invention is preferably 0.;! To 10% by weight, more preferably 0.5 to 5% by weight. is there. 0.1% by weight or more provides a proton relaxation rate enhancing effect, and 10% by weight or less suppresses viscosity and is suitable for injections and the like.
  • the intracellular residence type MRI contrast agent of the present invention includes components generally used in injections such as mannitol, in addition to the metal chelate complex of ⁇ 1> above, solvents such as water, alcohol, and physiological saline, Alternatively, MRI contrast agents can be made to contain more commonly used components.
  • compositions can be included within a range that does not adversely affect the properties of the intracellular residence type MRI contrast agent of the present invention! /,
  • a pharmaceutically acceptable carrier for example, as a pharmaceutically acceptable carrier.
  • excipients include organic excipients and inorganic excipients.
  • the intracellular residence type MRI contrast medium of the present invention is suitable for treatment of transplanted cells even when the form of the liquid agent is preferable, such as an injection, a catheter liquid, an oral liquid, a cell treatment liquid, and a tissue treatment liquid.
  • Cell treatment solution and tissue treatment solution are preferred.
  • the intracellular MRI contrast agent of the present invention is obtained by a method such as mixing the above-mentioned metal chelate complex as the main component and each of the other components such as the second component at room temperature and normal pressure. That's the power S.
  • the intracellular residence type MRI contrast agent of the present invention has the property that it is difficult to pass through the cell membrane of the metal chelate complex force that is the main component, and has the property that it is less likely to approach the cell membrane itself. Further, the intracellular residence type MRI contrast agent of the present invention stays in the cell for a long time when introduced into the cell, and after cell division, the intracellular residence type MRI contrast agent extends over almost the entire cell group. Was observed ( Figures 15 and 16).
  • the administration method for administering the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention to muscles, blood vessels, organs and other tissues includes oral administration, catheter administration, static administration.
  • Intravenous administration such as injection, intramuscular administration, transdermal administration, nasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, rectal administration, mucosal administration, inhalation, etc. can be mentioned. In terms of keeping constant, intravenous administration such as intravenous injection is preferable.
  • a method such as tail vein injection is also included.
  • a method for administering the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention to the target cells cells are immersed in a solution such as a cell treatment solution or a tissue treatment solution.
  • a solution such as a cell treatment solution or a tissue treatment solution.
  • the dosage forms of the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention include cell treatment solution and tissue treatment solution used for electoral mouth position, etc., as well as injection and catheter solution, Examples include oral liquid preparations, but are not necessarily limited thereto.
  • the dose can be adjusted as appropriate according to the type and number of target tissues and cells, the type of suspected disease, the degree of progression, the patient's age, sex, weight, etc., and is not necessarily limited. In general, however, the following amounts can be selected.
  • the amount of the lei complex is from 0. Olmg to 1000 mg, preferably from 0.1 mg to 1000 mg, more preferably from 0.1 mg to OOmg.
  • the Gd weight it is preferable to administer 0.001 mg to 10 mg per administration.
  • administer when administered directly into cells by methods such as microinjection, electoporation, and cell membrane fusion ribosome, administer so that 10 7 to 10 12 metal chelate complexes per cell enter. I prefer it.
  • the appropriate amount of the metal chelate complex is also distributed to the dividing cells during cell division, which is more reliable than the ability to follow the tissue regeneration process through the growth of cell transplantation. Because.
  • a proton relaxation rate enhancer, an MRI contrast agent, or an intracellular residence type MRI contrast agent can be obtained by in vivo or in vitro methods using microinjection, electroporation, or cell membrane fusion ribosome, etc. Introduce into cells to be transplanted
  • the state of the transplanted cells and their dividing cells can be observed over time by 4.7T (three-dimensional) MRI or the like.
  • MRI contrast agent When the proton relaxation rate enhancer of the present invention, MRI contrast agent, or intracellular residence type MRI contrast agent, etc. are introduced into cells, they are present over almost the entire cell group even after cell division. It has been observed (Figs. 15 and 16) that, for example, in regeneration therapy, it is possible to observe the growth of transplanted cells.
  • the DOTA introduction rate (%) for the PVA unit was correlated with the amount of jamain charged to the PVA unit.
  • the metal chelate complex of the present invention was produced as follows.
  • the manufacturing process is as shown in FIG.
  • 1, 1 'Carbonylbis 1H— as a dehydrating agent for the side chain OH group of linear PVA with a weight average molecular weight of 74800 (calculated from the degree of polymerization), saponification degree of 98.5% and Tg of 45 ° C
  • 1,3-propanediamin was introduced at an arbitrary ratio, and four samples (A to D) having different introduction rates were obtained.
  • the DOTA introduction rate into the PVA unit is determined by NMR measurement in terms of unit ratios: Sample A: 13 ⁇ 2%, Sample B: 7.5%, Sample C: 3.6%, Sample D : 12. 9%.
  • the metal chelate complex of the present invention was obtained by complexing gadolinium, which is the central metal, with DOTA.
  • FITC fluorescent label
  • Mn the number average molecular weight of PVA
  • Mw the weight average molecular weight
  • the Inversion Recovery method is a method in which the NMR spectrum is measured at an arbitrary time after irradiating an electromagnetic wave, and T is measured from the time change of the signal intensity. This IR method
  • the metal chelate complex of the present invention enhances the proton relaxation rate in a concentration-dependent manner, and the degree of relaxation can be arbitrarily set by adjusting the amount of metal chelate in the complex.
  • the relaxation rate that is, the reciprocal of the relaxation time, can be obtained by the following equation.
  • the metal chelate complex (sample D), which is a proton relaxation rate enhancer of the present invention, is more relaxed than Magnevist (registered trademark, Gd-DTPA, manufactured by Schering), which is a known contrast agent.
  • Magnevist registered trademark, Gd-DTPA, manufactured by Schering
  • T- that is, T 1 when the amount of Gd is OmM, is a force that is difficult to distinguish in FIG. It is.
  • the relationship between the concentration of the proton relaxation rate enhancer of the present invention in the present experimental example and the relaxation rate is as follows.
  • the cytotoxicity test using WST-1 is a known method, and is a commercially available test kit (Premix WST-1 Cell Proliferation Assay System: manufactured by Takara Bio Inc./Cell Proliferation Reagent WST-1: manufactured by Roche / W201 : Dojindo Laboratories Co., Ltd., etc.) and can be performed according to the instructions attached to the kit, etc.
  • the principle is as follows.
  • Tetrazolium salt is degraded to formazan dye by succinate tetrazolium reductase (EC 1.3.99.1), which is active only in living cells. Therefore, if the number of viable cells increases, the enzyme activity in the sample increases and the formazan dye increases. The formazan dye and the number of viable cells show a linear correlation. Therefore, the number of viable cells can be measured by measuring the absorbance of the dye solution by ELISA or the like and quantifying the formazan dye. That is, after adding a metal chelate complex or a metal chelate to the cell culture medium, the cytotoxicity can be confirmed by measuring the number of viable cells.
  • a metal chelate Magneticnevist (registered trademark, Gd-DTPA, manufactured by Schering)
  • a PBS solution containing the metal chelate complex of the present invention (sample B), which is a known MRI contrast medium for comparison
  • the cytotoxicity was confirmed by measuring the number of viable cells when each control PBS solution was administered to the medium in which NIH-3T3 cells were cultured, and the results are shown in Figs.
  • the vertical axis represents the UV value.
  • the chelate concentration of the solution containing the metal chelate or metal chelate complex is as shown in FIGS.
  • Cytotoxicity is measured by measuring the number of viable cells according to the above-described method when the culture solution containing the metal chelate complex of the present invention (samples A and B) is introduced into the cells by electroporation. confirmed. The results are shown in Figs. The vertical axis represents the UV value.
  • a PBS solution containing the metal chelate complex of the present invention (samples A, B, and C in Table 1) was imaged using MRI.
  • the brightness of each sampled image was measured by methods such as image processing.
  • FIG. 8 is a diagram showing the respective concentrations corresponding to the three metal chelate complexes in the MRI imaging diagrams of FIGS.
  • the vertical axis in Fig. 12 represents the luminance of each sample when the luminance of PBS is 1.
  • the metal chelate complex of the present invention (Sample A in Table 1) was introduced into NIH-3T3 cells by electopore positioning.
  • the agarose gel encapsulating the obtained cells was imaged using MRI.
  • the obtained image image processing as one of the luminance of Agarosugeru, Agarosu + cells (cells only), Agarosu + 10 3 cells transduced under the conditions of M (10_ 3 M), Agarosu + 10- 2 M of obtains the brightness of the cells (10- 2 M) was introduced under the conditions, and each Dara off of.
  • Samples were introduced into NIH-3T3 cells using the electoporation method, and changes over time in the amount of sample in the cells were measured using fluorescence measurements.
  • Sample D was used as the metal chelate complex sample of the present invention. The results are shown in FIG. In addition, the vertical axis
  • example D introduction into the cells was performed by the electopore method.
  • the sample was distributed evenly to a certain degree evenly to the individual split cells as well as not leaking out of the cell.
  • the sample is retained in the cells over time, and that it can be traced by MRI for a long time even after cell transplantation.
  • example D in Table 1 After the metal chelate complex of the present invention (sample D in Table 1) is introduced into NIH-3T3 cells by electo-poration, the cells are accumulated at the bottom of the test tube and sliced horizontally to include the bottom of the test tube. MRI imaging was performed. The results are shown in FIG.
  • NIH-3T3 cells into which no sample was introduced and a test tube containing only the medium were used.
  • the cells into which the metal chelate complex (sample D) was introduced showed an increase in brightness as compared with the comparison target. Since the increase in luminance depends on the sample contained in the cells, it was confirmed that the cells can be detected by MRI by using the metal chelate complex of the present invention.
  • the metal chelate complex of the present invention (sample D in Table 1) was introduced into NIH 3T3 cells by electopore positioning, and the cells were encapsulated in an agarose gel. The obtained gel was implanted subcutaneously in mice and MRI imaging was performed. The results are shown in FIG.
  • FIGS. 9, 10, 11, 15 and 16 the same drawings with higher resolution are shown as FIGS. 20, 21, 22, 23 and 24, respectively.
  • the present invention relates to a novel compound that can be used for diagnostic imaging, and changes over time in certain areas such as cells and organs by staying in a certain place without going back and forth between cell membranes. It is related to proton relaxation rate enhancers or MRI contrast agents that can be verified, and can be widely used in medical and research fields.
  • FIG. 1 is a scheme showing the production process of the metal chelate complex of the present invention.
  • FIG. 2 is a graph showing that the proton relaxation rate increases in proportion to the concentration of the metal chelate complex (polymer) in the solution and the amount of metal chelate in the complex.
  • FIG. 3 is a graph showing that the slope (relaxation) increases as the amount of metal introduced into the metal chelate complex increases.
  • FIG. 4 is a graph showing cell toxicity when a known metal chelate (Magnevist: registered trademark) is administered extracellularly.
  • FIG. 5 is a graph showing cytotoxicity when the metal chelate complex of the present invention is administered extracellularly.
  • FIG. 6 is a graph showing cytotoxicity when the metal chelate complex of the present invention (sample A) is administered into cells.
  • FIG. 7 is a graph showing cytotoxicity when the metal chelate complex of the present invention (sample B) is administered intracellularly.
  • FIG. 9 It is the figure which imaged the PBS solution of the metal chelate complex (sample A) of the present invention by MRI at different concentrations.
  • FIG. 13 A diagram showing the results of MRI measurement of cells labeled with the metal chelate complex of the present invention (sample A).
  • FIG. 14 A graph showing intracellular retention of the metal chelate complex of the present invention (sample D) administered into cells.
  • FIG. 15 is a view showing a bright field photograph two days after introduction of the metal chelate complex of the present invention (sample D) into cells.
  • FIG. 16 shows a fluorescent photograph two days after introduction of the metal chelate complex of the present invention (sample D) into cells.
  • FIG. 17 A graph showing the intracellular retention of the metal chelate complex together with the cell proliferation ability after administration of the metal chelate complex of the present invention (sample D) into the cell.
  • FIG. 18 This figure shows the results of MRI imaging after introducing samples into NIH-3T3 cells, accumulating cells at the bottom of the test tube, slicing horizontally to include the bottom of the test tube, and so on.
  • FIG. 4 is a view showing the results of MRI imaging after the metal chelate complex of the present invention (sample D) was introduced into cells and transplanted subcutaneously into mice.
  • FIG. 20 is a diagram in which the resolution is increased in FIG.
  • FIG. 21 is a diagram in which the resolution is increased in FIG.
  • FIG. 22 is a diagram with the resolution increased in FIG.
  • FIG. 23 is a diagram in which the resolution is increased in FIG.
  • FIG. 24 is a diagram in which the resolution is increased in FIG.

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Abstract

It is intended to provide a proton relaxation rate enhancing agent, an MRI contrast agent or an intracellular retained MRI contrast agent, which enables monitoring over time of a compound that can be stay at a fixed site for a long time without moving in and out of the cell membrane and the state of tissues in a fixed area, particularly the process of regeneration by cell transplantation. A metal chelate complex, characterized by comprising one or more metal chelates and polyvinyl alcohol as constituents, and a proton relaxation rate enhancing agent, an MRI contrast agent or an intracellular retained MRI contrast agent, characterized by comprising the same.

Description

明 細 書  Specification
金属キレート複合体及びプロトン緩和速度増強剤並びに MRI造影剤 技術分野  Metal chelate complex, proton relaxation rate enhancer, and MRI contrast agent
[0001] 本発明は、画像診断に用いることが可能な新規化合物に関するものであり、細胞膜 の内外で行き来することなぐ一定の箇所に滞在することによって、細胞や器官等の 一定エリアの経時的変化を検証可能な、プロトン緩和速度増強剤，又は MRI造影剤 に関するものである。特に、細胞内に滞在させ、再生治療過程等における細胞の追 跡を行うための、細胞内滞在型 MRI造影剤に関するものである。  TECHNICAL FIELD [0001] The present invention relates to a novel compound that can be used for diagnostic imaging, and changes over time in a certain area such as a cell or an organ by staying at a certain place without going back and forth between cell membranes. This relates to a proton relaxation rate enhancer or MRI contrast agent that can be verified. In particular, the present invention relates to an intracellular stay type MRI contrast agent for staying in a cell and tracking the cell in a regenerative treatment process or the like.
背景技術  Background art
[0002] 現代医療において、画像診断の果たす役割は大きいが、中でも非破壊的，非侵襲 的に生体の内部形態や内部機能を測定し、その結果を画像によって示すことのでき る画像診断方法が重要になっている。  [0002] In modern medicine, image diagnosis plays a major role, but there is an image diagnosis method that can measure the internal form and function of a living body non-destructively and non-invasively and show the result as an image. It has become important.
[0003] このような画像診断方法には、 X線診断方法，放射線核医学診断方法 (ポジトロン C[0003] Such diagnostic imaging methods include X-ray diagnostic methods, radionuclide diagnostic methods (Positron C
T : PET,シングルフオトン： SPECT) ,核磁気共鳴診断法（MRI) ,超音波 US等が 挙げられる。 T: PET, single photon (SPECT), nuclear magnetic resonance diagnosis (MRI), ultrasound US, etc.
中でも、 MRIは、軟部組織の解像力に優れ、任意の方向での撮像が可能で、 PET のような大力 Sかりも不要という利点があるため、汎用性が高い。  Above all, MRI is highly versatile because it has the advantages of superior soft tissue resolution, imaging in any direction, and the need for high-power measurement like PET.
[0004] MRIとは、核磁気共鳴 (NMR)現象を利用する方法であり、生体内に存在する水分 子中の水素原子（プロトン)から得られる MR信号を、画像化して用いる方法である。 測定対象中に存在するプロトンの、エネルギーレベル力 MRI装置から照射された 電磁波によって、一旦高くなつた後、元のエネルギーレベルに戻るまでの時間（緩和 時間）又はその際の速度 (緩和速度）の差，あるいは被験物中のプロトン密度の違!/、 によって、画像上にコントラストとして現れる。  [0004] MRI is a method that utilizes the nuclear magnetic resonance (NMR) phenomenon, and is a method that uses an MR signal obtained from hydrogen atoms (protons) in water molecules present in a living body. The energy level force of protons present in the measurement object The time (relaxation time) or speed (relaxation rate) until it returns to the original energy level after it is once raised by electromagnetic waves irradiated from the MRI apparatus Due to the difference or the difference in proton density in the test object, it appears as contrast on the image.
[0005] この MRIには、 MRI造影剤が用いられる場合がある。 MRI造影剤とは、 MRI等にお いて、プロトンの緩和時間を短くする作用を有する物質を用いて、コントラストが小さ い場合等に、より鮮明な画像を得るために用いられるものである。  [0005] An MRI contrast agent may be used for this MRI. The MRI contrast agent is used for obtaining a clearer image when the contrast is small using a substance having an action of shortening the proton relaxation time in MRI or the like.
現在用いられている MRI造影剤としては、ガドリニウム等に代表される金属を中心と したキレート化合物（金属キレート）が挙げられる。 Currently used MRI contrast agents are mainly metals such as gadolinium. And chelate compounds (metal chelates).
この金属キレートが、プロトンの緩和時間を短くする作用を有することから、被験物に おいて、金属キレートを用いた造影剤が滞在する領域と、滞在しない領域との間のコ ントラストが、より強調される。  Since this metal chelate has the action of shortening the relaxation time of protons, the contrast between the region where the contrast agent using the metal chelate stays and the region where it does not stay is more emphasized in the test object. Is done.
[0006] MRI造影剤としては、ォムニスキャン (登録商標， Gd-DTPA-BMA,第一製薬製 ) ,プロハンス（登録商標， Gd (HP— D03A) ,エーザィ製），マグネスコープ (登録 商標， Gd— DOTA,栄研又は田辺製薬製），マグネビスト(登録商標， Gd— DTPA ,シエーリング製）等の低分子化合物が市販されて!/、る。  [0006] As MRI contrast agents, Omniscan (registered trademark, Gd-DTPA-BMA, manufactured by Daiichi Pharmaceutical), Prohans (registered trademark, Gd (HP—D03A), manufactured by Eisai), Magnescope (registered trademark, Gd— Low molecular weight compounds such as DOTA, manufactured by Eiken or Tanabe Seiyaku) and Magnevist (registered trademark, Gd-DTPA, manufactured by Schering) are commercially available!
[0007] また、血流情報をより正確に得るために、上記の低分子造影剤の血中半減期を延長 させた、高分子結合型造影剤 (非特許文献;!〜 3等参照），癌をターゲットとした金属 内包ミセル，コントラストを高めるために多量の金属キレートを結合させうるようにした 、デンドリマー型の造影剤等が開発されている。  [0007] In addition, in order to obtain blood flow information more accurately, a polymer-bound contrast agent in which the blood half-life of the above-described low-molecular contrast agent is extended (Non-Patent Documents; see! -3 etc.), Metal-encapsulated micelles targeting cancer, dendrimer-type contrast agents that can bind a large amount of metal chelate to enhance contrast have been developed.
[0008] 例えば、下記非特許文献 1の造影剤は、ポリグルタミン酸 (PG)に結合させた、 Gd— DTPA, Gd— DOTAが記載されている。非特許文献 2, 3では、ポリエチレングリコ ール（PEG)に、 Gd— DTPA—システィン複合体や、 Gd— D〇3A等を結合させたも のが記載されている。  For example, the following contrast agent of Non-Patent Document 1 describes Gd-DTPA and Gd-DOTA bound to polyglutamic acid (PG). Non-Patent Documents 2 and 3 describe a product in which Gd-DTPA-cysteine complex, Gd-D03A, or the like is bound to polyethylene glycol (PEG).
[0009] し力、しながら、人為的に製造した化合物を用いた MRI造影剤は、生体にとっては異 物である。そのため、上記の非特許文献に記載のものは、全て"一度撮像した後には 、速やかに生体内で分解する"ようにデザインされており、生体投与後 1時間から 24 時間程度で生分解されてレ、る。  However, an MRI contrast agent using an artificially produced compound is different for a living body. For this reason, all of the above-mentioned non-patent documents are designed to “decompose quickly in vivo after taking an image once” and are biodegraded in about 1 to 24 hours after administration. Les.
[0010] 一方、失われた身体機能の回復手段として、臓器移植などに代わって、患者自身の 細胞を用いる細胞移植による再生療法が注目を集めて!/、る。  [0010] On the other hand, regenerative therapy using cell transplantation using the patient's own cells instead of organ transplantation has attracted attention as a means of recovering lost body functions!
臓器移植には、提供者不足や、移植後の拒絶反応，拒絶反応を抑制するための免 疫抑制剤による副作用等の問題点があるためである。  This is because organ transplantation has problems such as a shortage of donors, rejection after transplantation, and side effects caused by an immune suppressant to suppress rejection.
現在、ほぼ全ての組織，臓器の再生が臨床応用を目指して研究されており、皮膚， 軟骨，血管などの再生は、臨床応用に耐えうる結果が示されている力、その応用の 成果を確認する際にも、画像診断方法の果たす役割は、大きくなつてきている。  Currently, the regeneration of almost all tissues and organs is being studied for clinical application. The regeneration of skin, cartilage, blood vessels, etc. has been shown to be able to withstand clinical application, and the results of its application have been confirmed. In doing so, the role played by the diagnostic imaging method has been increasing.
[0011] しかし、上述の低分子化合物からなる造影剤は、小さいため細胞膜等も透過可能で 、生体に投与後も、自由に移動してしまい、必ずしも目的とする一定エリアの組織の 様子を追跡することができなレ、。 [0011] However, since the above-mentioned contrast agent composed of a low molecular weight compound is small, it can penetrate cell membranes and the like. Even after being administered to a living body, it is free to move and it is not always possible to track the state of the tissue in the target area.
また、上述の通り、高分子結合型造影剤ゃデンドリマー型造影剤等の比較的分子量 の大きいものも開発されているが、これらもまた、細胞膜との相互作用の防止効果が 十分で無いことから、やはり膜透過によって追跡対象とのズレが生じる可能性がある 。しかも、上述した様に、非特許文献に記載されている高分子結合型 MRI造影剤は いずれも、一回の撮像後には生分解されてしまうのが現状である。  In addition, as described above, polymers having a relatively large molecular weight such as a polymer-bonded contrast agent and a dendrimer type contrast agent have been developed, but these also have an insufficient effect of preventing interaction with the cell membrane. Also, there is a possibility that deviation from the tracking target may occur due to membrane permeation. Moreover, as described above, all of the polymer-bound MRI contrast agents described in non-patent literature are biodegraded after one imaging.
[0012] 従って、上述のいずれの MRI造影剤によっても、非常に長期間を要する組織再生過 程での、「経時的な作用機序 (移植細胞の増殖，分化，可塑性，融合等）を解明する ことは、できないというのが現状である。  [0012] Therefore, with any of the above-mentioned MRI contrast agents, “the mechanism of action over time (proliferation, differentiation, plasticity, fusion, etc. of transplanted cells) in the tissue regeneration process that requires a very long period of time is elucidated. The current situation is that you can't.
[0013] そこで、本発明者の一人が、血中半減期の長い高分子を探索した結果、ポリビュル アルコールを見レ、だした（非特許文献 4)。  [0013] Therefore, as a result of searching for a polymer having a long blood half-life by one of the present inventors, polybulol alcohol was found (Non-patent Document 4).
[0014] 非特許文献 l : Bioconjugate Chem.; (Article); 2004; 15(6); 1408-1415  [0014] Non-patent literature l: Bioconjugate Chem .; (Article); 2004; 15 (6); 1408-1415
非特許文献 2： Bioconjugate Chem.; (Article); 2004; 15(6); 1424-1430  Non-Patent Document 2: Bioconjugate Chem .; (Article); 2004; 15 (6); 1424-1430
非特許文献 3 : Journal of Alloys and Compounds 249 (1997) 185-190  Non-Patent Document 3: Journal of Alloys and Compounds 249 (1997) 185-190
非特許文献 4 : J. Pharm. Pharmacol., 1995， 47， 479-486.  Non-Patent Document 4: J. Pharm. Pharmacol., 1995, 47, 479-486.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0015] 本発明者等は、ポリビュルアルコールを用いた造影剤力、長期間安定に細胞内に留 まり、長期にわたる撮像が可能であるにもかかわらず、細胞毒性が極めて低いことを 見出し本発明に到達したものであって、その目的とするところは、細胞膜の内外で行 き来することなく、一定の箇所に滞在し続けることができ、一回切りの撮像に留まらず 、一定のエリアの組織の様子，特に細胞移植による再生過程を、経時的に観察する こと力 Sでき、尚かつ細胞毒性の極めて低い、金属キレート複合体，プロトン緩和速度 増強剤， MRI造影剤，又は細胞内滞在型 MRI造影剤を提供するにある。 [0015] The present inventors have found that the contrast agent power using polybulualcohol is stable in the cell for a long period of time, and is extremely low in cytotoxicity even though long-term imaging is possible. The invention has reached the invention, and the purpose of the invention is to stay in a certain place without going in and out of the cell membrane, and to stay in a certain area without being limited to one-time imaging. The ability to observe the tissue state of the tissue, especially the regeneration process by cell transplantation over time, and is extremely low in cytotoxicity, metal chelate complex, proton relaxation rate enhancer, MRI contrast agent, or intracellular residence To provide type MRI contrast media.
課題を解決するための手段  Means for solving the problem
[0016] 上述の目的は、下記第一の発明から第九の発明によって、達成される。 [0016] The above object is achieved by the following first to ninth inventions.
[0017] <第一の発明〉 1又は 2以上の金属キレートと、ポリビュルアルコールを構成成分として含むことを特 徴とする、金属キレート複合体である。 <First Invention> A metal chelate complex comprising one or more metal chelates and polybulal alcohol as constituent components.
[0018] <第二の発明〉 [0018] <Second invention>
金属キレートが、リンカ一を介してポリビュルアルコールに結合していることを特徴と する、第一の発明に記載の金属キレート複合体である。  The metal chelate complex according to the first invention, characterized in that the metal chelate is bound to polybulal alcohol via a linker.
[0019] <第三の発明〉 [0019] <Third invention>
リンカ一が、ジァミンであることを特徴とする、第二の発明に記載の金属キレート複合 体である。  The metal chelate complex according to the second invention, wherein the linker is diamine.
[0020] <第四の発明〉 [0020] <Fourth Invention>
キレート化剤が、 DTPA, DTP A無水物， DOTA, D03A, HP— D03A, DTPA BMAからなる群から選択される 1又は 2以上のキレート化剤であることを特徴とす る、第一乃至第三の発明のいずれかに記載の金属キレート複合体である。  The chelating agent is one or more chelating agents selected from the group consisting of DTPA, DTP A anhydride, DOTA, D03A, HP—D03A, DTPA BMA. The metal chelate complex according to any one of the three inventions.
[0021] <第五の発明〉 [0021] <Fifth Invention>
ポリビュルアルコールの重量平均分子量が 2000〜； 10万であることを特徴とする、第 一乃至第四の発明のいずれかに記載の金属キレート複合体である。  The metal chelate complex according to any one of the first to fourth inventions, wherein the polybulal alcohol has a weight average molecular weight of 2000 to 100,000.
[0022] <第六の発明〉 [0022] <Sixth Invention>
金属がガドリニウムであることを特徴とする、第一乃至第五の発明のいずれかに記載 の金属キレート複合体である。  The metal chelate complex according to any one of the first to fifth inventions, wherein the metal is gadolinium.
[0023] <第七の発明〉 [0023] <Seventh Invention>
第一乃至第六の発明のいずれかに記載の金属キレート複合体を含むことを特徴とす る、プロトン緩和速度増強剤である。  A proton relaxation rate enhancer comprising the metal chelate complex according to any one of the first to sixth inventions.
[0024] <第八の発明〉 <Eighth Invention>
第一乃至第六の発明のいずれかに記載の金属キレート複合体を含むことを特徴とす る、 MRI造影剤である。  An MRI contrast agent comprising the metal chelate complex according to any one of the first to sixth inventions.
[0025] <第九の発明〉 <Ninth Invention>
第一乃至第六の発明のいずれかに記載の金属キレート複合体を含むことを特徴とす る、細胞内滞在型 MRI造影剤である。  An intracellular stay-type MRI contrast agent comprising the metal chelate complex according to any one of the first to sixth inventions.
発明の効果 [0026] 本発明の化合物は、画像診断に用いることが可能であり、細胞膜の内外で行き来す ることなぐ一定のエリアに滞在することによって、細胞や器官等の一定エリア内の組 織の様子（疾患の有無，進行度等）を経時的に追跡可能な、プロトン緩和速度増強 剤，より具体的には MRI造影剤等に利用することが可能である。 The invention's effect [0026] The compound of the present invention can be used for diagnostic imaging. By staying in a certain area without going back and forth between cell membranes, the state of the organization in a certain area such as a cell or an organ. It can be used as a proton relaxation rate enhancer, more specifically an MRI contrast agent, etc. that can track the presence or absence of disease and the degree of progression over time.
特に、細胞内に導入した場合には、細胞内に滞在し続け、 1つの細胞内に複数導入 してある場合には、細胞増殖等の後も、増殖細胞に分配されることから、再生過程の 追跡が可能である。  In particular, when they are introduced into cells, they stay in the cells, and when they are introduced into a single cell, they are distributed to the proliferating cells after cell proliferation, etc. Can be tracked.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0027] < 1〉本発明の化合物 [0027] <1> Compound of the present invention
[0028] (構造） [0028] (Structure)
本発明の金属キレート複合体は、 1又は 2以上の金属キレートと、ポリビュルアルコー ル (以下、「PVA」と記載することがある。）を構成成分として含んでいる。  The metal chelate complex of the present invention contains one or more metal chelates and polybulal alcohol (hereinafter sometimes referred to as “PVA”) as constituent components.
[0029] (バリエーション） [0029] (Variation)
(1)金属キレート  (1) Metal chelate
(i)金属  (i) Metal
金属キレートに用いられる金属イオンとしては、各種の磁性金属が挙げられる。具体 的には、ガドリニウムイオン，セリウムイオン，サマリウムイオン，ユウピウムイオン，ジス プロシゥムイオン，ツリウムイオン，イツトリビゥムイオンなどのランタノイド系元素のィォ ン，クロミゥムイオン，モリブデニゥムイオン，タングステンイオンなどの 6族元素のィォ ン，マンガンなどの 7族元素のイオン，鉄イオン，ルテニウムイオン，ォスニゥムイオン などの 8族元素のイオン，コバルトイオン，ロジウムなどの 9族元素のイオン，ニッケル イオン，パラジウムイオンなどの 10族元素のイオン，銅イオンなどの 11族元素のィォ ン，亜鉛イオン，カドミウムイオンなどの 12族元素のイオンなどが挙げられる。  Examples of metal ions used for the metal chelate include various magnetic metals. Specifically, the six lanthanoid elements such as gadolinium ion, cerium ion, samarium ion, upium ion, dysprosium ion, thulium ion, and yttrium ion, chromium ion, molybdenum ion, tungsten ion, etc. Element ions, Group 7 element ions such as manganese, Iron ion, Ruthenium ion, Group 8 element ion such as osmium ion, Cobalt ion, Group 9 element ion such as rhodium, Nickel ion, Palladium ion, etc. 10 Ions of group elements, ions of group 11 elements such as copper ions, ions of group 12 elements such as zinc ions and cadmium ions.
これらの中では、キレート化剤の原子と配位する数が最も多ぐ錯体の安定性が高い などの点から、ガドリュウム（Gd)イオンを用いることが好ましい。ガドリュウムイオンを 用いた本発明の金属キレート複合体は、プロトンの緩和速度増強効果が最も高ぐ造 影剤としての効果が高レ、ためである。  Of these, gadmium (Gd) ions are preferably used from the viewpoint that the complex having the largest number of coordination with the chelating agent atom has the highest stability. This is because the metal chelate complex of the present invention using gadolinium ions is highly effective as an agent having the highest proton relaxation rate enhancement effect.
[0030] (ii)キレート化剤 金属キレートに用いられるキレート化剤（キレート配位子）としては、公知の DTPA (ジ エチレントリアミン五酢酸）， DTPA—BMA (ジエチレントリアミン五酢酸 ブチルメタ タリレート）， DTP A無水物， DOTA ( l , 4, 7, 10 テトラァザシクロドデカン 1 , 4, 7, 10 テトラアセテート）， D03A ( 1 , 4, 7, 10 テトラァザシクロドデカン 1 , 4, 7 トリアセテート）， HP— D〇3A (2 ヒドロキシプロピノレー 1 , 4, 7, 10 テトラァザ シクロドデカン— 1 , 4, 7 トリアセテート）等が挙げられるが、特に、臨床で使用され た実績が長いという点では、 DTPAが好ましく、キレートの安定性の点では、 DOTA が好ましい。 [0030] (ii) chelating agent Chelating agents (chelating ligands) used for metal chelates include the well-known DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid butyl methacrylate), DTP A anhydride, DOTA (l, 4, 7, 10 Tetraazacyclododecane 1, 4, 7, 10 Tetraacetate), D03A (1, 4, 7, 10 Tetraazacyclododecane 1, 4, 7 Triacetate), HP—D03A (2 Hydroxypro Pinole 1, 4, 7, 10 tetraazacyclododecane-1, 4, 7 triacetate), etc., but DTPA is particularly preferred because of its long track record of clinical use. Then DOTA is preferred.
従って、本発明の化合物を、本発明の、細胞内滞在型 MRI造影剤として用いる場合 には、特に、長期間の細胞追跡を必要とするため、安定性の高い DOTAが好ましい Therefore, when the compound of the present invention is used as the intracellular residence type MRI contrast agent of the present invention, DOTA having high stability is preferable because long-term cell tracking is required.
〇 Yes
[0031] (iii)金属キレート  [0031] (iii) Metal chelate
本発明の金属キレート複合体に用いられる金属キレートは、ォムニスキャン (登録商 標， Gd— DTPA— BMA,第一製薬製），プロハンス（登録商標， Gd (HP— D03A) ,エーザィ製），マグネスコープ (登録商標， Gd— DOTA,栄研又は田辺製薬製）， マグネビスト(登録商標， Gd— DTPA,シエーリング製）等の市販されているものを用 いること力 Sでさるが、常法により製造することもできる。具体的には、金属イオンとキレ 一ト化剤を、例えば水溶液中で常温，常圧化、 pH6〜6. 5で、 24時間混合する等の 方法により、製造することもできる。  The metal chelate used in the metal chelate complex of the present invention includes Omniscan (registered trademark, Gd—DTPA—BMA, manufactured by Daiichi Pharmaceutical), Prohans (registered trademark, Gd (HP—D03A), manufactured by Eisai), Magnescope (Registered trademark, Gd—DOTA, manufactured by Eiken or Tanabe Seiyaku), Magnevist (registered trademark, Gd—DTPA, manufactured by Schering), etc. Use force S. You can also. Specifically, the metal ion and the quenching agent can be produced by, for example, mixing in an aqueous solution at room temperature, normal pressure, pH 6 to 6.5 for 24 hours.
好ましい pHの範囲は、キレート化剤の種類にもよる力 一般に、キレート化が促進さ れとレ、う点で、上記のような pH値とすることが好まし!/、。  The preferred pH range depends on the type of chelating agent. Generally, it is preferable to set the pH value as described above in view of the fact that chelation is promoted.
[0032] (2) PVA [0032] (2) PVA
本発明の金属キレート複合体に用レ、られる P VAとは、ビュルアルコールをその構成 単位 (モノマー）とする公知の高分子であり、非常に親水性の高い合成樹脂である。 構成成分であるビュルアルコールは、分子構造的に不安定で、高分子の製造原料と しては適さないことから、通常は、まず酢酸ビュルを重合し、ポリ酢酸ビュルとした後、 酢酸エステル残基を加水分解によって水酸基とすることによって製造される。  The PVA used in the metal chelate complex of the present invention is a known polymer having butyl alcohol as its structural unit (monomer), and is a highly hydrophilic synthetic resin. The structural component, butyalcohol, is unstable in molecular structure and unsuitable as a raw material for polymer production. Therefore, usually, acetic acid bull is first polymerized into polyacetic acid bull, and then the acetate ester residue is obtained. It is produced by converting a group into a hydroxyl group by hydrolysis.
[0033] 本発明の金属キレート複合体に用いられる PVAとしては、直鎖状のものの他、一部 が分岐構造をとつているものや、架橋構造や環状構造をとるもの等が挙げられる。そ の性質は、本発明の、プロトン緩和速度増強剤あるいは MRI造影剤としての性質に 影響しない限り、特に制限されるものでは無いが、下記のものが好ましい。 [0033] PVA used in the metal chelate complex of the present invention may be a straight chain or a part thereof. Have a branched structure, or have a crosslinked structure or a cyclic structure. The property is not particularly limited as long as it does not affect the property of the present invention as a proton relaxation rate enhancer or an MRI contrast agent, but the following are preferable.
[0034] (i)重量平均分子量  [0034] (i) Weight average molecular weight
重量平均分子量は、細胞膜を往き来し難ぐ血中半減期がある程度長ぐ一定エリア あるいは細胞内に留まり易いとレ、う点で 2000以上が好ましぐ体内に蓄積し難!/、と!/ヽ う点で 10万以下のものが好ましい。より好ましくは 2万〜 5万，更に好ましくは、 3万〜 4万である。  The weight-average molecular weight is a certain area where the blood half-life is difficult to traverse the cell membrane, or if it is likely to stay in the cell, it is difficult to accumulate more than 2000 in the body! / 100,000 is preferable in terms of the point. More preferably, it is 20,000-50,000, More preferably, it is 30,000-40,000.
[0035] (ii)ケン化度  [0035] (ii) Saponification degree
ケン化度は、 85-99. 9%が好ましい。  The saponification degree is preferably 85-99.9%.
[0036] (iii)ガラス転移温度 (Tg)  [0036] (iii) Glass transition temperature (Tg)
ガラス転移温度は、 10〜80°Cが好ましい。  The glass transition temperature is preferably 10 to 80 ° C.
ガラス転移温度は、 DSCで測定する。  The glass transition temperature is measured by DSC.
[0037] (iv)溶液粘度  [0037] (iv) Solution viscosity
溶液粘度は、；!〜 lOOOOOcps力 S好ましい。  The solution viscosity is preferably !! ~ lOOOOOcps force S.
[0038] 尚、本発明の金属キレート複合体に用いられる PVAは、本発明の、プロトン緩和速 度増強剤あるいは MRI造影剤としての性質に悪影響を及ぼさなレ、範囲で、他の高分 子の構成単位を用いて、ブロック共重合その他の共重合物としても良ぐまた他の高 分子とブレンドしても良い。  [0038] It should be noted that PVA used in the metal chelate complex of the present invention has other high molecular weights within a range that does not adversely affect the properties of the present invention as a proton relaxation rate enhancer or an MRI contrast agent. These block units may be used as block copolymers or other copolymers, and may be blended with other high molecules.
また、一つの種類の PVAを単独で用いても良ぐ複数種の PVAをブレンドして用い ることあでさる。  It is also possible to blend and use multiple types of PVA, which can be used alone.
[0039] 共重合の場合の、ビュルアルコール単位（原料成分としては、酢酸ビュル）の含有比 率は、特に限定されないが、ある程度の血中半減期と親水性，及び細胞膜に近づき 難い性質等を確実に保持するためには、高分子を構成するモノマー単位中、ビュル アルコール単位として大凡 70%以上，好ましくは 80%以上，特に好ましくは 90%以 上である。  [0039] In the case of copolymerization, the content ratio of the bull alcohol unit (the raw material component is bull acetate) is not particularly limited, but it has a certain half-life in blood, hydrophilicity, and a property that makes it difficult to access the cell membrane. In order to hold it reliably, it is about 70% or more, preferably 80% or more, particularly preferably 90% or more as a butyl alcohol unit among the monomer units constituting the polymer.
[0040] (3)リンカ一  [0040] (3) Linker
本発明のキレート複合体は、 PVAへの金属キレートの導入率をコントロールし易い点 で、リンカ一を構成成分として含んでいることが好ましい。特に、金属キレートの導入 率を上げるためには、リンカ一を使用することが好ましい。本発明で言うリンカ一とは、 その両端において、 PVAと金属キレートのそれぞれと結合することにより、 PVAと金 属キレートを結合させることのできる物質である。 The chelate complex of the present invention is easy to control the rate of introduction of metal chelate into PVA Thus, it is preferable that a linker is included as a constituent component. In particular, in order to increase the introduction rate of the metal chelate, it is preferable to use a linker. The term “linker” as used in the present invention is a substance capable of binding PVA and metal chelate by binding to PVA and metal chelate at both ends.
リンカ一の構成成分としては、ジァミン，トリアミン等のポリアミン類，ジアルコール等の 多価アルコール類，ォキシカルボン酸，アミノ酸，ジイソシアナート等が挙げられる。 ジァミンの一端は、金属キレートの持つカルボキシル基と、アミド結合し、ジァミンの他 端は、カルボキシイミダゾール（CDI)等のイミダゾール等の縮合剤を用いて、 PVAの OH基とウレタン結合する。中でも、ジァミンが、特別な装置等を必要とせず、緩和な 反応条件で行えるなど、反応が簡単であるため好まし!/、。  Examples of the structural component of the linker include polyamines such as diamine and triamine, polyhydric alcohols such as dialcohol, oxycarboxylic acid, amino acid, diisocyanate and the like. One end of diamine forms an amide bond with the carboxyl group of the metal chelate, and the other end of diamine forms a urethane bond with the OH group of PVA using a condensing agent such as imidazole such as carboxyimidazole (CDI). Among them, diamine is preferred because it can be performed under mild reaction conditions without the need for special equipment and the like!
[0041] ジァミンとしては、エチレンジァミン，プロパンジァミン，ブチレンジァミン，へキサメチ レンジァミン，ジァミノデカン等の、炭化水素化合物のジァミンが挙げられる。  [0041] Examples of diamines include hydrocarbon compounds such as ethylenediamine, propanediamine, butylenediamine, hexamethylenediamine, diaminodecane and the like.
[0042] リンカ一の炭素数は特に限定されないが、；!〜 10力 リンクさせる金属キレートと PVA との間に、適度な空間を作り、金属キレートが多く結合でき、また水溶性を維持しなが ら金属キレートの導入率を上げるとレ、う点，更には生体材料とレ、う観点から好ましレ、。 特に、生体材料としては 6以下が好ましぐ炭素数が 2〜5がより好ましぐ最も好まし いのは、プロパンジァミン類等の炭素数 3のリンカ一であり、特に、 1 , 3—プロパンジ ァミンが好ましい。  [0042] The number of carbon atoms of the linker is not particularly limited, but! ~ 10 forces Create a suitable space between the metal chelate to be linked and the PVA, so that a lot of metal chelate can be bound, and water solubility should not be maintained. However, increasing the rate of introduction of metal chelates is preferred from the standpoint of using biomaterials. In particular, as a biomaterial, 6 or less is preferable, and 2 to 5 is more preferable, and the most preferable is a linker having 3 carbon atoms such as propanediamin, and in particular, 1,3-propanediol. Ammine is preferred.
[0043] (製法）  [0043] (Production method)
本発明の金属キレート複合体は、例えば PVAの OH基に、エステル化剤等を用いて 、直接、キレートの COOH基を結合させ、その後金属イオンを導入する等、公知の方 法によって製造することができる力 予め製造した金属キレートの COOH基を、 PVA の OH基と結合させても良い。尚、本発明の金属キレート複合体の製造過程の一例 を表すスキームを図 1に示す。  The metal chelate complex of the present invention can be produced by a known method, for example, by directly bonding the chelate COOH group to the OH group of PVA using an esterifying agent or the like and then introducing a metal ion. The ability to produce COOH groups of metal chelates prepared in advance may be combined with the OH groups of PVA. A scheme showing an example of the production process of the metal chelate complex of the present invention is shown in FIG.
[0044] 本発明の金属キレート複合体がリンカ一を構成成分として含む場合は、例えば、 PV Aに、リンカ一を介して、キレート化剤を結合させた後、キレート化剤の中に、金属ィ オンを導入する等の方法によって、得ること力 Sできる。 [0044] When the metal chelate complex of the present invention contains a linker as a constituent component, for example, after a chelating agent is bonded to PV A via the linker, the metal chelating agent contains a metal It is possible to obtain power S by introducing a method such as ION.
これら各々の結合，導入工程は、常法に従い行うことができる。 尚、金属イオンを、予めキレート化剤によって金属キレートとしてから、 PVAへの結合 に用いても良い。 Each of these coupling | bonding and introduction | transduction processes can be performed in accordance with a conventional method. The metal ion may be used as a metal chelate by a chelating agent in advance and then used for binding to PVA.
[0045] PVAのリンカ一への結合は、常法によって行うことができる力 例えばジァミンをリン カーとして用いる場合、下記のようにして行うことができる。  [0045] The binding of PVA to the linker can be performed as follows, for example, when diamine is used as the linker.
まず、 PVAを DMSO (ジメチルスルフォキシド）に溶解させる。さらに CDI (カルボキ シジイミダゾール）を添加し、禁水条件で 1日攪拌する。 1日後ジァミンを過剰量加え、 1日攪拌する。得られた反応溶液を水中で透析し、凍結乾燥により目的物を得る。  First, PVA is dissolved in DMSO (dimethyl sulfoxide). Add CDI (Carboxidiimidazole) and stir for 1 day under water-free conditions. After 1 day, add an excess amount of diamine and stir for 1 day. The obtained reaction solution is dialyzed in water and the desired product is obtained by lyophilization.
[0046] リンカ一へのキレート化剤の結合は、常法によって行うことができる力 例えば、ジアミ ンを用いる場合、下記のようにして行うこと力 Sできる。 [0046] Binding of the chelating agent to the linker can be performed by a conventional method. For example, when diamine is used, it can be performed as follows.
まず、 DMSO中で、キレート化剤に対して、 N—ヒドロキシスクシンイミドを加え、活性 化エステルを形成させ、ァミンと反応させる。得られた反応溶液を水中で透析し、凍 結乾燥により目的物を得る。  First, in DMSO, N-hydroxysuccinimide is added to the chelating agent to form an activated ester, which is reacted with amine. The obtained reaction solution is dialyzed in water and freeze-dried to obtain the desired product.
[0047] キレート化剤中への金属イオンの導入は、常法によって行うことができる力 S、例えば、 下記のようにして fiうことカできる。 [0047] The introduction of the metal ion into the chelating agent can be performed by a force S that can be performed by a conventional method, for example, as follows.
まず、キレート化剤と金属イオンを水中、至適 pHで攪拌させる。得られた反応溶液を 水中で透析し、凍結乾燥により目的物を得る。  First, a chelating agent and metal ions are stirred in water at an optimum pH. The obtained reaction solution is dialyzed in water and the desired product is obtained by lyophilization.
[0048] 予め作成した金属キレートのリンカ一への結合は、常法によって行うことができるが、 例えば、下記のようにして fiうこと力 Sできる。 [0048] The metal chelate prepared in advance can be bound to the linker by a conventional method. For example, it can be applied as follows.
まず、例えば、アルデヒド型の金属錯体を用いてァミンと反応させ、シッフベースを形 成させることで、リンカ一に結合させる方法等である。  First, for example, a method of binding to a linker by reacting with an amine using an aldehyde-type metal complex to form a Schiff base.
[0049] 例えば、リンカ一が、ジァミンを構成成分として含む場合は、脱水縮合剤と PVA側鎖 アルコールを反応し、活性エステル中間体を形成させ、ジァミンを加える。次に、他方 のァミン基を、キレート化剤のカルボキシノレ基とアミド結合させる。最後に、キレート化 剤の中に、金属イオンを導入する。 [0049] For example, when the linker contains diamine as a constituent component, the dehydration condensing agent and PVA side chain alcohol are reacted to form an active ester intermediate, and diamine is added. Next, the other amine group is amide-bonded to the carboxylate group of the chelating agent. Finally, a metal ion is introduced into the chelating agent.
これらの反応は、常法に従って行うことができ、例えば Bioconjugate Chem.; (Article) ( 2004; 15(4); P.841-849)に記載の方法等を用いることができる。  These reactions can be performed according to a conventional method, and for example, the method described in Bioconjugate Chem .; (Article) (2004; 15 (4); P.841-849) can be used.
[0050] (金属キレート導入比率） [0050] (Metal chelate introduction ratio)
リンカ一を用いた場合の PVAへのリンカ一導入比率は、リンカ一の添加量に相関す るため、添加量を変化させることによって、導入比率を調整可能である。 ビュルアルコールを 1つのユニットと考えた場合に、上述の方法で導入した場合の、 全ユニットに対してリンカ一が導入されたユニットの割合は、技術的には、数％〜90 %程度まで可能である力 十分なプロトン緩和速度増強効果を発揮し、かつ水溶性 を維持するという観点からは、 3%〜40%が好ましぐ更に好ましくは 5%〜30%程 度である。 When a linker is used, the ratio of the linker introduced into the PVA correlates with the amount of linker added. Therefore, the introduction ratio can be adjusted by changing the addition amount. Technically, the percentage of units in which the linker is introduced to all the units when technically considering bull alcohol as one unit and introducing it by the above method can range from several percent to 90%. From the viewpoint of exhibiting a sufficient proton relaxation rate enhancing effect and maintaining water solubility, 3% to 40% is preferable, and more preferably about 5% to 30%.
[0051] リンカ一に対するキレート化剤の導入比率は、導入作業時に十分量のキレート化剤 を配合すれば、ほぼ 100%である。  [0051] The introduction ratio of the chelating agent to the linker is almost 100% if a sufficient amount of the chelating agent is added during the introduction operation.
[0052] 金属イオンの、キレート化剤に対する導入比率は、種々の実験条件によって、調整 可能である力 S、金属イオン導入前の PVA—リンカ一一キレート化剤の複合体に対し て、常温，常圧， ρΗ6〜ρΗ6· 5の条件下、金属イオンを混合する場合には、凡そ 50[0052] The introduction ratio of the metal ion to the chelating agent is a force S that can be adjusted according to various experimental conditions, and the PVA-linker single chelating agent complex before introduction of the metal ion at room temperature, When mixing metal ions under normal pressure, ρΗ6 to ρΗ6 · 5, approximately 50
%〜100%のキレートに導入可能である。 % To 100% of the chelate can be introduced.
尚、リンカ一を用いず、直接 PVAに対して、キレート化剤あるいは金属キレートを導 入する場合の導入比率は、種々の実験条件によって、またキレート化剤あるいは金 属キレートの添加量によって調整可能である。この場合も、リンカ一を用いる場合と同 じぐ全ユニットに対して金属キレートが導入されたユニットの割合力 S、 1. 5〜40%で あること力 S好ましく、更に好ましくは 2. 5〜30%である。  The introduction ratio when introducing a chelating agent or metal chelate directly into PVA without using a linker can be adjusted by various experimental conditions and by the amount of chelating agent or metal chelate added. It is. In this case as well, the ratio force S of the unit in which the metal chelate is introduced to the entire unit is the same as in the case of using the linker S, 1.5 to 40%. The force S is preferable, and more preferably 2.5 to 30%.
[0053] (性質） [0053] (Properties)
本発明の金属キレート複合体は、細胞膜を通り抜け難い性質を有している。特に、 P VAのケン化度が高い場合は、細胞膜への吸着が少ないという性質を有しており、細 胞膜に近づくこと自体が少なレ、と!/、う利点を有して!/、る。  The metal chelate complex of the present invention has the property of hardly passing through the cell membrane. In particular, when the degree of saponification of PVA is high, it has the property that the adsorption to the cell membrane is small, and there is little advantage of approaching the cell membrane itself! RU
特に、 PVAのケン化度が高い場合は、細胞膜への吸着が少ないという性質を有して いる。  In particular, when the degree of saponification of PVA is high, it has the property of being less adsorbed on the cell membrane.
[0054] また、本発明の金属キレート複合体を細胞内に導入した場合、細胞***後も、細胞 群のほぼ全体に亘つて、金属キレート複合体の存在が観察された（図 15, 16)。つま り、***細胞にも金属キレート複合体が分配されることが確認された。  [0054] When the metal chelate complex of the present invention was introduced into cells, the presence of the metal chelate complex was observed over almost the entire cell group even after cell division (Figs. 15 and 16). . In other words, it was confirmed that the metal chelate complex was also distributed to dividing cells.
このことによって、例えば、再生治療等において、 MRI等の手段によって、移植細胞 の増殖の様子を観察可能であることが分かった。 [0055] (用途） As a result, it has been found that, for example, in regeneration therapy, the state of proliferation of transplanted cells can be observed by means such as MRI. [0055] (Application)
本発明の金属キレート複合体は、後述のプロトン緩和速度増強剤や、 MRI造影剤等 として利用することが可能である。特に、細胞膜を通り抜け難いため、細胞内滞在型 MRI造影剤として特に有用である。  The metal chelate complex of the present invention can be used as a later-described proton relaxation rate enhancer, an MRI contrast agent, or the like. In particular, it is particularly useful as an intracellular MRI contrast agent because it is difficult to pass through the cell membrane.
[0056] < 2〉本発明のプロトン緩和速度増強剤  <0056> Proton relaxation rate enhancer of the present invention
[0057] (主成分）  [0057] (Main component)
本発明のプロトン緩和速度増強剤は、上記 < 1〉の金属キレート複合体を含むことを 特徴とする。  The proton relaxation rate enhancer of the present invention is characterized by containing the metal chelate complex of <1> above.
[0058] (濃度） [0058] (Concentration)
本発明のプロトン緩和速度増強剤中の、上記 < 1〉の金属キレート複合体の濃度は 、 0. ；!〜 10重量％が好ましぐ更に好ましくは、 0. 5〜5重量％である。 0. 1重量％ 以上で、プロトン緩和速度増強効果が得られ、 10重量％以下では、粘性が抑えられ 、注射液等に好適であるからである。  The concentration of the metal chelate complex of the above <1> in the proton relaxation rate enhancer of the present invention is preferably from 0.5 to 10% by weight, and more preferably from 0.5 to 5% by weight. This is because when the amount is 1% by weight or more, an effect of enhancing the proton relaxation rate is obtained, and when the amount is 10% by weight or less, the viscosity is suppressed and it is suitable for an injection solution or the like.
[0059] (第 2成分） [0059] (Second component)
本発明のプロトン緩和速度増強剤は、上記 < 1〉の金属キレート複合体の他、水，ァ ルコール，生理食塩水等の溶媒の他、マンニトール等の、注射剤等に一般に用いら れる成分，あるいはプロトン緩和速度増強剤や、 MRI造影剤に、一般に用いられて いる成分を、更に含ませることができる。  In addition to the metal chelate complex of <1> above, the proton relaxation rate enhancer of the present invention is a component generally used in injections such as mannitol, in addition to solvents such as water, alcohol, and physiological saline, Alternatively, components generally used in proton relaxation rate enhancers and MRI contrast agents can be further included.
[0060] また、本発明のプロトン緩和速度増強剤としての性質に悪影響を及ぼさな!/、範囲で、 他の成分を含有させることができ、例えば薬学的に許容される担体として、賦形剤， 滑沢剤，結合剤，崩壊剤，安定剤，矯味矯臭剤，希釈剤，界面活性剤，乳化剤，可 溶化剤，吸収促進剤，保湿剤，吸着剤，充填剤，増量剤，付湿剤，防腐剤等の添加 剤を用いて周知の方法で製剤化することができる。  [0060] In addition, other components may be included within a range that does not adversely affect the properties of the present invention as a proton relaxation rate enhancer. For example, as a pharmaceutically acceptable carrier, an excipient , Lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, moisturizers, adsorbents, fillers, extenders, moisturizers , And can be formulated by known methods using additives such as preservatives.
[0061] 賦形剤としては、有機系賦形剤及び無機系賦形剤等が挙げられる。  [0061] Examples of excipients include organic excipients and inorganic excipients.
[0062] (剤形）  [0062] (Dosage form)
本発明のプロトン緩和速度増強剤は、注射剤，カテーテル用液剤，経口用液剤，細 胞処理液，組織処理液等の、液剤の形態が好ましぐ中でも、移植細胞を処理する 場合の細胞処理液，組織処理液が好まし!/、。 [0063] (製法） The proton relaxation rate enhancer of the present invention is a cell treatment for treating transplanted cells even when the form of the solution is preferable, such as injection, catheter solution, oral solution, cell treatment solution, tissue treatment solution, etc. Liquid, tissue treatment liquid is preferred! [0063] (Production method)
本発明のプロトン緩和速度増強剤は、上記の主成分である金属キレート複合体と、 上記の第 2成分等の他の成分各々を、常温，常圧下で混合する等の方法によって、 得ること力 Sでさる。  The proton relaxation rate enhancer of the present invention can be obtained by a method such as mixing the metal chelate complex as the main component and each of the other components such as the second component at room temperature and normal pressure. Touch with S.
[0064] (性質） [0064] (Properties)
本発明のプロトン緩和速度増強剤は、主成分である金属キレート複合体が、細胞膜 を通り抜け難い性質を有しており、細胞膜に近づくこと自体、少ないという性質を有し ている。  The proton relaxation rate enhancer of the present invention has the property that the metal chelate complex as the main component is difficult to pass through the cell membrane, and the property that it hardly approaches the cell membrane itself.
また、本発明のプロトン緩和速度増強剤を細胞内に導入した場合、細胞***後も、 細胞群のほぼ全体に亘つて、プロトン緩和速度増強剤の存在が観察された（図 15, 16)。  In addition, when the proton relaxation rate enhancer of the present invention was introduced into cells, the presence of the proton relaxation rate enhancer was observed over almost the entire cell group even after cell division (FIGS. 15 and 16).
このことによって、例えば、再生治療等において、 MRI等の手段によって、移植細胞 の増殖の様子を観察可能であることが分力、つた。  As a result, for example, in regenerative treatment, it was possible to observe the growth of transplanted cells by means such as MRI.
[0065] (用途） [0065] (Application)
本発明のプロトン緩和速度増強剤は、後述の MRI造影剤， 中でも、細胞内滞在型の The proton relaxation rate enhancer of the present invention is an MRI contrast agent described later, in particular, an intracellular residence type.
MRI造影剤として適して!/ヽる。 Suitable as an MRI contrast agent!
[0066] < 3 >本発明の MRI造影剤 [0066] <3> MRI contrast agent of the present invention
[0067] (主成分） [0067] (Main component)
本発明の MRI造影剤は、上記 < 1〉の金属キレート複合体を含むことを特徴とする。  The MRI contrast agent of the present invention comprises the metal chelate complex of <1> above.
[0068] (濃度） [0068] (Concentration)
本発明の MRI造影剤中の、上記 < 1〉の金属キレート複合体の濃度は、 0. ；!〜 10 重量％が好ましぐ更に好ましくは、 0. 5〜5重量％である。 0. 1重量％以上で、プロ トン緩和速度増強効果が得られ、 10重量％以下では、粘性が抑えられ、注射液等に 好適であるからである。  The concentration of the metal chelate complex of the above <1> in the MRI contrast agent of the present invention is preferably from 0.5 to 10% by weight, and more preferably from 0.5 to 5% by weight. This is because when the amount is 1% by weight or more, the effect of enhancing the proton relaxation rate is obtained, and when the amount is 10% by weight or less, the viscosity is suppressed, which is suitable for an injection solution or the like.
[0069] (第 2成分） [0069] (Second component)
本発明の MRI造影剤は、上記 < 1〉の金属キレート複合体の他、水，アルコール， 生理食塩水等の溶媒の他、マンニトール等の、注射剤等に一般に用いられる成分， あるいは MRI造影剤に、一般に用いられている成分を、更に含ませることができる。 [0070] また、本発明の MRI造影剤としての性質に悪影響を及ぼさない範囲で、他の成分を 含有させることができ、例えば薬学的に許容される担体として、賦形剤，滑沢剤，結 合剤，崩壊剤，安定剤，矯味矯臭剤，希釈剤，界面活性剤，乳化剤，可溶化剤，吸 収促進剤，保湿剤，吸着剤，充填剤，増量剤，付湿剤，防腐剤等の添加剤を用いて 周知の方法で製剤化することができる。 The MRI contrast agent of the present invention includes components generally used for injections such as mannitol in addition to the metal chelate complex of <1> above, solvents such as water, alcohol, and physiological saline, or MRI contrast agents In addition, generally used components can be further included. [0070] In addition, other components can be contained within a range that does not adversely affect the properties of the MRI contrast agent of the present invention. For example, as a pharmaceutically acceptable carrier, an excipient, a lubricant, Binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption promoters, humectants, adsorbents, fillers, extenders, moisturizers, preservatives It can be formulated by a known method using additives such as.
[0071] 賦形剤としては、有機系賦形剤及び無機系賦形剤等が挙げられる。  [0071] Examples of excipients include organic excipients and inorganic excipients.
[0072] (剤形）  [0072] (Dosage form)
本発明の MRI造影剤は、注射剤，カテーテル用液剤，経口用液剤，細胞処理液， 組織処理液等の、液剤の形態が好ましぐ中でも、移植細胞を処理する場合の細胞 処理液，組織処理液が好ましい。  The MRI contrast medium of the present invention is a cell treatment solution or tissue for treating transplanted cells, even though the form of the solution is preferable, such as an injection, a catheter solution, an oral solution, a cell treatment solution, a tissue treatment solution, etc. A treatment liquid is preferred.
[0073] (製法） [0073] (Manufacturing method)
本発明の MRI造影剤は、上記の主成分である金属キレート複合体と、上記第 2成分 等の他の成分各々を、常温，常圧下で混合する等の方法によって、得ること力 Sできる  The MRI contrast agent of the present invention can be obtained by a method such as mixing the metal chelate complex as the main component and each of the other components such as the second component at room temperature and normal pressure.
[0074] (性質） [0074] (Properties)
本発明の MRI造影剤は、主成分である金属キレート複合体が、細胞膜を通り抜け難 い性質を有しており、細胞膜に近づくこと自体、少ないという性質を有している。 また、本発明の MRI造影剤を細胞内に導入した場合、細胞***後も、細胞群のほぼ 全体に亘つて、 MRI造影剤の存在が観察された（図 15, 16)。  The MRI contrast agent of the present invention has the property that the metal chelate complex as the main component is difficult to pass through the cell membrane, and the property of approaching the cell membrane itself is small. In addition, when the MRI contrast agent of the present invention was introduced into cells, the presence of the MRI contrast agent was observed over almost the entire cell group even after cell division (FIGS. 15 and 16).
このことによって、例えば、再生治療等において、移植細胞の増殖の様子を観察する ことが可能となる。  This makes it possible to observe the state of transplanted cell proliferation in, for example, regenerative treatment.
[0075] (用途） [0075] (Application)
本発明の MRI造影剤は、特に、後述の細胞内滞在型の MRI造影剤として適してい  The MRI contrast agent of the present invention is particularly suitable as an intracellular stay type MRI contrast agent described later.
[0076] < 4〉本発明の細胞内滞在型 MRI造影剤 <0076> Intracellular MRI contrast agent of the present invention
[0077] (主成分） [0077] (Main component)
本発明の細胞内滞在型 MRI造影剤は、上記 < 1〉の金属キレート複合体を含むこと を特徴とする。 [0078] (濃度） The intracellular MRI contrast agent of the present invention comprises the metal chelate complex of <1> above. [0078] (Concentration)
本発明の細胞内滞在型 MRI造影剤中の、上記 < 1〉の金属キレート複合体の濃度 は、 0.；!〜 10重量％が好ましぐ更に好ましくは、 0. 5〜5重量％である。 0. 1重量 %以上で、プロトン緩和速度増強効果が得られ、 10重量％以下では、粘性が抑えら れ、注射液等に好適であるからである。  The concentration of the metal chelate complex of <1> above in the intracellular residence type MRI contrast agent of the present invention is preferably 0.;! To 10% by weight, more preferably 0.5 to 5% by weight. is there. 0.1% by weight or more provides a proton relaxation rate enhancing effect, and 10% by weight or less suppresses viscosity and is suitable for injections and the like.
[0079] (第 2成分） [0079] (Second component)
本発明の細胞内滞在型 MRI造影剤は、上記 < 1〉の金属キレート複合体の他、水， アルコール，生理食塩水等の溶媒の他、マンニトール等の、注射剤等に一般に用い られる成分，あるいは MRI造影剤に、一般に用いられている成分を、更に含ませるこ と力 Sできる。  The intracellular residence type MRI contrast agent of the present invention includes components generally used in injections such as mannitol, in addition to the metal chelate complex of <1> above, solvents such as water, alcohol, and physiological saline, Alternatively, MRI contrast agents can be made to contain more commonly used components.
[0080] また、本発明の細胞内滞在型 MRI造影剤としての性質に悪影響を及ぼさな!/、範囲 で、他の成分を含有させることができ、例えば薬学的に許容される担体として、賦形 剤，滑沢剤，結合剤，崩壊剤，安定剤，矯味矯臭剤，希釈剤，界面活性剤，乳化剤 ,可溶化剤，吸収促進剤，保湿剤，吸着剤，充填剤，増量剤，付湿剤，防腐剤等の 添加剤を用いて周知の方法で製剤化することができる。  [0080] In addition, other components can be included within a range that does not adversely affect the properties of the intracellular residence type MRI contrast agent of the present invention! /, For example, as a pharmaceutically acceptable carrier. Forming agent, lubricant, binder, disintegrant, stabilizer, flavoring agent, diluent, surfactant, emulsifier, solubilizer, absorption enhancer, moisturizer, adsorbent, filler, extender It can be formulated by known methods using additives such as wetting agents and preservatives.
[0081] 賦形剤としては、有機系賦形剤及び無機系賦形剤等が挙げられる。  [0081] Examples of excipients include organic excipients and inorganic excipients.
[0082] (剤形）  [0082] (Dosage form)
本発明の細胞内滞在型 MRI造影剤は、注射剤，カテーテル用液剤，経口用液剤， 細胞処理液，組織処理液等の、液剤の形態が好ましぐ中でも、移植細胞を処理す る場合の細胞処理液，組織処理液が好ましレ、。  The intracellular residence type MRI contrast medium of the present invention is suitable for treatment of transplanted cells even when the form of the liquid agent is preferable, such as an injection, a catheter liquid, an oral liquid, a cell treatment liquid, and a tissue treatment liquid. Cell treatment solution and tissue treatment solution are preferred.
[0083] (製法） [0083] (Manufacturing method)
本発明の細胞内滞在型 MRI造影剤は、上記の主成分である金属キレート複合体と、 上記の第 2成分等の他の成分各々を、常温，常圧下混合する等の方法によって、得 ること力 Sでさる。  The intracellular MRI contrast agent of the present invention is obtained by a method such as mixing the above-mentioned metal chelate complex as the main component and each of the other components such as the second component at room temperature and normal pressure. That's the power S.
[0084] (性質） [0084] (Property)
本発明の細胞内滞在型 MRI造影剤は、主成分である金属キレート複合体力 細胞 膜を通り抜け難い性質を有しており、細胞膜に近づくこと自体、少ないという性質を有 している。 また、本発明の細胞内滞在型 MRI造影剤は、細胞内に導入した際、細胞内に長期 間滞在し、細胞***後は、細胞群のほぼ全体に亘つて、細胞内滞在型 MRI造影剤 の存在が観察された（図 15, 16)。 The intracellular residence type MRI contrast agent of the present invention has the property that it is difficult to pass through the cell membrane of the metal chelate complex force that is the main component, and has the property that it is less likely to approach the cell membrane itself. Further, the intracellular residence type MRI contrast agent of the present invention stays in the cell for a long time when introduced into the cell, and after cell division, the intracellular residence type MRI contrast agent extends over almost the entire cell group. Was observed (Figures 15 and 16).
このことによって、例えば、再生治療等において、移植細胞の増殖の様子を観察する ことが可能となる。  This makes it possible to observe the state of transplanted cell proliferation in, for example, regenerative treatment.
[0085] < 5〉本発明のプロトン緩和速度増強剤， MRI造影剤，細胞内滞在型 MRI造影剤 の使用方法  [0085] <5> Use of the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention
[0086] (投与方法） [0086] (Method of administration)
本発明のプロトン緩和速度増強剤， MRI造影剤，細胞内滞在型 MRI造影剤を、筋 肉，血管，臓器その他の組織に投与する場合の投与方法としては、経口投与，カテ 一テル投与，静注等の静脈投与，筋肉内投与，経皮投与，経鼻投与，皮内投与，皮 下投与，腹腔内投与，直腸内投与，粘膜投与、吸入等が挙げられるが、安全かっ血 中濃度を一定に保つという点では、静注等の静脈投与が好ましい。  The administration method for administering the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention to muscles, blood vessels, organs and other tissues includes oral administration, catheter administration, static administration. Intravenous administration such as injection, intramuscular administration, transdermal administration, nasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, rectal administration, mucosal administration, inhalation, etc. can be mentioned. In terms of keeping constant, intravenous administration such as intravenous injection is preferable.
マウス等の実験動物が測定対象の場合には、尾静脈注射等の方法も挙げられる。  When an experimental animal such as a mouse is a measurement target, a method such as tail vein injection is also included.
[0087] 本発明のプロトン緩和速度増強剤， MRI造影剤，細胞内滞在型 MRI造影剤を、対 象細胞に投与する方法としては、細胞処理液，組織処理液等の溶液に細胞を浸積し て電気刺激を与えるエレクト口ポレーシヨンのほ力、、マイクロインジェクション，細胞膜 融合性リボソームを用いる方法等を用いることができる。  [0087] As a method for administering the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention to the target cells, cells are immersed in a solution such as a cell treatment solution or a tissue treatment solution. Thus, the power of electoral position that provides electrical stimulation, microinjection, a method using a cell membrane-fused ribosome, and the like can be used.
[0088] (投与形態）  [0088] (Dosage form)
本発明のプロトン緩和速度増強剤， MRI造影剤，細胞内滞在型 MRI造影剤の剤形 は、エレクト口ポレーシヨン等に用いる細胞処理液，組織処理液の形態のほか、注射 剤やカテーテル用液剤，経口用液剤等の形態が挙げられるが、必ずしもこれらに限 られるものでは無い。  The dosage forms of the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention include cell treatment solution and tissue treatment solution used for electoral mouth position, etc., as well as injection and catheter solution, Examples include oral liquid preparations, but are not necessarily limited thereto.
[0089] (投与量） [0089] (Dose)
投与量は、標的となる組織や細胞の種類，数，疑われている疾患の種類，進行度， 患者の年齢，性別，体重等により適宜調整することができ、必ずしも限定されるもので は無いが、一般に、下記の量が選択し得る。  The dose can be adjusted as appropriate according to the type and number of target tissues and cells, the type of suspected disease, the degree of progression, the patient's age, sex, weight, etc., and is not necessarily limited. In general, however, the following amounts can be selected.
経口，経皮，注射等によって、組織に投与する際には、 1回の投与量は、通常金属キ レー卜複合体量として、 0. Olmg〜； 1000mg、好ましくは 0. lmg~1000mg,より好 ましくは 0. lmg〜； !OOmgである。 When administered to tissues by oral, dermal, injection, etc., a single dose is usually a metal key. The amount of the lei complex is from 0. Olmg to 1000 mg, preferably from 0.1 mg to 1000 mg, more preferably from 0.1 mg to OOmg.
Gd重量としては、 1回の投与当たり、 0. OOlmgから 10mg投与することが好ましい。 また、マイクロインジェクション，エレクト口ポレーシヨン，細胞膜融合性リボソームを用 いる方法等によって、細胞内に直接投与する際には、 1細胞辺り 107〜1012個の金属 キレート複合体が入る様に投与することが好ましレ、。 As the Gd weight, it is preferable to administer 0.001 mg to 10 mg per administration. In addition, when administered directly into cells by methods such as microinjection, electoporation, and cell membrane fusion ribosome, administer so that 10 7 to 10 12 metal chelate complexes per cell enter. I prefer it.
この量を投与された細胞は、細胞***に際して、***細胞にも適当な量の金属キレ ート複合体が分配されるため、細胞移植の増殖による組織の再生課程の追跡力 よ り確実となるからである。  When cells are administered in this amount, the appropriate amount of the metal chelate complex is also distributed to the dividing cells during cell division, which is more reliable than the ability to follow the tissue regeneration process through the growth of cell transplantation. Because.
[0090] < 6〉本発明のプロトン緩和速度増強剤， MRI造影剤，細胞内滞在型 MRI造影剤 の、細胞移植における使用方法 [0090] <6> Use of the proton relaxation rate enhancer, MRI contrast agent, and intracellular residence type MRI contrast agent of the present invention in cell transplantation
細胞移植に際し、 in vivo,あるいは in vitroで、マイクロインジェクション，エレクトロポ レーシヨン，又は細胞膜融合性リボソームを用いる方法等によって、プロトン緩和速度 増強剤， MRI造影剤，又は細胞内滞在型 MRI造影剤を、移植する細胞に導入する For cell transplantation, a proton relaxation rate enhancer, an MRI contrast agent, or an intracellular residence type MRI contrast agent can be obtained by in vivo or in vitro methods using microinjection, electroporation, or cell membrane fusion ribosome, etc. Introduce into cells to be transplanted
〇 Yes
当該導入細胞を移植した後、 4. 7T (3次元） MRI等によって、移植細胞，及びその ***細胞の様子を、経時的に観察することができる。  After transplanting the introduced cells, the state of the transplanted cells and their dividing cells can be observed over time by 4.7T (three-dimensional) MRI or the like.
本発明のプロトン緩和速度増強剤， MRI造影剤，又は細胞内滞在型 MRI造影剤等 を、細胞内に導入した場合、細胞***後も、細胞群のほぼ全体に亘つて、それらが 存在していることが観察されており（図 15, 16)、例えば再生治療等において、移植 細胞の増殖の様子を観察することが可能となる。  When the proton relaxation rate enhancer of the present invention, MRI contrast agent, or intracellular residence type MRI contrast agent, etc. are introduced into cells, they are present over almost the entire cell group even after cell division. It has been observed (Figs. 15 and 16) that, for example, in regeneration therapy, it is possible to observe the growth of transplanted cells.
[0091] 以下、本発明を、実施例を挙げて説明するが、本発明はこれらに限られるものでは無 い。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
実施例 1  Example 1
[0092] [リンカ一結合量の確認] [0092] [Confirmation of linker-binding amount]
PVAユニットに対する DOTA導入率（％)は、 PVAユニットに対するジァミンの仕込 み量に相関していた。  The DOTA introduction rate (%) for the PVA unit was correlated with the amount of jamain charged to the PVA unit.
結果を、表 1に示す。 キレート化剤（DOTA)は、添加量が十分であれば、ほぼ 100%のリンカ一（ジァミン） に結合することから、表 1の結果は、 PVAへのリンカ一の結合量が、リンカ一の添カロ 量に相関することを示して!/、る。 The results are shown in Table 1. Since the chelating agent (DOTA) binds to almost 100% of the linker (diamin) if the amount added is sufficient, the results in Table 1 show that the amount of linker bound to PVA is the same as that of the linker. Show that it correlates with the amount of added calories!
実施例 2  Example 2
[0093] [ガドリニウム導入率の確認] [0093] [Confirmation of gadolinium introduction rate]
ガドリニウムをキレートに導入した際、およそ 5〜7割程度の割合で、安定して導入さ れることを確言忍した。  When gadolinium was introduced into the chelate, we confirmed that it was stably introduced at a rate of about 50 to 70%.
結果を、後述する表 1に示す。  The results are shown in Table 1 below.
実施例 3  Example 3
[0094] 〔プロトン緩和速度増強効果の確認 (測定溶液中）〕  [0094] [Confirmation of proton relaxation rate enhancement effect (in measurement solution)]
プロトン緩和速度増強効果力 PVAに導入されたリンカ一比率（ 金属キレート導入 比率)や、プロトン緩和速度増強中の金属キレート複合体濃度に比例することを、実 験により確認した。  Proton relaxation rate enhancement effect It was confirmed by experiments that the ratio of the linker introduced into PVA (metal chelate introduction rate) and the concentration of the metal chelate complex during the proton relaxation rate enhancement were proportional.
[0095] 金属キレート複合体の製造： [0095] Production of metal chelate complex:
本発明の金属キレート複合体を、下記の様にして製造した。  The metal chelate complex of the present invention was produced as follows.
尚、製造過程は、図 1に示した通りである。  The manufacturing process is as shown in FIG.
[0096] (ステップ 1) [0096] (Step 1)
重量平均分子量 74800 (重合度からの計算値），ケン化度 98. 5%, Tg45°Cの、直 鎖状 PVAの側鎖 OH基に対し、脱水剤である 1 , 1 ' カルボニルビス 1H—イミダ ゾールを用いて、 1 , 3—プロパンジァミンを任意の割合で導入し、導入率の異なる 4 種のサンプル (A〜D)を得た。  1, 1 'Carbonylbis 1H— as a dehydrating agent for the side chain OH group of linear PVA with a weight average molecular weight of 74800 (calculated from the degree of polymerization), saponification degree of 98.5% and Tg of 45 ° C Using imidazole, 1,3-propanediamin was introduced at an arbitrary ratio, and four samples (A to D) having different introduction rates were obtained.
下記表 1に示す通り PVAユニットへの DOTA導入率は、 NMR測定により、ュ ニット比で、サンプル A : 13· 2%,サンプル B : 7· 5%,サンプル C : 3· 6%,サンプル D : 12. 9%であった。  As shown in Table 1 below, the DOTA introduction rate into the PVA unit is determined by NMR measurement in terms of unit ratios: Sample A: 13 · 2%, Sample B: 7.5%, Sample C: 3.6%, Sample D : 12. 9%.
[0097] (ステップ 2) [0097] (Step 2)
次に、キレート化剤である 1 , 4, 7, 10 テトラァザシクロドデカン 1 , 4, 7, 10 テ トラアセテート（DOTA)を側鎖に導入した。  Next, 1, 4, 7, 10 tetraazacyclododecane 1, 4, 7, 10 tetraacetate (DOTA), a chelating agent, was introduced into the side chain.
[0098] (ステップ 3) 更に、中心金属であるガドリニウムを DOTAと錯形成させることで、本発明の金属キ レート複合体を得た。 [0098] (Step 3) Furthermore, the metal chelate complex of the present invention was obtained by complexing gadolinium, which is the central metal, with DOTA.
尚、サンプル Dに関しては、 DOTAの導入に先立って、測定用の蛍光標識（FITC) を添加し、 PVAに結合した複数のリンカ一（ジァミン）の一部に、 DOTAでは無ぐ FI TCを結合させた。  For sample D, prior to the introduction of DOTA, a fluorescent label (FITC) for measurement is added, and FITC, which is not available in DOTA, is bound to some of the multiple linkers (diamins) bound to PVA. I let you.
[0099] 尚、以下の試験で用いた金属キレート複合体の構成を下記の表 1に示す。  [0099] The structure of the metal chelate complex used in the following tests is shown in Table 1 below.
また、表中、 Mnは、 PVAの数平均分子量， Mwは重量平均分子量を表す。  In the table, Mn represents the number average molecular weight of PVA, and Mw represents the weight average molecular weight.
[0100] [表 1] [0100] [Table 1]
Figure imgf000021_0001
プロトン緩和時間の測定：
Figure imgf000021_0001
Measurement of proton relaxation time:
上記サンプル A〜Cの金属キレート複合体を用い、 795μ\<ΌΌ Οと 5 1の Η Οを混 合した測定用溶液中で、プロトン緩和時間の測定を行った。 Using the metal chelate complexes of Samples A to C above, proton relaxation time was measured in a measurement solution in which 795 μ \ and 51 Η were mixed.
、/則疋は、 Varian Gemini 300 J MR spectrometerを使用し、 Inversion Recovery法 によって fiつた。 , / Nozomi, using Varian Gemini 300 J MR spectrometer, Inversion Recovery method By fi tsuta.
Inversion Recovery法（反転回復（IR)法）とは、電磁波を照射後、任意の時間に NM Rスペクトルを測定し、信号強度の時間変化から Tを測定する方法である。この IR法  The Inversion Recovery method (inversion recovery (IR) method) is a method in which the NMR spectrum is measured at an arbitrary time after irradiating an electromagnetic wave, and T is measured from the time change of the signal intensity. This IR method
1  1
では、まず始めに 180° ノ ルスを印加する。次に、任意の時間に 90° ノ ルスを印加 し、得られる信号虽度を日寺間に対してプロットすることで Tを得ること力 Sできる。  First of all, apply 180 ° nol. Next, it is possible to obtain T by applying 90 ° noise at an arbitrary time and plotting the obtained signal intensity against Nichiji.
1  1
高分子濃度に対してプロトン緩和速度 (緩和時間の逆数)をプロットしたところ、上記 測定用溶液中の、金属キレート複合体（高分子)濃度や、金属キレート複合体中の金 属キレートの量に対して比例的に、プロトン緩和速度が増加していることが分かった（ 図 2)。  When the proton relaxation rate (reciprocal of relaxation time) was plotted against the polymer concentration, the concentration of metal chelate complex (polymer) and the amount of metal chelate in the metal chelate complex were measured. On the other hand, it was found that the proton relaxation rate increased proportionally (Figure 2).
これらの結果から、本発明の金属キレート複合体は、濃度依存的にプロトン緩和速度 を増強し、また複合体中の金属キレート量を調節することによって、緩和度を任意に 設定可能であることが分力、つた。  From these results, it can be seen that the metal chelate complex of the present invention enhances the proton relaxation rate in a concentration-dependent manner, and the degree of relaxation can be arbitrarily set by adjusting the amount of metal chelate in the complex. Component power, ivy.
[0102] 尚、緩和速度すなわち緩和時間の逆数は、次式によって求めることができる。 [0102] The relaxation rate, that is, the reciprocal of the relaxation time, can be obtained by the following equation.
T _1 ( = 1/T ) = 1/T +RX C T _1 (= 1 / T) = 1 / T + RX C
1 1 0  1 1 0
Τ：測定対象の緩和時間  Τ: Measurement target relaxation time
1  1
τ—1:測定対象の緩和速度 τ- 1 : Relaxation rate of measurement target
1  1
τ：物質固有の緩和時間  τ: Material specific relaxation time
0  0
C：プロトン緩和速度増強剤（又は MRI造影剤）濃度  C: Proton relaxation rate enhancer (or MRI contrast agent) concentration
R :緩和度（グラフの傾き）  R: degree of relaxation (slope of graph)
実施例 4  Example 4
[0103] 〔プロトン緩和速度増強効果に対する、 PVAの役割〕  [0103] [PVA's role in enhancing proton relaxation rate]
本発明の金属キレート複合体（サンプル D)と、公知の造影剤であるマグネビスト (登 録商標， Gd— DTPA,シエーリング製）とで、プロトン緩和速度を比較した。結果を図 3に k f^。  The proton relaxation rate was compared between the metal chelate complex of the present invention (Sample D) and Magnevist (registered trademark, Gd-DTPA, manufactured by Schering), which is a known contrast agent. The result is k f ^ in Fig. 3.
[0104] 図 3から、本発明のプロトン緩和速度増強剤である、金属キレート複合体（サンプル D )は、公知の造影剤であるマグネビスト（登録商標， Gd— DTPA,シエーリング製）より も、緩和度が高ぐ緩和速度増強効果が高いことが分力、つた。  [0104] From FIG. 3, the metal chelate complex (sample D), which is a proton relaxation rate enhancer of the present invention, is more relaxed than Magnevist (registered trademark, Gd-DTPA, manufactured by Schering), which is a known contrast agent. The higher the degree, the higher the relaxation rate enhancement effect.
[0105] 尚、 Gd量が OmMの場合の T— 即ち T 1は、図 3では判別が困難である力 0. 0639 である。 [0105] Note that T-, that is, T 1 when the amount of Gd is OmM, is a force that is difficult to distinguish in FIG. It is.
即ち、本実験例における本発明のプロトン緩和速度増強剤の濃度と、緩和速度の関 係、下記式の通りとなる。  That is, the relationship between the concentration of the proton relaxation rate enhancer of the present invention in the present experimental example and the relaxation rate is as follows.
[0106] Y=T _1 ( = 1/T ) = 1/T +RX C = 0. 0639 + 6. 6067 X X [0106] Y = T _1 (= 1 / T) = 1 / T + RX C = 0.0639 + 6. 6067 XX
1 1 0  1 1 0
実施例 5  Example 5
[0107] [細胞毒性試験]  [0107] [Cytotoxicity test]
本発明の金属キレート複合体の安全性を確認するため、テトラゾリゥム塩 (WST— 1) を用いた細胞毒性試験を実施した。  In order to confirm the safety of the metal chelate complex of the present invention, a cytotoxicity test using a tetrazolium salt (WST-1) was performed.
[0108] 〈細胞毒性試験方法〉  <Cytotoxicity test method>
WST— 1を用いた細胞毒性試験は、公知の方法であり、市販の試験キット（Premix WST-1 Cell Proliferation Assay System :タカラバイオ（株）製/ Cell Proliferation Rea gent WST-1 :ロッシュ製/ W201 :株式会社 同仁化学研究所製等）を用い、当該キッ トに付属の説明書等に従って行うことができる力 S、その原理は下記の通りである。  The cytotoxicity test using WST-1 is a known method, and is a commercially available test kit (Premix WST-1 Cell Proliferation Assay System: manufactured by Takara Bio Inc./Cell Proliferation Reagent WST-1: manufactured by Roche / W201 : Dojindo Laboratories Co., Ltd., etc.) and can be performed according to the instructions attached to the kit, etc. The principle is as follows.
[0109] テトラゾリゥム塩は、生存細胞にだけ活性のあるコハク酸塩テトラゾリゥム還元酵素 (EC 1.3.99.1)によって、ホルマザン色素に分解される。従って、生存細胞数が増加すれ ば、試料中の酵素活性が増加し、ホルマザン色素が増加することになる。ホルマザン 色素と生存細胞数とは直線的な相関を示す。従って、 ELISA法等によって色素溶液 の吸光度を測定し、ホルマザン色素を定量することによって、生存細胞数が測定でき る。つまり、細胞培養液に、金属キレート複合体や金属キレートを添加した後に、生 存細胞数を測定することによって、これらの細胞毒性を確認することができる。  [0109] Tetrazolium salt is degraded to formazan dye by succinate tetrazolium reductase (EC 1.3.99.1), which is active only in living cells. Therefore, if the number of viable cells increases, the enzyme activity in the sample increases and the formazan dye increases. The formazan dye and the number of viable cells show a linear correlation. Therefore, the number of viable cells can be measured by measuring the absorbance of the dye solution by ELISA or the like and quantifying the formazan dye. That is, after adding a metal chelate complex or a metal chelate to the cell culture medium, the cytotoxicity can be confirmed by measuring the number of viable cells.
[0110] 以下、この方法を用い、金属キレートや金属キレート複合体を、細胞の内'外に投与 した場合の細胞毒性を、各々確認した。その結果を、図 4, 5で表す。  [0110] In the following, this method was used to confirm cytotoxicity when a metal chelate or metal chelate complex was administered inside or outside the cell. The results are shown in FIGS.
[0111] 〈1 :細胞外投与による毒性試験〉  [0111] <1: Toxicity study by extracellular administration>
上述の方法に従い、比較対照となる公知の MRI造影剤である金属キレート（マグネビ スト（登録商標， Gd-DTPA,シエーリング製）又は本発明の金属キレート複合体（サ ンプル B)を含む PBS溶液，あるいは対照となる PBS溶液を、 NIH— 3T3細胞を培 養している培地に各々投与した場合の、生存細胞数を測定することによって、細胞毒 性を確認した。結果を図 4, 5に示す。尚、縦軸は UVの値を表す。 尚、金属キレート又は金属キレート複合体を含む溶液の、キレート濃度は、図 4, 5に 示す通りである。 According to the above-mentioned method, a metal chelate (Magnevist (registered trademark, Gd-DTPA, manufactured by Schering)) or a PBS solution containing the metal chelate complex of the present invention (sample B), which is a known MRI contrast medium for comparison, Alternatively, the cytotoxicity was confirmed by measuring the number of viable cells when each control PBS solution was administered to the medium in which NIH-3T3 cells were cultured, and the results are shown in Figs. The vertical axis represents the UV value. The chelate concentration of the solution containing the metal chelate or metal chelate complex is as shown in FIGS.
[0112] 図 4, 5から、本発明の金属キレート複合体は、投与 3日後も、無投与群と比べて生存 細胞数に代わりが無ぐ筋肉，血管，臓器その他の組織に投与した場合に、従来公 知の MRI造影剤と比較して、同等あるいはそれ以上に細胞毒性が無!/、ことが分かつ た。  [0112] From FIGS. 4 and 5, when the metal chelate complex of the present invention is administered to muscles, blood vessels, organs or other tissues that have no substitute for the number of viable cells compared to the non-administration group even 3 days after administration. Compared with the conventionally known MRI contrast agents, it has been found that there is no or no cytotoxicity.
[0113] 〈2 :細胞内投与による毒性試験〉  [0113] <2: Toxicity test by intracellular administration>
本発明の金属キレート複合体（サンプル A, B)を含有する培養液を、エレクトロボレ ーシヨンによって細胞内に導入した場合の、生存細胞数を、上述の方法に従って測 定することにより、細胞毒性を確認した。結果を図 6, 7に示す。尚、縦軸は UVの値を 表す。  Cytotoxicity is measured by measuring the number of viable cells according to the above-described method when the culture solution containing the metal chelate complex of the present invention (samples A and B) is introduced into the cells by electroporation. confirmed. The results are shown in Figs. The vertical axis represents the UV value.
[0114] 図 6, 7から、本発明の金属キレート複合体（サンプル A, B)は、いずれも、投与 3日 後も、無投与群と比べて生存細胞数に代わりが無ぐ細胞内に投与した場合にも、殆 ど細胞毒性が無レ、ことが分力、つた。  [0114] From Figs. 6 and 7, it can be seen that the metal chelate complex of the present invention (samples A and B) was not changed in the number of viable cells compared to the non-administered group even after 3 days of administration. Even when administered, it was almost non-cytotoxic.
実施例 6  Example 6
[0115] [MRIによる細胞内コントラスト増加の確認試験]  [0115] [Confirmation test of intracellular contrast increase by MRI]
本発明の金属キレート複合体（表 1のサンプル A, B, C)を含む PBS溶液を、 MRIを 用いて撮像した。撮像されたそれぞれのサンプルの画像の輝度を画像処理等の方 法によって測定した。  A PBS solution containing the metal chelate complex of the present invention (samples A, B, and C in Table 1) was imaged using MRI. The brightness of each sampled image was measured by methods such as image processing.
[0116] 結果を図 9〜； 11及び図 12に表す。  [0116] The results are shown in FIGS. 9 to 11 and FIG.
[0117] 尚、図 8は、図 9〜； 11の MRI撮像図中の、 3つの金属キレート複合体に対応するそれ ぞれの濃度を表す図である。  [0117] FIG. 8 is a diagram showing the respective concentrations corresponding to the three metal chelate complexes in the MRI imaging diagrams of FIGS.
また、図 12の縦軸は PBSの輝度を 1とした場合におけるそれぞれのサンプルの輝度 を表している。  The vertical axis in Fig. 12 represents the luminance of each sample when the luminance of PBS is 1.
[0118] 図 12から、本発明の金属キレート複合体（サンプル A, B, C)は、いずれも、サンプル 濃度の増加に応じて MRI画像のコントラストの増加が見られることが分力、つた。また、 金属キレートの導入率に依存してコントラストに差が見られることもわ力、つた。  [0118] From FIG. 12, it was found that the metal chelate complexes of the present invention (samples A, B, C) all showed that the contrast of the MRI image was increased as the sample concentration increased. In addition, the difference in contrast depending on the introduction rate of the metal chelate was seen.
実施例 Ί [0119] [MRIによる、造影剤ラベル化細胞の撮像] Example Ί [0119] Imaging of contrast-labeled cells by MRI
本発明の金属キレート複合体（表 1のサンプル A)を、エレクト口ポレーシヨンによって NIH— 3T3細胞内に導入した。得られた細胞を封入したァガロースゲルを、 MRIを 用いて撮像した。得られた画像を画像処理により、ァガロースゲルの輝度を 1として、 ァガロース +細胞（細胞のみ）、ァガロース + 10— 3Mの条件下で導入した細胞（10_3 M)、ァガロース + 10— 2Mの条件下で導入した細胞（10— 2M)の輝度を求め、各々ダラ フ化した。 The metal chelate complex of the present invention (Sample A in Table 1) was introduced into NIH-3T3 cells by electopore positioning. The agarose gel encapsulating the obtained cells was imaged using MRI. The obtained image image processing, as one of the luminance of Agarosugeru, Agarosu + cells (cells only), Agarosu + 10 3 cells transduced under the conditions of M (10_ 3 M), Agarosu + 10- 2 M of obtains the brightness of the cells (10- 2 M) was introduced under the conditions, and each Dara off of.
[0120] 図 13から、細胞内にサンプル Aを導入した場合、 MRIを用いて撮像可能であるとい うことが分かった。  [0120] From Fig. 13, it was found that when sample A was introduced into cells, imaging was possible using MRI.
実施例 8  Example 8
[0121] [細胞内滞在性の確認]  [0121] [Confirmation of intracellular residence]
NIH— 3T3細胞内にエレクト口ポレーシヨン法を用いてサンプルを導入し、細胞内の サンプル量の経時的変化を、蛍光測定を用いて測定した。本発明の金属キレート複 合体サンプルとしては、サンプル Dを用いた。結果を図 14に表す。尚、図中の縦軸 は、蛍光強度の値である。  Samples were introduced into NIH-3T3 cells using the electoporation method, and changes over time in the amount of sample in the cells were measured using fluorescence measurements. Sample D was used as the metal chelate complex sample of the present invention. The results are shown in FIG. In addition, the vertical axis | shaft in a figure is a value of fluorescence intensity.
[0122] 図 14から、細胞が増殖するにも関わらず、系中におけるサンプル量はほぼ一定の値 をとつた。これより、細胞の増殖過程において、サンプルが細胞外に漏れ出すことなく 、細胞内に留まっているということが分かった。つまり、時間が経過しても細胞内にサ ンプルが保持されており、細胞移植しても長期間 MRI等によって追跡が可能である ことを示唆している。  [0122] From FIG. 14, the amount of sample in the system was almost constant despite the cell growth. Thus, it was found that the sample remained in the cell without leaking out of the cell during the cell growth process. In other words, it is suggested that the sample is retained in the cells over time, and that it can be traced by MRI for a long time even after cell transplantation.
実施例 9  Example 9
[0123] [細胞内に取り込まれている様子の確認]  [0123] [Confirmation of cellular uptake]
本発明の金属キレート複合体（サンプル D)を用い、エレクト口ポレーシヨン法により細 胞内への導入を行った。導入 2日後の顕微鏡写真（明視野、蛍光）を示す（図 15, 16 )  Using the metal chelate complex of the present invention (sample D), introduction into the cells was performed by the electopore method. A micrograph (bright field, fluorescence) 2 days after introduction is shown (Fig. 15, 16)
[0124] 図 15, 16から、細胞内に導入後 2日経過した後でも、細胞内に安定にサンプルが存 在して!/、ると!/、うことが分かった。  [0124] From FIGS. 15 and 16, it was found that even after 2 days from introduction into the cell, the sample was stably present in the cell! /, And! /.
また、細胞が増殖しているにも関わらず、ほとんどすべての細胞が光っていることから 、サンプルが、細胞***の過程で、細胞外に漏れ出していないだけでなぐ個々の分 裂細胞にも、ある程度均等に、サンプルが分配されていることが分力、つた。 Also, because almost all cells are shining even though the cells are growing In the process of cell division, the sample was distributed evenly to a certain degree evenly to the individual split cells as well as not leaking out of the cell.
実施例 10  Example 10
[0125] [細胞内滞在性の確認 2]  [0125] [Confirmation of intracellular retention 2]
実施例 8と同様にして、本発明の金属キレート複合体（サンプル D)を細胞内に投与 した後の、金属キレート複合体の細胞内滞在性の経時的変化を、細胞の増殖性と共 に測定した結果を、図 17に示す。  In the same manner as in Example 8, the time-dependent change in intracellular retention of the metal chelate complex after administration of the metal chelate complex of the present invention (sample D) into the cells was observed together with the cell growth properties. Figure 17 shows the measurement results.
[0126] 図 17から、系中におけるサンプル量はほぼ一定の値をとる一方、細胞数は、サンプ ルを導入しいていない細胞と同様に、 日数経過に伴い確実に増殖していること力 数 値的に確認できた (破線)。これより、細胞の増殖過程において、サンプルが細胞外 に漏れ出すことなぐ細胞内に留まって!/、ると!/、うことが分力、つた。  [0126] From FIG. 17, it can be seen that the amount of sample in the system is almost constant, but the number of cells is proliferating with the passage of days, as in the case of cells not introduced with the sample. Value was confirmed (dashed line). As a result, during the cell growth process, the sample stayed inside the cell without leaking out of the cell! / ,!
つまり、時間が経過しても細胞内にサンプルが保持されており、細胞移植しても長期 間 MRI等によって追跡が可能であることを示唆している。  In other words, it is suggested that the sample is retained in the cells over time, and that it can be traced by MRI for a long time even after cell transplantation.
実施例 11  Example 11
[0127] [細胞の in vitroにおける MRI撮像] [0127] [MRI imaging of cells in vitro]
本発明の金属キレート複合体（表 1のサンプル D)を、エレクト口ポレーシヨンによって NIH— 3T3細胞内に導入後、試験管底部に細胞を集積させ、試験管底部を含むよ うに水平方向にスライスし、 MRI撮像を行った。結果を図 18に示す。  After the metal chelate complex of the present invention (sample D in Table 1) is introduced into NIH-3T3 cells by electo-poration, the cells are accumulated at the bottom of the test tube and sliced horizontally to include the bottom of the test tube. MRI imaging was performed. The results are shown in FIG.
比較対象として、サンプルを導入していない NIH— 3T3細胞，及び培地のみを入れ た試験管を用いた。  For comparison, NIH-3T3 cells into which no sample was introduced and a test tube containing only the medium were used.
[0128] 図 18から、金属キレート複合体（サンプル D)を導入した細胞は、比較対象に比べて 輝度の増加が見られた。輝度の上昇は細胞内に含まれるサンプルに依存するため、 本発明の金属キレート複合体を用いることによって、 MRIによる細胞の検出が出来る ことが確認できた。  [0128] From FIG. 18, the cells into which the metal chelate complex (sample D) was introduced showed an increase in brightness as compared with the comparison target. Since the increase in luminance depends on the sample contained in the cells, it was confirmed that the cells can be detected by MRI by using the metal chelate complex of the present invention.
実施例 12  Example 12
[0129] [細胞の in vivoにおける MRI撮像]  [0129] [MRI imaging of cells in vivo]
本発明の金属キレート複合体（表 1のサンプル D)を、エレクト口ポレーシヨンによって NIH 3T3細胞内に導入後、ァガロースゲル内に細胞を封入した。 得られたゲルをマウス皮下に移植し、 MRI撮像を行った。結果を図 19に示す。 The metal chelate complex of the present invention (sample D in Table 1) was introduced into NIH 3T3 cells by electopore positioning, and the cells were encapsulated in an agarose gel. The obtained gel was implanted subcutaneously in mice and MRI imaging was performed. The results are shown in FIG.
比較対象として、ァガロースゲルのみを、同マウスの別の部位 (皮下）に移植した。  For comparison, only agarose gel was transplanted to another site (subcutaneous) of the same mouse.
[0130] 図 19から、金属キレート複合体（サンプル D)を導入した細胞が内包されているゲル の部分で、輝度の上昇が見られた。 [0130] From FIG. 19, an increase in luminance was observed in the gel portion in which the cells into which the metal chelate complex (sample D) was introduced were encapsulated.
これより、 in vivoにおいても、 MRIを用いて細胞の追跡が可能であることが確認できた  This confirms that cells can be traced using MRI even in vivo.
[0131] 尚、図 9, 10, 11 , 15, 16について、同じ図面の、解像度を上げたものを、それぞれ 図 20, 21 , 22, 23, 24として、記載する。 [0131] For FIGS. 9, 10, 11, 15 and 16, the same drawings with higher resolution are shown as FIGS. 20, 21, 22, 23 and 24, respectively.
産業上の利用可能性  Industrial applicability
[0132] 本発明は、画像診断に用いることが可能な新規化合物に関するものであり、細胞膜 の内外で行き来することなぐ一定の箇所に滞在することによって、細胞や器官等の 一定エリアの経時的変化を検証可能な、プロトン緩和速度増強剤，又は MRI造影剤 に関するものであり、医療現場や研究現場での幅広い利用が可能である。 [0132] The present invention relates to a novel compound that can be used for diagnostic imaging, and changes over time in certain areas such as cells and organs by staying in a certain place without going back and forth between cell membranes. It is related to proton relaxation rate enhancers or MRI contrast agents that can be verified, and can be widely used in medical and research fields.
図面の簡単な説明  Brief Description of Drawings
[0133] [図 1]本発明の金属キレート複合体の製造過程を表すスキームである。  FIG. 1 is a scheme showing the production process of the metal chelate complex of the present invention.
[図 2]溶液中の、金属キレート複合体 (高分子)濃度や、複合体中の金属キレート量が 多くなるのに比例して、プロトン緩和速度が増加することを示す図である。  FIG. 2 is a graph showing that the proton relaxation rate increases in proportion to the concentration of the metal chelate complex (polymer) in the solution and the amount of metal chelate in the complex.
[図 3]金属キレート複合体中の金属導入量が増加するに従って、傾き (緩和度）が大 きくなることを示す図である。  FIG. 3 is a graph showing that the slope (relaxation) increases as the amount of metal introduced into the metal chelate complex increases.
[図 4]公知の金属キレート（マグネビスト：登録商標）を、細胞外に投与した場合の細 胞毒性を示す図である。  FIG. 4 is a graph showing cell toxicity when a known metal chelate (Magnevist: registered trademark) is administered extracellularly.
[図 5]本発明の金属キレート複合体を、細胞外に投与した場合の細胞毒性を示す図 である。  FIG. 5 is a graph showing cytotoxicity when the metal chelate complex of the present invention is administered extracellularly.
[図 6]本発明の金属キレート複合体 (サンプル A)を、細胞内に投与した場合の細胞 毒性を示す図である。  FIG. 6 is a graph showing cytotoxicity when the metal chelate complex of the present invention (sample A) is administered into cells.
[図 7]本発明の金属キレート複合体 (サンプル B)を、細胞内に投与した場合の細胞 毒性を示す図である。  FIG. 7 is a graph showing cytotoxicity when the metal chelate complex of the present invention (sample B) is administered intracellularly.
[図 8]図 9〜； 11の MRI撮像図中の、 3つの金属キレート複合体に対応するそれぞれ の濃度を表す図である。 [Figure 8] Figures 9 to 11; each corresponding to three metal chelate complexes in the MRI images of 11 FIG.
園 9]本発明の金属キレート複合体（サンプル A)の PBS溶液を、異なる濃度で MRI により撮像した図である。 FIG. 9] It is the figure which imaged the PBS solution of the metal chelate complex (sample A) of the present invention by MRI at different concentrations.
園 10]本発明の金属キレート複合体（サンプル B)の PBS溶液を、異なる濃度で MRI により撮像した図である。 [Sen10] These are images of PBS solutions of the metal chelate complex of the present invention (sample B) taken at different concentrations by MRI.
園 11]本発明の金属キレート複合体（サンプル C)の PBS溶液を、異なる濃度で MRI により撮像した図である。 11] These are images of PBS solutions of the metal chelate complex of the present invention (sample C) taken at different concentrations by MRI.
園 12]PBSの輝度を 1とした場合のそれぞれの輝度を表す図である。 12] It is a diagram showing the respective luminances when the luminance of PBS is 1.
園 13]細胞を本発明の金属キレート複合体 (サンプル A)でラベル化したものを MRI で測定した結果を示す図である。 FIG. 13] A diagram showing the results of MRI measurement of cells labeled with the metal chelate complex of the present invention (sample A).
園 14]細胞内に投与した、本発明の金属キレート複合体 (サンプル D)の、細胞内滞 在性を示す図である。 FIG. 14] A graph showing intracellular retention of the metal chelate complex of the present invention (sample D) administered into cells.
[図 15]細胞内への、本発明の金属キレート複合体（サンプル D)の導入 2日後の、明 視野写真を示す図である。  FIG. 15 is a view showing a bright field photograph two days after introduction of the metal chelate complex of the present invention (sample D) into cells.
[図 16]細胞内への、本発明の金属キレート複合体（サンプル D)の導入 2日後の、蛍 光写真を示す図である。  FIG. 16 shows a fluorescent photograph two days after introduction of the metal chelate complex of the present invention (sample D) into cells.
園 17]本発明の金属キレート複合体 (サンプル D)を細胞内に投与した後の、金属キ レート複合体の細胞内滞在性を、細胞の増殖性と共に示す図である。 FIG. 17] A graph showing the intracellular retention of the metal chelate complex together with the cell proliferation ability after administration of the metal chelate complex of the present invention (sample D) into the cell.
園 18]NIH— 3T3細胞内にサンプルを導入後、試験管底部に細胞を集積させ、試 験管底部を含むように水平方向にスライスし、 MRI撮像を行った結果を示す図である 園 19]本発明の金属キレート複合体（サンプル D)を細胞内に導入し、マウス皮下に 移植後、 MRIにより撮像した結果を示す図である。 Fig. 18] This figure shows the results of MRI imaging after introducing samples into NIH-3T3 cells, accumulating cells at the bottom of the test tube, slicing horizontally to include the bottom of the test tube, and so on. FIG. 4 is a view showing the results of MRI imaging after the metal chelate complex of the present invention (sample D) was introduced into cells and transplanted subcutaneously into mice.
[図 20]図 9の、解像度を上げた図である。  FIG. 20 is a diagram in which the resolution is increased in FIG.
[図 21]図 10の、解像度を上げた図である。  FIG. 21 is a diagram in which the resolution is increased in FIG.
[図 22]図 11の、解像度を上げた図である。  FIG. 22 is a diagram with the resolution increased in FIG.
[図 23]図 15の、解像度を上げた図である。  FIG. 23 is a diagram in which the resolution is increased in FIG.
[図 24]図 16の、解像度を上げた図である。  FIG. 24 is a diagram in which the resolution is increased in FIG.

Claims

請求の範囲 The scope of the claims
[1] 1又は 2以上の金属キレートと、ポリビュルアルコールを構成成分として含むことを特 徴とする、金属キレート複合体。  [1] A metal chelate complex comprising one or more metal chelates and polybulal alcohol as constituent components.
[2] 金属キレートが、リンカ一を介してポリビュルアルコールに結合していることを特徴と する、請求項 1記載の金属キレート複合体。 [2] The metal chelate complex according to [1], wherein the metal chelate is bound to polybulal alcohol via a linker.
[3] リンカ一が、ジァミンであることを特徴とする、請求項 2記載の金属キレート複合体。 [3] The metal chelate complex according to [2], wherein the linker is diamine.
[4] キレート化剤が、 DTPA, DTP A無水物， DOTA, D03A, HP— D03A, DTPA [4] Chelating agent is DTPA, DTP A anhydride, DOTA, D03A, HP— D03A, DTPA
BMAからなる群から選択される 1又は 2以上のキレート化剤であることを特徴とす る、請求項 1乃至 3のいずれかに記載の金属キレート複合体。  4. The metal chelate complex according to claim 1, wherein the metal chelate complex is one or more chelating agents selected from the group consisting of BMA.
[5] ポリビュルアルコールの重量平均分子量が 2000〜； 10万であることを特徴とする、請 求項 1乃至 4のいずれかに記載の金属キレート複合体。 [5] The metal chelate complex according to any one of claims 1 to 4, wherein the polybulal alcohol has a weight average molecular weight of 2000 to 100,000.
[6] 金属がガドリニウムであることを特徴とする、請求項 1乃至 5のいずれかに記載の金属 キレート複合体。 [6] The metal chelate complex according to any one of [1] to [5], wherein the metal is gadolinium.
[7] 請求項 1乃至 6記載のいずれかに記載の金属キレート複合体を含むことを特徴とする [7] Including the metal chelate complex according to any one of claims 1 to 6.
、プロトン緩和速度増強剤。 , Proton relaxation rate enhancer.
[8] 請求項 1乃至 6記載のいずれかに記載の金属キレート複合体を含むことを特徴とする[8] Including the metal chelate complex according to any one of claims 1 to 6.
、 MRI造影剤。 MRI contrast agent.
[9] 請求項 1乃至 6記載のいずれかに記載の金属キレート複合体を含むことを特徴とする 、細胞内滞在型 MRI造影剤。  [9] An intracellular stay-type MRI contrast agent comprising the metal chelate complex according to any one of [1] to [6].
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