WO2024058065A1 - Heat induction type drug delivery system - Google Patents

Heat induction type drug delivery system Download PDF

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WO2024058065A1
WO2024058065A1 PCT/JP2023/032806 JP2023032806W WO2024058065A1 WO 2024058065 A1 WO2024058065 A1 WO 2024058065A1 JP 2023032806 W JP2023032806 W JP 2023032806W WO 2024058065 A1 WO2024058065 A1 WO 2024058065A1
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temperature
polymer
particles
responsive polymer
functional group
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PCT/JP2023/032806
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French (fr)
Japanese (ja)
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健二郎 花岡
創太 山田
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慶應義塾
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a drug delivery composition and a drug delivery system.
  • U.S. Pat. No. 5,001,301 discloses magnetically responsive compositions for delivering bioactive substances consisting of particles that can be injected into blood vessels. This technology transports biologically active substances by magnetically guiding magnetic particles to the affected area.
  • Patent Document 2 discloses a drug that is composed of an organic compound or an inorganic compound and is characterized by having magnetism due to side chain modification and/or crosslinking between side chains.
  • U.S. Pat. No. 5,002,003 discloses a temperature-sensitive liposome comprising at least one phosphatidylcholine, at least one phosphatidylglycerol, and at least one lysolipid, the liposome having a gel-liquid phase transition temperature of about 39.0°C to about 45°C. Disclosed are temperature-sensitive liposomes having a temperature-sensitive liposome.
  • Non-Patent Document 1 discloses that polypeptide nanoparticles encapsulating a thermoresponsive drug are administered into the veins of mice, and the target tissue is heated, thereby increasing the delivery efficiency of the drug in the nanoparticles to the target tissue. This is disclosed.
  • the problem to be solved by the present invention is to provide a drug delivery composition and a drug delivery system in which the effect of drug retention at a target site by heating persists even after the heating is stopped.
  • the present invention includes the embodiments described below.
  • Item 1 first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group; a second particle containing a second temperature-responsive polymer and a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer; Contains and By heating, the first reactive functional group of the first polymer and the second reactive functional group of the second polymer react, and a crosslink is formed between the first particle and the second particle.
  • a composition for drug delivery A composition for drug delivery.
  • the first temperature-responsive polymer has a first lower critical melting temperature; the second temperature-responsive polymer has a second lower critical melting temperature; The first temperature-responsive polymer and the second temperature-responsive polymer become hydrophobic by heating at a temperature equal to or higher than the higher of the first lower critical melting temperature and the second lower critical melting temperature.
  • Each of the first particles and the second particles is a micelle, and each of the first temperature-responsive polymer and the second temperature-responsive polymer includes a hydrophilic segment and a hydrophobic segment.
  • Each of the hydrophilic segments of the first temperature-responsive polymer and the second temperature-responsive polymer is a poly(meth)acrylamide derivative, a polyvinylamide derivative, a poly(meth)acrylate derivative, a polyvinyl methyl ether, a cellulose derivative, Item 2.
  • the drug delivery composition according to item 1, comprising a polypeptide, poly(vinyl)caprolactam, a copolymer of polyethylene glycol and polypropylene glycol, or a copolymer of two or more thereof.
  • Section 5. The drug delivery composition according to Item 1, wherein the first particles and the second particles are hydrophilic at 37°C or lower and hydrophobic at 38°C or higher.
  • Section 6 In the state before heating, the main chain length of the second polymer is shorter than the main chain length of the second temperature-responsive polymer, and in the state after heating, part or all of the second polymer is shorter than the main chain length of the second temperature-responsive polymer.
  • Item 2 The drug delivery composition according to Item 1, wherein the length of the main chain of the molecule is longer than the length of the main chain of the second temperature-responsive polymer.
  • Item 2 The composition for drug delivery according to Item 1, wherein the first reactive functional group and the second reactive functional group form a covalent bond by the heating.
  • Section 8. Item 7. The drug delivery composition according to any one of Items 1 to 6, wherein the drug delivery composition further contains a drug.
  • Section 9 Any one of items 1 to 6, wherein the drug delivery composition further includes a poorly water-soluble drug, and the poorly water-soluble drug is incorporated into one or both of the first particles and the second particles.
  • Item 10 first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group; a second particle containing a second temperature-responsive polymer and a second polymer having a reactive functional group capable of reacting with the first reactive functional group of the first polymer and a second reactive functional group;
  • a drug delivery system comprising:
  • the drug encapsulated in the particles is retained by the cross-linked body of the first particle and the second particle that is irreversibly formed by heating.
  • the action of the drug induced at the site can be sustained.
  • FIG. 2 is a schematic diagram showing changes in the structure of micelles when a composition containing the first particles and second particles of FIG. 1(B) is heated.
  • FIG. 2 is a schematic diagram showing a state in which the first particles and second particles of FIG. 1(B) are administered into blood and retained as a crosslinked body in a target tissue. Change in micelle transmittance with temperature.
  • A DBCO micelles
  • B Az micelles. Aggregation behavior of micelles depending on temperature.
  • A DBCO micelles
  • B Az micelles
  • C a mixture containing equal amounts of DBCO micelles and Az micelles. Aggregation behavior of micelles when present in serum. Accumulation of micelles in tumor tissue of tumor-bearing model mice. Changes in tumor volume in tumor-bearing model mice.
  • PBS PBS solution
  • Dox Free Dox
  • Dox+warming Administration of free Dox, with heating treatment
  • DBCO+Az+warming Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment
  • DBCO@Dox +Az Administration of DBCO micelles and Az micelles encapsulating Dox
  • DBCO@Dox+Az+Heating Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment Survival period of tumor-bearing model mice.
  • PBS PBS solution
  • Dox Free Dox
  • Dox+warming Administration of free Dox, with heating treatment
  • DBCO+Az+warming Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment
  • DBCO@Dox +Az Administration of DBCO micelles and Az micelles encapsulating Dox
  • DBCO@Dox+Az+Heating Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment
  • PBS PBS solution
  • Dox Free Dox
  • Dox+warming Administration of free Dox, with heating treatment
  • DBCO+Az+warming Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment
  • DBCO@Dox +Az Administration of DBCO micelles and Az micelles encapsulating Dox
  • DBCO@Dox+Az+Heating Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment. Changes in weight of tumor-bearing model mice.
  • PBS PBS solution
  • Dox Free Dox
  • Dox+warming Administration of free Dox, with heating treatment
  • DBCO+Az+warming Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment
  • DBCO@Dox +Az Administration of DBCO micelles and Az micelles encapsulating Dox
  • DBCO@Dox+Az+Heating Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment
  • (meth)acrylic acid refers to acrylic acid, methacrylic acid, or both.
  • hydrophilicity refers to the property of dissolving in water when mixed with the same amount of water
  • hydrophobic refers to the property of not dissolving in water when mixed with the same amount of water
  • having temperature responsiveness refers to the fact that the solubility in water changes after a certain temperature due to heating.
  • temperature-responsive polymer refers to a polymer whose solubility in water changes when heated above a certain temperature.
  • the lower critical solution temperature refers to the temperature below which a polymer (particles in the case of particle LCST) dissolves in water to form a transparent solution; At higher temperatures, polymers become insolubilized, becoming cloudy or forming precipitates, and phase separation occurs.
  • polymer refers to a compound having a structural unit formed by bonding multiple monomers (ie, monomers), and can be used interchangeably with “polymer”.
  • active agent refers to any compound that is to be delivered to a specific site within a subject.
  • poorly water-soluble drug means a drug with a solubility in water of 1,000 ⁇ g/mL or less.
  • the poorly water-soluble drug is not to be construed as limiting as long as it is a therapeutically effective active ingredient or a prophylactically effective active ingredient.
  • the solubility of the poorly water-soluble drug in water is preferably 100 ⁇ g/mL or less, more preferably 10 ⁇ g/mL or less, even more preferably 1 ⁇ g/mL or less.
  • segment is a repeating unit of a monomer in a block copolymer, and can be used interchangeably with “block.”
  • first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group a second particle containing a second temperature-responsive polymer and a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer; Contains and By heating, the first reactive functional group of the first polymer and the second reactive functional group of the second polymer react, and a crosslink is formed between the first particle and the second particle.
  • a composition for drug delivery is provided.
  • the first temperature-responsive polymer of the first particles is not particularly limited, but includes poly(meth)acrylamide derivatives, polyvinylamide derivatives, poly(meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly( (vinyl) caprolactam, a copolymer of polyethylene glycol and polypropylene glycol, or a copolymer of two or more of these.
  • poly(meth)acrylamide derivatives include polyN-substituted (meth)acrylamide.
  • polyN-substituted (meth)acrylamide include N-cyclopropyl (meth)acrylamide, N-ethyl (meth)acrylamide, N-isopropyl (meth)acrylamide, Nn-propyl (meth)acrylamide, N- Methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide, N,N-diethylacrylamide, N-acryloylpiperidine, N-acryloylpyrrolidine, N-tetrahydrofurylacrylamide, etc.
  • Examples include polymers obtained by polymerizing.
  • poly(meth)acrylamide derivatives can be obtained by copolymerizing multiple types of N-substituted (meth)acrylamide listed above, or by copolymerizing N-substituted (meth)acrylamide and unsubstituted (meth)acrylamide.
  • a copolymer obtained by copolymerizing other monomers may also be used.
  • polyvinylamide derivatives include polymers obtained by polymerizing N-vinylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, N-vinyl-N-methylformamide, N-vinylpropionamide, etc. Can be mentioned. Preferred are N-vinylacetamide and N-vinyl-N-methylacetamide. Further, the polyvinylamide derivative may be a copolymer obtained by copolymerizing a plurality of types of vinylamide monomers listed above, or by copolymerizing the vinylamide monomer and other monomers.
  • cellulose derivatives include alkyl-substituted cellulose derivatives such as methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and hydroxyethylmethylcellulose, as well as compounds obtained by esterifying some or all of the hydroxyl groups of cellulose with organic or inorganic acids. can be mentioned.
  • poly(meth)acrylate derivatives include poly[2-(2-ethoxyethoxy)ethyl acrylate] (PEEO2A), poly[2-(2-methoxyethoxy)ethyl methacrylate] (PMEO2MA), poly(methacrylic acid) 2-[2-(2-methoxyethoxy)ethoxy]ethyl) (PMEO3MA), poly[oligomethacrylic acid (ethylene glycol)] (POEGMA), poly(2-hydroxypropyl acrylate) (PHPA), and poly( Examples include 2-hydroxyethyl methacrylate (PHEMA).
  • PEEO2A poly[2-(2-ethoxyethoxy)ethyl acrylate]
  • PMEO2MA poly[2-(2-methoxyethoxy)ethyl methacrylate]
  • PMEO3MA poly(methacrylic acid) 2-[2-(2-methoxyethoxy)ethoxy]ethyl)
  • POEGMA poly[oligomethacrylic acid
  • polypeptides include, but are not limited to, polypeptides obtained by polymerizing one or more of amino acids such as glutamic acid, aspartic acid, asparagine, lysine, glutamine, cysteine, alanine, leucine, and arginine. . Amino acids can be used alone or in combination of two or more. Examples of polypeptides include elastin-like polypeptides, polylysine, polyglutamine, and polyarginine.
  • Elastin-like polypeptide refers to a polypeptide that, like elastin, has the property of aggregating at a certain temperature or higher.
  • the term "elastin-like polypeptide" is used in many documents, and many documents also explain what kind of amino acid sequence it has to exhibit temperature sensitivity. It has been reported in Accordingly, one skilled in the art can select an appropriate elastin-like polypeptide for use in the present invention.
  • Specific examples of elastin-like polypeptides include formula (I): Xaa1-Xaa2-Gly-Val-Pro [wherein Xaa1 represents Gly or Ala and Xaa2 represents any amino acid].
  • Examples include peptides consisting of repeating amino acid sequences represented by the following.
  • Xaa1 and Xaa2 in formula (I) may represent different amino acids each time they are repeated, they preferably represent the same amino acid.
  • a preferred specific example of the amino acid sequence represented by formula (I) is Ala-Val-Gly-Val-Pro (AVGVP).
  • the number of repetitions of the amino acid sequence represented by formula (I) is not particularly limited, but if the number of repetitions is too large, the gel may become cloudy, so the number of repetitions is preferably 30 to 60, and 40 to 60. It is more preferable to set it to 50.
  • the polypeptide may be modified with a functional group such as a carboxyl group or an amino group on the side chain of an amino acid constituting the polypeptide.
  • the first temperature-responsive polymer is (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized; It may be a polymer having repeating units, or (ii) a copolymer having repeating units derived from two or more monomers, which is obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. (iii) one or more monomers that constitute a temperature-responsive polymer when polymerized, and one or two monomers that constitute a polymer that does not exhibit temperature-responsiveness even when polymerized; A copolymer formed by polymerizing the above may also be used.
  • “on polymerization” and “even if polymerization” refer to the case where one type of monomer is polymerized.
  • polymers that do not exhibit temperature responsiveness even when polymerized include poly(meth)acrylic esters, copolymers of (meth)acrylic esters and (meth)acrylic acid, polyethylene oxide, polyalkylene oxide, and polymalic acid. , polyaspartic acid, polyglutamic acid, polylysine, polysaccharide, polyvinylpyrrolidone, polyvinyl alcohol, or a segment derived from a derivative thereof.
  • the copolymer may be a block polymer, an alternating polymer, or a random polymer. Preferably, it is a polymer.
  • the first temperature-responsive polymer when the first temperature-responsive polymer is a block copolymer, the first temperature-responsive polymer is polymerized with a block formed by polymerizing monomers that constitute the temperature-responsive polymer when polymerized. It may also have a block formed by polymerizing monomers constituting a polymer that does not exhibit temperature responsiveness.
  • the first temperature-responsive polymer is a block formed by polymerizing an N-substituted (meth)acrylamide such as N-isopropyl (meth)acrylamide, and a block formed by polymerizing an unsubstituted (meth)acrylamide. It is a temperature-responsive polymer containing a block.
  • the first temperature-responsive polymer is a block copolymer having a hydrophilic segment and a hydrophobic segment, and the first temperature-responsive polymer is coated on the surface of the first micelle.
  • a hydrophilic segment of the molecule is arranged and a hydrophobic segment of the first temperature-responsive polymer is arranged inside the first micelle.
  • the hydrophilic segment is, for example, the above-mentioned (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized, and the repeats derived from the one type of monomer. (ii) A copolymer having repeating units derived from two or more monomers, which is obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. or (iii) one or more monomers that constitute a temperature-responsive polymer when polymerized and one or two or more monomers that constitute a polymer that does not exhibit temperature responsiveness when polymerized. It may be a copolymer obtained by polymerizing these.
  • monomer for example, N-isopropyl (meth)acrylamide
  • polymers that can be used to form micelles for drug delivery can be used, such as poly(meth)acrylates, poly(meth)acrylamides, lactides, lactones. , poly( ⁇ -benzyl L-alpartate), poly( ⁇ -benzyl L-glutamate), poly( ⁇ -substituted aspartate), poly( ⁇ -substituted glutamate), hydrophobic polyamino acids such as poly(L-leucine) , poly(L-valine), poly(L-phenylalanine)), polystyrene, polyamide, polyester, polyalkylene oxide, and the like, but are not limited to these.
  • the first temperature-responsive polymer includes a hydrophilic segment formed by polymerizing N-substituted (meth)acrylamide, a hydrophilic segment formed by polymerizing unsubstituted (meth)acrylamide, It has a hydrophobic segment made of poly(meth)acrylic acid ester.
  • the ratio of the number of monomers that form a temperature-responsive polymer when polymerized to the total number of monomers constituting the first temperature-responsive polymer is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more. % or more, or 100%.
  • those skilled in the art can appropriately select monomers constituting the first temperature-responsive polymer.
  • the first temperature-responsive polymer has a lower critical solution temperature (LCST).
  • LCST critical solution temperature
  • Those skilled in the art can select a temperature-responsive polymer having a required LCST depending on the intended use, required functions, and the like. Since the drug delivery composition of the present invention is expected to be used in humans or non-human animals, the LCST should be higher than the animal's body temperature, and disadvantages such as protein denaturation within the animal's body should be avoided as much as possible. It is preferable that the temperature is such that it can be used.
  • the lower critical solution temperature (LCST) of the first temperature-responsive polymer is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and more preferably in the range of 40 to 42°C. It is more preferable to fall within this range.
  • the first temperature-responsive polymer is a block copolymer having a segment that exhibits a lower critical solution temperature (LCST).
  • LCST critical solution temperature
  • Such a block copolymer is not particularly limited, but because polymerization is easy, it preferably has a structural unit derived from a monomer having a vinyl group, and more preferably has a structure having an acryloyl group or a methacryloyl group.
  • Examples of the monomer having a vinyl group include (meth)acrylamide, vinylamide, (meth)acrylic acid ester, and acrylonitrile.
  • the molecular weight of the first temperature-responsive polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa.
  • the concentration of the first temperature-responsive polymer in the first particles is preferably in the range of 50 wt% to 99 wt% from the viewpoint of controlling the temperature responsiveness of the first particles and the reaction with the second particles.
  • the first polymer of the first particle has a polymer chain portion and a first reactive functional group that binds to the polymer chain.
  • the polymer chains of the first polymer may be formed from the monomers described above with respect to the first temperature-responsive polymer.
  • the polymer chain of the first polymer is a temperature-responsive polymer
  • the polymer chain of the first polymer is a polymer (e.g., poly(metal)) constituting the first temperature-responsive polymer.
  • the polymer chain of the first polymer is derived from (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized; or (ii) a copolymer having repeating units derived from two or more monomers, which are obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized.
  • one type of monomer for example, N-isopropyl (meth)acrylamide
  • a copolymer having repeating units derived from two or more monomers which are obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized.
  • one or more monomers that form a temperature-responsive polymer when polymerized, and one or two monomers that form a polymer that does not show temperature-responsiveness when polymerized may be a copolymer obtained by polymerizing two or more species, and (iv) it does not contain any structural units derived from monomers that constitute a temperature-responsive polymer when polymerized, and does not exhibit temperature-responsiveness even when polymerized. It may be a polymer obtained by polymerizing one or more monomers constituting the polymer.
  • the copolymer may be a block polymer, an alternating polymer, or a random polymer. It is preferable that there be.
  • the first polymer when the first polymer is a block copolymer, the first polymer may have a block formed by polymerizing a monomer that constitutes a temperature-responsive polymer when polymerized, and a block formed by polymerizing a monomer that constitutes a polymer that does not exhibit temperature responsiveness when polymerized.
  • the first polymer is a temperature-responsive polymer that includes a block formed by polymerizing an N-substituted (meth)acrylamide such as N-isopropyl(meth)acrylamide, and a block formed by polymerizing an unsubstituted (meth)acrylamide.
  • the first polymer is a block copolymer having a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment of the first polymer is on the surface of the first micelle.
  • the hydrophobic segment of the first polymer is arranged inside the first micelle.
  • hydrophilic block and the hydrophobic block are as described for the first temperature-responsive polymer.
  • the chain length of the main chain of the first polymer is the same as or longer than the chain length of the main chain of the first temperature-responsive polymer. is preferable in that it is exposed on the surface of the first particle.
  • the chain length of the backbone of the first polymer is the same as the chain length of the backbone of the first temperature-responsive polymer at any temperature within the range of 5 to 50°C. It's longer than that.
  • the chain length of the main chain of the first polymer is the same as or longer than the chain length of the main chain of the first temperature-responsive polymer over the entire range of 5 to 50°C.
  • the first reactive functional group of the first polymer of the first particle reacts with the second reactive functional group of the second polymer of the second particle by heating, and the first reactive functional group of the first polymer of the first particle reacts with the second reactive functional group of the second polymer of the second particle. It is not particularly limited as long as it is a functional group that forms a crosslink between the particle and the second particle.
  • Such a pair of first and second reactive functional groups is known from click chemistry (H. C. Kolb, M. G. Finn & K. B. Sharpless: Angew. Chem., Int. Ed., 40 , 2004 (2001).) Pairs of reactive functional groups that produce click reactions can be used. Click reactions include strain-promoted azide-alkyne cycloaddition (SPAAC), which is a reaction between an alkyne and azide, and strain-promoted reverse electron request Diels-Alder, which is a reaction between an electron-deficient heterocycle and a strained C-C multiple bond compound. reaction (SPIEDAC), etc.
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • Diels-Alder which is a reaction between an electron-deficient heterocycle and a strained C-C multiple bond compound. reaction (SPIEDAC), etc.
  • Alkynes used in SPAAC include dibenzylcyclooctyne (DBCO), cyclooctyne difluoride (DIFO), bicyclononine (BCN), dibenzocyclooctyne (DIBO), and diarylazacycloocthinone (BARAC).
  • Examples include octyne derivatives. For example, by attaching a cyclooctyne derivative reagent to a polymer forming a polymer chain of the first polymer, the first reactive functional group, which is a cyclooctyne group, can be introduced into the first particle. .
  • Azides used in SPAAC include sodium azide, trimethylsilyl azide, diphenylphosphate azide, tetrabutylammonium azide, tetramethylguanidinium azide, azidoiodinane, 4-acetami2-azido-1,3-dimethylimidazo Examples include azide-introducing reagents such as linium hexafluorophosphate dobenzenesulfonic acid azide.
  • a second reactive functional group which is an azide group, can be introduced into the second particle by reacting an azide-introducing reactant with a polymer that forms a polymer chain of the second polymer. .
  • Electron-deficient heterocycles used in SPIEDAC include tetrazine and triazine.
  • strained C-C multiple bond compounds used in SPIEDAC include norbornene, trans-cyclooctene, and cyclooctyne.
  • the first reactive functional group of the first polymer of the first particle is activated by the second reactive functional group of the second polymer of the second particle at a temperature higher than the body temperature of the animal to which the first particle is administered.
  • it is a functional group that reacts with a reactive functional group.
  • the first reactive functional group of the first polymer of the first particle is activated by the first reactive functional group of the second polymer of the second particle at a temperature higher than the LCST temperature of the first temperature-responsive polymer.
  • it is a functional group that reacts with the terminal second reactive functional group.
  • the molecular weight of the first polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa.
  • the concentration of the first polymer in the first particles is preferably in the range of 1 wt% to 50 wt% from the viewpoint of controlling the temperature responsiveness of the first particles and the reaction with the second particles.
  • the first particles are preferably micelles.
  • the first particles are more preferably micelles that can be loaded with drugs.
  • the first particles are more preferably micelles that can be filled with an oil-in-water type drug that is poorly water-soluble at 20°C.
  • the first particles preferably have a lower critical solution temperature (LCST).
  • the LCST of the first particle can be determined by one skilled in the art by selecting the first temperature-responsive polymer, selecting the first polymer, and selecting the amount of those polymers in the first particle. Can be set.
  • the LCST of the first particles can be evaluated by measuring the visible light transmittance of an aqueous solution containing the particles while increasing the temperature, and from the change in the transmittance.
  • the LCST of the first particles is preferably a temperature that is higher than the animal's body temperature and that can avoid disadvantages in the animal's body such as protein denaturation as much as possible.
  • the LCST of the first particles is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and even more preferably in the range of 40 to 42°C.
  • the particle size of the first particles is preferably in the range of 10 nm to 500 nm, more preferably 30 nm to 200 nm, at a temperature below the LCST of the first particles. It is preferable that the particle size of the first particles is 10 nm or more in terms of drug encapsulation and particle stability. It is preferable that the particle size of the first particles is 500 nm or less in that the particles are delivered to the target tissue in the living body and the particles are prevented from being trapped in other non-target organs.
  • the polymer constituting the second temperature-responsive polymer of the second particle may be, but is not limited to, the same polymer as described for the first temperature-responsive polymer of the first particle. can.
  • the second temperature-responsive polymer of the second particle and the first temperature-responsive polymer of the first particle may be the same or different.
  • the second temperature-responsive polymer of the second particle and the first temperature-responsive polymer of the first particle are of the same type (e.g. poly(meth)acrylamide derivative), but the monomer composition ratio is different. and/or the length of the main chain may be different.
  • each of the first temperature-responsive polymer of the first particle and the second temperature-responsive polymer of the second particle is a poly(meth)acrylamide derivative, a polyvinylamide derivative, a polyvinylamide derivative, or Temperature-responsive polymers such as (meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly(vinyl)caprolactam, copolymers of polyethylene glycol and polypropylene glycol, or copolymers of two or more of these. include.
  • the second temperature-responsive polymer includes (i) one type of monomer that, when polymerized, constitutes the temperature-responsive polymer (for example, N-isopropyl (meth)acrylamide); ) may be a polymer having repeating units derived from the one type of monomer, or (ii) may be a polymer formed by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. It may be a copolymer having repeating units derived from two or more monomers, or (iii) one or more monomers that constitute a temperature-responsive polymer when polymerized, and a polymer that is temperature-responsive even when polymerized.
  • one type of monomer that, when polymerized, constitutes the temperature-responsive polymer for example, N-isopropyl (meth)acrylamide
  • ) may be a polymer having repeating units derived from the one type of monomer, or (ii) may be a polymer formed by polymerizing two or more monomers that constitute
  • It may also be a copolymer obtained by polymerizing one or more monomers constituting a polymer that exhibits no properties.
  • “on polymerization” and “even if polymerization” refer to the case where one type of monomer is polymerized.
  • the copolymer may be a block polymer, an alternating polymer, or a random polymer. Preferably, it is a polymer.
  • the second temperature-responsive polymer when the second temperature-responsive polymer is a block copolymer, the second temperature-responsive polymer is polymerized with a block formed by polymerizing monomers that constitute the temperature-responsive polymer when polymerized. It may also have a block formed by polymerizing monomers constituting a polymer that does not exhibit temperature responsiveness.
  • the second temperature-responsive polymer is a block formed by polymerizing an N-substituted (meth)acrylamide such as N-isopropyl (meth)acrylamide, and a block formed by polymerizing an unsubstituted (meth)acrylamide. It is a temperature-responsive polymer containing blocks.
  • the second temperature-responsive polymer is a block copolymer having a hydrophilic segment and a hydrophobic segment, and the second temperature-responsive polymer is attached to the surface of the second micelle.
  • a hydrophilic segment of the molecule is arranged and a hydrophobic segment of the second temperature-responsive polymer is arranged inside the second micelle.
  • hydrophilic block and the hydrophobic block are as described for the first temperature-responsive polymer.
  • the ratio of the number of monomers that form a temperature-responsive polymer when polymerized to the total number of monomers constituting the second temperature-responsive polymer is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more. % or more, or 100%.
  • those skilled in the art can appropriately select monomers constituting the second temperature-responsive polymer.
  • the second temperature-responsive polymer has a lower critical solution temperature (LCST).
  • LCST critical solution temperature
  • the drug delivery composition of the present invention is expected to be used in humans or non-human animals (e.g., mammals such as mice, rats, guinea pigs, rabbits, etc.)
  • the LCST is equal to or higher than the animal's body temperature, and The temperature is preferably such that disadvantages such as protein denaturation within the animal's body can be avoided as much as possible.
  • the lower critical solution temperature (LCST) of the second temperature-responsive polymer is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and more preferably in the range of 40 to 42°C. It is more preferable to fall within this range.
  • the LCST of the first temperature-responsive polymer and the LCST of the second temperature-responsive polymer may be the same or different. If the LCST of the first temperature-responsive polymer and the LCST of the second temperature-responsive polymer are different, the difference is within ⁇ 2°C in terms of matching the temperature sensitivities of the first and second particles. It is preferable that
  • the second temperature-responsive polymer is a block copolymer having a segment that exhibits a lower critical solution temperature (LCST).
  • LCST critical solution temperature
  • Such a block copolymer is not particularly limited, but because polymerization is easy, it preferably has a structural unit derived from a monomer having a vinyl group, and more preferably has a structure having an acryloyl group or a methacryloyl group.
  • Examples of the monomer having a vinyl group include (meth)acrylamide, vinylamide, (meth)acrylic acid ester, and acrylonitrile.
  • the molecular weight of the second temperature-responsive polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa.
  • the concentration of the second temperature-responsive polymer in the second particles is preferably in the range of 50 wt% to 99 wt% from the viewpoint of controlling the temperature responsiveness of the second particles and the reaction with the first particles.
  • the second polymer of the second particle has a polymer chain portion and a second reactive functional group that binds to the polymer chain.
  • the polymer chains of the second polymer may be formed from the monomers described above with respect to the first temperature-responsive polymer.
  • the polymer chain of the second polymer may be composed of the same type of polymer as the polymer constituting the first temperature-responsive polymer (e.g., poly(meth)acrylamide derivatives, polyvinylamide derivatives, poly(meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly(vinyl)caprolactam, copolymers of polyethylene glycol and polypropylene glycol, or copolymers of two or more of these, etc.), or may be composed of a different type of polymer.
  • poly(meth)acrylamide derivatives e.g., poly(meth)acrylamide derivatives, polyvinylamide derivatives, poly(meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly(vinyl)caprolactam, copolymers of polyethylene glycol and polypropylene glycol, or copolymers of two or more of these, etc.
  • the polymer chain of the second polymer is derived from (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized; or (ii) a copolymer having repeating units derived from two or more monomers, which are obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized.
  • one type of monomer for example, N-isopropyl (meth)acrylamide
  • a copolymer having repeating units derived from two or more monomers which are obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized.
  • one or more monomers that form a temperature-responsive polymer when polymerized, and one or two monomers that form a polymer that does not show temperature-responsiveness when polymerized may be a copolymer obtained by polymerizing two or more species, and (iv) it does not contain any structural units derived from monomers that constitute a temperature-responsive polymer when polymerized, and does not exhibit temperature-responsiveness even when polymerized. It may be a polymer obtained by polymerizing one or more monomers constituting the polymer.
  • the polymer chains of the second polymer are structural units derived from monomers that constitute a temperature-responsive polymer when polymerized. It is preferable to use a polymer obtained by polymerizing one or more types of monomers constituting a polymer that does not contain , and does not exhibit temperature responsiveness even when polymerized.
  • the second reactivity of the second polymer is such that the main chain length of the second polymer is shorter than that of the second temperature-responsive polymer before heating. This is preferable in that it prevents the functional group from being exposed to the surface of the second particle.
  • the chain length of the main chain of the second polymer is shorter than the chain length of the main chain of the second temperature-responsive polymer at a temperature below the second LCST of the second temperature-responsive polymer. It is preferable.
  • the chain length of the main chain of the second polymer is longer than the chain length of a part or all of the second temperature-responsive polymer in the state after heating. This is preferable in that the reactive functional group of is exposed on the surface of the second particle.
  • the chain length of the main chain of the second polymer is greater than the chain length of the main chain of the second temperature-responsive polymer at a temperature higher than the second LCST of the second temperature-responsive polymer. Preferably long.
  • the chain length of the backbone of the second polymer is greater than the chain length of the backbone of the second temperature-responsive polymer at any temperature within the range of 5 to 37°C. The first longer than the chain length of the main chain of the temperature-responsive polymer.
  • the chain length of the backbone of the second polymer is shorter than the chain length of the backbone of the second temperature-responsive polymer over the entire range of 5 to 37°C, and between 38 and 37°C. In the entire range of 50°C, preferably in the entire range of 38 to 45°C, more preferably in the entire range of 40 to 42°C, it is longer than the chain length of the main chain of the first temperature-responsive polymer.
  • the reactive functional group at the end of the second polymer of the second particle is as described above.
  • the molecular weight of the second polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa.
  • the concentration of the second polymer in the second particles is preferably in the range of 1 wt% to 50 wt% from the viewpoint of controlling the temperature responsiveness of the second particles and the reaction with the first particles.
  • the second particles are preferably micelles.
  • the second particles are more preferably micelles that can be loaded with drugs.
  • the second particles are more preferably micelles that can be filled with an oil-in-water type drug that is poorly water-soluble at 20°C.
  • the second particles preferably have a lower critical solution temperature (LCST).
  • the LCST of the second particle can be determined by one skilled in the art by selecting the second temperature-responsive polymer, selecting the second polymer, and selecting the amount of those polymers in the second particle. Can be set.
  • the LCST of the second particles can be evaluated by measuring the visible light transmittance of an aqueous solution containing the particles while increasing the temperature, and from the change in the transmittance.
  • the LCST of the second particles is preferably a temperature that is equal to or higher than the animal's body temperature and that can avoid disadvantages in the animal's body such as protein denaturation as much as possible.
  • the LCST of the second particles is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and even more preferably in the range of 40 to 42°C.
  • the LCST of the first particle and the LCST of the second particle may be the same or different. If the LCST of the first particle and the LCST of the second particle are different, the difference is preferably within ⁇ 2° C. in terms of adjusting the temperature sensitivity of the particles.
  • the particle size of the second particles is preferably in the range of 10 nm to 500 nm, more preferably 30 nm to 200 nm, at a temperature below the LCST of the second particles. It is preferable that the particle size of the second particles is 10 nm or more in terms of drug encapsulation and particle stability. It is preferable that the particle size of the second particles is 500 nm or less in order to deliver the particles to the target tissue in the living body and prevent the particles from being trapped in other non-target organs.
  • the first particle and the second particle are hydrophilic at 37°C or lower and hydrophobic at 38°C or higher.
  • hydrophilic at 37°C or lower means that it is sufficient to be hydrophilic at any temperature within the range of 37°C or lower
  • hydrophobic at 38°C or higher means , 38°C or higher.
  • the first particle and the second particle are hydrophilic at any temperature in the range of 5°C to 37°C and hydrophobic at any temperature in the range of 38°C to 50°C. It is gender.
  • the first particle and the second particle are hydrophilic over the entire range of 5°C to 37°C and hydrophobic over the entire range of 38°C to 50°C.
  • the drug delivery composition has a first particle having a lower critical solution temperature, a second particle having a second lower critical solution temperature, and a first particle having a lower critical solution temperature.
  • the first particles and the second particles become hydrophobic and contract.
  • part or all of the second reactive functional group of the second polymer in the second particle is exposed to the second particle, and the first reactive functional group of the first polymer is exposed to the second particle. This makes it easier to react.
  • the first reactivity of the first polymer is increased by heating to a temperature higher than the higher of the first lower critical melting temperature and the second lower critical melting temperature.
  • the functional group and the second reactive functional group of the second polymer react to form a crosslink between the first particle and the second particle.
  • the temperature at which the first reactive functional group of the first polymer and the second reactive functional group of the second polymer start the reaction is preferably in the range of 38 to 50°C, and 40 to 42°C. It is more preferable to be in the range of °C.
  • the first reactive functional group of the first polymer and the second reactive functional group of the second polymer form a covalent bond by heating.
  • the covalent bond irreversibly crosslinks the first particle and the second particle. Therefore, even after heating is stopped, the crosslinked body is maintained.
  • the drug delivery composition further comprises a drug.
  • a drug Preferably, it is incorporated into one or both of the first particles and the second particles.
  • the first particles and/or the second particles encapsulating the drug are encapsulated in one or both of the first particles and the second particles before heating (for example, at 20° C.).
  • drugs include, but are not limited to, antitumor agents, antibacterial agents, antifungal agents, antiparasitic agents, antiallergic agents, antiinflammatory agents, and antiviral agents.
  • the drug may be an active agent.
  • the drug preferably includes a poorly water-soluble drug.
  • poorly water-soluble drugs include: Imipramine, tofisopam, gamma oryzanol, sulpiride, thiamine disulfide, naproxen, carbamazepine, indomethacin, fentanyl, huperzine A, levodopa, clozapine, diclofenac, fexofenadine, meclozine, paroxetine, tizanidine, risperidone, olanzapine, ziprasidone, rivastigmine, naloxone, naltrexone , drugs for the nervous system and sense organs such as ibuprofen; Varnidipine hydrochloride, nicardipine hydrochloride, indenolol hydrochloride, amosralol hydrochloride, amosralol hydrochloride, isosorbide nitrate
  • poorly water-soluble drugs may form acid adduct salts, and such salts are also included in the poorly water-soluble drugs herein as long as they are pharmaceutically acceptable salts.
  • poorly water-soluble drugs include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, as well as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, and succinic acid.
  • acid addition salts with organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, para-toluenesulfonic acid, and glutamic acid.
  • the amount of drug in the first particles and/or second particles is not particularly limited, but it is preferable that the drug loading capacity (weight of drug/weight of particles containing drug) is large.
  • compositions for drug delivery may be administered by any suitable route, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intradermal intraarticular, intraventricular, intranasal, pulmonary inhalation. , oral administration, and other suitable routes of administration known to those skilled in the art.
  • the subject is a human or non-human animal, and the non-human animal is preferably a mammal such as a mouse, rat, guinea pig, or rabbit.
  • a drug delivery composition administered to a subject is delivered to a target tissue or target site within the subject's body.
  • Tissues that can be treated using this method include, but are not limited to, lung, liver, kidney, bone, soft tissue, muscle, adrenal tissue, and breast. Tissues that can be treated include both cancerous, diseased, or damaged tissue and, if desired, healthy tissue.
  • a person skilled in the art can easily determine the dosage and administration period of a drug to be administered to a subject using the drug delivery composition of the present invention.
  • the drug is suitably administered intravenously over a period of about 1 minute to several hours, such as 2, 3, 4, 6, 24 hours or more.
  • the target tissue of the subject or the target site therein may be heated before and/or during and/or after administration of the drug delivery composition of the present invention.
  • the target tissue is first heated (eg, 10-30 minutes) and the drug delivery composition of the invention is delivered into the subject as soon as possible after heating.
  • a drug delivery composition of the invention is delivered to a subject and the target tissue is heated as soon as possible after administration.
  • Any suitable heating device for heating the target tissue may be used to warm the target tissue or target region therein, such as the use of radiofrequency radiation, ultrasound (high intensity focused ultrasound), Any method that generates external or internal radiation, such as the use of microwave radiation, the use of microwave radiation, infrared radiation, light, and radioactive isotopes, electric and magnetic fields, and/or combinations of the above, in which heat is generated.
  • the sources include:
  • Heating the target tissue or site to a temperature above the higher of the first LCST of the first particle and the second LCST of the second particle causes the first particle and the second particle to become hydrophobic.
  • the first reactive functional group of the first polymer reacts with the second reactive functional group of the second polymer, and a crosslink is formed between the first particle and the second particle. is formed.
  • the drug incorporated in the first particle and/or the second particle is released from the first particle and/or the second particle.
  • the formation of an irreversible cross-linked network between the first and second particles allows the drug to continue to act at the target tissue or site even after heating has ceased.
  • the first particle contains a first temperature-responsive polymer, a first polymer having a first reactive functional group, and a second temperature-responsive polymer.
  • a second particle containing a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer is provided.
  • the first particles and the second particles are as described for the drug delivery composition of the first aspect.
  • the drug delivery system may further comprise a drug as described with respect to the drug delivery composition of the first aspect.
  • Such a combination of first particles and second particles can be used to deliver a drug to a target site and to sustain the action of the drug at the target site.
  • a first particle containing a first temperature-responsive polymer and a first polymer having a reactive functional group; a second temperature-responsive polymer; A kit for drug delivery comprising a second particle containing a second polymer having a reactive functional group capable of reacting with a first reactive functional group of the first polymer.
  • a kit for drug delivery comprising a second particle containing a second polymer having a reactive functional group capable of reacting with a first reactive functional group of the first polymer.
  • Such a combination of first particles and second particles can be used to deliver a drug to a target site and to sustain the action of the drug at the target site.
  • a method for treating a disease using the drug delivery composition, drug delivery system, or drug delivery kit of the above-described aspects of the present invention Each step, administration route, subject, target tissue, or target site in the treatment method is as described for the method of using the drug delivery composition of the first aspect.
  • the drug delivery compositions, drug delivery systems, and drug delivery kits according to the embodiments of the present invention described above can be used in clinical medicine for image diagnosis techniques (such as MRI and CT) that visualize affected areas and devices that locally heat the inside of the body (
  • image diagnosis techniques such as MRI and CT
  • a hyperthermia device such as a hyperthermia device
  • drugs can be efficiently delivered to the target tissue or site.
  • the first temperature-responsive polymer 11 has hydrophilicity at room temperature (around 20° C.).
  • the first temperature-responsive polymer 11 becomes hydrophobic when heated to a temperature equal to or higher than the lower critical solution temperature (LCST).
  • LCST lower critical solution temperature
  • the second temperature-responsive polymer 21 also becomes hydrophobic when heated to a temperature equal to or higher than the lower critical solution temperature (LCST), and returns to hydrophilicity when cooled.
  • the first particle 10 contains a first temperature-responsive polymer 11 and a first polymer 12 having a first reactive functional group 14 at the end. do.
  • the second particle 20 has a second temperature-responsive polymer 21 and a second reactive functional group 24 capable of reacting with the first reactive functional group 14 of the first polymer 12 in the chain. and a second polymer 22.
  • the first polymer 12 has a portion of the polymer chain 13 and a reactive functional group 14 that binds to the polymer chain 13
  • the second polymer 22 has a portion of the polymer chain 23 and a reactive functional group 14 that binds to the polymer chain 13. It has a reactive functional group 24 that binds to a molecular chain 23.
  • the polymer chain 13 of the first polymer 12 is also formed from a temperature-responsive polymer, and the monomers constituting the polymer chain 13 are of the same type as the monomers constituting the first temperature-responsive polymer 11. It may be different or different.
  • the chain length of the main chain of the first polymer 12 is longer than the chain length of the main chain of the first temperature-responsive polymer 11, and the reactive functional group 14 is attached to the first particle 10. exposed on the surface.
  • the polymer chain 23 of the second polymer 22 is not a temperature-responsive polymer.
  • the chain length of the main chain of the second polymer 22 is shorter than the chain length of the main chain of the second temperature-responsive polymer 21, and the reactive functional group 24 is attached to the second particle 20. It is not exposed on the surface of the temperature-responsive polymer 21, but is buried in the hydration layer formed by the hydrophilic segment of the temperature-responsive polymer 21.
  • first temperature-responsive polymer 11 and the second temperature-responsive polymer 21 are the same and have the same LCST. It is assumed that the polymer chains 13 of 12 become hydrophobic and contract.
  • the first temperature of the first particle 10 increases. Since the temperature-responsive polymer 11, the polymer chain 13 of the first polymer 12, and the second temperature-responsive polymer 21 of the second particle 20 change from hydrophilic to hydrophobic, the first particle 10 and the second particles 20 both change to be hydrophobic.
  • the first temperature-responsive polymer 11 of the first particle 10, the polymer chain 13 of the first polymer 12, and the second temperature-responsive polymer 21 of the second particle 20 are heated to a temperature higher than the LCST.
  • the chain length of the main chain of the second temperature-responsive polymer 21 of the second particle 20 becomes shorter, the chain length of the polymer chain 23 of the second polymer 22 becomes relatively shorter than the second temperature-responsive polymer 21.
  • the length of the main chain of the second temperature-responsive polymer 21 of the particle 20 is longer than that of the second particle 20, and the reactive functional group 24 is exposed on the surface of the second particle 20.
  • the individual particles exist individually dispersed before heating ( Figure 2 left).
  • the temperature of the first particles 10 and the second particles 20 becomes equal to or higher than the LCST of the first temperature-responsive polymer 11 and the second temperature-responsive polymer 21 due to heating, the first particles 10 and the second particles 20
  • the particles 20 become hydrophobic, hydrophobic interaction occurs between molecules, and the first particles 10 and the second particles 20 aggregate (center of FIG. 2).
  • the reactive functional groups 14 of the first polymer 12 and the reactive functional groups 24 of the second polymer 22 react, and the first particles 10 and the second particles 20 are heated.
  • a crosslink is formed between the first particle 10 and the second particle 20, resulting in a crosslinked body 30 (FIG. 2 right).
  • the drug delivery composition 1 containing the first particles 10 and the second particles 20 when administered to the blood vessel 40 of a human or non-human animal, the first particles 10 and the second particles Particles 20 pass through the blood vessels and travel throughout the body.
  • Blood vessel 40 branches into a peripheral blood vessel 41 within target tissue (or target site thereof) 42 .
  • target tissue 42 When the target tissue 42 is heated, the first particles 10 and the second particles 20 that have been delivered to the target tissue 42 through the peripheral blood vessels 41 come together due to hydrophobic interaction, and further the first particles 10 and the second particles 20 are brought together by hydrophobic interaction.
  • a crosslink is formed between the two particles 20.
  • the drug incorporated into one or both of the first particles 10 and the second particles 20 is released from the drug-loaded particles 10, 20.
  • the drug delivery composition of the illustrated embodiment the drug is retained by the crosslinked body 30 of the first particles 10 and the second particles 20 that are irreversibly formed by heating.
  • the drug is retained in the target tissue or target site even after the treatment, and the action of the drug in the target tissue or site is improved compared to the case where irreversible crosslinking does not occur between the first particles 10 and the second particles 20. last.
  • Example 1 Synthesis of polymer 1. Synthesis of temperature-responsive diblock copolymer Hydrophilic polymer 1, a random copolymer, was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). . 4-cyano-4-[[(dodecylthio)carbonothioyl]thio]pentanoic acid (CDTPA) as a chain transfer agent, 4,4'-azobis(4-cyanovaleric acid) (V-501) as an azo polymerization initiator was used.
  • RAFT reversible addition-fragmentation chain transfer
  • NIPAAm 3411 mg, 30.1 mmol
  • AAm 89.30 mg, 1.26 mmol
  • CDTPA 158.4 mg, 0.393 mmol
  • V-501 11.0 mg, 0.039 mmol
  • the reaction solution was added dropwise to diethyl ether, and the precipitate was filtered and dried under reduced pressure to obtain a pale yellow solid.
  • polymers with different copolymerization ratios and molecular weights were synthesized by changing the amounts of reagents.
  • NIPAAm (1896 mg, 16.8 mmol), AAm (103.6 mg, 1.46 mmol), and CDTPA (52.5 mg, 0.13 mmol) were dissolved in 6.1 mL of 1,4-dioxane, and V-501 (11.0 mg, 0.039 mmol) Hydrophilic polymer 2 was synthesized by adding and performing the same operation as above.
  • NIPAAm 4466 mg, 39.5 mmol
  • AAm 534.3 mg, 7.52 mmol
  • CDTPA (158.0 mg, 0.392 mmol) were dissolved in 15.7 mL of 1,4-dioxane, and V-501 (11.0 mg, 0.039 mmol) was dissolved.
  • Hydrophilic polymer 3 was synthesized by adding and performing the same operation as above.
  • amphiphilic polymers 4, 5, and 6, which are diblock copolymers were synthesized by RAFT polymerization of butyl methacrylate (BMA) to the synthesized hydrophilic polymers 1, 2, and 3, respectively.
  • the molecular weights of amphiphilic polymers 4, 5, and 6 were approximately 12 kDa, 19 kDa, and 17 kDa, respectively.
  • a hydrophilic polymer and 50 equivalents of BMA were dissolved in 1,4-dioxane, and dissolved oxygen was removed by bubbling with argon gas for 30 minutes. Then, 0.2 equivalents of V-501 were added, heated to 74°C, and stirred for 24 hours. The reaction solution was dialyzed against methanol for 3 days and dried under reduced pressure to obtain a pale yellow solid.
  • the reaction solution was dialyzed against methanol for 3 days and dried under reduced pressure to obtain DBCO-introduced temperature-responsive diblock copolymer 7 as a pale yellow solid.
  • the molecular weight of copolymer 7 was approximately 17 kDa.
  • Hydrophilic polymer 8 and 50 equivalents of BMA were dissolved in 1,4-dioxane, and dissolved oxygen was removed by bubbling with argon gas for 30 minutes. Then, 0.2 equivalents of V-501 were added, heated to 74°C, and stirred for 24 hours. The reaction solution was dialyzed against dimethyl sulfoxide for 3 days to obtain amphiphilic polymer 9, which is a diblock copolymer. Amphiphilic polymer 9, 5 equivalents of sodium azide, and 5 equivalents of ammonium chloride were reacted in dimethyl sulfoxide at 50°C for 24 hours. The reaction solution was dialyzed against water for 3 days and freeze-dried to obtain Az-introduced diblock copolymer 10 as a pale yellow solid. The molecular weight of copolymer 7 was approximately 11 kDa.
  • Example 2 Preparation of polymer micelles 1.
  • DBCO micelles contain the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) (Examples 3, 4, 5), and the anticancer drug.
  • doxorubicin (Dox) (Examples 6 and 7), or the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine (DiR) (Example 6) , 7) were enclosed at the time of production.
  • DiI-encapsulated DBCO micelles 9.61 mg of temperature-responsive diblock copolymer 4, 5.39 mg of DBCO-incorporated temperature-responsive diblock copolymer 7, and 0.20 mg of DiI were mixed in 2.0 mL of dimethylacetamide. The mixture was stirred overnight at 20°C. This polymer solution was added dropwise to 6.0 mL of water for injection that was being stirred at 750 rpm at 0.1 mL/min using a syringe pump, dialyzed against water for 20 hours at 20°C, and then poured into a syringe with a pore size of 0.45 ⁇ m. DBCO micelles were obtained by filtering.
  • Dox-encapsulated DBCO micelles 12.0 mg of temperature-responsive diblock copolymer 4, 7.96 mg of DBCO-introduced temperature-responsive diblock copolymer 7, and 2.0 mg of Dox neutralized with triethylamine were combined into 4.0 Stirred in mL of dimethylacetamide at 20°C overnight.
  • Dox-encapsulated DBCO micelles were obtained by the same operation as above.
  • DiI-encapsulated DBCO micelles DiR was used instead of DiI as the encapsulation dye, and a control without Dox (DBCO micelles without Dox encapsulated) was produced under the same conditions.
  • Example 3 Temperature responsiveness of micelles
  • the transmittance of micelles (1.0 mg/mL) in phosphate buffered saline (PBS) at a measurement wavelength of 750 nm was measured using an absorbance meter while increasing the temperature at 1.0°C/min. .
  • PBS phosphate buffered saline
  • Figure 4(A) shows the change in transmittance of DBCO micelles due to temperature. If the temperature at which the transmittance decreases by 50% is defined as the LCST, the LCST was approximately 39.5°C.
  • FIG. 4(B) shows the change in transmittance of Az micelles depending on the temperature, and the LCST was about 40.0°C.
  • Example 4 Crosslinking of micelles by heating 1. Measurement of particle size of micelles depending on temperature The particle size of micelles (1.0 mg/mL) in PBS was measured by dynamic light scattering while repeatedly heating and cooling. The low temperature was 32°C, and the temperature was raised to 37°C, which is below the LCST of the micelles, or 47°C, which is above the LCST.
  • Figure 5(A) shows the change in particle size of DBCO micelles depending on temperature. Even when heated to 37°C, which is below the LCST, the particle size of the DBCO micelles did not change.
  • FIG. 5(B) shows the change in particle size of Az micelles depending on temperature. When heated at 37°C and 47°C, changes similar to those of DBCO micelles were observed in Az micelles.
  • FIG. 5(C) shows the measurement results obtained by mixing equal amounts of DBCO micelles and Az micelles. Unlike the results for each individual micelle, the increase in particle size upon heating to 47°C was maintained even after cooling to 32°C, indicating that the change in particle size upon heating was irreversible. .
  • Example 5 Temperature-responsive crosslinking control of micelles in serum
  • Figure 6 shows the results of evaluating the ability of micelles to form crosslinks in PBS containing 70% fetal bovine serum as an environment simulating in vivo. Equal amounts of DBCO micelles and Az micelles were mixed, and each cycle consisted of heating and cooling for 10 minutes. After repeating this cycle for two cycles, the particle size of the particles in the mixture was measured using a dynamic light scattering method. It was 240 ⁇ 50 nm when heated to 37°C, which is the physiological body temperature, and 1922 ⁇ 70 nm when heated to 42°C, which is clinically applicable as thermotherapy.
  • Example 6 Effect of heating on the accumulation of micelles in tumor tissue
  • the effect of heating on the accumulation of micelles on the target tissue was evaluated using the tumor tissue of a tumor-bearing model mouse as the target tissue.
  • a mouse model was created by subcutaneously transplanting a mouse colon-derived cancer cell line (Colon-26) into the right hind limb of a BALB/c mouse.
  • a PBS solution containing equal amounts of Dox-encapsulated DBCO micelles and DiD-encapsulated Az micelles was administered into the tail vein (120 mg polymer/kg body weight, 200 ⁇ L/mouse).
  • the micelle accumulation rate increased dramatically by more than 10 times by heating (two bars on the left side of Figure 7).
  • Example 7 Anti-tumor effect of combined administration of micelles and heating The therapeutic effect of delivering micelles encapsulating an anti-cancer drug to the tumor tissue of tumor-bearing model mice was evaluated. Similarly to Example 6, a PBS solution containing equal amounts of Dox-encapsulated DBCO micelles and DiD-encapsulated Az micelles was administered to tumor-bearing model mice (3.0 mg Dox/kg body weight, 200 ⁇ L/mouse). ) and locally heated the tumor area. In addition, comparisons were made with a group administered with PBS as a vehicle control and a group administered with free Dox without using micelles.
  • the antitumor effect of heating is common, as exemplified by cancer hyperthermia therapy.
  • the antitumor effect was smaller in the group that combined Dox administration and heating, indicating that micelles played an important role in cancer treatment in this experiment.
  • the antitumor effect was smaller in the group in which Dox was not encapsulated in DBCO micelles than in the group in which Dox was encapsulated in DBCO micelles. Since there was a difference depending on the presence or absence of Dox encapsulation in micelles, it was suggested that the accumulation of Dox, an anticancer agent, is largely involved in improving the antitumor effect due to crosslink formation.
  • Time course of survival rate Figure 9 shows the results of analyzing the survival period of tumor-bearing model mice for 60 days after treatment using the Kaplan-Meier method. This survival rate is the result of euthanasia with a humane endpoint based on tumor volume (>2000 mm 3 ). Survival time was significantly prolonged in the group that received Dox-encapsulated DBCO micelles and Az micelles in combination with heating compared to the other groups. In 7 out of 10 animals treated with this treatment, the tumor had shrunk to an unmeasurable size (less than 1 mm in diameter) by the 24th day, and the tumor could not be confirmed even on the 100th day. It was determined that the patient had a complete success. Thereafter, as a result of continuing the experiment, as shown in FIG. 10, the survival rate of the mice in the group in which Dox-encapsulated DBCO micelles and Az micelles were administered in combination with heating was maintained until 360 days later.
  • SYMBOLS 1 Composition for drug delivery, 10... First particle, 11... First temperature-responsive polymer, 12... First polymer, 14... First reactive functional group, 20... Second particle , 21...Second temperature-responsive polymer, 22...Second polymer, 24...Second reactive functional group.

Abstract

A drug delivery composition comprising: a first particle that includes a first temperature responsive macromolecule and a first macromolecule having a first reactive functional group; and a second particle that includes a second temperature responsive macromolecule and a second macromolecule having a second reactive functional group that can react with the first reactive functional group of the first macromolecule, wherein, when heat is applied, the first reactive functional group of the first macromolecule and the second reactive functional group of the second macromolecule react with each other to form a crosslink between the first particle and the second particle.

Description

熱誘導型薬物送達システムHeat-induced drug delivery system
　本発明は、薬物送達用組成物及び薬物送達システムに関する。 The present invention relates to a drug delivery composition and a drug delivery system.
　体内に注入された薬物を患部に送達する技術が知られている。 There are known techniques for delivering drugs injected into the body to the affected area.
　例えば、特許文献１は血管に注入可能な粒子から成る生物活性物質を運搬するための磁気応答組成物を開示している。この技術では、磁性を有する粒子を磁力で患部に誘導することにより、生物活性物質を運搬している。 For example, U.S. Pat. No. 5,001,301 discloses magnetically responsive compositions for delivering bioactive substances consisting of particles that can be injected into blood vessels. This technology transports biologically active substances by magnetically guiding magnetic particles to the affected area.
　特許文献２は、有機化合物または無機化合物から構成され、側鎖の修飾または／ 及び側鎖間の架橋により磁性を有することを特徴とする薬を開示している。 Patent Document 2 discloses a drug that is composed of an organic compound or an inorganic compound and is characterized by having magnetism due to side chain modification and/or crosslinking between side chains.
　特許文献３は、少なくとも１つのホスファチジルコリン、少なくとも１つのホスファチジルグリセロールおよび少なくとも１つのリゾ脂質を含む温度感受性リポソームであって、該リポソームは約３９．０℃～約４５℃のゲル－液相転移温度を有する、温度感受性リポソームを開示している。 U.S. Pat. No. 5,002,003 discloses a temperature-sensitive liposome comprising at least one phosphatidylcholine, at least one phosphatidylglycerol, and at least one lysolipid, the liposome having a gel-liquid phase transition temperature of about 39.0°C to about 45°C. Disclosed are temperature-sensitive liposomes having a temperature-sensitive liposome.
　非特許文献１は、熱応答性を有する薬剤封入したポリペプチドナノ粒子をマウスの静脈に投与するとともに、標的組織を加温することにより、ナノ粒子中の薬剤の標的組織への送達効率を高めることを開示している。 Non-Patent Document 1 discloses that polypeptide nanoparticles encapsulating a thermoresponsive drug are administered into the veins of mice, and the target tissue is heated, thereby increasing the delivery efficiency of the drug in the nanoparticles to the target tissue. This is disclosed.
日本国特開2001-10978Japanese Patent Publication 2001-10978 WO2008/001851WO2008/001851 WO2009/062398WO2009/062398
　特許文献１及び２に記載の技術では、磁力による引力は一過的であるため、外部の磁石を除去した場合には薬剤の誘導の効果が持続しない。特許文献３に記載の温度感受性リポソームは、加温によってリポソームからの薬剤放出を引き起こすものであり、薬剤を含有するリポソームが患部に誘導されるわけでない。非特許文献１の技術では、ナノ粒子は一定の温度範囲内のみで可逆的な温度応答性を示し、効果は一過的である。 In the techniques described in Patent Documents 1 and 2, the attractive force caused by the magnetic force is temporary, so the effect of inducing the drug does not persist when the external magnet is removed. The temperature-sensitive liposome described in Patent Document 3 causes drug release from the liposome by heating, and the drug-containing liposome is not guided to the affected area. In the technique of Non-Patent Document 1, nanoparticles exhibit reversible temperature responsiveness only within a certain temperature range, and the effect is temporary.
　本発明が解決すべき課題は、加温による標的部位での薬物保持の効果が加温を停止した後でも持続する薬物送達用組成物および薬物送達システムを提供することにある。 The problem to be solved by the present invention is to provide a drug delivery composition and a drug delivery system in which the effect of drug retention at a target site by heating persists even after the heating is stopped.
　本発明は、以下に記載の実施形態を包含する。 The present invention includes the embodiments described below.
　項１．第１の温度応答性高分子と、第１の反応性官能基を有する第１の高分子とを含有する第１の粒子と、
　第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と反応可能な第２の反応性官能基を有する第２の高分子とを含有する第２の粒子と
を含有し、
　加熱により、第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基が反応し、第１の粒子と第２の粒子の間に架橋が形成される、薬物送達用組成物。 Item 1. first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group;
a second particle containing a second temperature-responsive polymer and a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer; Contains and
By heating, the first reactive functional group of the first polymer and the second reactive functional group of the second polymer react, and a crosslink is formed between the first particle and the second particle. A composition for drug delivery.
　項２．第１の温度応答性高分子が第１の下限臨界溶解温度を有し、
　第２の温度応答性高分子が第２の下限臨界溶解温度を有し、
　第１の下限臨界溶解温度及び第２の下限臨界溶解温度のうちの高い方の温度以上の温度での加熱により、第１の温度応答性高分子及び第２の温度応答性高分子が疎水性となって収縮する項１に記載の薬物送達用組成物。 Item 2. the first temperature-responsive polymer has a first lower critical melting temperature;
the second temperature-responsive polymer has a second lower critical melting temperature;
The first temperature-responsive polymer and the second temperature-responsive polymer become hydrophobic by heating at a temperature equal to or higher than the higher of the first lower critical melting temperature and the second lower critical melting temperature. Item 2. The composition for drug delivery according to item 1, which contracts as follows.
　項３．前記第１の粒子及び第２の粒子の各々がミセルであり、第１の温度応答性高分子及び第２の温度応答性高分子の各々が、親水性セグメントと疎水性セグメントとを備える項１に記載の薬物送達用組成物。 Section 3. Item 1: Each of the first particles and the second particles is a micelle, and each of the first temperature-responsive polymer and the second temperature-responsive polymer includes a hydrophilic segment and a hydrophobic segment. A composition for drug delivery according to.
　項４．第１の温度応答性高分子及び第２の温度応答性高分子のそれぞれの親水性セグメントが、ポリ（メタ）アクリルアミド誘導体、ポリビニルアミド誘導体、ポリ（メタ）アクリレート誘導体、ポリビニルメチルエーテル、セルロース誘導体、ポリペプチド、ポリ（ビニル）カプロラクタム、ポリエチレングリコールとポリプロピレングリコールの共重合体、またはこれらの２種以上の共重合体を含む項１に記載の薬物送達用組成物。 Section 4. Each of the hydrophilic segments of the first temperature-responsive polymer and the second temperature-responsive polymer is a poly(meth)acrylamide derivative, a polyvinylamide derivative, a poly(meth)acrylate derivative, a polyvinyl methyl ether, a cellulose derivative, Item 2. The drug delivery composition according to item 1, comprising a polypeptide, poly(vinyl)caprolactam, a copolymer of polyethylene glycol and polypropylene glycol, or a copolymer of two or more thereof.
　項５．前記第１の粒子及び前記第２の粒子が、３７℃以下で親水性であり、かつ３８℃以上で疎水性である、項１に記載の薬物送達用組成物。 Section 5. Item 2. The drug delivery composition according to Item 1, wherein the first particles and the second particles are hydrophilic at 37°C or lower and hydrophobic at 38°C or higher.
　項６．前記加熱前の状態では第２の高分子の主鎖の長さは第２の温度応答性高分子の主鎖の長さよりも短く、前記加熱後の状態では一部又は全部の第２の高分子の主鎖の長さが第２の温度応答性高分子の主鎖の長さよりも長くなる、項１に記載の薬物送達用組成物。 Section 6. In the state before heating, the main chain length of the second polymer is shorter than the main chain length of the second temperature-responsive polymer, and in the state after heating, part or all of the second polymer is shorter than the main chain length of the second temperature-responsive polymer. Item 2. The drug delivery composition according to Item 1, wherein the length of the main chain of the molecule is longer than the length of the main chain of the second temperature-responsive polymer.
　項７．第１の反応性官能基と第２の反応性官能基とが、前記加熱により共有結合を形成する項１に記載の薬物送達用組成物。 Section 7. Item 2. The composition for drug delivery according to Item 1, wherein the first reactive functional group and the second reactive functional group form a covalent bond by the heating.
　項８．前記薬物送達用組成物が薬物をさらに含む項１～６のいずれか一項に記載の薬物送達用組成物。 Section 8. Item 7. The drug delivery composition according to any one of Items 1 to 6, wherein the drug delivery composition further contains a drug.
　項９．前記薬物送達用組成物が難水溶性薬物をさらに含み、前記難水溶性薬物は第１の粒子及び第２の粒子のうちの一方または両方に組み込まれている項１～６のいずれか一項に記載の薬物送達用組成物。 Section 9. Any one of items 1 to 6, wherein the drug delivery composition further includes a poorly water-soluble drug, and the poorly water-soluble drug is incorporated into one or both of the first particles and the second particles. A composition for drug delivery according to.
　項１０．第１の温度応答性高分子と、第１の反応性官能基を有する第１の高分子とを含有する第１の粒子と、
　第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と第２の反応可能な反応性官能基を有する第２の高分子とを含有する第２の粒子とを備えた、薬物送達システム。 Item 10. first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group;
a second particle containing a second temperature-responsive polymer and a second polymer having a reactive functional group capable of reacting with the first reactive functional group of the first polymer and a second reactive functional group; A drug delivery system comprising:
　本発明によれば、加熱により不可逆的に形成された第１の粒子と第２の粒子の架橋体により粒子に封入した薬物が保持されるため、加熱後も標的部位において薬物が保持され、標的部位に誘導された薬物の作用を持続させることができる。 According to the present invention, the drug encapsulated in the particles is retained by the cross-linked body of the first particle and the second particle that is irreversibly formed by heating. The action of the drug induced at the site can be sustained.
（Ａ）温度応答性高分子の挙動を示す模式図。（Ｂ）第１の粒子と第２の粒子の構成及び作用を説明する模式図。(A) Schematic diagram showing the behavior of a temperature-responsive polymer. (B) A schematic diagram illustrating the structure and action of the first particle and the second particle. 図１（Ｂ）の第１の粒子と第２の粒子を含有する組成物を加熱したときのミセルの構造の変化を示す模式図。FIG. 2 is a schematic diagram showing changes in the structure of micelles when a composition containing the first particles and second particles of FIG. 1(B) is heated. 図１（Ｂ）の第１の粒子と第２の粒子が血液内に投与され、標的組織にて架橋体として保持された状態を示す模式図。FIG. 2 is a schematic diagram showing a state in which the first particles and second particles of FIG. 1(B) are administered into blood and retained as a crosslinked body in a target tissue. 温度によるミセルの透過率の変化。（Ａ）DBCOミセル、（Ｂ）Azミセル。Change in micelle transmittance with temperature. (A) DBCO micelles, (B) Az micelles. 温度によるミセルの凝集挙動。（Ａ）DBCOミセル、（Ｂ）Azミセル、（Ｃ）DBCOミセルとAzミセルを等量含む混合物。Aggregation behavior of micelles depending on temperature. (A) DBCO micelles, (B) Az micelles, (C) a mixture containing equal amounts of DBCO micelles and Az micelles. 血清中に存在する場合のミセルの凝集挙動。Aggregation behavior of micelles when present in serum. 担癌モデルマウスの腫瘍組織へのミセルの集積。Accumulation of micelles in tumor tissue of tumor-bearing model mice. 担癌モデルマウスの腫瘍体積の推移。PBS: PBS溶液、Dox:遊離Dox、Dox+加温:遊離Doxの投与，加温処理あり、DBCO+Az+加温:Doxを封入しないDBCOミセルとAzミセルの投与，加温処理あり、DBCO@Dox+Az:Doxを封入したDBCOミセルとAzミセルの投与、 DBCO@Dox+Az+加温:Doxを封入したDBCOミセルとAzミセルの投与，加温処理ありChanges in tumor volume in tumor-bearing model mice. PBS: PBS solution, Dox: Free Dox, Dox+warming: Administration of free Dox, with heating treatment, DBCO+Az+warming: Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment, DBCO@Dox +Az: Administration of DBCO micelles and Az micelles encapsulating Dox, DBCO@Dox+Az+Heating: Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment 担癌モデルマウスの生存期間。PBS: PBS溶液、Dox:遊離Dox、Dox+加温:遊離Doxの投与，加温処理あり、DBCO+Az+加温:Doxを封入しないDBCOミセルとAzミセルの投与，加温処理あり、DBCO@Dox+Az:Doxを封入したDBCOミセルとAzミセルの投与、 DBCO@Dox+Az+加温:Doxを封入したDBCOミセルとAzミセルの投与，加温処理ありSurvival period of tumor-bearing model mice. PBS: PBS solution, Dox: Free Dox, Dox+warming: Administration of free Dox, with heating treatment, DBCO+Az+warming: Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment, DBCO@Dox +Az: Administration of DBCO micelles and Az micelles encapsulating Dox, DBCO@Dox+Az+Heating: Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment 図９の実験を続けた後の担癌モデルマウスの生存期間。PBS: PBS溶液、Dox:遊離Dox、Dox+加温:遊離Doxの投与，加温処理あり、DBCO+Az+加温:Doxを封入しないDBCOミセルとAzミセルの投与，加温処理あり、DBCO@Dox+Az:Doxを封入したDBCOミセルとAzミセルの投与、 DBCO@Dox+Az+加温:Doxを封入したDBCOミセルとAzミセルの投与，加温処理あり。The survival period of the tumor-bearing model mouse after continuing the experiment shown in FIG. 9. PBS: PBS solution, Dox: Free Dox, Dox+warming: Administration of free Dox, with heating treatment, DBCO+Az+warming: Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment, DBCO@Dox +Az: Administration of DBCO micelles and Az micelles encapsulating Dox, DBCO@Dox+Az+Heating: Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment. 担癌モデルマウスの体重の推移。PBS: PBS溶液、Dox:遊離Dox、Dox+加温:遊離Doxの投与，加温処理あり、DBCO+Az+加温:Doxを封入しないDBCOミセルとAzミセルの投与，加温処理あり、DBCO@Dox+Az:Doxを封入したDBCOミセルとAzミセルの投与、 DBCO@Dox+Az+加温:Doxを封入したDBCOミセルとAzミセルの投与，加温処理ありChanges in weight of tumor-bearing model mice. PBS: PBS solution, Dox: Free Dox, Dox+warming: Administration of free Dox, with heating treatment, DBCO+Az+warming: Administration of DBCO micelles and Az micelles without Dox encapsulated, with heating treatment, DBCO@Dox +Az: Administration of DBCO micelles and Az micelles encapsulating Dox, DBCO@Dox+Az+Heating: Administration of DBCO micelles and Az micelles encapsulating Dox, with heating treatment
　本明細書において、単数形（a, an, the）は、本明細書で別途明示がある場合または文脈上明らかに矛盾する場合を除き、単数と複数を含むものとする。
　本明細書において、「含有する（comprise）」は、「実質的にのみからなる（consist essentially of）」、及び「のみからなる（consist of）」も包含する概念である。
　本明細書に段階的に記載されている数値範囲において、ある段階の数値範囲の上限値又は下限値は、他の段階の数値範囲の上限値又は下限値と任意に組み合わせることができる。また、本明細書に記載されている数値範囲において、その数値範囲の上限値又は下限値は、実施例に示されている値又は実施例から一義的に導き出せる値に置き換えてもよい。更に、本明細書において、「～」で結ばれた数値は、「～」の前後の数値を下限値及び上限値として含む数値範囲を意味する。 As used herein, the singular forms (a, an, the) shall include the singular and the plural unless the context clearly dictates otherwise.
As used herein, "comprise" is a concept that includes "consist essentially of" and "consist of".
In the numerical ranges described stepwise in this specification, the upper limit or lower limit of the numerical range of one step can be arbitrarily combined with the upper limit or lower limit of the numerical range of another step. Further, in the numerical ranges described in this specification, the upper limit or lower limit of the numerical range may be replaced with a value shown in the Examples or a value that can be uniquely derived from the Examples. Further, in this specification, numerical values connected by "~" mean a numerical range that includes the numerical values before and after the "~" as lower and upper limits.
　本明細書において、「（メタ）アクリル酸」とは、アクリル酸、メタクリル酸、又はその両方を指す。 As used herein, "(meth)acrylic acid" refers to acrylic acid, methacrylic acid, or both.
　本明細書において、「親水性」とは、同量の水と混ぜたときに水に溶解する性質を指し、「疎水性」とは、同量の水と混ぜたときに水に溶解しない性質を指す。 In this specification, "hydrophilicity" refers to the property of dissolving in water when mixed with the same amount of water, and "hydrophobic" refers to the property of not dissolving in water when mixed with the same amount of water. refers to
　本明細書において、「温度応答性」を有するとは、加温によって、ある温度を境に水に対する溶解性が変化することを指す。 In this specification, "having temperature responsiveness" refers to the fact that the solubility in water changes after a certain temperature due to heating.
　本明細書において、「温度応答性高分子」とは、加温によって、ある温度を境に水に対する溶解性が変化する高分子を指す。 In this specification, the term "temperature-responsive polymer" refers to a polymer whose solubility in water changes when heated above a certain temperature.
　本明細書において、下限臨界溶解温度（ＬＣＳＴ）とは、この温度よりも低い温度では高分子（粒子のＬＣＳＴの場合は、粒子）が水に溶解して透明の溶液になるが、この温度よりも高い温度では高分子が不溶化して白濁するか沈殿が生じ、相分離する温度である。 In this specification, the lower critical solution temperature (LCST) refers to the temperature below which a polymer (particles in the case of particle LCST) dissolves in water to form a transparent solution; At higher temperatures, polymers become insolubilized, becoming cloudy or forming precipitates, and phase separation occurs.
　本明細書において、「高分子」は、複数の単量体（すなわちモノマー）の結合により形成された構造単位を有する化合物を指し、「ポリマー」と互換的に使用することができる。 As used herein, "polymer" refers to a compound having a structural unit formed by bonding multiple monomers (ie, monomers), and can be used interchangeably with "polymer".
　本明細書において、「活性薬剤」は、対象内の特定の部位に送達されるべき任意の化合物を指す。 As used herein, "active agent" refers to any compound that is to be delivered to a specific site within a subject.
　本明細書において、「難水溶性薬物」は、水への溶解度が１，０００μｇ／ｍＬ以下の薬物を意味する。難水溶性薬物は、治療学的に有効な活性成分、あるいは予防学的に有効な活性成分であれば制限的に解釈されない。難水溶性薬物の水への溶解度は、好ましくは１００μｇ／ｍＬ以下、更に好ましくは１０μｇ／ｍＬ以下、より更に好ましくは１μｇ／ｍＬ以下である。 As used herein, "poorly water-soluble drug" means a drug with a solubility in water of 1,000 μg/mL or less. The poorly water-soluble drug is not to be construed as limiting as long as it is a therapeutically effective active ingredient or a prophylactically effective active ingredient. The solubility of the poorly water-soluble drug in water is preferably 100 μg/mL or less, more preferably 10 μg/mL or less, even more preferably 1 μg/mL or less.
　本明細書において、「セグメント」は、ブロック共重合体中の単量体の繰り返し単位であり、「ブロック」と互換的に使用することができる。 As used herein, "segment" is a repeating unit of a monomer in a block copolymer, and can be used interchangeably with "block."
　以下、本発明の実施形態を図面を参照しながら説明する。
　本発明の第１態様において、第１の温度応答性高分子と、第１の反応性官能基を有する第１の高分子とを含有する第１の粒子と、
　第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と反応可能な第２の反応性官能基を有する第２の高分子とを含有する第２の粒子と
を含有し、
　加熱により、第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基が反応し、第１の粒子と第２の粒子の間に架橋が形成される、薬物送達用組成物が提供される。 Embodiments of the present invention will be described below with reference to the drawings.
In the first aspect of the present invention, first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group;
a second particle containing a second temperature-responsive polymer and a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer; Contains and
By heating, the first reactive functional group of the first polymer and the second reactive functional group of the second polymer react, and a crosslink is formed between the first particle and the second particle. A composition for drug delivery is provided.
　第１の粒子の第１の温度応答性高分子は、特に限定されないが、ポリ（メタ）アクリルアミド誘導体、ポリビニルアミド誘導体、ポリ（メタ）アクリレート誘導体、ポリビニルメチルエーテル、セルロース誘導体、ポリペプチド、ポリ（ビニル）カプロラクタム、ポリエチレングリコールとポリプロピレングリコールの共重合体、またはこれらの２種以上の共重合体などの温度応答性高分子を含む。 The first temperature-responsive polymer of the first particles is not particularly limited, but includes poly(meth)acrylamide derivatives, polyvinylamide derivatives, poly(meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly( (vinyl) caprolactam, a copolymer of polyethylene glycol and polypropylene glycol, or a copolymer of two or more of these.
　ポリ（メタ）アクリルアミド誘導体としては、例えば、ポリＮ－置換(メタ)アクリルアミドが挙げられる。ポリＮ－置換（メタ）アクリルアミドとしては、例えば、Ｎ－シクロプロピル（メタ）アクリルアミド、Ｎ－エチル（メタ）アクリルアミド、Ｎ－イソプロピル（メタ）アクリルアミド、Ｎ－ｎ－プロピル（メタ）アクリルアミド、Ｎ－メチル－Ｎ－エチルアクリルアミド、Ｎ－メチル－Ｎ－イソプロピルアクリルアミド、Ｎ－メチル－Ｎ－ｎ－プロピルアクリルアミド、Ｎ，Ｎ－ジエチルアクリルアミド、Ｎ－アクリロイルピペリジン、Ｎ－アクリロイルピロリジン、Ｎ－テトラヒドロフリルアクリルアミド等を重合して得られるポリマーが挙げられる。また、ポリ（メタ）アクリルアミド誘導体は、複数種類の上記に列挙したＮ－置換（メタ）アクリルアミドを共重合したり、Ｎ－置換（メタ）アクリルアミドと、非置換の（メタ）アクリルアミドを始めとするその他のモノマーを共重合して得られる共重合体であってもよい。 Examples of poly(meth)acrylamide derivatives include polyN-substituted (meth)acrylamide. Examples of the polyN-substituted (meth)acrylamide include N-cyclopropyl (meth)acrylamide, N-ethyl (meth)acrylamide, N-isopropyl (meth)acrylamide, Nn-propyl (meth)acrylamide, N- Methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide, N,N-diethylacrylamide, N-acryloylpiperidine, N-acryloylpyrrolidine, N-tetrahydrofurylacrylamide, etc. Examples include polymers obtained by polymerizing. In addition, poly(meth)acrylamide derivatives can be obtained by copolymerizing multiple types of N-substituted (meth)acrylamide listed above, or by copolymerizing N-substituted (meth)acrylamide and unsubstituted (meth)acrylamide. A copolymer obtained by copolymerizing other monomers may also be used.
　ポリビニルアミド誘導体としては、例えば、Ｎ－ビニルホルムアミド、Ｎ－ビニルアセトアミド、Ｎ－ビニル－Ｎ－メチルアセトアミド、Ｎ－ビニル－Ｎ－メチルホルムアミド、Ｎ－ビニルプロピオンアミド等を重合して得られるポリマーが挙げられる。好ましくはＮ－ビニルアセトアミド、Ｎ－ビニル－Ｎ－メチルアセトアミドである。また、ポリビニルアミド誘導体は、複数種類の上記に列挙したビニルアミドモノマーを共重合したり、該ビニルアミドモノマーとその他のモノマーを共重合して得られる共重合体であってもよい。 Examples of polyvinylamide derivatives include polymers obtained by polymerizing N-vinylformamide, N-vinylacetamide, N-vinyl-N-methylacetamide, N-vinyl-N-methylformamide, N-vinylpropionamide, etc. Can be mentioned. Preferred are N-vinylacetamide and N-vinyl-N-methylacetamide. Further, the polyvinylamide derivative may be a copolymer obtained by copolymerizing a plurality of types of vinylamide monomers listed above, or by copolymerizing the vinylamide monomer and other monomers.
　セルロース誘導体としては、例えば、メチルセルロース、エチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ヒドロキシエチルメチルセルロースなどのアルキル置換セルロース誘導体、ならびにセルロースのヒドロキシル基の一部又は全部を有機酸または無機酸でエステル化した化合物が挙げられる。 Examples of cellulose derivatives include alkyl-substituted cellulose derivatives such as methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and hydroxyethylmethylcellulose, as well as compounds obtained by esterifying some or all of the hydroxyl groups of cellulose with organic or inorganic acids. can be mentioned.
　ポリ（メタ）アクリレート誘導体としては、例えばポリ［アクリル酸２－（２－エトキシエトキシ）エチル］（ＰＥＥＯ２Ａ）、ポリ［メタクリル酸２－（２－メトキシエトキシ）エチル］（ＰＭＥＯ２ＭＡ）、ポリ（メタクリル酸２－［２－（２－メトキシエトキシ）エトキシ］エチル）（ＰＭＥＯ３ＭＡ）、ポリ［オリゴメタクリル酸（エチレングリコール）］（ＰＯＥＧＭＡ）、ポリ（アクリル酸２－ヒドロキシプロピル）（ＰＨＰＡ）、および、ポリ（メタクリル酸２－ヒドロキシエチル）（ＰＨＥＭＡ）などが挙げられる。 Examples of poly(meth)acrylate derivatives include poly[2-(2-ethoxyethoxy)ethyl acrylate] (PEEO2A), poly[2-(2-methoxyethoxy)ethyl methacrylate] (PMEO2MA), poly(methacrylic acid) 2-[2-(2-methoxyethoxy)ethoxy]ethyl) (PMEO3MA), poly[oligomethacrylic acid (ethylene glycol)] (POEGMA), poly(2-hydroxypropyl acrylate) (PHPA), and poly( Examples include 2-hydroxyethyl methacrylate (PHEMA).
　ポリペプチドとしては、グルタミン酸、アスパラギン酸、アスパラギン、リジン、グルタミン、システイン、アラニン、ロイシン、アルギニンなどのアミノ酸のうちの１種又は２種以上を重合してなるポリペプチドが挙げられるがこれに限定されない。アミノ酸は、１種単独で又は２種以上を組み合わせて使用することができる。ポリペプチドとしては、例えばエラスチン様ポリペプチド、ポリリジン、ポリグルタミン、及びポリアルギニンが挙げられる。 Examples of polypeptides include, but are not limited to, polypeptides obtained by polymerizing one or more of amino acids such as glutamic acid, aspartic acid, asparagine, lysine, glutamine, cysteine, alanine, leucine, and arginine. . Amino acids can be used alone or in combination of two or more. Examples of polypeptides include elastin-like polypeptides, polylysine, polyglutamine, and polyarginine.
　エラスチン様ポリペプチドとは、エラスチンのように一定温度以上になると凝集する性質を持つポリペプチドをいう。「エラスチン様ポリペプチド（Elastin-like polypeptide）」という用語は多くの文献において使用されている用語であり、また、どのようなアミノ酸配列を有すれば温度感受性を示すようになるかも、多くの文献において報告されている。従って、当業者は、本発明において使用する適切なエラスチン様ポリペプチドを選択することができる。エラスチン様ポリペプチドの具体例としては、式(I)： Xaa1-Xaa2-Gly-Val-Pro〔式中、Xaa1はGly又はAlaを表し、Xaa2は任意のアミノ酸を表す。〕で表されるアミノ酸配列の繰り返し配列からなるペプチドを挙げることができる。式(I)におけるXaa1及びXaa2は繰り返しごとに異なるアミノ酸を表してもよいが、同じアミノ酸を表すことが好ましい。式(I)で表されるアミノ酸配列の好ましい具体例としては、Ala-Val-Gly-Val-Pro（AVGVP）を挙げることができる。式(I)で表されるアミノ酸配列の繰り返し回数は特に制限はないが、繰り返し回数が多いとゲルが白濁する可能性があるため、繰り返し回数は、30～60とするのが好ましく、40～50とするのがより好ましい。 Elastin-like polypeptide refers to a polypeptide that, like elastin, has the property of aggregating at a certain temperature or higher. The term "elastin-like polypeptide" is used in many documents, and many documents also explain what kind of amino acid sequence it has to exhibit temperature sensitivity. It has been reported in Accordingly, one skilled in the art can select an appropriate elastin-like polypeptide for use in the present invention. Specific examples of elastin-like polypeptides include formula (I): Xaa1-Xaa2-Gly-Val-Pro [wherein Xaa1 represents Gly or Ala and Xaa2 represents any amino acid]. ] Examples include peptides consisting of repeating amino acid sequences represented by the following. Although Xaa1 and Xaa2 in formula (I) may represent different amino acids each time they are repeated, they preferably represent the same amino acid. A preferred specific example of the amino acid sequence represented by formula (I) is Ala-Val-Gly-Val-Pro (AVGVP). The number of repetitions of the amino acid sequence represented by formula (I) is not particularly limited, but if the number of repetitions is too large, the gel may become cloudy, so the number of repetitions is preferably 30 to 60, and 40 to 60. It is more preferable to set it to 50.
　ポリペプチドは、ポリペプチドを構成するアミノ酸の側鎖のカルボキシル基、アミノ基などの官能基が修飾されていてもよい。 The polypeptide may be modified with a functional group such as a carboxyl group or an amino group on the side chain of an amino acid constituting the polypeptide.
　第１の温度応答性高分子は、（ｉ）重合すると温度応答性のポリマーを構成する１種類のモノマー（例えばＮ－イソプロピル（メタ）アクリルアミド）が重合されてなる、該１種類のモノマー由来の繰り返し単位を有する高分子であってもよいし、（ｉｉ）重合すると温度応答性のポリマーを構成するモノマーが２種以上重合されてなる、該２種以上のモノマー由来の繰り返し単位を有する共重合体であってもよいし、（ｉｉｉ）重合すると温度応答性のポリマーを構成するモノマー１種または２種以上と、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上とが重合されてなる共重合体であってもよい。なお、「重合すると」及び「重合しても」とは、そのモノマー１種類を重合した場合を指す。 The first temperature-responsive polymer is (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized; It may be a polymer having repeating units, or (ii) a copolymer having repeating units derived from two or more monomers, which is obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. (iii) one or more monomers that constitute a temperature-responsive polymer when polymerized, and one or two monomers that constitute a polymer that does not exhibit temperature-responsiveness even when polymerized; A copolymer formed by polymerizing the above may also be used. In addition, "on polymerization" and "even if polymerization" refer to the case where one type of monomer is polymerized.
　重合しても温度応答性を示さないポリマーの例としては、ポリ（メタ）アクリル酸エステル、（メタ）アクリル酸エステルと（メタ）アクリル酸の共重合体、ポリエチレンオキシド、ポリアルキレンオキシド、ポリリンゴ酸、ポリアスパラギン酸、ポリグルタミン酸、ポリリシン、ポリサッカライド、ポリビニルピロリドン、ポリビニルアルコール、又はこれらの誘導体由来のセグメントなどが挙げられるがこれらに限定されない。 Examples of polymers that do not exhibit temperature responsiveness even when polymerized include poly(meth)acrylic esters, copolymers of (meth)acrylic esters and (meth)acrylic acid, polyethylene oxide, polyalkylene oxide, and polymalic acid. , polyaspartic acid, polyglutamic acid, polylysine, polysaccharide, polyvinylpyrrolidone, polyvinyl alcohol, or a segment derived from a derivative thereof.
　第１の温度応答性高分子が共重合体である場合、共重合体はブロック重合体、交互重合体、ランダム重合体のいずれであってもよいが、温度応答性の制御の点で、ブロック重合体であることが好ましい。 When the first temperature-responsive polymer is a copolymer, the copolymer may be a block polymer, an alternating polymer, or a random polymer. Preferably, it is a polymer.
　例えば、第１の温度応答性高分子がブロック共重合体である場合、第１の温度応答性高分子は、重合すると温度応答性のポリマーを構成するモノマーを重合してなるブロックと、重合しても温度応答性を示さないポリマーを構成するモノマーを重合してなるブロックとを有してもよい。特定の好ましい実施形態の第１の温度応答性高分子は、Ｎ－イソプロピル（メタ）アクリルアミドなどのＮ－置換（メタ）アクリルアミドを重合してなるブロックと非置換の（メタ）アクリルアミドを重合してなるブロックとを含む温度応答性高分子である。 For example, when the first temperature-responsive polymer is a block copolymer, the first temperature-responsive polymer is polymerized with a block formed by polymerizing monomers that constitute the temperature-responsive polymer when polymerized. It may also have a block formed by polymerizing monomers constituting a polymer that does not exhibit temperature responsiveness. In a specific preferred embodiment, the first temperature-responsive polymer is a block formed by polymerizing an N-substituted (meth)acrylamide such as N-isopropyl (meth)acrylamide, and a block formed by polymerizing an unsubstituted (meth)acrylamide. It is a temperature-responsive polymer containing a block.
　第１の粒子がミセルである場合、第１の温度応答性高分子は親水性セグメントと疎水性セグメントとを有するブロック共重合体であり、第１のミセルの表面に第１の温度応答性高分子の親水性セグメントが配置され、第１のミセルの内部に第１の温度応答性高分子の疎水性セグメントが配置されることが好ましい。 When the first particles are micelles, the first temperature-responsive polymer is a block copolymer having a hydrophilic segment and a hydrophobic segment, and the first temperature-responsive polymer is coated on the surface of the first micelle. Preferably, a hydrophilic segment of the molecule is arranged and a hydrophobic segment of the first temperature-responsive polymer is arranged inside the first micelle.
　親水性セグメントは、例えば、上述の（ｉ）重合すると温度応答性のポリマーを構成する１種類のモノマー（例えばＮ－イソプロピル（メタ）アクリルアミド）が重合されてなる、該１種類のモノマー由来の繰り返し単位を有する高分子であってもよいし、（ｉｉ）重合すると温度応答性のポリマーを構成するモノマーが２種以上重合されてなる、該２種以上のモノマー由来の繰り返し単位を有する共重合体であってもよいし、（ｉｉｉ）重合すると温度応答性のポリマーを構成するモノマー１種または２種以上と、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上とが重合されてなる共重合体であってよい。 The hydrophilic segment is, for example, the above-mentioned (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized, and the repeats derived from the one type of monomer. (ii) A copolymer having repeating units derived from two or more monomers, which is obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. or (iii) one or more monomers that constitute a temperature-responsive polymer when polymerized and one or two or more monomers that constitute a polymer that does not exhibit temperature responsiveness when polymerized. It may be a copolymer obtained by polymerizing these.
　疎水性セグメントとしては、薬物送達用のミセルを形成するのに使用することのできるポリマーを使用することができ、例えば、ポリ（メタ）アクリル酸エステル、ポリ（メタ）アクリル酸アミド、ラクチド、ラクトン、ポリ（β－ベンジル  Ｌ－アルパルテート）、ポリ（γ－ベンジル  Ｌ－グルタメート）、ポリ（β－置換アスパルテート）、ポリ（γ－置換グルタメート）、疎水性ポリアミノ酸（例えばポリ（Ｌ－ロイシン）、ポリ（Ｌ－バリン）、ポリ（Ｌ－フェニルアラニン））、ポリスチレン、ポリアミド、ポリエステル、ポリアルキレンオキシドなどが挙げられるがこれらに限定されない。 As hydrophobic segments, polymers that can be used to form micelles for drug delivery can be used, such as poly(meth)acrylates, poly(meth)acrylamides, lactides, lactones. , poly(β-benzyl L-alpartate), poly(γ-benzyl L-glutamate), poly(β-substituted aspartate), poly(γ-substituted glutamate), hydrophobic polyamino acids such as poly(L-leucine) , poly(L-valine), poly(L-phenylalanine)), polystyrene, polyamide, polyester, polyalkylene oxide, and the like, but are not limited to these.
　特定の実施形態において、第１の温度応答性高分子は、Ｎ－置換（メタ）アクリルアミドを重合してなる親水性セグメントと、非置換の（メタ）アクリルアミドを重合してなる親水性セグメントと、ポリ（メタ）アクリル酸エステルからなる疎水性セグメントとを有する。 In a specific embodiment, the first temperature-responsive polymer includes a hydrophilic segment formed by polymerizing N-substituted (meth)acrylamide, a hydrophilic segment formed by polymerizing unsubstituted (meth)acrylamide, It has a hydrophobic segment made of poly(meth)acrylic acid ester.
　第１の温度応答性高分子を構成するモノマーの総数中の、重合すると温度応答性ポリマーを有するモノマーの数の割合は、例えば５０％以上、６０％以上、７０％以上、８０％以上、９０％以上、又は１００％である。適切な温度応答性を示す第１の温度応答性高分子を合成するために、当業者は第１の温度応答性高分子を構成するモノマーを適宜選択することができる。 The ratio of the number of monomers that form a temperature-responsive polymer when polymerized to the total number of monomers constituting the first temperature-responsive polymer is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more. % or more, or 100%. In order to synthesize a first temperature-responsive polymer that exhibits appropriate temperature responsiveness, those skilled in the art can appropriately select monomers constituting the first temperature-responsive polymer.
　いくつかの実施形態では、第１の温度応答性高分子は、下限臨界溶解温度（ＬＣＳＴ）を有する。当業者には、用途や要求する機能などに応じて、必要なＬＣＳＴを有する温度応答性高分子を選択することができる。本発明の薬物送達用組成物は、ヒト又は非ヒト動物への使用が想定されるため、ＬＣＳＴは動物の体温以上であって、かつタンパク質の変性などの動物の体内での不利益を極力回避できる温度であることが好ましい。例えば、第１の温度応答性高分子の下限臨界溶解温度（ＬＣＳＴ）は、３８～５０℃の範囲にあることが好ましく、３８～４５℃の範囲にあることがより好ましく、４０～４２℃の範囲にあることがより好ましい。 In some embodiments, the first temperature-responsive polymer has a lower critical solution temperature (LCST). Those skilled in the art can select a temperature-responsive polymer having a required LCST depending on the intended use, required functions, and the like. Since the drug delivery composition of the present invention is expected to be used in humans or non-human animals, the LCST should be higher than the animal's body temperature, and disadvantages such as protein denaturation within the animal's body should be avoided as much as possible. It is preferable that the temperature is such that it can be used. For example, the lower critical solution temperature (LCST) of the first temperature-responsive polymer is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and more preferably in the range of 40 to 42°C. It is more preferable to fall within this range.
　いくつかの実施形態では、第１の温度応答性高分子は、下限臨界溶解温度（ＬＣＳＴ）を示すセグメントを有するブロック共重合体である。かかるブロック共重合体としては、特に限定はないが、重合が容易なことから、ビニル基を有するモノマー由来の構成単位を有することが好ましく、アクリロイル基あるいはメタクリロイル基を有する構造を有することがより好ましい。ビニル基を有するモノマーとしては、（メタ）アクリルアミド、ビニルアミド、（メタ）アクリル酸エステル、アクリロニトリルなどが挙げられる。 In some embodiments, the first temperature-responsive polymer is a block copolymer having a segment that exhibits a lower critical solution temperature (LCST). Such a block copolymer is not particularly limited, but because polymerization is easy, it preferably has a structural unit derived from a monomer having a vinyl group, and more preferably has a structure having an acryloyl group or a methacryloyl group. . Examples of the monomer having a vinyl group include (meth)acrylamide, vinylamide, (meth)acrylic acid ester, and acrylonitrile.
　例えば、下限臨界溶解温度（ＬＣＳＴ）を示すセグメントの繰返し単位（以下、「ＬＣＳＴ繰返し単位」ということがある）を構成するモノマーの例としては、例えば、Ｎ－エチルアクリルアミド（ＬＣＳＴ＝７２℃）、Ｎ－シクロプロピルアクリルアミド（ＬＣＳＴ＝４６℃）、Ｎ－イソプロピルアクリルアミド（ＬＣＳＴ＝３２℃）、Ｎ－ｎ－プロピルメタクリルアミド（ＬＣＳＴ＝２２℃）、Ｎ－テトラヒドロフルフリルアクリルアミド（ＬＣＳＴ＝２８℃）、Ｎ－エトキシエチルアクリルアミド（ＬＣＳＴ＝３５℃）、Ｎ，Ｎ－ジエチルアクリルアミド（ＬＣＳＴ＝３２℃）、Ｎ－シクロプロピルメタクリルアミド（ＬＣＳＴ＝５９℃）、Ｎ－イソプロピルメタクリルアミド（ＬＣＳＴ＝４４℃）、Ｎ－ｎ－プロピルメタクリルアミド（ＬＣＳＴ＝２８℃）、Ｎ－テトラヒドロフルフリルメタクリルアミド（ＬＣＳＴ＝３５℃）、Ｎ－メチル－Ｎ－エチルアクリルアミド（ＬＣＳＴ＝５６℃）、Ｎ－メチル－Ｎ－イソプロピルアクリルアミド（ＬＣＳＴ＝２３℃）、Ｎ－メチル－Ｎ－ｎ－プロピルアクリルアミド（ＬＣＳＴ＝２０℃）、およびＮ，Ｎ－ジメチルアミノエチルメタクリレート（ＬＣＳＴ＝４７℃）等が挙げられる。 For example, examples of monomers constituting the repeating unit of the segment exhibiting the lower critical solution temperature (LCST) (hereinafter sometimes referred to as "LCST repeating unit") include N-ethyl acrylamide (LCST=72°C), N-cyclopropylacrylamide (LCST = 46°C), N-isopropylacrylamide (LCST = 32°C), N-n-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfurylacrylamide (LCST = 28°C), N-ethoxyethyl acrylamide (LCST=35°C), N,N-diethylacrylamide (LCST=32°C), N-cyclopropylmethacrylamide (LCST=59°C), N-isopropylmethacrylamide (LCST=44°C), N-n-propyl methacrylamide (LCST=28°C), N-tetrahydrofurfuryl methacrylamide (LCST=35°C), N-methyl-N-ethylacrylamide (LCST=56°C), N-methyl-N-isopropyl Examples include acrylamide (LCST=23°C), N-methyl-Nn-propylacrylamide (LCST=20°C), and N,N-dimethylaminoethyl methacrylate (LCST=47°C).
　第１の温度応答性高分子の分子量は特に限定されないが、例えば２ｋＤａ～１００ｋＤａである。第１の粒子における第１の温度応答性高分子の濃度は、第１の粒子の温度応答性と第２の粒子との反応の制御の点で、５０ｗｔ％～９９ｗｔ％の範囲が好ましい。 The molecular weight of the first temperature-responsive polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa. The concentration of the first temperature-responsive polymer in the first particles is preferably in the range of 50 wt% to 99 wt% from the viewpoint of controlling the temperature responsiveness of the first particles and the reaction with the second particles.
　第１の粒子の第１の高分子は、高分子鎖の部分と、高分子鎖と結合する第１の反応性官能基とを有する。第１の高分子の高分子鎖は、第１の温度応答性高分子に関して上述したモノマーから形成してもよい。 The first polymer of the first particle has a polymer chain portion and a first reactive functional group that binds to the polymer chain. The polymer chains of the first polymer may be formed from the monomers described above with respect to the first temperature-responsive polymer.
　第１の高分子の高分子鎖を、温度応答性高分子とする場合、第１の高分子の高分子鎖は、第１の温度応答性高分子を構成する重合体（例えば、ポリ（メタ）アクリルアミド誘導体、ポリビニルアミド誘導体、ポリ（メタ）アクリレート誘導体、ポリビニルメチルエーテル、セルロース誘導体、ポリペプチド、ポリ（ビニル）カプロラクタム、ポリエチレングリコールとポリプロピレングリコールの共重合体、またはこれらの２種以上の共重合体など）と同じ種類の重合体から構成されてもよく、異なる種類の重合体から構成されてもよい。 When the polymer chain of the first polymer is a temperature-responsive polymer, the polymer chain of the first polymer is a polymer (e.g., poly(metal)) constituting the first temperature-responsive polymer. ) Acrylamide derivatives, polyvinylamide derivatives, poly(meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly(vinyl)caprolactam, copolymers of polyethylene glycol and polypropylene glycol, or copolymers of two or more of these It may be composed of the same type of polymer as the polymer (coalescence, etc.), or it may be composed of a different type of polymer.
　第１の高分子の高分子鎖は、（ｉ）重合すると温度応答性のポリマーを構成する１種類のモノマー（例えばＮ－イソプロピル（メタ）アクリルアミド）が重合されてなる、該１種類のモノマー由来の繰り返し単位を有する高分子であってもよいし、（ｉｉ）重合すると温度応答性のポリマーを構成するモノマーが２種以上重合されてなる、該２種以上のモノマー由来の繰り返し単位を有する共重合体であってもよいし、（ｉｉｉ）重合すると温度応答性のポリマーを構成するモノマー１種または２種以上と、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上とが重合されてなる共重合体であってもよいし、（ｉｖ）重合すると温度応答性のポリマーを構成するモノマー由来の構成単位を含まない、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上が重合されてなる重合体であってもよい。 The polymer chain of the first polymer is derived from (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized; or (ii) a copolymer having repeating units derived from two or more monomers, which are obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. (iii) one or more monomers that form a temperature-responsive polymer when polymerized, and one or two monomers that form a polymer that does not show temperature-responsiveness when polymerized; It may be a copolymer obtained by polymerizing two or more species, and (iv) it does not contain any structural units derived from monomers that constitute a temperature-responsive polymer when polymerized, and does not exhibit temperature-responsiveness even when polymerized. It may be a polymer obtained by polymerizing one or more monomers constituting the polymer.
　第１の高分子が共重合体である場合、共重合体はブロック重合体、交互重合体、ランダム重合体のいずれであってもよいが、温度応答性の制御の点で、ブロック重合体であることが好ましい。 When the first polymer is a copolymer, the copolymer may be a block polymer, an alternating polymer, or a random polymer. It is preferable that there be.
　例えば、第１の高分子がブロック共重合体である場合、第１の高分子は、重合すると温度応答性のポリマーを構成するモノマーを重合してなるブロックと、重合しても温度応答性を示さないポリマーを構成するモノマーを重合してなるブロックとを有してもよい。特定の好ましい実施形態の第１の高分子は、Ｎ－イソプロピル（メタ）アクリルアミドなどのＮ－置換（メタ）アクリルアミドを重合してなるブロックと非置換の（メタ）アクリルアミドを重合してなるブロックとを含む温度応答性高分子である。 For example, when the first polymer is a block copolymer, the first polymer may have a block formed by polymerizing a monomer that constitutes a temperature-responsive polymer when polymerized, and a block formed by polymerizing a monomer that constitutes a polymer that does not exhibit temperature responsiveness when polymerized. In a particular preferred embodiment, the first polymer is a temperature-responsive polymer that includes a block formed by polymerizing an N-substituted (meth)acrylamide such as N-isopropyl(meth)acrylamide, and a block formed by polymerizing an unsubstituted (meth)acrylamide.
　第１の粒子がミセルである場合、第１の高分子は親水性セグメントと疎水性セグメントとを有するブロック共重合体であり、第１のミセルの表面に第１の高分子の親水性セグメントが配置され、第１のミセルの内部に第１の高分子の疎水性セグメントが配置されることが好ましい。 When the first particle is a micelle, the first polymer is a block copolymer having a hydrophilic segment and a hydrophobic segment, and the hydrophilic segment of the first polymer is on the surface of the first micelle. Preferably, the hydrophobic segment of the first polymer is arranged inside the first micelle.
　親水性ブロック及び疎水性ブロックの例は、第１の温度応答性高分子に関して説明した通りである。 Examples of the hydrophilic block and the hydrophobic block are as described for the first temperature-responsive polymer.
　第１の高分子の主鎖の鎖長は、第１の温度応答性高分子の主鎖の鎖長と同一かそれよりも長いことが、第１の高分子の第１の反応性官能基を第１の粒子の表面に露出させる点で好ましい。特定の実施形態では、第１の高分子の主鎖の鎖長は、５～５０℃の範囲内のいずれかの温度において、第１の温度応答性高分子の主鎖の鎖長と同一かそれよりも長い。特定の実施形態では、第１の高分子の主鎖の鎖長は、５～５０℃の全範囲において、第１の温度応答性高分子の主鎖の鎖長と同一かそれよりも長い。 The chain length of the main chain of the first polymer is the same as or longer than the chain length of the main chain of the first temperature-responsive polymer. is preferable in that it is exposed on the surface of the first particle. In certain embodiments, the chain length of the backbone of the first polymer is the same as the chain length of the backbone of the first temperature-responsive polymer at any temperature within the range of 5 to 50°C. It's longer than that. In certain embodiments, the chain length of the main chain of the first polymer is the same as or longer than the chain length of the main chain of the first temperature-responsive polymer over the entire range of 5 to 50°C.
　第１の粒子の第１の高分子の第１の反応性官能基は、加温により第２の粒子の第２の高分子の第２の反応性官能基と反応して、第１の粒子と第２の粒子の間に架橋を形成する官能基であれば特に限定されない。 The first reactive functional group of the first polymer of the first particle reacts with the second reactive functional group of the second polymer of the second particle by heating, and the first reactive functional group of the first polymer of the first particle reacts with the second reactive functional group of the second polymer of the second particle. It is not particularly limited as long as it is a functional group that forms a crosslink between the particle and the second particle.
　そのような第１及び第２の反応性官能基のペアとしては、クリックケミストリー（H. C. Kolb, M. G. Finn & K. B. Sharpless : Angew. Chem., Int. Ed., 40, 2004（2001）.）の公知のクリック反応を生じさせる反応性官能基のペアを使用することができる。クリック反応としては、アルキンとアジドの反応である歪み促進型アジド-アルキン付加環化反応（SPAAC）、電子不足な複素環と歪C-C多重結合化合物の反応である歪み促進逆電子要請型Diels-Alder反応（SPIEDAC）などが挙げられる。 Such a pair of first and second reactive functional groups is known from click chemistry (H. C. Kolb, M. G. Finn & K. B. Sharpless: Angew. Chem., Int. Ed., 40 , 2004 (2001).) Pairs of reactive functional groups that produce click reactions can be used. Click reactions include strain-promoted azide-alkyne cycloaddition (SPAAC), which is a reaction between an alkyne and azide, and strain-promoted reverse electron request Diels-Alder, which is a reaction between an electron-deficient heterocycle and a strained C-C multiple bond compound. reaction (SPIEDAC), etc.
　SPAACに使用されるアルキンとしては、ジベンジルシクロオクチン(DBCO)、二フッ化シクロオクチン（DIFO）、ビシクロノニン（BCN）、ジベンゾシクロオクチン（DIBO）、二アリールアザシクロオクチノン(BARAC)などのシクロオクチン誘導体が挙げられる。例えば、第１の高分子の高分子鎖を形成する高分子に、シクロオクチン誘導体試薬を取り付けることにより、第１の粒子にシクロオクチン基である第１の反応性官能基を導入することができる。 Alkynes used in SPAAC include dibenzylcyclooctyne (DBCO), cyclooctyne difluoride (DIFO), bicyclononine (BCN), dibenzocyclooctyne (DIBO), and diarylazacycloocthinone (BARAC). Examples include octyne derivatives. For example, by attaching a cyclooctyne derivative reagent to a polymer forming a polymer chain of the first polymer, the first reactive functional group, which is a cyclooctyne group, can be introduced into the first particle. .
　SPAACに使用されるアジドとしては、アジ化ナトリウム、トリメチルシリルアジド、ジフェニルリン酸アジド、テトラブチルアンモニウムアジド、テトラメチルグアニジニウムアジド、アジドヨージナン、4-アセトアミ2-アジド-1,3-ジメチルイミダゾリニウムヘキサフルオロリン酸塩ドベンゼンスルホン酸アジドなどのアジド導入反応剤が挙げられる。例えば、第２の高分子の高分子鎖を形成する高分子に、アジド導入反応剤を反応させることにより、第２の粒子にアジド基である第２の反応性官能基を導入することができる。 Azides used in SPAAC include sodium azide, trimethylsilyl azide, diphenylphosphate azide, tetrabutylammonium azide, tetramethylguanidinium azide, azidoiodinane, 4-acetami2-azido-1,3-dimethylimidazo Examples include azide-introducing reagents such as linium hexafluorophosphate dobenzenesulfonic acid azide. For example, a second reactive functional group, which is an azide group, can be introduced into the second particle by reacting an azide-introducing reactant with a polymer that forms a polymer chain of the second polymer. .
　SPIEDACに使用される電子不足な複素環としては、テトラジンやトリアジンなどが挙げられる。 Electron-deficient heterocycles used in SPIEDAC include tetrazine and triazine.
　SPIEDACに使用される歪C-C多重結合化合物としては、ノルボルネンやtrans-シクロオクテン、シクロオクチンなどが挙げられる。 Examples of strained C-C multiple bond compounds used in SPIEDAC include norbornene, trans-cyclooctene, and cyclooctyne.
　第１の粒子の第１の高分子の第１の反応性官能基は、第１の粒子が投与される動物の体温よりも高い温度で第２の粒子の第２の高分子の第２の反応性官能基と反応する官能基であることが好ましい。 The first reactive functional group of the first polymer of the first particle is activated by the second reactive functional group of the second polymer of the second particle at a temperature higher than the body temperature of the animal to which the first particle is administered. Preferably, it is a functional group that reacts with a reactive functional group.
　また、第１の粒子の第１の高分子の第１の反応性官能基は、第１の温度応答性高分子のＬＣＳＴの温度よりも高い温度で第２の粒子の第２の高分子の末端の第２の反応性官能基と反応する官能基であることが好ましい。 Further, the first reactive functional group of the first polymer of the first particle is activated by the first reactive functional group of the second polymer of the second particle at a temperature higher than the LCST temperature of the first temperature-responsive polymer. Preferably, it is a functional group that reacts with the terminal second reactive functional group.
　第１の高分子の分子量は特に限定されないが、例えば２ｋＤａ～１００ｋＤａである。 The molecular weight of the first polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa.
　第１の粒子における第１の高分子の濃度は、第１の粒子の温度応答性と第２の粒子との反応の制御の点で、１ｗｔ％～５０ｗｔ％の範囲が好ましい。 The concentration of the first polymer in the first particles is preferably in the range of 1 wt% to 50 wt% from the viewpoint of controlling the temperature responsiveness of the first particles and the reaction with the second particles.
　第１の粒子は好ましくはミセルである。第１の粒子は、より好ましくは薬物を内部に充填できるミセルである。第１の粒子は、より好ましくは、２０℃で水中油滴型の、水難溶性薬物を内部に充填できるミセルである。 The first particles are preferably micelles. The first particles are more preferably micelles that can be loaded with drugs. The first particles are more preferably micelles that can be filled with an oil-in-water type drug that is poorly water-soluble at 20°C.
　第１の粒子は、好ましくは下限臨界溶解温度（ＬＣＳＴ）を有する。第１の粒子のＬＣＳＴは、第１の温度応答性高分子の選択、第１の高分子の選択、及び第１の粒子中のそれらの高分子の量の選択により、当業者には適切に設定することができる。第１の粒子のＬＣＳＴは、粒子を含む水溶液の可視光における透過率を昇温しながら測定し、その透過率の変化から評価することができる。 The first particles preferably have a lower critical solution temperature (LCST). The LCST of the first particle can be determined by one skilled in the art by selecting the first temperature-responsive polymer, selecting the first polymer, and selecting the amount of those polymers in the first particle. Can be set. The LCST of the first particles can be evaluated by measuring the visible light transmittance of an aqueous solution containing the particles while increasing the temperature, and from the change in the transmittance.
　第１の粒子のＬＣＳＴは、動物の体温以上であって、かつタンパク質の変性などの動物の体内での不利益を極力回避できる温度であることが好ましい。例えば、第１の粒子のＬＣＳＴは、３８～５０℃の範囲にあることが好ましく、３８～４５℃の範囲にあることがより好ましく、４０～４２℃の範囲にあることがより好ましい。 The LCST of the first particles is preferably a temperature that is higher than the animal's body temperature and that can avoid disadvantages in the animal's body such as protein denaturation as much as possible. For example, the LCST of the first particles is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and even more preferably in the range of 40 to 42°C.
　第１の粒子の粒径は、第１の粒子のＬＣＳＴ未満の温度で、１０ｎｍ～５００ｎｍの範囲であることが好ましく、より好ましくは３０ｎｍ～２００ｎｍである。第１の粒子の粒径が１０ｎｍ以上であると、薬物の封入及び粒子の安定性の点で好ましい。第１の粒子の粒径が５００ｎｍ以下であると、粒子を生体内の標的組織へ送達し、標的としない他の臓器に粒子がトラップされることを防ぐ点で好ましい。 The particle size of the first particles is preferably in the range of 10 nm to 500 nm, more preferably 30 nm to 200 nm, at a temperature below the LCST of the first particles. It is preferable that the particle size of the first particles is 10 nm or more in terms of drug encapsulation and particle stability. It is preferable that the particle size of the first particles is 500 nm or less in that the particles are delivered to the target tissue in the living body and the particles are prevented from being trapped in other non-target organs.
　第２の粒子の第２の温度応答性高分子を構成する高分子、特に限定されないが、第１の粒子の第１の温度応答性高分子に関して説明したのと同じ高分子を使用することができる。 The polymer constituting the second temperature-responsive polymer of the second particle may be, but is not limited to, the same polymer as described for the first temperature-responsive polymer of the first particle. can.
　第２の粒子の第２の温度応答性高分子と第１の粒子の第１の温度応答性高分子は、同一であってもよいし、異なっていてもよい。或いは、第２の粒子の第２の温度応答性高分子と第１の粒子の第１の温度応答性高分子は、同じ種類（例　ポリ（メタ）アクリルアミド誘導体）であるが、モノマーの組成比および／または主鎖の長さが異なっていてもよい。 The second temperature-responsive polymer of the second particle and the first temperature-responsive polymer of the first particle may be the same or different. Alternatively, the second temperature-responsive polymer of the second particle and the first temperature-responsive polymer of the first particle are of the same type (e.g. poly(meth)acrylamide derivative), but the monomer composition ratio is different. and/or the length of the main chain may be different.
　いくつかの実施形態において、第１の粒子の第１の温度応答性高分子及び第２の粒子の第２の温度応答性高分子のそれぞれが、ポリ（メタ）アクリルアミド誘導体、ポリビニルアミド誘導体、ポリ（メタ）アクリレート誘導体、ポリビニルメチルエーテル、セルロース誘導体、ポリペプチド、ポリ（ビニル）カプロラクタム、ポリエチレングリコールとポリプロピレングリコールの共重合体、またはこれらの２種以上の共重合体などの温度応答性高分子を含む。 In some embodiments, each of the first temperature-responsive polymer of the first particle and the second temperature-responsive polymer of the second particle is a poly(meth)acrylamide derivative, a polyvinylamide derivative, a polyvinylamide derivative, or Temperature-responsive polymers such as (meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly(vinyl)caprolactam, copolymers of polyethylene glycol and polypropylene glycol, or copolymers of two or more of these. include.
　第２の温度応答性高分子は、第１の温度応答性高分子に関して上述したように、（ｉ）重合すると温度応答性のポリマーを構成する１種類のモノマー（例えばＮ－イソプロピル（メタ）アクリルアミド）が重合されてなる、該１種類のモノマー由来の繰り返し単位を有する高分子であってもよいし、（ｉｉ）重合すると温度応答性のポリマーを構成するモノマーが２種以上重合されてなる、該２種以上のモノマー由来の繰り返し単位を有する共重合体であってもよいし、（ｉｉｉ）重合すると温度応答性のポリマーを構成するモノマー１種または２種以上と、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上とが重合されてなる共重合体であってもよい。なお、「重合すると」及び「重合しても」とは、そのモノマー１種類を重合した場合を指す。 As described above with respect to the first temperature-responsive polymer, the second temperature-responsive polymer includes (i) one type of monomer that, when polymerized, constitutes the temperature-responsive polymer (for example, N-isopropyl (meth)acrylamide); ) may be a polymer having repeating units derived from the one type of monomer, or (ii) may be a polymer formed by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. It may be a copolymer having repeating units derived from two or more monomers, or (iii) one or more monomers that constitute a temperature-responsive polymer when polymerized, and a polymer that is temperature-responsive even when polymerized. It may also be a copolymer obtained by polymerizing one or more monomers constituting a polymer that exhibits no properties. In addition, "on polymerization" and "even if polymerization" refer to the case where one type of monomer is polymerized.
　第２の温度応答性高分子が共重合体である場合、共重合体はブロック重合体、交互重合体、ランダム重合体のいずれであってもよいが、温度応答性の制御の点で、ブロック重合体であることが好ましい。 When the second temperature-responsive polymer is a copolymer, the copolymer may be a block polymer, an alternating polymer, or a random polymer. Preferably, it is a polymer.
　例えば、第２の温度応答性高分子がブロック共重合体である場合、第２の温度応答性高分子は、重合すると温度応答性のポリマーを構成するモノマーを重合してなるブロックと、重合しても温度応答性を示さないポリマーを構成するモノマーを重合してなるブロックとを有してもよい。特定の好ましい実施形態の第２の温度応答性高分子は、Ｎ－イソプロピル（メタ）アクリルアミドなどのＮ－置換（メタ）アクリルアミドを重合してなるブロックと非置換の（メタ）アクリルアミドを重合してなるブロックとを含む温度応答性高分子である。 For example, when the second temperature-responsive polymer is a block copolymer, the second temperature-responsive polymer is polymerized with a block formed by polymerizing monomers that constitute the temperature-responsive polymer when polymerized. It may also have a block formed by polymerizing monomers constituting a polymer that does not exhibit temperature responsiveness. In a specific preferred embodiment, the second temperature-responsive polymer is a block formed by polymerizing an N-substituted (meth)acrylamide such as N-isopropyl (meth)acrylamide, and a block formed by polymerizing an unsubstituted (meth)acrylamide. It is a temperature-responsive polymer containing blocks.
　第２の粒子がミセルである場合、第２の温度応答性高分子は親水性セグメントと疎水性セグメントとを有するブロック共重合体であり、第２のミセルの表面に第２の温度応答性高分子の親水性セグメントが配置され、第２のミセルの内部に第２の温度応答性高分子の疎水性セグメントが配置されることが好ましい。 When the second particles are micelles, the second temperature-responsive polymer is a block copolymer having a hydrophilic segment and a hydrophobic segment, and the second temperature-responsive polymer is attached to the surface of the second micelle. Preferably, a hydrophilic segment of the molecule is arranged and a hydrophobic segment of the second temperature-responsive polymer is arranged inside the second micelle.
　親水性ブロック及び疎水性ブロックの例は、第１の温度応答性高分子に関して説明した通りである。 Examples of the hydrophilic block and the hydrophobic block are as described for the first temperature-responsive polymer.
　第２の温度応答性高分子を構成するモノマーの総数中の、重合すると温度応答性ポリマーを有するモノマーの数の割合は、例えば５０％以上、６０％以上、７０％以上、８０％以上、９０％以上、又は１００％である。適切な温度応答性を示す第２の温度応答性高分子を合成するために、当業者は第２の温度応答性高分子を構成するモノマーを適宜選択することができる。 The ratio of the number of monomers that form a temperature-responsive polymer when polymerized to the total number of monomers constituting the second temperature-responsive polymer is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more. % or more, or 100%. In order to synthesize a second temperature-responsive polymer that exhibits appropriate temperature responsiveness, those skilled in the art can appropriately select monomers constituting the second temperature-responsive polymer.
　いくつかの実施形態では、第２の温度応答性高分子は、下限臨界溶解温度（ＬＣＳＴ）を有する。当業者には、用途や要求する機能などに応じて、必要なＬＣＳＴを有する温度応答性高分子を選択することができる。本発明の薬物送達用組成物は、ヒト又は非ヒト動物（例えば、マウス、ラット、モルモット、ウサギなどの哺乳動物）への使用が想定されるため、ＬＣＳＴは動物の体温以上であって、かつタンパク質の変性などの動物の体内での不利益を極力回避できる温度であることが好ましい。例えば、第２の温度応答性高分子の下限臨界溶解温度（ＬＣＳＴ）は、３８～５０℃の範囲にあることが好ましく、３８～４５℃の範囲にあることがより好ましく、４０～４２℃の範囲にあることがより好ましい。 In some embodiments, the second temperature-responsive polymer has a lower critical solution temperature (LCST). Those skilled in the art can select a temperature-responsive polymer having a required LCST depending on the intended use, required functions, and the like. Since the drug delivery composition of the present invention is expected to be used in humans or non-human animals (e.g., mammals such as mice, rats, guinea pigs, rabbits, etc.), the LCST is equal to or higher than the animal's body temperature, and The temperature is preferably such that disadvantages such as protein denaturation within the animal's body can be avoided as much as possible. For example, the lower critical solution temperature (LCST) of the second temperature-responsive polymer is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and more preferably in the range of 40 to 42°C. It is more preferable to fall within this range.
　第１の温度応答性高分子のＬＣＳＴと、第２の温度応答性高分子のＬＣＳＴは、同じであっても異なっていてもよい。第１の温度応答性高分子のＬＣＳＴと、第２の温度応答性高分子のＬＣＳＴが異なる場合、第１の粒子と第２の粒子の温度感受性を合わせる点で、その差は±２℃以内であることが好ましい。 The LCST of the first temperature-responsive polymer and the LCST of the second temperature-responsive polymer may be the same or different. If the LCST of the first temperature-responsive polymer and the LCST of the second temperature-responsive polymer are different, the difference is within ±2°C in terms of matching the temperature sensitivities of the first and second particles. It is preferable that
　いくつかの実施形態では、第２の温度応答性高分子は、下限臨界溶解温度（ＬＣＳＴ）を示すセグメントを有するブロック共重合体である。かかるブロック共重合体としては、特に限定はないが、重合が容易なことから、ビニル基を有するモノマー由来の構成単位を有することが好ましく、アクリロイル基あるいはメタクリロイル基を有する構造を有することがより好ましい。ビニル基を有するモノマーとしては、（メタ）アクリルアミド、ビニルアミド、（メタ）アクリル酸エステル、アクリロニトリルなどが挙げられる。 In some embodiments, the second temperature-responsive polymer is a block copolymer having a segment that exhibits a lower critical solution temperature (LCST). Such a block copolymer is not particularly limited, but because polymerization is easy, it preferably has a structural unit derived from a monomer having a vinyl group, and more preferably has a structure having an acryloyl group or a methacryloyl group. . Examples of the monomer having a vinyl group include (meth)acrylamide, vinylamide, (meth)acrylic acid ester, and acrylonitrile.
　例えば、下限臨界溶解温度（ＬＣＳＴ）を示すセグメントの繰返し単位（以下、「ＬＣＳＴ繰返し単位」ということがある）を構成するモノマーの例としては、例えば、Ｎ－エチルアクリルアミド（ＬＣＳＴ＝７２℃）、Ｎ－シクロプロピルアクリルアミド（ＬＣＳＴ＝４６℃）、Ｎ－イソプロピルアクリルアミド（ＬＣＳＴ＝３２℃）、Ｎ－ｎ－プロピルメタクリルアミド（ＬＣＳＴ＝２２℃）、Ｎ－テトラヒドロフルフリルアクリルアミド（ＬＣＳＴ＝２８℃）、Ｎ－エトキシエチルアクリルアミド（ＬＣＳＴ＝３５℃）、Ｎ，Ｎ－ジエチルアクリルアミド（ＬＣＳＴ＝３２℃）、Ｎ－シクロプロピルメタクリルアミド（ＬＣＳＴ＝５９℃）、Ｎ－イソプロピルメタクリルアミド（ＬＣＳＴ＝４４℃）、Ｎ－ｎ－プロピルメタクリルアミド（ＬＣＳＴ＝２８℃）、Ｎ－テトラヒドロフルフリルメタクリルアミド（ＬＣＳＴ＝３５℃）、Ｎ－メチル－Ｎ－エチルアクリルアミド（ＬＣＳＴ＝５６℃）、Ｎ－メチル－Ｎ－イソプロピルアクリルアミド（ＬＣＳＴ＝２３℃）、Ｎ－メチル－Ｎ－ｎ－プロピルアクリルアミド（ＬＣＳＴ＝２０℃）、およびＮ，Ｎ－ジメチルアミノエチルメタクリレート（ＬＣＳＴ＝４７℃）等が挙げられる。 For example, examples of monomers constituting the repeating unit of the segment exhibiting the lower critical solution temperature (LCST) (hereinafter sometimes referred to as "LCST repeating unit") include N-ethyl acrylamide (LCST=72°C), N-cyclopropylacrylamide (LCST = 46°C), N-isopropylacrylamide (LCST = 32°C), N-n-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfurylacrylamide (LCST = 28°C), N-ethoxyethyl acrylamide (LCST=35°C), N,N-diethylacrylamide (LCST=32°C), N-cyclopropylmethacrylamide (LCST=59°C), N-isopropylmethacrylamide (LCST=44°C), N-n-propyl methacrylamide (LCST=28°C), N-tetrahydrofurfuryl methacrylamide (LCST=35°C), N-methyl-N-ethylacrylamide (LCST=56°C), N-methyl-N-isopropyl Examples include acrylamide (LCST=23°C), N-methyl-Nn-propylacrylamide (LCST=20°C), and N,N-dimethylaminoethyl methacrylate (LCST=47°C).
　第２の温度応答性高分子の分子量は特に限定されないが、例えば２ｋＤａ～１００ｋＤａである。第２の粒子における第２の温度応答性高分子の濃度は、第２の粒子の温度応答性と第１の粒子との反応の制御の点で、５０ｗｔ％～９９ｗｔ％の範囲が好ましい。 The molecular weight of the second temperature-responsive polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa. The concentration of the second temperature-responsive polymer in the second particles is preferably in the range of 50 wt% to 99 wt% from the viewpoint of controlling the temperature responsiveness of the second particles and the reaction with the first particles.
　第２の粒子の第２の高分子は、高分子鎖の部分と、高分子鎖と結合する第２の反応性官能基とを有する。第２の高分子の高分子鎖は、第１の温度応答性高分子に関して上述したモノマーから形成してもよい。 The second polymer of the second particle has a polymer chain portion and a second reactive functional group that binds to the polymer chain. The polymer chains of the second polymer may be formed from the monomers described above with respect to the first temperature-responsive polymer.
　第２の高分子の高分子鎖を、温度応答性高分子とする場合、第２の高分子の高分子鎖は、第１の温度応答性高分子を構成する重合体（例えば、ポリ（メタ）アクリルアミド誘導体、ポリビニルアミド誘導体、ポリ（メタ）アクリレート誘導体、ポリビニルメチルエーテル、セルロース誘導体、ポリペプチド、ポリ（ビニル）カプロラクタム、ポリエチレングリコールとポリプロピレングリコールの共重合体、またはこれらの２種以上の共重合体など）と同じ種類の重合体から構成されてもよく、異なる種類の重合体から構成されてもよい。 When the polymer chain of the second polymer is a temperature-responsive polymer, the polymer chain of the second polymer may be composed of the same type of polymer as the polymer constituting the first temperature-responsive polymer (e.g., poly(meth)acrylamide derivatives, polyvinylamide derivatives, poly(meth)acrylate derivatives, polyvinyl methyl ether, cellulose derivatives, polypeptides, poly(vinyl)caprolactam, copolymers of polyethylene glycol and polypropylene glycol, or copolymers of two or more of these, etc.), or may be composed of a different type of polymer.
　第２の高分子の高分子鎖は、（ｉ）重合すると温度応答性のポリマーを構成する１種類のモノマー（例えばＮ－イソプロピル（メタ）アクリルアミド）が重合されてなる、該１種類のモノマー由来の繰り返し単位を有する高分子であってもよいし、（ｉｉ）重合すると温度応答性のポリマーを構成するモノマーが２種以上重合されてなる、該２種以上のモノマー由来の繰り返し単位を有する共重合体であってもよいし、（ｉｉｉ）重合すると温度応答性のポリマーを構成するモノマー１種または２種以上と、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上とが重合されてなる共重合体であってもよいし、（ｉｖ）重合すると温度応答性のポリマーを構成するモノマー由来の構成単位を含まない、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上が重合されてなる重合体であってもよい。 The polymer chain of the second polymer is derived from (i) one type of monomer (for example, N-isopropyl (meth)acrylamide) that constitutes a temperature-responsive polymer when polymerized; or (ii) a copolymer having repeating units derived from two or more monomers, which are obtained by polymerizing two or more monomers that constitute a temperature-responsive polymer when polymerized. (iii) one or more monomers that form a temperature-responsive polymer when polymerized, and one or two monomers that form a polymer that does not show temperature-responsiveness when polymerized; It may be a copolymer obtained by polymerizing two or more species, and (iv) it does not contain any structural units derived from monomers that constitute a temperature-responsive polymer when polymerized, and does not exhibit temperature-responsiveness even when polymerized. It may be a polymer obtained by polymerizing one or more monomers constituting the polymer.
　第２の粒子の加熱後の第２の高分子の鎖長の収縮を抑制する点で、第２の高分子の高分子鎖は、重合すると温度応答性のポリマーを構成するモノマー由来の構成単位を含まない、重合しても温度応答性を示さないポリマーを構成するモノマー１種または２種以上が重合されてなる重合体であることが好ましい。 In terms of suppressing the contraction of the chain length of the second polymer after heating the second particles, the polymer chains of the second polymer are structural units derived from monomers that constitute a temperature-responsive polymer when polymerized. It is preferable to use a polymer obtained by polymerizing one or more types of monomers constituting a polymer that does not contain , and does not exhibit temperature responsiveness even when polymerized.
　第２の高分子の主鎖の鎖長は、加温前の状態では第２の温度応答性高分子の主鎖の鎖長よりも短いことが、第２の高分子の第２の反応性官能基が第２の粒子の表面へ露出することを防ぐ点で好ましい。特に、第２の高分子の主鎖の鎖長は、第２の温度応答性高分子の第２のＬＣＳＴ未満の温度で、第２の温度応答性高分子の主鎖の鎖長よりも短いことが好ましい。また、第２の高分子の主鎖の鎖長は、加温後の状態では、一部または全部の第２の温度応答性高分子の主鎖の鎖長よりも長くなることが、第２の反応性官能基を第２の粒子の表面に露出させる点で好ましい。特に、第２の高分子の主鎖の鎖長は、第２の温度応答性高分子の第２のＬＣＳＴよりも高い温度で、第２の温度応答性高分子の主鎖の鎖長よりも長いことが好ましい。特定の実施形態では、第２の高分子の主鎖の鎖長は、５～３７℃の範囲内のいずれかの温度において第２の第２の温度応答性高分子の主鎖の鎖長よりも短く、３８～５０℃の範囲内のいずれかの温度、好ましくは３８～４５℃の範囲内のいずれかの温度、より好ましくは４０～４２℃の範囲内のいずれかの温度において、第１の温度応答性高分子の主鎖の鎖長と同一よりも長い。特定の実施形態では、第２の高分子の主鎖の鎖長は、５～３７℃の全範囲において第２の第２の温度応答性高分子の主鎖の鎖長よりも短く、３８～５０℃の全範囲、好ましくは３８～４５℃の全範囲、より好ましくは４０～４２℃の全範囲において、第１の温度応答性高分子の主鎖の鎖長と同一よりも長い。 The second reactivity of the second polymer is such that the main chain length of the second polymer is shorter than that of the second temperature-responsive polymer before heating. This is preferable in that it prevents the functional group from being exposed to the surface of the second particle. In particular, the chain length of the main chain of the second polymer is shorter than the chain length of the main chain of the second temperature-responsive polymer at a temperature below the second LCST of the second temperature-responsive polymer. It is preferable. Further, the chain length of the main chain of the second polymer is longer than the chain length of a part or all of the second temperature-responsive polymer in the state after heating. This is preferable in that the reactive functional group of is exposed on the surface of the second particle. In particular, the chain length of the main chain of the second polymer is greater than the chain length of the main chain of the second temperature-responsive polymer at a temperature higher than the second LCST of the second temperature-responsive polymer. Preferably long. In certain embodiments, the chain length of the backbone of the second polymer is greater than the chain length of the backbone of the second temperature-responsive polymer at any temperature within the range of 5 to 37°C. The first longer than the chain length of the main chain of the temperature-responsive polymer. In certain embodiments, the chain length of the backbone of the second polymer is shorter than the chain length of the backbone of the second temperature-responsive polymer over the entire range of 5 to 37°C, and between 38 and 37°C. In the entire range of 50°C, preferably in the entire range of 38 to 45°C, more preferably in the entire range of 40 to 42°C, it is longer than the chain length of the main chain of the first temperature-responsive polymer.
　第２の粒子の第２の高分子の末端の反応性官能基については、上述した通りである。 The reactive functional group at the end of the second polymer of the second particle is as described above.
　第２の高分子の分子量は特に限定されないが、例えば２ｋＤａ～１００ｋＤａである。 The molecular weight of the second polymer is not particularly limited, but is, for example, 2 kDa to 100 kDa.
　第２の粒子における第２の高分子の濃度は、第２の粒子の温度応答性と第１の粒子との反応の制御の点で、１ｗｔ％～５０ｗｔ％の範囲が好ましい。 The concentration of the second polymer in the second particles is preferably in the range of 1 wt% to 50 wt% from the viewpoint of controlling the temperature responsiveness of the second particles and the reaction with the first particles.
　第２の粒子は好ましくはミセルである。第２の粒子は、より好ましくは薬物を内部に充填できるミセルである。第２の粒子は、より好ましくは、２０℃で水中油滴型の、水難溶性薬物を内部に充填できるミセルである。 The second particles are preferably micelles. The second particles are more preferably micelles that can be loaded with drugs. The second particles are more preferably micelles that can be filled with an oil-in-water type drug that is poorly water-soluble at 20°C.
　第２の粒子は、好ましくは下限臨界溶解温度（ＬＣＳＴ）を有する。第２の粒子のＬＣＳＴは、第２の温度応答性高分子の選択、第２の高分子の選択、及び第２の粒子中のそれらの高分子の量の選択により、当業者には適切に設定することができる。第２の粒子のＬＣＳＴは、粒子を含む水溶液の可視光における透過率を昇温しながら測定し、その透過率の変化から評価することができる。 The second particles preferably have a lower critical solution temperature (LCST). The LCST of the second particle can be determined by one skilled in the art by selecting the second temperature-responsive polymer, selecting the second polymer, and selecting the amount of those polymers in the second particle. Can be set. The LCST of the second particles can be evaluated by measuring the visible light transmittance of an aqueous solution containing the particles while increasing the temperature, and from the change in the transmittance.
　第２の粒子のＬＣＳＴは、動物の体温以上であって、かつタンパク質の変性などの動物の体内での不利益を極力回避できる温度であることが好ましい。例えば、第２の粒子のＬＣＳＴは、３８～５０℃の範囲にあることが好ましく、３８～４５℃の範囲にあることがより好ましく、４０～４２℃の範囲にあることがより好ましい。 The LCST of the second particles is preferably a temperature that is equal to or higher than the animal's body temperature and that can avoid disadvantages in the animal's body such as protein denaturation as much as possible. For example, the LCST of the second particles is preferably in the range of 38 to 50°C, more preferably in the range of 38 to 45°C, and even more preferably in the range of 40 to 42°C.
　第１の粒子のＬＣＳＴと、第２の粒子のＬＣＳＴは、同じであっても異なっていてもよい。第１の粒子のＬＣＳＴと、第２の粒子のＬＣＳＴが異なる場合、粒子の温度感受性を合わせる点で、その差は±２℃以内であることが好ましい。 The LCST of the first particle and the LCST of the second particle may be the same or different. If the LCST of the first particle and the LCST of the second particle are different, the difference is preferably within ±2° C. in terms of adjusting the temperature sensitivity of the particles.
　第２の粒子の粒径は、第２の粒子のＬＣＳＴ未満の温度で、１０ｎｍ～５００ｎｍの範囲であることが好ましく、より好ましくは３０ｎｍ～２００ｎｍである。第２の粒子の粒径が１０ｎｍ以上であると、薬物の封入及び粒子の安定性の点で好ましい。第２の粒子の粒径が５００ｎｍ以下であると、粒子を生体内の標的組織へ送達し、標的としない他の臓器に粒子がトラップされることを防ぐ点で好ましい。 The particle size of the second particles is preferably in the range of 10 nm to 500 nm, more preferably 30 nm to 200 nm, at a temperature below the LCST of the second particles. It is preferable that the particle size of the second particles is 10 nm or more in terms of drug encapsulation and particle stability. It is preferable that the particle size of the second particles is 500 nm or less in order to deliver the particles to the target tissue in the living body and prevent the particles from being trapped in other non-target organs.
　いくつかの実施形態において、第１の粒子及び第２の粒子が、３７℃以下で親水性であり、かつ３８℃以上で疎水性である。ここで、「３７℃以下で親水性である」とは、３７℃以下の範囲のいずれかの温度で親水性であればよいという意味であり、「３８℃以上で疎水性である」とは、３８℃以上の範囲のいずれかの温度で疎水性であればよいという意味である。特定の実施形態では、第１の粒子及び第２の粒子が、５℃～３７℃の範囲のいずれかの温度で親水性であり、かつ３８℃～５０℃の範囲のいずれかの温度で疎水性である。別の特定の実施形態では、第１の粒子及び第２の粒子が、５℃～３７℃の全範囲で親水性であり、かつ３８℃～５０℃の全範囲で疎水性である。 In some embodiments, the first particle and the second particle are hydrophilic at 37°C or lower and hydrophobic at 38°C or higher. Here, "hydrophilic at 37°C or lower" means that it is sufficient to be hydrophilic at any temperature within the range of 37°C or lower, and "hydrophobic at 38°C or higher" means , 38°C or higher. In certain embodiments, the first particle and the second particle are hydrophilic at any temperature in the range of 5°C to 37°C and hydrophobic at any temperature in the range of 38°C to 50°C. It is gender. In another specific embodiment, the first particle and the second particle are hydrophilic over the entire range of 5°C to 37°C and hydrophobic over the entire range of 38°C to 50°C.
　いくつかの実施形態において、薬物送達用組成物は、第１の粒子が下限臨界溶解温度を有し、第２の粒子が第２の下限臨界溶解温度を有し、第１の下限臨界溶解温度及び第２の下限臨界溶解温度のうちの高い方の温度以上の温度での加熱により、第１の粒子及び第２の粒子が疎水性となって収縮する。これにより、第２の粒子中の第２の高分子の第２の反応性官能基の一部または全部が第２の粒子に露出し、第１の高分子の第１の反応性官能基との反応がしやすい状態となる。次に、第１の下限臨界溶解温度及び第２の下限臨界溶解温度のうちの高い方の温度以上の温度よりもさらに高い温度に加熱することで、第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基が反応し、第１の粒子と第２の粒子の間に架橋が形成される。 In some embodiments, the drug delivery composition has a first particle having a lower critical solution temperature, a second particle having a second lower critical solution temperature, and a first particle having a lower critical solution temperature. By heating at a temperature equal to or higher than the higher of the second lower limit critical melting temperature, the first particles and the second particles become hydrophobic and contract. As a result, part or all of the second reactive functional group of the second polymer in the second particle is exposed to the second particle, and the first reactive functional group of the first polymer is exposed to the second particle. This makes it easier to react. Next, the first reactivity of the first polymer is increased by heating to a temperature higher than the higher of the first lower critical melting temperature and the second lower critical melting temperature. The functional group and the second reactive functional group of the second polymer react to form a crosslink between the first particle and the second particle.
　第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基が反応を開始する温度は、３８～５０℃の範囲にあることが好ましく、４０～４２℃の範囲にあることがより好ましい。 The temperature at which the first reactive functional group of the first polymer and the second reactive functional group of the second polymer start the reaction is preferably in the range of 38 to 50°C, and 40 to 42°C. It is more preferable to be in the range of °C.
　いくつかの実施形態では、第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基とが、加熱により共有結合を形成する。共有結合により、第１の粒子と第２の粒子とが不可逆的に架橋する。このため、加熱を停止した後も、架橋体は維持される。 In some embodiments, the first reactive functional group of the first polymer and the second reactive functional group of the second polymer form a covalent bond by heating. The covalent bond irreversibly crosslinks the first particle and the second particle. Therefore, even after heating is stopped, the crosslinked body is maintained.
　いくつかの実施形態において、薬物送達用組成物は薬物をさらに含む。第１の粒子及び第２の粒子のうちの一方または両方に組み込まれていることが好ましい。薬物を封入した第１の粒子及び／又は第２の粒子は、加熱前の状態（例えば２０℃）では第１の粒子及び第２の粒子のうちの一方または両方に封入される。 In some embodiments, the drug delivery composition further comprises a drug. Preferably, it is incorporated into one or both of the first particles and the second particles. The first particles and/or the second particles encapsulating the drug are encapsulated in one or both of the first particles and the second particles before heating (for example, at 20° C.).
　薬物としては抗腫瘍剤、抗菌剤、抗真菌剤、抗寄生虫薬、抗アレルギー薬、抗炎症剤、抗ウイルス剤などが挙げられるがこれらに限定されない。 Examples of drugs include, but are not limited to, antitumor agents, antibacterial agents, antifungal agents, antiparasitic agents, antiallergic agents, antiinflammatory agents, and antiviral agents.
　薬物は、活性薬剤であってもよい。 The drug may be an active agent.
　薬物は、好ましくは難水溶性薬物を含む。難水溶性薬物として例えば、
イミプラミン、トフィソパム、ガンマオリザノール、スルピリド、チアミンジスルフィド、ナプロキセン、カルバマゼピン、インドメタシン、フェンタニル、フペルジンＡ、レボドパ、クロザピン、ジクロフェナク、フェキソフェナジン、メクロジン、パロキセチン、チザニジン、リスペリドン、オランザピン、ジプラシドン、リバスチグミン、ナロキソン、ナルトレキソン、イブプロフェン等の神経系及び感覚器官用医薬；
塩酸バルニジピン、塩酸ニカルジピン、塩酸インデノロール、塩酸アモスラロール、塩酸アモスラロール、硝酸イソソルビド、硝酸グアネチジン、グリベンクラミド、スコポラミン、フェロジピン、ファモチジン、フェノフィブラート、フロセミド、エナラプリル、ニフェジピン、フロセミド、スピロノラクトン、レセルピン、メトプロロール、デキサメタゾン、トリアムシノロンアセトニド、ヒドロコルチゾン、リシノプリル、ピロカルピン、ダナゾール、プレドニゾロン、テオフィリン、ケトプロフェン、塩酸タムスロシン、プロゲステロン、プロスタグランジン、エストロゲン、エストラジオール、プロブコール、クロフィブラート、グリベンクラミド、フェノフィブラート、ラロキシフェン、アロプリノール、アミオダロン、ベナゼプリル、カルシトリオール、カンデサルタン、エプロサルタン、デスモプレシン、レボチロキシン、アトルバスタチン、ロバスタチン、メトラゾン、モメタゾン、ナブメトン、オメプラゾール、プロパフェノン、キナプリル、シンバスタチン、レボノルゲストレル、ノレチステロン、テノキシカム、ピロキシカム、ブチルフタリド、ＧＷ６４７１、ＣＯＸ－ＩＩ阻害剤、アロマターゼ阻害剤、ペプチド医薬等の循環器・呼吸器・ホルモン剤等の個々の器官系医薬品；
ビタミンＡ、ビタミンＫ、ベラプロストナトリウム、補酵素Ｑ１０、レスベラトロール等の代謝性医薬品；
アムサクリン、イクサベピロン、イホスファミド、エストラムスチン、エトポシド、エポチロン、テニポシド、カルボプラチン、オキサリプラチン、カンプトテシン、イリノテカン、シクロホスファミド、カルムスチン、ビンブラスチン、ビンクリスチン、ビノレルビン、トポテカン、カバジタキセル、ドセタキセル、ラロタキセル、パクリタキセル、メトトレキサート、ブスルファン、メルカプトプリン、ビンデシン、メイタンシン、ハリントンニン、ホモハリントンニン、マイトマイシン、ブレオマイシン、タネスピマイシン、アクチノマイシンＤ、エピルビシン、ＳＮ－３８、ゲムシタビン、シスプラチン、タネスピマイシン、イダルビシン、ダウノルビシン、ドキソルビシン、カペシタビン、フルダラビン、クラドリビン、ボルテゾミブ、カルフィルゾミブ、イキサゾミブ、レトロゾール、ミトキサントロン、クルクミン、ビンクリスチン、ボリコナゾール、ポサコナゾール、イクサベピロン、カンプトテシン、ダサチニブ、ボスチニブ、ポナチニブ、ニロチニブ、フルオロウラシル、シタラビン、エリブリン、トラベクチン、フルベストラント、レトロゾール、テモゾロミド、タモキシフェン、レナリドマイド、イクサベピロン、メトトレキサート、ペメトレキセド、エンザルタミド、アビラテロン、ベンダムスチン、オラパリブ、コンブレタスタチンこれらの塩、およびこれらの水和物等の抗腫瘍剤；
シクロスポリン、テムシロリムス、タクロリムス、シロリムス、エベロリムス、アザチオプリン等の免疫抑制剤をはじめとする組織細胞機能用医薬品；
ラパマイシン、プリカマイシン、バンコマイシン、アンホテリシンＢ、ドキシサイクリン、クロラムフェニコール、クラリスロマイシン、リファンピシン、ケトコナゾール、イトラコナゾール、フルコナゾール、ボリコナゾール、ポサコナゾール、カスポファンギン、ミカファンギン、グリセオフルビン、ナイスタチン、ナリジクス酸、パモ酸ピルビニウム、シクロスポリンＡ、クルクミンをはじめとする病原生物に対する医薬品；
アシクロビル等をはじめとする抗ウイルス薬が挙げられる。 The drug preferably includes a poorly water-soluble drug. Examples of poorly water-soluble drugs include:
Imipramine, tofisopam, gamma oryzanol, sulpiride, thiamine disulfide, naproxen, carbamazepine, indomethacin, fentanyl, huperzine A, levodopa, clozapine, diclofenac, fexofenadine, meclozine, paroxetine, tizanidine, risperidone, olanzapine, ziprasidone, rivastigmine, naloxone, naltrexone , drugs for the nervous system and sense organs such as ibuprofen;
Varnidipine hydrochloride, nicardipine hydrochloride, indenolol hydrochloride, amosralol hydrochloride, amosralol hydrochloride, isosorbide nitrate, guanethidine nitrate, glibenclamide, scopolamine, felodipine, famotidine, fenofibrate, furosemide, enalapril, nifedipine, furosemide, spironolactone, reserpine, metoprolol, dexamethasone, triamcinolone acetate G Nido, hydrocortisone, lisinopril, pilocarpine, danazol, prednisolone, theophylline, ketoprofen, tamsulosin hydrochloride, progesterone, prostaglandins, estrogen, estradiol, probucol, clofibrate, glibenclamide, fenofibrate, raloxifene, allopurinol, amiodarone, benazepril, calcitriol, Candesartan, eprosartan, desmopressin, levothyroxine, atorvastatin, lovastatin, metolazone, mometasone, nabumetone, omeprazole, propafenone, quinapril, simvastatin, levonorgestrel, norethisterone, tenoxicam, piroxicam, butylphthalide, GW6471, COX-II inhibitor, aromatase inhibitor Individual organ system medicines such as cardiovascular, respiratory, and hormonal medicines, such as drugs and peptide medicines;
Metabolic drugs such as vitamin A, vitamin K, beraprost sodium, coenzyme Q10, resveratrol;
Amsacrine, ixabepilone, ifosfamide, estramustine, etoposide, epothilone, teniposide, carboplatin, oxaliplatin, camptothecin, irinotecan, cyclophosphamide, carmustine, vinblastine, vincristine, vinorelbine, topotecan, cabazitaxel, docetaxel, larotaxel, paclitaxel, methotrexate, Busulfan, mercaptopurine, vindesine, maytansine, harringtonine, homoharringtonine, mitomycin, bleomycin, tanespimycin, actinomycin D, epirubicin, SN-38, gemcitabine, cisplatin, tanesspimycin, idarubicin, daunorubicin, doxorubicin, capecitabine, fludarabine, cladribine, bortezomib, carfilzomib, ixazomib, letrozole, mitoxantrone, curcumin, vincristine, voriconazole, posaconazole, ixabepilone, camptothecin, dasatinib, bosutinib, ponatinib, nilotinib, fluorouracil, cytarabine, eribulin, travectin, fulvestrant, retro anti-tumor agents such as Zole, temozolomide, tamoxifen, lenalidomide, ixabepilone, methotrexate, pemetrexed, enzalutamide, abiraterone, bendamustine, olaparib, combretastatin, salts thereof, and hydrates thereof;
Pharmaceuticals for tissue cell function, including immunosuppressants such as cyclosporine, temsirolimus, tacrolimus, sirolimus, everolimus, and azathioprine;
Rapamycin, pricamycin, vancomycin, amphotericin B, doxycycline, chloramphenicol, clarithromycin, rifampicin, ketoconazole, itraconazole, fluconazole, voriconazole, posaconazole, caspofungin, micafungin, griseofulvin, nystatin, nalidixic acid, pyrvinium pamoate, cyclosporine A , medicines against pathogenic organisms including curcumin;
Examples include antiviral drugs such as acyclovir.
　難水溶性薬物は、酸付加物塩を形成する場合もあり、かかる塩も製薬学的に許容される塩である限りにおいて本明細書における難水溶性薬物に包含される。具体的には、難水溶性薬物の、塩酸、臭化水素酸、ヨウ化水素酸、硫酸、硝酸、リン酸等の無機酸や、ギ酸、酢酸、プロピオン酸、シュウ酸、マロン酸、コハク酸、フマル酸、マレイン酸、乳酸、リンゴ酸、クエン酸、酒石酸、炭酸、ピクリン酸、メタンスルホン酸、パラトルエンスルホン酸、グルタミン酸等の有機酸との酸付加塩が挙げられる。 Poorly water-soluble drugs may form acid adduct salts, and such salts are also included in the poorly water-soluble drugs herein as long as they are pharmaceutically acceptable salts. Specifically, poorly water-soluble drugs include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, as well as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, and succinic acid. , acid addition salts with organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, para-toluenesulfonic acid, and glutamic acid.
　第１の粒子及び／又は第２の粒子中の薬物の量は特に限定されないが、薬物負荷容量（薬物の重量／薬物を含む粒子の重量）が大きいことが好ましい。 The amount of drug in the first particles and/or second particles is not particularly limited, but it is preferable that the drug loading capacity (weight of drug/weight of particles containing drug) is large.
　次に、第１の態様の薬物送達用組成物の使用方法について説明する。 Next, a method of using the drug delivery composition of the first embodiment will be explained.
　薬物送達用組成物は、任意の適切な経路、例えば、静脈内投与、動脈内投与、筋肉内投与、腹腔内投与、皮下、皮内関節内、クモ膜下脳室内、鼻内噴霧、肺吸入、経口投与および当業者に公知の他の適切な投与経路を用いて、対象に投与することができる。対象はヒトまたは非ヒト動物であり、非ヒト動物はマウス、ラット、モルモット、ウサギなどの哺乳動物であることが好ましい。対象に投与された薬物送達用組成物は、対象の体内の標的組織や、その中の標的部位に送達される。本方法を用いて処置可能な組織としては、肺、肝臓、腎臓、骨、軟組織、筋肉、副腎組織、および胸部が挙げられるがこれらに限定されない。処置可能な組織には、癌組織、病変組織、または損傷組織と、所望であれば、健常組織の両方が含まれる。 Compositions for drug delivery may be administered by any suitable route, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intradermal intraarticular, intraventricular, intranasal, pulmonary inhalation. , oral administration, and other suitable routes of administration known to those skilled in the art. The subject is a human or non-human animal, and the non-human animal is preferably a mammal such as a mouse, rat, guinea pig, or rabbit. A drug delivery composition administered to a subject is delivered to a target tissue or target site within the subject's body. Tissues that can be treated using this method include, but are not limited to, lung, liver, kidney, bone, soft tissue, muscle, adrenal tissue, and breast. Tissues that can be treated include both cancerous, diseased, or damaged tissue and, if desired, healthy tissue.
　本発明の薬物送達用組成物を用いて対象に投与する薬物の投与量及び投与期間は、当業者であれば容易に決定できる。例えば、薬物は、約１分～数時間、例えば、２、３、４、６、２４時間またはそれ以上にわたって適切に静脈内投与される。 A person skilled in the art can easily determine the dosage and administration period of a drug to be administered to a subject using the drug delivery composition of the present invention. For example, the drug is suitably administered intravenously over a period of about 1 minute to several hours, such as 2, 3, 4, 6, 24 hours or more.
　対象の標的組織またはその中の標的部位は、本発明の薬物送達用組成物の投与前および／または投与中および／または投与後に加熱してもよい。１つの実施形態では、上記標的組織がまず加熱され（例えば、１０～３０分）、本発明の薬物送達用組成物は、その加熱後できるだけすぐに対象内に送達される。他の実施形態では、本発明の薬物送達用組成物が対象に送達され、上記標的組織は、その投与後できるだけすぐに加熱される。 The target tissue of the subject or the target site therein may be heated before and/or during and/or after administration of the drug delivery composition of the present invention. In one embodiment, the target tissue is first heated (eg, 10-30 minutes) and the drug delivery composition of the invention is delivered into the subject as soon as possible after heating. In other embodiments, a drug delivery composition of the invention is delivered to a subject and the target tissue is heated as soon as possible after administration.
　標的組織またはその中の標的部位の加温には上記標的組織を加熱する任意の適切な加熱装置を用いてもよく、例えば、高周波照射の利用、超音波（高密度焦点式超音波であってもよい）の利用、マイクロ波放射の利用、赤外線放射、光、ならびに放射性同位体、電場および磁場および／または上記の組み合わせにより熱が発生されるもの等、外部もしくは内部から受ける放射を発生する任意の発生源が挙げられる。 Any suitable heating device for heating the target tissue may be used to warm the target tissue or target region therein, such as the use of radiofrequency radiation, ultrasound (high intensity focused ultrasound), Any method that generates external or internal radiation, such as the use of microwave radiation, the use of microwave radiation, infrared radiation, light, and radioactive isotopes, electric and magnetic fields, and/or combinations of the above, in which heat is generated. The sources include:
　標的組織又は標的部位を、第１の粒子の第１のＬＣＳＴ及び第２の粒子の第２のＬＣＳＴのうちの高い方を超える温度まで加熱すると、第１の粒子及び第２の粒子が疎水性となって収縮し、第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基が反応し、第１の粒子と第２の粒子の間に架橋が形成される。第１の粒子及び／又は第２の粒子に組み込まれていていた薬物は、第１の粒子及び／又は第２の粒子から放出される。第１及び第２の粒子間で不可逆的な架橋ネットワークを形成することにより、加温を止めた後も標的組織又は標的部位での薬剤の作用が持続する。 Heating the target tissue or site to a temperature above the higher of the first LCST of the first particle and the second LCST of the second particle causes the first particle and the second particle to become hydrophobic. The first reactive functional group of the first polymer reacts with the second reactive functional group of the second polymer, and a crosslink is formed between the first particle and the second particle. is formed. The drug incorporated in the first particle and/or the second particle is released from the first particle and/or the second particle. The formation of an irreversible cross-linked network between the first and second particles allows the drug to continue to act at the target tissue or site even after heating has ceased.
　本発明の第２態様において、第１の温度応答性高分子と、第１反応性官能基を有する第１の高分子とを含有する第１の粒子と、第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と反応可能な第２の反応性官能基を有する第２の高分子とを含有する第２の粒子とを備えた、薬物送達システムが提供される。 In the second aspect of the present invention, the first particle contains a first temperature-responsive polymer, a first polymer having a first reactive functional group, and a second temperature-responsive polymer. , a second particle containing a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer. provided.
　第１の粒子及び第２の粒子については、第１態様の薬物送達用組成物に関して説明した通りである。薬物送達システムは、第１態様の薬物送達用組成物に関して説明した薬物をさらに備えてもよい。かかる第１の粒子及び第２の粒子の組み合わせを用いて、薬物を標的部位に送達し、かつ標的部位における薬物の作用を持続させることができる。 The first particles and the second particles are as described for the drug delivery composition of the first aspect. The drug delivery system may further comprise a drug as described with respect to the drug delivery composition of the first aspect. Such a combination of first particles and second particles can be used to deliver a drug to a target site and to sustain the action of the drug at the target site.
　本発明の第３態様において、第１の温度応答性高分子と、反応性官能基を有する第１の高分子とを含有する第１の粒子と、第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と反応可能な反応性官能基を有する第２の高分子とを含有する第２の粒子とを備えた、薬物送達用キットが提供される。かかる第１の粒子及び第２の粒子の組み合わせを用いて、薬物を標的部位に送達し、かつ標的部位における薬物の作用を持続させることができる。
　本発明の第４態様において、上述の本発明の態様の薬物送達用組成物、薬物送達システム、又は薬物送達キットを用いた疾患の治療方法が提供される。治療方法における各工程、投与経路、対象、標的組織又は標的部位については、第１の態様の薬物送達用組成物の使用方法について説明した通りである。 In a third aspect of the present invention, a first particle containing a first temperature-responsive polymer and a first polymer having a reactive functional group; a second temperature-responsive polymer; A kit for drug delivery is provided, comprising a second particle containing a second polymer having a reactive functional group capable of reacting with a first reactive functional group of the first polymer. Such a combination of first particles and second particles can be used to deliver a drug to a target site and to sustain the action of the drug at the target site.
In a fourth aspect of the present invention, there is provided a method for treating a disease using the drug delivery composition, drug delivery system, or drug delivery kit of the above-described aspects of the present invention. Each step, administration route, subject, target tissue, or target site in the treatment method is as described for the method of using the drug delivery composition of the first aspect.
　上述の本発明の態様の薬物送達用組成物、薬物送達システム、及び薬物送達キットは、臨床医療において、患部を可視化する画像診断技術（MRIやCTなど）や、体内を局所加温する装置（ハイパーサーミア装置など）と併用することで、標的組織又は標的部位に薬剤を効率的に届けることができる。 The drug delivery compositions, drug delivery systems, and drug delivery kits according to the embodiments of the present invention described above can be used in clinical medicine for image diagnosis techniques (such as MRI and CT) that visualize affected areas and devices that locally heat the inside of the body ( When used in conjunction with a hyperthermia device (such as a hyperthermia device), drugs can be efficiently delivered to the target tissue or site.
　ここで発明の理解を容易にするために、図を参照しながら本発明の特定の実施形態について説明するが、本発明はかかる実施形態に限定されるわけではない。 Here, in order to facilitate understanding of the invention, specific embodiments of the invention will be described with reference to the drawings, but the invention is not limited to such embodiments.
　図１（Ａ）に模式的に示すと、図示した実施形態において、第１の温度応答性高分子１１は、室温（２０℃前後）で親水性を有する。第１の温度応答性高分子１１は、加温により、下限臨界溶液温度（ＬＣＳＴ）以上の温度になると疎水性となる。第１の温度応答性高分子１１を冷却すると、親水性に戻る。図示はしないが、第２の温度応答性高分子２１も、加温により、下限臨界溶液温度（ＬＣＳＴ）以上の温度になると疎水性となり、冷却により、親水性に戻る。 As schematically shown in FIG. 1(A), in the illustrated embodiment, the first temperature-responsive polymer 11 has hydrophilicity at room temperature (around 20° C.). The first temperature-responsive polymer 11 becomes hydrophobic when heated to a temperature equal to or higher than the lower critical solution temperature (LCST). When the first temperature-responsive polymer 11 is cooled, it returns to hydrophilicity. Although not shown, the second temperature-responsive polymer 21 also becomes hydrophobic when heated to a temperature equal to or higher than the lower critical solution temperature (LCST), and returns to hydrophilicity when cooled.
　図１（Ｂ）に示されるように、第１の粒子１０は、第１の温度応答性高分子１１と、末端に第１の反応性官能基１４を有する第１の高分子１２とを含有する。第２の粒子２０は、第２の温度応答性高分子２１と、鎖中に第１の高分子１２の第１の反応性官能基１４と反応可能な第２の反応性官能基２４を有する第２の高分子２２とを含有する。第１の高分子１２は、高分子鎖１３の部分と、高分子鎖１３と結合する反応性官能基１４とを有し、第２の高分子２２は、高分子鎖２３の部分と、高分子鎖２３と結合する反応性官能基２４とを有する。 As shown in FIG. 1(B), the first particle 10 contains a first temperature-responsive polymer 11 and a first polymer 12 having a first reactive functional group 14 at the end. do. The second particle 20 has a second temperature-responsive polymer 21 and a second reactive functional group 24 capable of reacting with the first reactive functional group 14 of the first polymer 12 in the chain. and a second polymer 22. The first polymer 12 has a portion of the polymer chain 13 and a reactive functional group 14 that binds to the polymer chain 13, and the second polymer 22 has a portion of the polymer chain 23 and a reactive functional group 14 that binds to the polymer chain 13. It has a reactive functional group 24 that binds to a molecular chain 23.
　第１の高分子１２の高分子鎖１３も温度応答性高分子から形成されており、高分子鎖１３を構成するモノマーは、第１の温度応答性高分子１１を構成するモノマーと同一種であっても異なっていてもよい。加温前の状態において、第１の高分子１２の主鎖の鎖長は第１の温度応答性高分子１１の主鎖の鎖長よりも長く、反応性官能基１４は第１の粒子１０の表面に露出している。 The polymer chain 13 of the first polymer 12 is also formed from a temperature-responsive polymer, and the monomers constituting the polymer chain 13 are of the same type as the monomers constituting the first temperature-responsive polymer 11. It may be different or different. In the state before heating, the chain length of the main chain of the first polymer 12 is longer than the chain length of the main chain of the first temperature-responsive polymer 11, and the reactive functional group 14 is attached to the first particle 10. exposed on the surface.
　一方、第２の高分子２２の高分子鎖２３は、温度応答性高分子ではない。加温前の状態において、第２の高分子２２の主鎖の鎖長は第２の温度応答性高分子２１の主鎖の鎖長よりも短く、反応性官能基２４は第２の粒子２０の表面には露出せず、温度応答性高分子２１の親水性セグメントが形成する水和層内に埋もれている。 On the other hand, the polymer chain 23 of the second polymer 22 is not a temperature-responsive polymer. In the state before heating, the chain length of the main chain of the second polymer 22 is shorter than the chain length of the main chain of the second temperature-responsive polymer 21, and the reactive functional group 24 is attached to the second particle 20. It is not exposed on the surface of the temperature-responsive polymer 21, but is buried in the hydration layer formed by the hydrophilic segment of the temperature-responsive polymer 21.
　便宜上、図示した実施形態では、第１の温度応答性高分子１１と第２の温度応答性高分子２１は同一であり、同一のＬＣＳＴを有するものとし、このＬＣＳＴ以上において、第１の高分子１２の高分子鎖１３は疎水性となり収縮するものとする。 For convenience, in the illustrated embodiment, it is assumed that the first temperature-responsive polymer 11 and the second temperature-responsive polymer 21 are the same and have the same LCST. It is assumed that the polymer chains 13 of 12 become hydrophobic and contract.
　加熱により第１の粒子１０及び第２の粒子２０の温度が第１の温度応答性高分子１１及び第２の温度応答性高分子２１のＬＣＳＴ以上になると、第１の粒子１０の第１の温度応答性高分子１１、第１の高分子１２の高分子鎖１３、第２の粒子２０の第２の温度応答性高分子２１が親水性から疎水性に変化するため、第１の粒子１０及び第２の粒子２０はいずれも疎水性に変化する。第１の粒子１０の第１の温度応答性高分子１１、第１の高分子１２の高分子鎖１３、第２の粒子２０の第２の温度応答性高分子２１はＬＣＳＴ以上への加温により収縮する。このとき、第２の粒子２０の第２の温度応答性高分子２１の主鎖の鎖長が短くなるために、相対的に第２の高分子２２の高分子鎖２３の鎖長が第２の粒子２０の第２の温度応答性高分子２１の主鎖の鎖長よりも長くなり、反応性官能基２４が第２の粒子２０の表面に露出する。 When the temperature of the first particles 10 and the second particles 20 becomes higher than the LCST of the first temperature-responsive polymer 11 and the second temperature-responsive polymer 21 due to heating, the first temperature of the first particle 10 increases. Since the temperature-responsive polymer 11, the polymer chain 13 of the first polymer 12, and the second temperature-responsive polymer 21 of the second particle 20 change from hydrophilic to hydrophobic, the first particle 10 and the second particles 20 both change to be hydrophobic. The first temperature-responsive polymer 11 of the first particle 10, the polymer chain 13 of the first polymer 12, and the second temperature-responsive polymer 21 of the second particle 20 are heated to a temperature higher than the LCST. Shrinks due to At this time, since the chain length of the main chain of the second temperature-responsive polymer 21 of the second particle 20 becomes shorter, the chain length of the polymer chain 23 of the second polymer 22 becomes relatively shorter than the second temperature-responsive polymer 21. The length of the main chain of the second temperature-responsive polymer 21 of the particle 20 is longer than that of the second particle 20, and the reactive functional group 24 is exposed on the surface of the second particle 20.
　図２に示されるように、第１の粒子１０及び第２の粒子２０を含有する薬物送達用組成物１では、加温前の状態では個々の粒子が個別に分散して存在している（図２左）。加温により第１の粒子１０及び第２の粒子２０の温度が第１の温度応答性高分子１１及び第２の温度応答性高分子２１のＬＣＳＴ以上になると、第１の粒子１０及び第２の粒子２０が疎水性となり、分子間に疎水性相互性作用が生じ、第１の粒子１０及び第２の粒子２０が集合する（図２中央）。次に、さらに第１の粒子１０及び第２の粒子２０を加温すると、第１の高分子１２の反応性官能基１４と第２の高分子２２の反応性官能基２４が反応し、第１の粒子１０と第２の粒子２０の間に架橋が形成され、架橋体３０となる（図２右）。 As shown in FIG. 2, in the drug delivery composition 1 containing the first particles 10 and the second particles 20, the individual particles exist individually dispersed before heating ( Figure 2 left). When the temperature of the first particles 10 and the second particles 20 becomes equal to or higher than the LCST of the first temperature-responsive polymer 11 and the second temperature-responsive polymer 21 due to heating, the first particles 10 and the second particles 20 The particles 20 become hydrophobic, hydrophobic interaction occurs between molecules, and the first particles 10 and the second particles 20 aggregate (center of FIG. 2). Next, when the first particles 10 and the second particles 20 are further heated, the reactive functional groups 14 of the first polymer 12 and the reactive functional groups 24 of the second polymer 22 react, and the first particles 10 and the second particles 20 are heated. A crosslink is formed between the first particle 10 and the second particle 20, resulting in a crosslinked body 30 (FIG. 2 right).
　このような第１の粒子１０及び第２の粒子２０の構成及び作用を利用して、標的組織又は標的部位への薬物の誘導及び標的組織又は標的部位へ送達された薬物の保持を達成することができる。 Utilizing the configuration and action of the first particles 10 and the second particles 20, it is possible to guide the drug to the target tissue or target site and to retain the drug delivered to the target tissue or target site. Can be done.
　図３に示されるように、第１の粒子１０及び第２の粒子２０を含有する薬物送達用組成物１をヒト又は非ヒト動物の血管４０に投与すると、第１の粒子１０及び第２の粒子２０は血管内を通過し、全身へ移動する。血管４０は分岐し、標的組織（又はその標的部位）４２内の末梢血管４１に到る。標的組織４２を加温すると、末梢血管４１を通って標的組織４２に送達された第１の粒子１０及び第２の粒子２０が疎水性相互作用により集合し、さらには第１の粒子１０と第２の粒子２０の間に架橋を形成する。第１の粒子１０及び第２の粒子２０のうちの一方または両方に組み込まれた薬物は、薬物を組み込んだ粒子１０，２０から放出される。加温を停止した場合にも、第１の粒子１０と第２の粒子２０の間の架橋は不可逆的に形成されるため、薬物は、架橋体３０中の薬物を組み込んだ粒子１０，２０から放出され続ける。このため、図示した実施形態の薬物送達用組成物を用いることにより、加熱により不可逆的に形成された第１の粒子１０と第２の粒子２０の架橋体３０により薬物が保持されるため、加熱後も標的組織又は標的部位に薬物が保持され、第１の粒子１０と第２の粒子２０の間に不可逆的な架橋が生じない場合と比較して、標的組織又は標的部位における薬物の作用が持続する。 As shown in FIG. 3, when the drug delivery composition 1 containing the first particles 10 and the second particles 20 is administered to the blood vessel 40 of a human or non-human animal, the first particles 10 and the second particles Particles 20 pass through the blood vessels and travel throughout the body. Blood vessel 40 branches into a peripheral blood vessel 41 within target tissue (or target site thereof) 42 . When the target tissue 42 is heated, the first particles 10 and the second particles 20 that have been delivered to the target tissue 42 through the peripheral blood vessels 41 come together due to hydrophobic interaction, and further the first particles 10 and the second particles 20 are brought together by hydrophobic interaction. A crosslink is formed between the two particles 20. The drug incorporated into one or both of the first particles 10 and the second particles 20 is released from the drug-loaded particles 10, 20. Even when heating is stopped, the crosslinks between the first particles 10 and the second particles 20 are irreversibly formed, so the drug is removed from the drug-incorporated particles 10 and 20 in the crosslinked body 30. continues to be released. Therefore, by using the drug delivery composition of the illustrated embodiment, the drug is retained by the crosslinked body 30 of the first particles 10 and the second particles 20 that are irreversibly formed by heating. The drug is retained in the target tissue or target site even after the treatment, and the action of the drug in the target tissue or site is improved compared to the case where irreversible crosslinking does not occur between the first particles 10 and the second particles 20. last.
　本明細書中に引用されているすべての特許出願および文献の開示は、それらの全体が参照により本明細書に組み込まれるものとする。 The disclosures of all patent applications and documents cited herein are incorporated by reference in their entirety.
　以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されない。 The present invention will be described in more detail with reference to Examples below, but the present invention is not limited thereto.
実施例１　高分子の合成
１．温度応答性ジブロック共重合体の合成
　N-イソプロピルアクリルアミド(NIPAAm)、アクリルアミド (AAm) を可逆的付加開裂連鎖移動（RAFT）重合することでランダム共重合体である親水性高分子1を合成した。連鎖移動剤として4-シアノ-4-[[(ドデシルチオ)カルボノチオイル]チオ]ペンタン酸 (CDTPA) 、アゾ重合開始剤として4,4'-アゾビス(4-シアノ吉草酸) (V-501) を用いた。NIPAAm (3411 mg, 30.1 mmol) とAAm (89.30 mg, 1.26 mmol)、及びCDTPA (158.4 mg, 0.393 mmol) を10.5 mLの1,4-ジオキサンに溶解し、アルゴンガスで30分バブリングし、溶存酸素を除去した。その後、V-501 (11.0 mg, 0.039 mmol) を添加し、74℃に加温して16時間撹拌した。反応溶液をジエチルエーテルに滴下し、沈殿物をろ過し、減圧乾燥することで薄い黄色の固体を得た。また、試薬量を変えて共重合比率および分子量の異なる高分子を合成した。NIPAAm (1896 mg, 16.8 mmol)、AAm (103.6 mg, 1.46 mmol)、及びCDTPA (52.5 mg, 0.13 mmol) を6.1 mLの1,4-ジオキサンに溶解し、V-501 (11.0 mg, 0.039 mmol) を添加して上記と同様の操作を行うことで、親水性高分子2を合成した。NIPAAm (4466 mg, 39.5 mmol)、AAm (534.3 mg, 7.52 mmol)、CDTPA (158.0 mg, 0.392 mmol) を15.7 mLの1,4-ジオキサンに溶解し、V-501 (11.0 mg, 0.039 mmol) を添加して上記と同様の操作を行うことで、親水性高分子3を合成した。続いて、合成した親水性高分子1、2、3にメタクリル酸ブチル (BMA) をRAFT重合することで、ジブロック共重合体である両親媒性高分子4、5、6をそれぞれ合成した。両親媒性高分子4、5、6の分子量はそれぞれ約12kDa, 19kDa, 17kDaであった。親水性高分子と50当量のBMAを1,4-ジオキサンに溶解し、アルゴンガスで30分バブリングし、溶存酸素を除去した。その後、0.2当量のV-501を添加し、74℃に加温して24時間撹拌した。反応溶液をメタノールに対して3日間透析し、減圧乾燥することで薄い黄色の固体を得た。 Example 1 Synthesis of polymer 1. Synthesis of temperature-responsive diblock copolymer Hydrophilic polymer 1, a random copolymer, was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). . 4-cyano-4-[[(dodecylthio)carbonothioyl]thio]pentanoic acid (CDTPA) as a chain transfer agent, 4,4'-azobis(4-cyanovaleric acid) (V-501) as an azo polymerization initiator was used. NIPAAm (3411 mg, 30.1 mmol), AAm (89.30 mg, 1.26 mmol), and CDTPA (158.4 mg, 0.393 mmol) were dissolved in 10.5 mL of 1,4-dioxane and bubbled with argon gas for 30 minutes to remove dissolved oxygen. was removed. Then, V-501 (11.0 mg, 0.039 mmol) was added, heated to 74°C, and stirred for 16 hours. The reaction solution was added dropwise to diethyl ether, and the precipitate was filtered and dried under reduced pressure to obtain a pale yellow solid. Furthermore, polymers with different copolymerization ratios and molecular weights were synthesized by changing the amounts of reagents. NIPAAm (1896 mg, 16.8 mmol), AAm (103.6 mg, 1.46 mmol), and CDTPA (52.5 mg, 0.13 mmol) were dissolved in 6.1 mL of 1,4-dioxane, and V-501 (11.0 mg, 0.039 mmol) Hydrophilic polymer 2 was synthesized by adding and performing the same operation as above. NIPAAm (4466 mg, 39.5 mmol), AAm (534.3 mg, 7.52 mmol), CDTPA (158.0 mg, 0.392 mmol) were dissolved in 15.7 mL of 1,4-dioxane, and V-501 (11.0 mg, 0.039 mmol) was dissolved. Hydrophilic polymer 3 was synthesized by adding and performing the same operation as above. Next, amphiphilic polymers 4, 5, and 6, which are diblock copolymers, were synthesized by RAFT polymerization of butyl methacrylate (BMA) to the synthesized hydrophilic polymers 1, 2, and 3, respectively. The molecular weights of amphiphilic polymers 4, 5, and 6 were approximately 12 kDa, 19 kDa, and 17 kDa, respectively. A hydrophilic polymer and 50 equivalents of BMA were dissolved in 1,4-dioxane, and dissolved oxygen was removed by bubbling with argon gas for 30 minutes. Then, 0.2 equivalents of V-501 were added, heated to 74°C, and stirred for 24 hours. The reaction solution was dialyzed against methanol for 3 days and dried under reduced pressure to obtain a pale yellow solid.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
２．DBCO導入温度応答性ジブロック共重合体の合成
　200 mgの両親媒性高分子6を3 mLのテトラヒドロフランに溶解して、Sulfo DBCO-amine (Click Chemistry Tools社，200 mg, 6.0 mmol)、4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモルホリニウムクロリド (32.6 mg, 0.12 mmol)、トリエチルアミン(11.9 mg, 0.12 mmol) を加え、4℃で16時間撹拌後、25℃で16時間撹拌した。反応溶液をメタノールに対して3日間透析し、減圧乾燥することで薄い黄色の固体としてDBCO導入温度応答性ジブロック共重合体7を得た。共重合体7の分子量は約17kDaであった。 2. Synthesis of DBCO-introduced temperature-responsive diblock copolymer 200 mg of amphiphilic polymer 6 was dissolved in 3 mL of tetrahydrofuran, and Sulfo DBCO-amine (Click Chemistry Tools, 200 mg, 6.0 mmol), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (32.6 mg, 0.12 mmol) and triethylamine (11.9 mg, 0.12 mmol) were added, and the mixture was incubated at 4°C for 16 min. After stirring for an hour, the mixture was stirred at 25°C for 16 hours. The reaction solution was dialyzed against methanol for 3 days and dried under reduced pressure to obtain DBCO-introduced temperature-responsive diblock copolymer 7 as a pale yellow solid. The molecular weight of copolymer 7 was approximately 17 kDa.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
３．Az導入ジブロック共重合体の合成
　アクリル酸グリシジル (348.7 mg, 2.72 mmol) とAAm (451.3 mg, 6.35 mmol)、及びCDTPA (33.3 mg, 0.083 mmol) を9.1 mLのジメチルスルホキシドに溶解し、アルゴンガスで30分バブリングし、溶存酸素を除去した。その後、V-501 (2.31 mg, 0.0083 mmol) を添加し、74℃に加温して16時間撹拌した。反応溶液をアセトンに滴下し、沈殿物をろ過し、減圧乾燥することで薄い黄色の固体として親水性高分子8を得た。親水性高分子8と50当量のBMAを1,4-ジオキサンに溶解し、アルゴンガスで30分バブリングし、溶存酸素を除去した。その後、0.2当量のV-501を添加し、74℃に加温して24時間撹拌した。反応溶液をジメチルスルホキシドに対して3日間透析し、ジブロック共重合体である両親媒性高分子9を得た。両親媒性高分子9と5当量のアジ化ナトリウム、5当量の塩化アンモニウムをジメチルスルホキシド中で、50℃で24時間反応させた。反応溶液を水に対して3日間透析し、凍結乾燥することで薄い黄色の固体としてAz導入ジブロック共重合体10を得た。共重合体7の分子量は約11kDaであった。 3. Synthesis of Az-introduced diblock copolymer Glycidyl acrylate (348.7 mg, 2.72 mmol), AAm (451.3 mg, 6.35 mmol), and CDTPA (33.3 mg, 0.083 mmol) were dissolved in 9.1 mL of dimethyl sulfoxide, and argon gas was added. Bubbling was performed for 30 minutes to remove dissolved oxygen. Then, V-501 (2.31 mg, 0.0083 mmol) was added, heated to 74°C, and stirred for 16 hours. The reaction solution was added dropwise to acetone, and the precipitate was filtered and dried under reduced pressure to obtain hydrophilic polymer 8 as a pale yellow solid. Hydrophilic polymer 8 and 50 equivalents of BMA were dissolved in 1,4-dioxane, and dissolved oxygen was removed by bubbling with argon gas for 30 minutes. Then, 0.2 equivalents of V-501 were added, heated to 74°C, and stirred for 24 hours. The reaction solution was dialyzed against dimethyl sulfoxide for 3 days to obtain amphiphilic polymer 9, which is a diblock copolymer. Amphiphilic polymer 9, 5 equivalents of sodium azide, and 5 equivalents of ammonium chloride were reacted in dimethyl sulfoxide at 50°C for 24 hours. The reaction solution was dialyzed against water for 3 days and freeze-dried to obtain Az-introduced diblock copolymer 10 as a pale yellow solid. The molecular weight of copolymer 7 was approximately 11 kDa.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
実施例２　高分子ミセルの調製
１．DBCOミセルの作製
　DBCOミセルには、蛍光色素である1,1’-ジオクタデシル-3,3,3’,3’-テトラメチルインドカルボシアニン (DiI) （実施例３，４，５）、抗癌剤であるドキソルビシン (Dox) （実施例６，７）、もしくは蛍光色素である1,1’-ジオクタデシル-3,3,3’,3’-テトラメチルインドトリカルボシアニン (DiR) （実施例６，７）を作製時に封入した。　DiI封入DBCOミセルにおいては、9.61 mgの温度応答性ジブロック共重合体4と、5.39 mgのDBCO導入温度応答性ジブロック共重合体7と、0.20 mg のDiIとを、2.0 mLのジメチルアセトアミド中において20℃で一晩撹拌した。この高分子溶液をシリンジポンプを用いて0.1 mL/minで750 rpmで撹拌している6.0 mLの注射用水に対して滴下し、水に対して20℃で20時間透析し、孔径0.45 μmのシリンジフィルターでろ過することで、DBCOミセルを得た。
　Dox封入DBCOミセルにおいては、12.0 mgの温度応答性ジブロック共重合体4と、7.96 mgのDBCO導入温度応答性ジブロック共重合体7と、トリエチルアミンで中和した2.0 mgのDoxとを、4.0 mLのジメチルアセトアミド中において20℃で一晩撹拌した。上記と同様の操作により、Dox封入DBCOミセルを得た。
　上記DiI封入DBCOミセルの作製において、封入色素としてDiIの代わりにDiRを用い、他は同じ条件で、Doxを含まないコントロール（Doxを封入しないDBCOミセル）を作製した。 Example 2 Preparation of polymer micelles 1. Preparation of DBCO micelles DBCO micelles contain the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) (Examples 3, 4, 5), and the anticancer drug. doxorubicin (Dox) (Examples 6 and 7), or the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine (DiR) (Example 6) , 7) were enclosed at the time of production. For DiI-encapsulated DBCO micelles, 9.61 mg of temperature-responsive diblock copolymer 4, 5.39 mg of DBCO-incorporated temperature-responsive diblock copolymer 7, and 0.20 mg of DiI were mixed in 2.0 mL of dimethylacetamide. The mixture was stirred overnight at 20°C. This polymer solution was added dropwise to 6.0 mL of water for injection that was being stirred at 750 rpm at 0.1 mL/min using a syringe pump, dialyzed against water for 20 hours at 20°C, and then poured into a syringe with a pore size of 0.45 μm. DBCO micelles were obtained by filtering.
In the Dox-encapsulated DBCO micelles, 12.0 mg of temperature-responsive diblock copolymer 4, 7.96 mg of DBCO-introduced temperature-responsive diblock copolymer 7, and 2.0 mg of Dox neutralized with triethylamine were combined into 4.0 Stirred in mL of dimethylacetamide at 20°C overnight. Dox-encapsulated DBCO micelles were obtained by the same operation as above.
In the production of the DiI-encapsulated DBCO micelles, DiR was used instead of DiI as the encapsulation dye, and a control without Dox (DBCO micelles without Dox encapsulated) was produced under the same conditions.
２．Azミセルの作製
　Azミセルには、蛍光色素である1,1’-ジオクタデシル-3,3,3’,3’-テトラメチルインドジカルボシアニン (DiD) を作製時に封入した。18.6 mgの温度応答性ジブロック共重合体5と、1.45 mgのAz導入ジブロック共重合体10と、0.20 mg のDiDとを、2.7 mLのジメチルアセトアミド中において20℃で一晩撹拌した。上記と同様の操作により、Azミセルを得た。 2. Preparation of Az micelles The fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine (DiD) was encapsulated in the Az micelles during preparation. 18.6 mg of temperature-responsive diblock copolymer 5, 1.45 mg of Az-introduced diblock copolymer 10, and 0.20 mg of DiD were stirred overnight at 20°C in 2.7 mL of dimethylacetamide. Az micelles were obtained by the same procedure as above.
３．Ctrlミセルの作製
　上記Azミセルの作製において、ミセル作製時の高分子として温度応答性ジブロック共重合体5のみを用い、他は同じ条件で、アジドを含まないコントロール（Ctrlミセル）を作製した。 3. Preparation of Ctrl micelles In the preparation of the Az micelles described above, only temperature-responsive diblock copolymer 5 was used as the polymer during micelle preparation, and a control (Ctrl micelle) containing no azide was prepared under the same conditions.
実施例３　ミセルの温度応答性
　リン酸緩衝生理食塩水 (PBS) 中におけるミセル (1.0 mg/mL) の測定波長750 nmにおける透過率を、1.0℃/minで昇温しながら吸光度計で測定した。ブランクにはPBSを用いた。DBCOミセルの温度による透過率の変化を示したのが図４（Ａ）であり、透過率が50％減少したときの温度をＬＣＳＴと定義すると、ＬＣＳＴは約39.5℃であった。また、Azミセルの温度による透過率の変化を示したのが図４（Ｂ）であり、ＬＣＳＴは約40.0℃であった。 Example 3 Temperature responsiveness of micelles The transmittance of micelles (1.0 mg/mL) in phosphate buffered saline (PBS) at a measurement wavelength of 750 nm was measured using an absorbance meter while increasing the temperature at 1.0°C/min. . PBS was used as a blank. Figure 4(A) shows the change in transmittance of DBCO micelles due to temperature. If the temperature at which the transmittance decreases by 50% is defined as the LCST, the LCST was approximately 39.5°C. Further, FIG. 4(B) shows the change in transmittance of Az micelles depending on the temperature, and the LCST was about 40.0°C.
実施例４　加温によるミセルの架橋形成
１．温度によるミセルの粒子径測定
　PBS中におけるミセル (1.0 mg/mL) の粒子径を、加温と冷却を繰り返しながら動的光散乱法で測定した。32℃を低温として、ミセルのＬＣＳＴ以下である37℃もしくはＬＣＳＴ以上である47℃に加温した。DBCOミセルの温度による粒子径の変化を示したのが図５（Ａ）である。ＬＣＳＴ以下である37℃に加温してもDBCOミセルの粒子径は変化しなかった。一方、ＬＣＳＴ以上である47℃に加温したときにはDBCOミセルの粒子径は増加したが、その後32℃に冷却すると粒子径は元に戻り、加温時の粒子径変化は可逆的であった。Azミセルの温度による粒子径の変化を示したのが図５（Ｂ）である。37℃及び47℃のそれぞれで加温した場合、AzミセルでもDBCOミセルと同様の変化が確認された。
　DBCOミセルとAzミセルを等量混合して測定した結果が図５（Ｃ）である。それぞれ単独のミセルの結果とは異なり、47℃に加温したときの粒子径の増加は、その後32℃に冷却しても保たれており、加温時の粒子径変化は不可逆的であった。 Example 4 Crosslinking of micelles by heating 1. Measurement of particle size of micelles depending on temperature The particle size of micelles (1.0 mg/mL) in PBS was measured by dynamic light scattering while repeatedly heating and cooling. The low temperature was 32°C, and the temperature was raised to 37°C, which is below the LCST of the micelles, or 47°C, which is above the LCST. Figure 5(A) shows the change in particle size of DBCO micelles depending on temperature. Even when heated to 37°C, which is below the LCST, the particle size of the DBCO micelles did not change. On the other hand, the particle size of DBCO micelles increased when heated to 47°C, which is higher than the LCST, but the particle size returned to its original size when it was subsequently cooled to 32°C, and the particle size change upon heating was reversible. FIG. 5(B) shows the change in particle size of Az micelles depending on temperature. When heated at 37°C and 47°C, changes similar to those of DBCO micelles were observed in Az micelles.
FIG. 5(C) shows the measurement results obtained by mixing equal amounts of DBCO micelles and Az micelles. Unlike the results for each individual micelle, the increase in particle size upon heating to 47°C was maintained even after cooling to 32°C, indicating that the change in particle size upon heating was irreversible. .
２．ミセルの凝集体の蛍光測定
　さらに、このときの不可逆的な凝集体を、遠心分離で回収してジメチルスルホキシドで溶解し、蛍光光度計で蛍光測定すると、DBCOミセルに封入したDiIおよびAzミセルに封入したDiDが共に検出された。各色素の蛍光強度から、DBCOミセルのAzミセルに対する存在比率は1.00±0.13 (平均±標準偏差) と算出され、DBCOミセルとAzミセルはおよそ1:1の比率で凝集していた。
　これらの結果から、DBCOミセルとAzミセルを混合してＬＣＳＴ以上に加温することで不可逆的な架橋を形成できることが確認された。 2. Fluorescence measurement of micelle aggregates Furthermore, the irreversible aggregates at this time were recovered by centrifugation, dissolved in dimethyl sulfoxide, and fluorescence measured using a fluorometer. Both DiDs were detected. From the fluorescence intensity of each dye, the abundance ratio of DBCO micelles to Az micelles was calculated to be 1.00±0.13 (mean±standard deviation), and DBCO micelles and Az micelles were aggregated at a ratio of approximately 1:1.
From these results, it was confirmed that irreversible crosslinking could be formed by mixing DBCO micelles and Az micelles and heating the mixture above the LCST.
実施例５　血清中におけるミセルの温度応答性架橋制御
　生体内を模した環境として、ウシ胎児血清を70％含有するPBS中におけるミセルの架橋形成能を評価した結果が図６である。DBCOミセルとAzミセルを等量混合し、各10分間の加温と冷却を1サイクルとして、これを2サイクル繰り返した後の混合物中の粒子の粒子径を動的光散乱法で測定した。生理的体温である37℃に加温したときには240±50 nmであり、温熱療法として臨床適用可能な42℃に加温したときには1922±70 nmであった。加温前の混合物中の粒子の粒子径 (129±0.87 nm) （図６の左側のバー）と比較すると、37℃に加温したときの増加率（加温後の粒子径／加温前の粒子径）は2倍以下と軽微であったのに対し（図６の中央のバー）、42℃に加温したときの増加率は約15倍と非常に大きかった（図６の右側のバー）。この結果より、血清存在下でもDBCOミセルとAzミセルの架橋を温度によって制御可能であることが示された。 Example 5 Temperature-responsive crosslinking control of micelles in serum Figure 6 shows the results of evaluating the ability of micelles to form crosslinks in PBS containing 70% fetal bovine serum as an environment simulating in vivo. Equal amounts of DBCO micelles and Az micelles were mixed, and each cycle consisted of heating and cooling for 10 minutes. After repeating this cycle for two cycles, the particle size of the particles in the mixture was measured using a dynamic light scattering method. It was 240 ± 50 nm when heated to 37°C, which is the physiological body temperature, and 1922 ± 70 nm when heated to 42°C, which is clinically applicable as thermotherapy. Compared to the particle size of the particles in the mixture before heating (129±0.87 nm) (left bar in Figure 6), the increase rate when heated to 37°C (particle size after heating/before heating) (particle size) was slight, less than double (center bar in Figure 6), but when heated to 42°C, the increase rate was approximately 15 times, which was very large (bar on the right side of Figure 6). bar). This result showed that crosslinking between DBCO micelles and Az micelles can be controlled by temperature even in the presence of serum.
実施例６　加温によるミセルの腫瘍組織への集積効果
　担癌モデルマウスの腫瘍組織を標的組織として、加温によるミセルの標的組織への集積効果を評価した。BALB/cマウスの右後肢にマウス結腸由来がん細胞株 (Colon-26) を皮下移植してモデルマウスを作製した。腫瘍体積が50～100 mm3に達したときに、Doxを封入したDBCOミセルとDiDを封入したAzミセルとを等量混合したPBS溶液を尾静脈内投与した (120 mg polymer /kg body weight、200 μL/mouse)。投与直後から30分間、充電式カイロ（XY-101，ZeRay社）を用いて42℃で腫瘍部を局所加温した。投与48時間後に腫瘍組織を回収してホモジナイズし、組織溶解液中のDiDの蛍光を蛍光光度計で測定した。投与量から算出したミセルの集積率 (%ID/g = % injected dose /g tissue) を示したのが図７である。DBCOミセルとAzミセルの投与群において、ミセルの集積率は加温によって10倍以上と劇的に増大した（図７の左側２つのバー）。一方で、Azミセルの代わりに、アジドを含まないCtrlミセルを投与した群においては、加温の有無による集積率に大きな差は認められなかった（図７の右側２つのバー）。このように、加温によるDBCOミセルとAzミセルとの架橋形成によって、加温組織へのミセルの集積効率を高めることに成功した。 Example 6 Effect of heating on the accumulation of micelles in tumor tissue The effect of heating on the accumulation of micelles on the target tissue was evaluated using the tumor tissue of a tumor-bearing model mouse as the target tissue. A mouse model was created by subcutaneously transplanting a mouse colon-derived cancer cell line (Colon-26) into the right hind limb of a BALB/c mouse. When the tumor volume reached 50 to 100 mm3 , a PBS solution containing equal amounts of Dox-encapsulated DBCO micelles and DiD-encapsulated Az micelles was administered into the tail vein (120 mg polymer/kg body weight, 200 μL/mouse). Immediately after administration, the tumor site was locally heated at 42°C for 30 minutes using a rechargeable body warmer (XY-101, ZeRay). 48 hours after administration, tumor tissue was collected and homogenized, and the fluorescence of DiD in the tissue lysate was measured using a fluorometer. FIG. 7 shows the micelle accumulation rate (%ID/g = % injected dose/g tissue) calculated from the dose. In the groups treated with DBCO micelles and Az micelles, the micelle accumulation rate increased dramatically by more than 10 times by heating (two bars on the left side of Figure 7). On the other hand, in the group in which azide-free Ctrl micelles were administered instead of Az micelles, there was no significant difference in the accumulation rate depending on the presence or absence of heating (two bars on the right side of Figure 7). In this way, we succeeded in increasing the efficiency of micelle accumulation in the heated tissue by forming crosslinks between DBCO micelles and Az micelles through heating.
実施例７　ミセル投与と加温の併用による抗腫瘍効果
　担癌モデルマウスの腫瘍組織に対して抗癌剤を封入したミセルを届けることによる治療効果を評価した。実施例６と同様に、担癌モデルマウスに対してDoxを封入したDBCOミセルとDiDを封入したAzミセルとを等量混合したPBS溶液を投与 (3.0 mg Dox /kg body weight、200 μL/mouse) し、腫瘍部を局所加温した。また、溶媒対照としてPBSを投与した群や、ミセルを用いずに遊離のDoxを投与した群と比較した。 Example 7 Anti-tumor effect of combined administration of micelles and heating The therapeutic effect of delivering micelles encapsulating an anti-cancer drug to the tumor tissue of tumor-bearing model mice was evaluated. Similarly to Example 6, a PBS solution containing equal amounts of Dox-encapsulated DBCO micelles and DiD-encapsulated Az micelles was administered to tumor-bearing model mice (3.0 mg Dox/kg body weight, 200 μL/mouse). ) and locally heated the tumor area. In addition, comparisons were made with a group administered with PBS as a vehicle control and a group administered with free Dox without using micelles.
１．腫瘍の経時的な増殖
　処置日を0日目として、隔日で腫瘍体積を測定した結果が図８である（各群8～10匹）。PBS投与群を非治療群として比較すると、Dox封入DBCOミセルとAzミセルの投与によって腫瘍の増殖が抑制された。さらに、これらミセルの投与に加えて局所加温を併用することで腫瘍の増殖が劇的に抑制され、投与後22日間にわたり腫瘍はほとんど増殖していなかった。このように、加温によるDBCOミセルとAzミセルとの架橋形成によって、Dox封入ミセルの抗腫瘍効果を大きく向上させることに成功した。
　加温の併用による抗腫瘍効果の増大は、Dox投与群においても認められた。加温による抗腫瘍効果は、がん温熱療法に代表されるように一般的なものである。しかし、Doxミセル投与と加温を併用した群と比較すると、Dox投与と加温を併用した群の方が抗腫瘍効果は小さく、本実験のがん治療においてミセルが重要な役割を果たしていた。
　また、ミセル投与と加温を併用した群間で比較すると、DBCOミセルにDoxを封入した群よりも、Doxを封入していない群の方が抗腫瘍効果は小さかった。ミセルへのDox封入の有無によって差が生じたことから、架橋形成による抗腫瘍効果の向上には、抗癌剤であるDoxの集積が大きく関与していると示唆された。 1. Growth of tumor over time The tumor volume was measured every other day, with the treatment day being day 0. Figure 8 shows the results (8 to 10 animals in each group). When the PBS-treated group was compared with the non-treated group, tumor growth was suppressed by administration of Dox-encapsulated DBCO micelles and Az micelles. Furthermore, by administering these micelles in combination with local heating, tumor growth was dramatically suppressed, with almost no tumor growth for 22 days after administration. In this way, we succeeded in significantly improving the antitumor effect of Dox-encapsulated micelles by forming crosslinks between DBCO micelles and Az micelles by heating.
Increased antitumor effects due to the combined use of heating were also observed in the Dox-administered group. The antitumor effect of heating is common, as exemplified by cancer hyperthermia therapy. However, compared to the group that combined Dox administration and heating, the antitumor effect was smaller in the group that combined Dox administration and heating, indicating that micelles played an important role in cancer treatment in this experiment.
Furthermore, when comparing the groups in which micelles were administered in combination with heating, the antitumor effect was smaller in the group in which Dox was not encapsulated in DBCO micelles than in the group in which Dox was encapsulated in DBCO micelles. Since there was a difference depending on the presence or absence of Dox encapsulation in micelles, it was suggested that the accumulation of Dox, an anticancer agent, is largely involved in improving the antitumor effect due to crosslink formation.
２．生存率の時間推移
　処置から60日間の担癌モデルマウスの生存期間をカプランマイヤー法により分析した結果が図９である。この生存率は、腫瘍体積を指標に設定した人道的エンドポイント (＞2000 mm3) で安楽死処置をした結果である。Dox封入DBCOミセルとAzミセルの投与と加温を併用した群で、生存期間が他群と比べて大きく延長された。この処置を行った10匹中のうち7匹は、腫瘍が24日目までに測定不能な大きさ (直径1 mm以下) に縮小しており、100日目においても腫瘍を確認できなかったため、完全奏功と判断することができた。その後、実験を続けた結果、図１０に示すように、Dox封入DBCOミセルとAzミセルの投与と加温を併用した群のマウスは３６０日後まで生存率を維持できた。 2. Time course of survival rate Figure 9 shows the results of analyzing the survival period of tumor-bearing model mice for 60 days after treatment using the Kaplan-Meier method. This survival rate is the result of euthanasia with a humane endpoint based on tumor volume (>2000 mm 3 ). Survival time was significantly prolonged in the group that received Dox-encapsulated DBCO micelles and Az micelles in combination with heating compared to the other groups. In 7 out of 10 animals treated with this treatment, the tumor had shrunk to an unmeasurable size (less than 1 mm in diameter) by the 24th day, and the tumor could not be confirmed even on the 100th day. It was determined that the patient had a complete success. Thereafter, as a result of continuing the experiment, as shown in FIG. 10, the survival rate of the mice in the group in which Dox-encapsulated DBCO micelles and Az micelles were administered in combination with heating was maintained until 360 days later.
３．全身状態の経時的な変化
　処置日から隔日で体重を測定した結果が図１１である（各群8～10匹）。DBCOミセルとAzミセルの投与によって体重が減少することはなく、異常行動も観察されなかった。本発明のミセルに大きな毒性がないことが分かった。 3. Changes in general condition over time Figure 11 shows the results of body weight measurements every other day from the day of treatment (8 to 10 animals in each group). Administration of DBCO micelles and Az micelles did not cause weight loss, and no abnormal behavior was observed. It was found that the micelles of the present invention have no significant toxicity.
　１…薬物送達用組成物、１０…第１の粒子、１１…第１の温度応答性高分子、１２…第１の高分子、１４…第１の反応性官能基、２０…第２の粒子、２１…第２の温度応答性高分子、２２…第２の高分子、２４…第２の反応性官能基。 DESCRIPTION OF SYMBOLS 1... Composition for drug delivery, 10... First particle, 11... First temperature-responsive polymer, 12... First polymer, 14... First reactive functional group, 20... Second particle , 21...Second temperature-responsive polymer, 22...Second polymer, 24...Second reactive functional group.

Claims (10)

  1. 　第１の温度応答性高分子と、第１の反応性官能基を有する第１の高分子とを含有する第１の粒子と、
    　第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と反応可能な第２の反応性官能基を有する第２の高分子とを含有する第２の粒子と
    を含有し、
    　加熱により、第１の高分子の第１の反応性官能基と第２の高分子の第２の反応性官能基が反応し、第１の粒子と第２の粒子の間に架橋が形成される、薬物送達用組成物。     first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group;
    a second particle containing a second temperature-responsive polymer and a second polymer having a second reactive functional group capable of reacting with the first reactive functional group of the first polymer; Contains and
    By heating, the first reactive functional group of the first polymer and the second reactive functional group of the second polymer react, and a crosslink is formed between the first particle and the second particle. A composition for drug delivery.
  2. 　第１の温度応答性高分子が第１の下限臨界溶解温度を有し、
    　第２の温度応答性高分子が第２の下限臨界溶解温度を有し、
    　第１の下限臨界溶解温度及び第２の下限臨界溶解温度のうちの高い方の温度以上の温度での加熱により、第１の温度応答性高分子及び第２の温度応答性高分子が疎水性となって収縮する請求項１に記載の薬物送達用組成物。     the first temperature-responsive polymer has a first lower critical melting temperature;
    the second temperature-responsive polymer has a second lower critical melting temperature;
    The first temperature-responsive polymer and the second temperature-responsive polymer become hydrophobic by heating at a temperature equal to or higher than the higher of the first lower critical melting temperature and the second lower critical melting temperature. The composition for drug delivery according to claim 1, which contracts as follows.
  3. 　前記第１の粒子及び第２の粒子の各々がミセルであり、第１の温度応答性高分子及び第２の温度応答性高分子の各々が、親水性セグメントと疎水性セグメントとを備える請求項１に記載の薬物送達用組成物。 Each of the first particles and the second particles is a micelle, and each of the first temperature-responsive polymer and the second temperature-responsive polymer includes a hydrophilic segment and a hydrophobic segment. 1. The composition for drug delivery according to 1.
  4. 　第１の温度応答性高分子及び第２の温度応答性高分子のそれぞれの親水性セグメントが、ポリ（メタ）アクリルアミド誘導体、ポリビニルアミド誘導体、ポリ（メタ）アクリレート誘導体、ポリビニルメチルエーテル、セルロース誘導体、ポリペプチド、ポリ（ビニル）カプロラクタム、ポリエチレングリコールとポリプロピレングリコールの共重合体、またはこれらの２種以上の共重合体を含む請求項１に記載の薬物送達用組成物。 Each of the hydrophilic segments of the first temperature-responsive polymer and the second temperature-responsive polymer is a poly(meth)acrylamide derivative, a polyvinylamide derivative, a poly(meth)acrylate derivative, a polyvinyl methyl ether, a cellulose derivative, The drug delivery composition according to claim 1, comprising a polypeptide, poly(vinyl)caprolactam, a copolymer of polyethylene glycol and polypropylene glycol, or a copolymer of two or more thereof.
  5. 　前記第１の粒子及び前記第２の粒子が、３７℃以下で親水性であり、かつ３８℃以上で疎水性である、請求項１に記載の薬物送達用組成物。 The composition for drug delivery according to claim 1, wherein the first particles and the second particles are hydrophilic at 37°C or lower and hydrophobic at 38°C or higher.
  6. 　前記加熱前の状態では第２の高分子の主鎖の長さは第２の温度応答性高分子の主鎖の長さよりも短く、前記加熱後の状態では一部又は全部の第２の高分子の主鎖の長さが第２の温度応答性高分子の主鎖の長さよりも長くなる、請求項１に記載の薬物送達用組成物。 In the state before heating, the main chain length of the second polymer is shorter than the main chain length of the second temperature-responsive polymer, and in the state after heating, part or all of the second polymer is shorter than the main chain length of the second temperature-responsive polymer. The composition for drug delivery according to claim 1, wherein the length of the main chain of the molecule is longer than the length of the main chain of the second temperature-responsive polymer.
  7. 　第１の反応性官能基と第２の反応性官能基とが、前記加熱により共有結合を形成する請求項１に記載の薬物送達用組成物。 The composition for drug delivery according to claim 1, wherein the first reactive functional group and the second reactive functional group form a covalent bond by the heating.
  8. 　前記薬物送達用組成物が薬物をさらに含む請求項１～６のいずれか一項に記載の薬物送達用組成物。 The drug delivery composition according to any one of claims 1 to 6, wherein the drug delivery composition further contains a drug.
  9. 　前記薬物送達用組成物が難水溶性薬物をさらに含み、前記難水溶性薬物は第１の粒子及び第２の粒子のうちの一方または両方に組み込まれている請求項１～６のいずれか一項に記載の薬物送達用組成物。 Any one of claims 1 to 6, wherein the drug delivery composition further comprises a poorly water-soluble drug, and the poorly water-soluble drug is incorporated into one or both of the first particles and the second particles. The drug delivery composition described in Section 1.
  10. 　第１の温度応答性高分子と、第１の反応性官能基を有する第１の高分子とを含有する第１の粒子と、
    　第２の温度応答性高分子と、前記第１の高分子の第１の反応性官能基と第２の反応可能な反応性官能基を有する第２の高分子とを含有する第２の粒子とを備えた、薬物送達システム。     first particles containing a first temperature-responsive polymer and a first polymer having a first reactive functional group;
    a second particle containing a second temperature-responsive polymer and a second polymer having a reactive functional group capable of reacting with the first reactive functional group of the first polymer and a second reactive functional group; A drug delivery system comprising:
PCT/JP2023/032806 2022-09-16 2023-09-08 Heat induction type drug delivery system WO2024058065A1 (en)

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