WO2017188257A1 - Methylated polyrotaxane and synthesis method for same - Google Patents

Methylated polyrotaxane and synthesis method for same Download PDF

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WO2017188257A1
WO2017188257A1 PCT/JP2017/016390 JP2017016390W WO2017188257A1 WO 2017188257 A1 WO2017188257 A1 WO 2017188257A1 JP 2017016390 W JP2017016390 W JP 2017016390W WO 2017188257 A1 WO2017188257 A1 WO 2017188257A1
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cyclodextrin
polyrotaxane compound
acid
decomposable
polyrotaxane
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PCT/JP2017/016390
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French (fr)
Japanese (ja)
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由井　伸彦
篤志 田村
慶 西田
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国立大学法人 東京医科歯科大学
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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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/40—Cyclodextrins; Derivatives thereof

Definitions

  • the present invention relates to an acid-decomposable polyrotaxane compound having a plurality of methylated cyclic molecules and a synthesis method thereof. More specifically, the present invention relates to an acid-decomposable polyrotaxane compound having a plurality of methylated ⁇ -cyclodextrins, and a method for synthesizing the same. The present invention also relates to the use of an acid-decomposable polyrotaxane compound having a plurality of methylated cyclic molecules as an autophagy-inducing agent and as a therapeutic agent for diseases.
  • a polyrotaxane is a compound having a structure in which a linear molecule (also referred to as the main axis) serving as an axis penetrates a plurality of macrocyclic molecules (rings).
  • the macrocycle cannot be removed from the shaft due to an obstacle.
  • the bulky sites at both ends are called stoppers or caps and end groups. When there is no stopper or when the bulk is insufficient even if there is a stopper, the ring and the shaft may be separated, which is called pseudo-rotaxane.
  • Patent Document 1 discloses an acid-decomposable polyrotaxane containing ⁇ -cyclodextrin as a cyclic molecule. This acid-decomposable polyrotaxane can be decomposed in the intracellular environment and release the penetrated ⁇ -cyclodextrin locally in the cell. Since conventional free ⁇ -cyclodextrin mainly acts on cell membranes, acid-degradable polyrotaxane is being studied as a compound capable of changing the site of action of ⁇ -cyclodextrin and applied to disease treatment. .
  • Non-Patent Document 1 discloses the substance inclusion action by ⁇ -cyclodextrin. It is known that the substance inclusion action by ⁇ -cyclodextrin is changed by chemical modification to ⁇ -cyclodextrin.
  • ⁇ -cyclodextrin derivatives derivatives obtained by methylating hydroxyl groups (methylated ⁇ -cyclodextrin) are known to have the highest binding constants (complex stability constants) with various compounds such as cholesterol and adamantane.
  • An acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclic molecules and a linear molecule having a terminal group.
  • the polyrotaxane compound according to [1] which degrades in an intracellular acidic environment.
  • O-triphenylmethyl group with or without substituents at the terminal group, S-triphenylmethyl group with or without substituents, and N-triphenyl with or without substituents The polyrotaxane compound according to any one of [1] to [10], which is selected from the group consisting of methyl groups.
  • polyrotaxane compound according to any one of [1] to [11], wherein the terminal group is linked to the linear molecule via a peptide bond, carbamate bond, ester bond, or ether bond.
  • a method for producing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules comprising the step of reacting an acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide ,Production method.
  • a method for producing an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclic molecules the step of obtaining a poloxamer having primary amino groups at both ends of the poloxamer, the poloxamer and ⁇ -cyclodextrin; To obtain a pseudopolyrotaxane, capping both ends of the pseudopolyrotaxane with N-tritylglycine, and reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide. ,Production method.
  • a composition for inducing autophagy in a cell comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
  • a pharmaceutical composition for treating cancer comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
  • the pharmaceutical composition according to [16], wherein the cancer is a cancer resistant to apoptosis.
  • To treat or prevent a disease caused by intracellular cholesterol accumulation or a disease caused by dysfunction of autophagy comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules Pharmaceutical composition.
  • a pharmaceutical composition for treating or preventing Niemann-Pick disease type C comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
  • NPC Niemann-Pick disease type C
  • a method for inducing autophagy in a cell comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
  • a method for treating cancer comprising administering to a subject an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
  • the method of [21] wherein the cancer is a cancer resistant to apoptosis.
  • an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing Niemann-Pick disease type C (NPC).
  • NPC Niemann-Pick disease type C
  • An acid-decomposable polyrotaxane compound comprising ⁇ -cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule.
  • Niemann-Pick disease C containing an acid-decomposable polyrotaxane compound containing ⁇ -cyclodextrin having a substituent as a cyclic molecule and a poloxamer having a terminal N-triphenylmethyl group as a linear molecule.
  • an acid-decomposable polyrotaxane compound including a plurality of methylated cyclic molecules and a linear molecule having a terminal group.
  • Such a polyrotaxane compound can be decomposed in an acidic environment to release a plurality of methylated cyclic molecules.
  • FIG. 3 shows a reaction scheme for introducing a methyl group into ⁇ -cyclodextrin of polyrotaxane. It is the figure which showed the result of size chromatography. Since PRX and Me-PRX have a higher elution time than ⁇ -CD and have a higher molecular weight, synthesis is confirmed. It is the figure which showed the result of the proton nuclear magnetic resonance spectrum of Me-PRX. It is the figure which showed the result of the size chromatography of HEE-PRX. It is the figure which showed the result of the proton nuclear magnetic resonance spectrum of Me-PRX.
  • FIG. 3 is a graph showing the N-trityl group elimination rate of Me-PRX.
  • FIG. 3 is a diagram showing the N-trityl group elimination rate of HEE-PRX. It is the figure which showed the result of the size exclusion chromatography analysis of HEE-PRX. Depending on the elimination rate of the N-trityl group, the peak derived from HEE-PRX decreased and the peak derived from ⁇ -CD increased. It is the figure which showed the measurement result of the transmittance
  • FIG. 6 is a graph showing the cell viability of HeLa cells treated with Me-P103, HEE-PRX, and Me- ⁇ -CD. It is a graph showing the cell viability when cells were treated with Me-PRX and Me- ⁇ -CD in the presence of 3-methyladenine, an autophagy inhibitor.
  • the present inventors produced an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules by reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide.
  • Polyrotaxane (PRX) compound Rotaxane is a macromolecule in which a linear molecule penetrates and a bulky part is bonded to both ends of the linear molecule so that the ring cannot be removed from the shaft due to steric hindrance. .
  • one linear molecule penetrates the ring of a plurality of macrocyclic molecules.
  • the linear molecule and cyclic molecule used in the present invention are not particularly limited, and examples of the linear molecule include polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol (poloxamer), polyethylene imine, and polyamino acid. And one or more selected from the group consisting of polymethyl vinyl ether.
  • the average molecular weight of the linear molecule is preferably 1000 to 20000, particularly 2000 to 10,000, or 4000 to 7000. For example, a poloxamer having a molecular weight of about 5000 can be used.
  • the cyclic molecule is preferably ⁇ , ⁇ or ⁇ -cyclodextrin, but may have a similar cyclic structure, such as cyclic polyether, cyclic polyester, cyclic Examples include polyether amines and cyclic polyamines.
  • a preferable cyclic molecule from the viewpoint of cholesterol inclusion ability is ⁇ - or ⁇ -cyclodextrin, and ⁇ -cyclodextrin is particularly preferable.
  • the cyclic molecule contained in the polyrotaxane according to the present invention has a methyl group.
  • the cyclic molecule is preferably ⁇ -cyclodextrin, and the hydroxyl group of ⁇ -cyclodextrin is methylated.
  • ⁇ -cyclodextrin has, for example, 1 to 21, preferably 6 to 21 methyl groups per molecule.
  • the cyclic molecule may have a substituent other than a methyl group.
  • substituents examples include 2-hydroxyethoxyethyl (HEE) group, hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, hydroxyethoxyethyl group, N, N-dimethylaminoethyl group (referred to as DMAE group).
  • water-soluble polymers such as carboxyl groups, primary amino groups, or polyethylene glycol, protein molecules such as transferrin, and peptide molecules such as oligoarginine. These groups may be directly bonded to the cyclic molecule or may be bonded via a linker.
  • the linker is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the linker may be a carbamate ester bond (—O—CO—NH—), an ester bond (—O—CO—), a carbonate bond (— O-CO-O-), ether bond (-O-) and the like.
  • a combination of a linear molecule and a cyclic molecule a combination of ⁇ -cyclodextrin and poloxamer is preferable.
  • the synthesis of polyrotaxane by a combination of ⁇ -cyclodextrin and poloxamer is also disclosed in Patent Document 1 described above, the contents of which are also incorporated herein by reference.
  • the ratio of the number of molecules of linear molecules to the number of molecules of cyclic molecules is 1: 4 to 1:50, more preferably 1: 8 to 1:20, for example, 1:10 to 1:15. The ratio is used. That is, preferably 4 to 50 cyclic molecules are included in one molecule of the linear molecule, more preferably 8 to 20 cyclic molecules, for example, 10 to 15 cyclic molecules may be included.
  • terminal groups also referred to as bulky substituents used in the present invention include O-triphenylmethyl (O-Trt) group, S-triphenylmethyl (S-Trt) group, N-triphenylmethyl ( N-Trt) group, but not limited thereto.
  • the terminal group may be a substituted O-triphenylmethyl group, S-triphenylmethyl group, N-triphenylmethyl group or the like.
  • an N-triphenylmethyl group is used.
  • the N-Trt group is decomposed in a weakly acidic environment, the polyrotaxane skeleton is destroyed, and a cyclic molecule such as ⁇ -CD is released.
  • the end group can be linked to the linear molecule via a peptide bond, a carbamate bond, an ester bond, an ether bond or the like, but preferably a peptide bond is used. Therefore, one embodiment of the present invention relates to an acid-decomposable polyrotaxane compound containing ⁇ -cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule.
  • the substituent that ⁇ -cyclodextrin has is preferably a methyl group, but is not limited, and examples thereof include 2-hydroxyethoxyethyl (HEE) group, hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, hydroxyethoxyethyl.
  • HEE 2-hydroxyethoxyethyl
  • carboxyl group sometimes referred to as DMAE group
  • carboxyl group sometimes referred to as DMAE group
  • carboxyl group primary amino group
  • water-soluble polymer such as polyethylene glycol
  • protein molecule such as transferrin
  • peptide molecule such as oligoarginine It may be.
  • These groups may be directly bonded to the cyclic molecule or may be bonded via a linker.
  • the linker is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the linker may be a carbamate ester bond (—O—CO—NH—), an ester bond (—O—CO—), a carbonate bond (— O-CO-O-), ether bond (-O-) and the like.
  • the polyrotaxane compound according to the present invention may have a structure in which a terminal group is linked to a linear molecule via an acid-decomposable bond.
  • the acid-decomposable bond include, but are not limited to, an acetal bond, a ketal bond, a disulfide bond, an ester bond, an orthoester bond, a vinyl ether bond, a hydrazide bond, and an amide bond. These may be used individually by 1 type and may use 2 or more types together.
  • the terminal group for example, N-tritylglycine, a group having one or more benzene rings, a group having one or more tert-butyls, and the like can be used, but are not limited thereto.
  • Examples of the group having one or more benzene rings include a benzyloxycarbonyl (Z) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyl ester (OBz) group, and the like.
  • Examples of the group having tertiary butyl include tertiary butylcarbonyl (Boc) group and amino acid tertiary butyl ester (OBu group).
  • the acid-decomposable bond and the end group do not need to be directly linked, and may be linked via a linker moiety known to those skilled in the art.
  • the number average molecular weight of the polyrotaxane according to the present invention is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably about 10,000 to 100,000.
  • the linear molecule is a poloxamer including polyethylene glycol (PEG) and polypropylene glycol (PPG), the cyclic molecule is ⁇ -cyclodextrin, and the end group is an N-triphenylmethyl group. It is particularly preferred that the end group is linked to the linear molecule via a peptide bond.
  • the molecular weight of the poloxamer can be 4000 to 7000 (eg, about 5000), and the number of ⁇ -cyclodextrin per linear molecule can be 10 to 15 (eg, about 12).
  • the polyrotaxane compound according to the present invention includes a polyrotaxane having the chemical structure shown below:
  • m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer (here, since 3 repeating units of polypropylene glycol are described in parentheses, “m / 3” is described). M need not be a multiple of 3).
  • n is an integer indicating the number of polyethylene glycol repeating units.
  • x is an integer indicating the number of ⁇ -cyclodextrin.
  • R represents a substituent and is bonded to ⁇ -cyclodextrin through a chemical bond L.
  • ⁇ -cyclodextrin is shown as a cyclic structure in parentheses.
  • substituents for example, a methyl group
  • the chemical bond L may not be interposed.
  • Z represents a capping molecule (for example, an N-triphenylmethyl group), and is bonded to both ends of the main polymer through a chemical bond Y (for example, a peptide bond).
  • the chemical bond Y may not be interposed.
  • the polyrotaxane compound according to the present invention is an acid-decomposable polyrotaxane compound that decomposes in an acidic environment. For example, it degrades in an acidic environment at pH 4.0 to 6.0.
  • an N-Trt group is used as a terminal group
  • the N-Trt group is decomposed in a weakly acidic environment
  • the polyrotaxane skeleton is destroyed, and a cyclic molecule such as ⁇ -CD is released.
  • vesicles such as lysosomes and late endosomes exist in eukaryotic cells including humans, and the lumens of these vesicles are acidified.
  • the pH of the lysosomal lumen is around 5. Therefore, the polyrotaxane compound according to the present invention can be decomposed by being taken into these vesicles.
  • the polyrotaxane compound according to the present invention releases a cyclic molecule such as ⁇ -CD upon decomposition.
  • ⁇ -cyclodextrin when ⁇ -cyclodextrin is released in lysosomes, it can include cholesterol present in lysosomes, thereby causing Niemann-Pick disease type C, which is caused by excessive accumulation of cholesterol in lysosomes. Lysosomal disease can be treated or prevented.
  • one embodiment of the present invention relates to a pharmaceutical composition for treating a disease caused by intracellular cholesterol accumulation, comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  • a pharmaceutical composition for treating a disease caused by intracellular cholesterol accumulation comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  • one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human.
  • the present invention relates to a method for treating or preventing Niemann-Pick disease type C (NPC). Furthermore, one aspect of the present invention is the use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing Neimanpick disease type C (NPC). Also related.
  • lysosomal disease more specifically Gaucher disease (Gaucher disease), Niemann-Pick disease type A (Niemann-Pick disease type A), Niemann-Pick disease type B ( Niemann-Pick disease type B), Niemann-Pick disease type C (Niemann-Pick disease type C), GM1 gangliosidosis, GM2 gangliosidosis (sometimes referred to as “Tay-Sachs Sandhoff type AB”), Krabbe.
  • Gaucher disease Gaucher disease
  • Niemann-Pick disease type A Niemann-Pick disease type A
  • Niemann-Pick disease type B Niemann-Pick disease type B
  • Niemann-Pick disease type C Niemann-Pick disease type C
  • GM1 gangliosidosis GM2 gangliosidosis (sometimes referred to as “Tay-Sachs Sandhoff type AB”)
  • Krabbe GM1 gangliosidosis
  • one embodiment of the present invention relates to a pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC) comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  • one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human.
  • the present invention relates to a method for treating or preventing Niemann-Pick disease type C (NPC).
  • one aspect of the present invention is the use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing Neimanpick disease type C (NPC). Also related. Further, as described above, one embodiment of the present invention is an acid-decomposable product including ⁇ -cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at a terminal as a linear molecule. Also related to the polyrotaxane compound.
  • one embodiment of the present invention contains an acid-decomposable polyrotaxane compound containing ⁇ -cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule.
  • the present invention also relates to a pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC).
  • NPC Niemann-Pick disease type C
  • Autophagy can be induced in cells by acting an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  • the cyclodextrin is preferably ⁇ -cyclodextrin. Accordingly, one aspect of the present invention relates to a composition for inducing autophagy in a cell, comprising an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
  • one embodiment of the present invention relates to a method for inducing autophagy in a cell containing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. Furthermore, one aspect of the present invention also relates to the use of an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for inducing autophagy in a cell.
  • the methylated polyrotaxane according to the present invention can induce autophagic cell death in cells. It is known to those skilled in the art that autophagic cell death can be used to induce cell death in cancer cells.
  • one embodiment of the present invention is a pharmaceutical composition for treating cancer, preferably treating apoptosis-resistant cancer, comprising an acid-degradable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  • the present invention relates to a pharmaceutical composition.
  • one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human.
  • one aspect of the present invention also relates to the use of an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating cancer.
  • one embodiment of the present invention is a pharmaceutical composition for treating or preventing a disease caused by autophagy dysfunction, which comprises an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  • one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human.
  • the present invention relates to a method for treating or preventing a disease caused by autophagy dysfunction. Furthermore, one aspect of the present invention is the use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing a disease resulting from autophagy dysfunction. Also related. Examples of diseases caused by autophagy dysfunction include the above-mentioned lysosomal diseases, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.
  • An acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules can be produced by a method comprising a step of reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide.
  • one embodiment of the production method according to the present invention includes a step of obtaining a poloxamer having primary amino groups at both ends of the poloxamer (step A), and reacting the poloxamer with ⁇ -cyclodextrin to produce a pseudopolyrotaxane.
  • Step B capping both ends of the pseudopolyrotaxane with N-tritylglycine (step C), and reacting acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide (Step D).
  • a polyrotaxane dissolved in dehydrated dimethyl sulfoxide can be used.
  • the reaction between the acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide can be carried out by stirring for about 30 minutes to 3 hours, preferably about 1 hour. Stirring is preferably performed vigorously.
  • the number of methyl group modifications can be controlled by the equivalent of methyl iodide during the reaction.
  • compositions The polyrotaxane compound according to the present invention can be used as an active ingredient in a pharmaceutical composition used for the treatment or prevention of the above-mentioned diseases. Therefore, one embodiment of the present invention relates to a pharmaceutical composition used for treatment or prevention of diseases.
  • the other components in the pharmaceutical composition according to the present invention are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include pharmaceutically acceptable carriers. There is no restriction
  • the pharmaceutical composition according to the present invention is water-soluble at around body temperature, for example, 34 ° C to 42 ° C, more preferably 35 ° C to 38 ° C or 37 ° C.
  • an injection Solution, suspension, solid agent for use, etc.
  • inhaled powders for example, a pH regulator, a buffer, a stabilizer, a tonicity agent, a local anesthetic, etc. are added to the polyrotaxane compound according to the present invention, and subcutaneous, intramuscular, intravenous, etc. are added by a conventional method.
  • An injection for internal use can be produced.
  • the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like.
  • Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like.
  • Examples of the isotonic agent include sodium chloride and glucose.
  • Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.
  • the administration method of the pharmaceutical composition according to the present invention is not particularly limited, and for example, either local administration or systemic administration can be selected according to the dosage form of the pharmaceutical composition, the patient's condition, and the like.
  • local administration includes intracerebroventricular administration.
  • the subject of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs, cats and the like. However, it is preferably a human.
  • the dosage of the pharmaceutical composition according to the present invention is not particularly limited, and can be appropriately selected depending on the dosage form, the age and weight of the administration subject, the degree of desired effect, and the like.
  • the administration time of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be administered prophylactically to a patient susceptible to the above-mentioned diseases, It may be administered therapeutically to patients presenting with symptoms. Moreover, there is no restriction
  • Pluronic P103 manufactured by ADEKA, trade name: Adekapluronic P-103; polyethylene glycol (hereinafter sometimes referred to as “PEG”) and polypropylene glycol (hereinafter referred to as “PPG”) as linear polymers.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • the Pluronic P103 reaction solution was added dropwise to the eggplant-shaped flask and stirred at room temperature for 24 hours. After the reaction, an unreacted product was removed by dialysis against methanol (manufactured by Kanto Chemical Co., Inc.) in addition to a dialysis membrane (manufactured by Spectrum) having a molecular weight cut off of 3500. By concentrating with a rotary evaporator, 7.22 g of Pluronic P103 having a primary amino group at both ends (hereinafter sometimes referred to as “P103-NH 2 ”) was obtained.
  • ⁇ -CD ⁇ -cyclodextrin
  • both ends of the pseudopolyrotaxane with N-tritylglycine (manufactured by Sigma-Aldrich) as follows, both ends of a linear polymer penetrating a plurality of ⁇ -CDs are subjected to an acidic pH environment.
  • a polyrotaxane hereinafter sometimes referred to as “PRX” having a bulky substituent that is eliminated by the reaction was obtained.
  • N-tritylglycine 276 mg of N-Hydroxysuccinimide (Acros Organics), Ethyl-3- (3-dimethylaminopropyl) carbimide, Hydrochloride (Tokyo Chemical Industry Co., Ltd., N 6.46 mL of N-dimethylformamide (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 3 hours.
  • N-dimethylformamide manufactured by Wako Pure Chemical Industries, Ltd.
  • To the pseudopolyrotaxane 17.2 mL of ultrapure water, 8.62 mL of methanol, and the N-tritylglycine reaction solution were added and stirred at room temperature for 24 hours. After the reaction, the resulting precipitate was collected by centrifugation.
  • the precipitate obtained in the order of methanol, N, N-dimethylformamide and ultrapure water was washed to remove unreacted substances.
  • the recovered solid was freeze-dried to obtain 1.79 g of polyrotaxane having its end capped with N-tritylglycine.
  • a methyl (hereinafter sometimes referred to as “Me”) group is introduced into ⁇ -CD of the polyrotaxane as follows (FIG. 1), and methylated polyrotaxane (hereinafter referred to as “Me-PRX”).
  • Me-PRX methylated polyrotaxane
  • m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer.
  • n is an integer indicating the number of polyethylene glycol repeating units.
  • x is an integer indicating the number of ⁇ -cyclodextrin.
  • ⁇ -cyclodextrin is shown as a cyclic structure in parentheses. For convenience of illustration, only one substituent on ⁇ -cyclodextrin is shown, but the number of substituents may be plural.
  • the Me-PRX was subjected to size exclusion chromatography measurement using 10 mM LiBr-containing dimethylsulfoxide as an eluent, and proton nuclear magnetic resonance spectrum measurement at 400 MHz measured in heavy water (manufactured by Kanto Chemical Co., Inc.).
  • Pluronic P123 Sigma-Aldrich; average molecular weight 5800
  • Pluronic P105 Densiichi Kogyo Seiyaku, trade name: Epan U-105; average
  • Pluronic P84 manufactured by ADEKA, trade name: Adeka Pluronic P-84; average molecular weight 4200
  • Pluronic L121 manufactured by Sigma-Aldrich; average molecular weight 4400 was synthesized as a main axis polymer.
  • the calculated ⁇ -CD penetration number, Me group modification number, and molecular weight from the results of proton nuclear magnetic resonance spectrum are shown in Table 1 below.
  • HEE hydroxyethoxyethyl
  • m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer.
  • n is an integer indicating the number of polyethylene glycol repeating units.
  • x is an integer indicating the number of ⁇ -cyclodextrin.
  • ⁇ -cyclodextrin is shown as a cyclic structure in parentheses. For convenience of illustration, only one substituent on ⁇ -cyclodextrin is shown, but the number of substituents may be plural.
  • the recovered aqueous solution was lyophilized to obtain a polyrotaxane (HEE-PRX) in which hydroxyethoxyethyl groups were introduced into ⁇ -CD and the pluronic ends were capped with N-tritylglycine.
  • HEE-PRX polyrotaxane
  • the HEE-PRX was subjected to size exclusion chromatography measurement using 10 mM LiBr-containing dimethylsulfoxide as an eluent, and proton nuclear magnetic resonance spectrum measurement at 400 MHz measured in heavy water (manufactured by Kanto Chemical Co., Inc.).
  • Example 2 pH acid decomposability> 10 mg of 12.2Me-P103 is dissolved in 2 mL of 10 mM acetate buffered saline (pH 5.0) or 10 mM phosphate buffered saline (pH 7.4) (hereinafter sometimes referred to as “PBS”). It was allowed to stand at 37 ° C. for a predetermined time (1, 3, 6, 12, 24, 48 hours). 100 ⁇ L was collected every hour and mixed with 200 ⁇ L of 50 mM carbonate buffer (pH 9.0) and 300 ⁇ L of acetonitrile (manufactured by Kanto Chemical Co., Inc.).
  • Desorption rate (%) ⁇ (Absorption intensity of Me-PRX solution at each pH and time) / (Absorption intensity when N-trityl group is completely eliminated) ⁇ ⁇ 100
  • 12.2Me-P103 had 60.9% of N-trityl group eliminated at 3 hours at pH 5.0 and 100% at 24 hours, while 48 hours at pH 7.4. The desorption rate was 10.5%. From the results shown in FIG. 7, the peak derived from 12.2Me-P103 decreased and the peak derived from ⁇ -CD increased according to the elimination rate of the N-trityl group. From the above results, it was found that Me-PRX is stable under physiological pH conditions, but terminal N-trityl is eliminated and the polyrotaxane structure is destroyed under an acidic pH environment. From the results shown in FIG.
  • HEE-PRX had 66.9% N-trityl groups eliminated at 3 hours at pH 5.0 and 98.8% at 24 hours, whereas 48 hours at pH 7.4. The desorption rate was 7.33%. From the results shown in FIG. 9, the peak derived from HEE-PRX decreased and the peak derived from ⁇ -CD increased according to the elimination rate of the N-trityl group. From the above, it has been clarified that HEE-PRX, like Me-PRX, loses terminal N-trityl under an acidic pH environment and collapses the polyrotaxane structure.
  • Example 3 Evaluation of temperature response> Each concentration of Me-PRX (Table 1) was dissolved in 1 mL of 10 mM phosphate buffer (pH 7.4) in a quartz cell. Using a UV / Vis spectrophotometer (manufactured by JASCO Corporation), transmittance measurement at 600 nm (temperature increase temperature: 1.0 ° C./min) was performed. The results are shown in FIG.
  • FIG. 10 shows that the temperature (LCST) at which the transmittance of each Me-PRX having a different main-axis polymer is 50% at 2.5 mg / mL is 13.8 Me-P105 (46.9 ° C.)> 14.3 Me. ⁇ P123 (40.0 ° C.)> 12.2 Me-P103 (39.1 ° C.)> 15.3 Me-P84 (33.1 ° C.)> 15.2 Me-L121 (30.6 ° C.) Show. Although each pluronic (poloxamer) has a different weight fraction of the PPG moiety in the molecular chain, it was revealed that the LCST decreases as the PPG weight fraction increases.
  • Me-PRX using pluronic P123 having different methyl group modification number as the axial polymer, 6.3Me-P123 is 43.0 ° C, 14.1Me-P123 is 36.5 ° C, and 21.0Me-P123 is 35 It was found that the LCST decreased as the number of methyl group introduction increased. It has been shown that Me-PRX can adjust the phase transition temperature by the constituent components.
  • Example 4 Induction evaluation of autophagy> HeLa cells stably expressing GFP-LC3 were seeded in a 35 mm glass bottom dish at a concentration of 1.0 ⁇ 10 4 cells / dish and incubated for 1 day. The following sample was added to the medium so as to be 1 mM in terms of ⁇ -cyclodextrin concentration. After 24 hours, confocal microscopy was performed, and the number of GFP-positive autophagosomes in the cells was counted. Further, the same experiment as described above was performed in a state where 3-methyladenine (manufactured by Merck), an autophagy inhibitor, was added to the medium at a concentration of 10 mM. The results are shown in FIG.
  • Me- ⁇ -CD Methyl- ⁇ -cyclodextrin
  • Example 5 Evaluation of cell death> HeLa cells were seeded on a 96-well plate at 0.5 ⁇ 10 4 cells / well and incubated for one day. The following samples were added to each concentration medium in terms of ⁇ -cyclodextrin concentration. After 48 hours, CellCounting Kit-8 (manufactured by Dojindo Laboratories) was added and incubated at 37 ° C. for 1.5 hours. The cell viability was evaluated by measuring absorbance at 450 nm with a microplate reader (Thermo Fisher Scientific). The results are shown in FIG.
  • the cells treated with 12.2Me-P103 had a significantly lower cell viability than the cells treated with HEE-PRX or Me- ⁇ -CD. In other words, cell death was strongly induced. This result is considered because 12.2Me-P103 acted by releasing methylated ⁇ -CD locally in the cell. In the presence of 3-methyladenine, cells treated with Me- ⁇ -CD decreased in cell viability, whereas cells treated with 12.2Me-P103 increased cell viability. In other words, 12.2Me-P103 is considered to suppress the induction of cell death under autophagy inhibition. From the above results, it became clear that Me-PRX has an effect of inducing autophagy cell death.
  • Example 6 Evaluation of cell death induction on apoptosis-resistant cells> Bax / Bak DKO MEF cells in which a pro-apoptotic protein Bax / Bak was knocked out as anti-apoptotic cells were seeded on a 96-well plate at 0.2 ⁇ 10 4 cells / well and incubated for one day.
  • zVAD-FMK which is a caspase inhibitor
  • Example 1 (1) 12.2Me-P103 (produced in Example 1) (2) Me- ⁇ -CD (3) 2,6-di-O-methyl- ⁇ -cyclodextrin (hereinafter sometimes referred to as “DM- ⁇ -CD”; compound represented by the following structural formula (2); manufactured by Sigma-Aldrich ; Product number H0513) Structural formula (2)
  • Example 7 Cholesterol-reducing action in NPC disease cells> Using healthy human-derived skin fibroblasts (hereinafter referred to as “NHF”, obtained from C Common Institute), Neiman-Pick disease type C patient-derived skin fibroblasts (hereinafter referred to as “NPC1”, obtained from Corell Institute) The NPC1, which was examined for intracellular cholesterol accumulation as follows, was seeded in a glass bottom dish (cell number: 1 ⁇ 10 4 cells / dish) and cultured at 37 ° C. for 24 hours. 10 ⁇ M to 1000 ⁇ M was added in terms of concentration, and further cultured at 37 ° C. for 24 hours.
  • NPC1 Neiman-Pick disease type C patient-derived skin fibroblasts
  • HEE-PRX prepared in Example 1 100 ⁇ M (converted to ⁇ -CD concentration)
  • HEE-PRX 1000 ⁇ M (converted to ⁇ -CD concentration) 4.2Me-P103 (prepared in Example 1) 100 ⁇ M (converted to ⁇ -CD concentration) (5) 12.2Me-P103 1000 ⁇ M (converted to ⁇ -CD concentration)
  • an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules is produced by a production method including a step of reacting an acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide. It was done.
  • an acid-decomposable polyrotaxane compound containing a plurality of methylated ⁇ -cyclodextrin molecules is used to treat or prevent lysosomal diseases such as Niemann-Pick disease type C caused by excessive accumulation of cholesterol in lysosomes, It can be useful in the treatment of cancer, particularly cancer that is resistant to apoptosis.

Abstract

The present invention provides an acid-cleavable polyrotaxane compound that includes: a plurality of methylated annular molecules; and a straight-chain molecule that has a terminal group. The acid-cleavable polyrotaxane compound that includes a plurality of methylated annular molecules can be produced by means of a production method that includes a step for reacting an acid-cleavable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide. The acid-cleavable polyrotaxane compound that includes a plurality of methylated annular molecules can be used as a therapeutic agent for diseases such as lysosomal diseases and cancer.

Description

メチル化ポリロタキサンおよびその合成方法Methylated polyrotaxane and method for synthesizing the same 関連出願の相互参照Cross-reference of related applications
　本願は、特願２０１６－９０１２１号（出願日：２０１６年４月２８日）の優先権の利益を享受する出願であり、これは引用することによりその全体が本明細書に取り込まれる。 This application is an application that enjoys the benefit of the priority of Japanese Patent Application No. 2016-90121 (filing date: April 28, 2016), which is incorporated herein by reference in its entirety.
　本発明は、メチル化された複数の環状分子を有する、酸分解性のポリロタキサン化合物、およびその合成方法に関する。より詳細には、メチル化された複数のβ－シクロデキストリンを有する、酸分解性のポリロタキサン化合物、およびその合成方法に関する。また、メチル化された複数の環状分子を有する、酸分解性のポリロタキサン化合物のオートファジー誘導剤としての使用、および疾患の治療薬としての使用にも関する。 The present invention relates to an acid-decomposable polyrotaxane compound having a plurality of methylated cyclic molecules and a synthesis method thereof. More specifically, the present invention relates to an acid-decomposable polyrotaxane compound having a plurality of methylated β-cyclodextrins, and a method for synthesizing the same. The present invention also relates to the use of an acid-decomposable polyrotaxane compound having a plurality of methylated cyclic molecules as an autophagy-inducing agent and as a therapeutic agent for diseases.
　ポリロタキサンは、軸となる直鎖状の分子（主軸とも言う）が複数の大環状分子（リング）を貫通した構造を有する化合物であり、軸の両末端に嵩高い部位を結合させることで、立体障害により大環状分子が軸から抜けなくなっている。両末端の嵩高い部位はストッパーまたはキャップ、末端基と呼ばれる。ストッパーがない場合や、ストッパーがあっても嵩高さが不十分な場合は、リングと軸が分かれることがあり、擬ロタキサンと呼ばれる。 A polyrotaxane is a compound having a structure in which a linear molecule (also referred to as the main axis) serving as an axis penetrates a plurality of macrocyclic molecules (rings). The macrocycle cannot be removed from the shaft due to an obstacle. The bulky sites at both ends are called stoppers or caps and end groups. When there is no stopper or when the bulk is insufficient even if there is a stopper, the ring and the shaft may be separated, which is called pseudo-rotaxane.
　国際公開パンフレットＷＯ２０１５／０２５８１５（特許文献１）には、β－シクロデキストリンを環状分子として含有する酸分解性ポリロタキサンが開示されている。この酸分解性ポリロタキサンは、細胞内環境で分解し、貫通していたβ－シクロデキストリンを細胞内局所で放出することが可能である。従来的な遊離β－シクロデキストリンは主に細胞膜と作用することから、酸分解性ポリロタキサンはβ－シクロデキストリンの作用部位を変化させることが可能な化合物として、疾患治療への応用が検討されている。上記ＷＯ２０１５／０２５８１５では、細胞のリソソームにコレステロールが蓄積するニーマンピック病Ｃ型に対する治療効果が試験され、酸分解性ポリロタキサンは従来のβ－シクロデキストリンよりも低濃度でコレステロールの蓄積を改善することが報告されている。 International Publication Pamphlet WO2015 / 025815 (Patent Document 1) discloses an acid-decomposable polyrotaxane containing β-cyclodextrin as a cyclic molecule. This acid-decomposable polyrotaxane can be decomposed in the intracellular environment and release the penetrated β-cyclodextrin locally in the cell. Since conventional free β-cyclodextrin mainly acts on cell membranes, acid-degradable polyrotaxane is being studied as a compound capable of changing the site of action of β-cyclodextrin and applied to disease treatment. . In the above WO2015 / 025815, the therapeutic effect on Niemann-Pick disease type C in which cholesterol accumulates in lysosomes of cells is tested, and acid-degradable polyrotaxane can improve cholesterol accumulation at a lower concentration than conventional β-cyclodextrin. It has been reported.
　β－シクロデキストリンによる物質包接作用は、β－シクロデキストリンへの化学修飾によって変化することが知られている。様々なβ－シクロデキストリン誘導体の中でも、水酸基をメチル化した誘導体（メチル化β－シクロデキストリン）はコレステロールやアダマンタン等、種々の化合物との結合定数（錯安定度定数）が最も高いことが知られている（非特許文献１）。よって、メチル化β－シクロデキストリンを環状分子として有する酸分解性ポリロタキサンは上記ニーマンピック病Ｃ型に対する治療効果を高めることや、新たな細胞機能を誘導することが期待されるものの、メチル化β-シクロデキストリンを含有する酸分解性ポリロタキサンの合成は、これまで達成されていなかった。 It is known that the substance inclusion action by β-cyclodextrin is changed by chemical modification to β-cyclodextrin. Among various β-cyclodextrin derivatives, derivatives obtained by methylating hydroxyl groups (methylated β-cyclodextrin) are known to have the highest binding constants (complex stability constants) with various compounds such as cholesterol and adamantane. (Non-Patent Document 1). Therefore, although an acid-decomposable polyrotaxane having methylated β-cyclodextrin as a cyclic molecule is expected to enhance the therapeutic effect on the above-mentioned Niemann-Pick disease type C and induce a new cell function, The synthesis of acid-decomposable polyrotaxanes containing cyclodextrins has not been achieved so far.
国際公開第２０１５／０２５８１５号International Publication No. 2015/025815
　本発明は、メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物を提供することを目的の一つとする。また、本発明は、メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物の合成方法を提供することを目的の一つとする。さらに、本発明は、メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物を含有する、疾患の治療剤を提供することを別の目的の一つとする。 An object of the present invention is to provide an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules. Another object of the present invention is to provide a method for synthesizing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules. Another object of the present invention is to provide a therapeutic agent for diseases containing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules.
　本発明の態様は以下の事項に関する。
［１］メチル化された複数の環状分子と、末端基を有する直鎖状分子とを含む、酸分解性のポリロタキサン化合物。
［２］細胞内の酸性環境下で分解する、［１］記載のポリロタキサン化合物。
［３］ｐＨ４．０～６．０の酸性環境下で分解する、［１］または［２］記載のポリロタキサン化合物。
［４］環状分子がβ－シクロデキストリンである、［１］～［３］のいずれか一項記載のポリロタキサン化合物。
［５］β－シクロデキストリンの水酸基がメチル化されている、［４］記載のポリロタキサン化合物。
［６］β－シクロデキストリンが１分子あたり６～２１個のメチル基を有する、［４］または［５］記載のポリロタキサン化合物。
［７］直鎖状分子がポリエチレングリコール（ＰＥＧ）および／またはポリプロピレングリコール（ＰＰＧ）を含む、［１］～［６］のいずれか一項記載のポリロタキサン化合物。
［８］直鎖状分子がポロキサマーを含む、［１］～［７］のいずれか一項記載のポリロタキサン化合物。
［９］直鎖状分子の分子量が４０００～７０００である、［１］～［８］のいずれか一項記載のポリロタキサン化合物。
［１０］直鎖状分子と環状分子との分子数の比率が１：１０～１：１５であることを特徴とする、［１］～［９］のいずれか一項記載のポリロタキサン化合物。
［１１］末端基が、置換基を有する又は有さないＯ－トリフェニルメチル基、置換基を有する又は有さないＳ－トリフェニルメチル基、および置換基を有する又は有さないＮ－トリフェニルメチル基から成る群より選択される、［１］～［１０］のいずれか一項記載のポリロタキサン化合物。
［１２］末端基が直鎖状分子にペプチド結合、カーバメート結合、エステル結合、またはエーテル結合を介して連結されている、［１］～［１１］のいずれか一項記載のポリロタキサン化合物。
［１３］メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物の製造方法であって、粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程を含む、製造方法。
［１４］メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物の製造方法であって、ポロキサマーの両末端に一級アミノ基を有するポロキサマーを得る工程、前記ポロキサマーとβ－シクロデキストリンとを反応させて擬ポリロタキサンを得る工程、前記擬ポリロタキサンの両末端をＮ－トリチルグリシンでキャッピングする工程、および粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程を含む、製造方法。
［１５］メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞においてオートファジーを誘導するための組成物。
［１６］メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、癌を治療するための医薬組成物。
［１７］癌がアポトーシス耐性の癌である、［１６］記載の医薬組成物。
［１８］メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞内のコレステロール蓄積に起因する疾患またはオートファジーの機能障害に起因する疾患を治療または予防するための医薬組成物。
［１９］メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物。
［２０］メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞においてオートファジーを誘導するための方法。
［２１］対象に対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、癌を治療するための方法。
［２２］癌がアポトーシス耐性の癌である、［２１］記載の方法。
［２３］対象に対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、細胞内のコレステロール蓄積に起因する疾患またはオートファジーの機能障害に起因する疾患を治療または予防するための方法。
［２４］対象に対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための方法。
［２５］細胞においてオートファジーを誘導するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用。
［２６］癌を治療するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用。
［２７］癌がアポトーシス耐性の癌である、［２６］記載の使用。
［２８］細胞内のコレステロール蓄積に起因する疾患またはオートファジーの機能障害に起因する疾患を治療または予防するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用。
［２９］ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用。
［３０］環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物。
［３１］環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物を含有する、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物。 Aspects of the present invention relate to the following items.
[1] An acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclic molecules and a linear molecule having a terminal group.
[2] The polyrotaxane compound according to [1], which degrades in an intracellular acidic environment.
[3] The polyrotaxane compound according to [1] or [2], which decomposes in an acidic environment at pH 4.0 to 6.0.
[4] The polyrotaxane compound according to any one of [1] to [3], wherein the cyclic molecule is β-cyclodextrin.
[5] The polyrotaxane compound according to [4], wherein the hydroxyl group of β-cyclodextrin is methylated.
[6] The polyrotaxane compound according to [4] or [5], wherein β-cyclodextrin has 6 to 21 methyl groups per molecule.
[7] The polyrotaxane compound according to any one of [1] to [6], wherein the linear molecule includes polyethylene glycol (PEG) and / or polypropylene glycol (PPG).
[8] The polyrotaxane compound according to any one of [1] to [7], wherein the linear molecule includes a poloxamer.
[9] The polyrotaxane compound according to any one of [1] to [8], wherein the linear molecule has a molecular weight of 4000 to 7000.
[10] The polyrotaxane compound according to any one of [1] to [9], wherein the ratio of the number of molecules of linear molecules to cyclic molecules is 1:10 to 1:15.
[11] O-triphenylmethyl group with or without substituents at the terminal group, S-triphenylmethyl group with or without substituents, and N-triphenyl with or without substituents The polyrotaxane compound according to any one of [1] to [10], which is selected from the group consisting of methyl groups.
[12] The polyrotaxane compound according to any one of [1] to [11], wherein the terminal group is linked to the linear molecule via a peptide bond, carbamate bond, ester bond, or ether bond.
[13] A method for producing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules, comprising the step of reacting an acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide ,Production method.
[14] A method for producing an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclic molecules, the step of obtaining a poloxamer having primary amino groups at both ends of the poloxamer, the poloxamer and β-cyclodextrin; To obtain a pseudopolyrotaxane, capping both ends of the pseudopolyrotaxane with N-tritylglycine, and reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide. ,Production method.
[15] A composition for inducing autophagy in a cell, comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
[16] A pharmaceutical composition for treating cancer, comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
[17] The pharmaceutical composition according to [16], wherein the cancer is a cancer resistant to apoptosis.
[18] To treat or prevent a disease caused by intracellular cholesterol accumulation or a disease caused by dysfunction of autophagy, comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules Pharmaceutical composition.
[19] A pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC) comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
[20] A method for inducing autophagy in a cell, comprising an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
[21] A method for treating cancer, comprising administering to a subject an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
[22] The method of [21], wherein the cancer is a cancer resistant to apoptosis.
[23] Due to a disease caused by intracellular cholesterol accumulation or autophagy dysfunction comprising a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject A method for treating or preventing a disease.
[24] A method for treating or preventing Niemann-Pick type C (NPC) comprising administering to a subject an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules.
[25] Use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for inducing autophagy in a cell.
[26] Use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating cancer.
[27] The use according to [26], wherein the cancer is a cancer resistant to apoptosis.
[28] Acid-degradable comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing a disease caused by intracellular cholesterol accumulation or a disease caused by autophagy dysfunction Use of polyrotaxane compounds.
[29] Use of an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing Niemann-Pick disease type C (NPC).
[30] An acid-decomposable polyrotaxane compound comprising β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule.
[31] Niemann-Pick disease C containing an acid-decomposable polyrotaxane compound containing β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having a terminal N-triphenylmethyl group as a linear molecule. A pharmaceutical composition for treating or preventing a type (NPC).
　本発明の一態様によると、メチル化された複数の環状分子と、末端基を有する直鎖状分子とを含む、酸分解性のポリロタキサン化合物が提供される。このようなポリロタキサン化合物は、酸性環境下で分解し、メチル化された複数の環状分子を放出することができる。 According to one embodiment of the present invention, an acid-decomposable polyrotaxane compound including a plurality of methylated cyclic molecules and a linear molecule having a terminal group is provided. Such a polyrotaxane compound can be decomposed in an acidic environment to release a plurality of methylated cyclic molecules.
ポリロタキサンのβ－シクロデキストリンにメチル基を導入するための反応スキームを示した図である。FIG. 3 shows a reaction scheme for introducing a methyl group into β-cyclodextrin of polyrotaxane. サイズクロマトグラフィーの結果を示した図である。ＰＲＸおよびＭｅ－ＰＲＸは溶出時間がβ－ＣＤよりも早く、高分子量化していることから、合成が確認される。It is the figure which showed the result of size chromatography. Since PRX and Me-PRX have a higher elution time than β-CD and have a higher molecular weight, synthesis is confirmed. Ｍｅ－ＰＲＸのプロトン核磁気共鳴スペクトルの結果を示した図である。It is the figure which showed the result of the proton nuclear magnetic resonance spectrum of Me-PRX. ＨＥＥ－ＰＲＸのサイズクロマトグラフィーの結果を示した図である。It is the figure which showed the result of the size chromatography of HEE-PRX. Ｍｅ－ＰＲＸのプロトン核磁気共鳴スペクトルの結果を示した図である。It is the figure which showed the result of the proton nuclear magnetic resonance spectrum of Me-PRX. Ｍｅ－ＰＲＸのＮ－トリチル基脱離率を示した図である。FIG. 3 is a graph showing the N-trityl group elimination rate of Me-PRX. Ｍｅ－ＰＲＸのサイズクロマトグラフィーの結果を示した図である。Ｎ－トリチル基の脱離率に応じて１２．２Ｍｅ－Ｐ１０３に由来したピークが減少し、β－ＣＤ由来のピークが増加した。It is the figure which showed the result of the size chromatography of Me-PRX. Depending on the elimination rate of the N-trityl group, the peak derived from 12.2Me-P103 decreased and the peak derived from β-CD increased. ＨＥＥ－ＰＲＸのＮ－トリチル基脱離率を示した図である。FIG. 3 is a diagram showing the N-trityl group elimination rate of HEE-PRX. ＨＥＥ－ＰＲＸのサイズ排除クロマトグラフィー分析の結果を示した図である。Ｎ－トリチル基の脱離率に応じてＨＥＥ－ＰＲＸに由来したピークが減少し、β－ＣＤ由来のピークが増加した。It is the figure which showed the result of the size exclusion chromatography analysis of HEE-PRX. Depending on the elimination rate of the N-trityl group, the peak derived from HEE-PRX decreased and the peak derived from β-CD increased. さまざまな濃度の各種Ｍｅ－ＰＲＸの６００ｎｍにおける透過率の測定結果を示した図である。It is the figure which showed the measurement result of the transmittance | permeability in 600 nm of various Me-PRX of various density | concentrations. ３－メチルアデニン（３－ＭＡ）の存在下および非存在下においてＤＭ－β－ＣＤまたはＭｅ－ＰＲＸで処置した細胞中のＧＦＰ陽性オートファゴソームを示した写真（左）と、３－メチルアデニン（３－ＭＡ）の存在下および非存在下においてＤＭ－β－ＣＤまたはＭｅ－ＰＲＸで処置した細胞中のＧＦＰ陽性のオートファゴソーム数を計測した結果を示すグラフ（右）である。A photograph (left) showing GFP-positive autophagosomes in cells treated with DM-β-CD or Me-PRX in the presence and absence of 3-methyladenine (3-MA), and 3-methyladenine ( 3 is a graph (right) showing the results of counting the number of GFP-positive autophagosomes in cells treated with DM-β-CD or Me-PRX in the presence and absence of 3-MA). Ｍｅ－Ｐ１０３、ＨＥＥ－ＰＲＸ、およびＭｅ－β－ＣＤで処置したＨｅＬａ細胞の細胞生存率を示したグラフである。FIG. 6 is a graph showing the cell viability of HeLa cells treated with Me-P103, HEE-PRX, and Me-β-CD. オートファジー阻害剤である３－メチルアデニンの存在下で細胞をＭｅ－ＰＲＸとＭｅ－β－ＣＤで処置した場合の細胞生存率を示したグラフである。It is a graph showing the cell viability when cells were treated with Me-PRX and Me-β-CD in the presence of 3-methyladenine, an autophagy inhibitor. Ｃａｓｐａｓｅ阻害剤であるｚＶＡＤ－ＦＭＫの存在下で、Ｂａｘ／Ｂａｋ　ＤＫＯ　ＭＥＦ細胞をＭｅ－ＰＲＸ、Ｍｅ－β－ＣＤ、またはＤＭ－β－ＣＤにより処置した場合の細胞の写真（左）と、細胞生存率を示したグラフ（右）である。Photographs of cells (left) of Bax / Bak DKO MEF cells treated with Me-PRX, Me-β-CD, or DM-β-CD in the presence of the caspase inhibitor zVAD-FMK, and cells It is the graph (right) which showed survival rate. ＨＰ－β－ＣＤまたはＰＲＸで処置したニーマン・ピック病Ｃ型患者由来皮膚繊維芽細胞におけるコレステロールの局在を観察した結果を示す写真である。It is a photograph showing the result of observing the localization of cholesterol in Nermann-Pick disease type C patient-derived dermal fibroblasts treated with HP-β-CD or PRX.
　以下に、本発明を詳細に説明する。本発明者らは、粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させることにより、メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物を製造した。 Hereinafter, the present invention will be described in detail. The present inventors produced an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules by reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide.
ポリロタキサン（ＰＲＸ）化合物
　ロタキサンは、大環状分子を直鎖状分子が貫通し、直鎖状分子の両末端に嵩高い部位を結合させることで、立体障害でリングが軸から抜けなくなったものである。ポリロタキサンでは、複数の大環状分子の環内を１本の直鎖状分子が貫いている。 Polyrotaxane (PRX) compound Rotaxane is a macromolecule in which a linear molecule penetrates and a bulky part is bonded to both ends of the linear molecule so that the ring cannot be removed from the shaft due to steric hindrance. . In polyrotaxane, one linear molecule penetrates the ring of a plurality of macrocyclic molecules.
　本発明において用いられる直鎖状分子や環状分子は特に限定されないが、直鎖状分子としては、ポリエチレングリコール、ポリプロピレングリコール、ポリエチレングリコールとポリプロピレングリコールとの共重合体（ポロキサマー）、ポリエチレンイミン、ポリアミノ酸、及びポリメチルビニルエーテルからなる群より選ばれる一種又は二種以上であることが好ましい。また、直鎖状分子の平均分子量は１０００～２００００、特に２０００～１００００あるいは４０００～７０００であることが好ましく、例えば、分子量およそ５０００のポロキサマーが使用されうる。 The linear molecule and cyclic molecule used in the present invention are not particularly limited, and examples of the linear molecule include polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol (poloxamer), polyethylene imine, and polyamino acid. And one or more selected from the group consisting of polymethyl vinyl ether. The average molecular weight of the linear molecule is preferably 1000 to 20000, particularly 2000 to 10,000, or 4000 to 7000. For example, a poloxamer having a molecular weight of about 5000 can be used.
　環状分子としては、α、β又はγ－シクロデキストリンであることが好ましいが、これと類似の環状構造を持つものであってもよく、そのような環状構造としては環状ポリエーテル、環状ポリエステル、環状ポリエーテルアミン、環状ポリアミン等が挙げられる。コレステロール包接能の観点から好ましい環状分子は、β－またはγ－シクロデキストリンであり、β－シクロデキストリンが特に好ましい。 The cyclic molecule is preferably α, β or γ-cyclodextrin, but may have a similar cyclic structure, such as cyclic polyether, cyclic polyester, cyclic Examples include polyether amines and cyclic polyamines. A preferable cyclic molecule from the viewpoint of cholesterol inclusion ability is β- or γ-cyclodextrin, and β-cyclodextrin is particularly preferable.
　本発明に係るポリロタキサンに含まれる環状分子はメチル基を有する。環状分子は、好ましくはβ－シクロデキストリンであり、β－シクロデキストリンの水酸基がメチル化されている。β－シクロデキストリンは、例えば、１分子あたり１～２１個、好ましくは６～２１個のメチル基を有する。環状分子は、メチル基以外の置換基を有していても良い。他の置換基としては、例えば、２－ヒドロキシエトキシエチル（ＨＥＥ）基、ヒドロキシエチル基、ヒドロキシプロピル基、ヒドロキシブチル基、ヒドロキシエトキシエチル基、Ｎ，Ｎ－ジメチルアミノエチル基（ＤＭＡＥ基と称することもある）、カルボキシル基、一級アミノ基、若しくはポリエチレングリコールなどの水溶性高分子や、トランスフェリンなどのタンパク質分子、オリゴアルギニンなどのペプチド分子などが挙げられる。これらの基は、環状分子に直接結合していても、リンカーを介して結合していても良い。リンカーとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、カルバミン酸エステル結合（－Ｏ－ＣＯ－ＮＨ－）、エステル結合（－Ｏ－ＣＯ－）、カルボネート結合（－Ｏ－ＣＯ－Ｏ－）、エーテル結合（－Ｏ－）などが挙げられる。 The cyclic molecule contained in the polyrotaxane according to the present invention has a methyl group. The cyclic molecule is preferably β-cyclodextrin, and the hydroxyl group of β-cyclodextrin is methylated. β-cyclodextrin has, for example, 1 to 21, preferably 6 to 21 methyl groups per molecule. The cyclic molecule may have a substituent other than a methyl group. Examples of other substituents include 2-hydroxyethoxyethyl (HEE) group, hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, hydroxyethoxyethyl group, N, N-dimethylaminoethyl group (referred to as DMAE group). And water-soluble polymers such as carboxyl groups, primary amino groups, or polyethylene glycol, protein molecules such as transferrin, and peptide molecules such as oligoarginine. These groups may be directly bonded to the cyclic molecule or may be bonded via a linker. The linker is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the linker may be a carbamate ester bond (—O—CO—NH—), an ester bond (—O—CO—), a carbonate bond (— O-CO-O-), ether bond (-O-) and the like.
　直鎖状分子と環状分子の組み合わせとしては、β－シクロデキストリンとポロキサマーとの組合せが好ましい。なお、β－シクロデキストリンとポロキサマーとの組合せによるポリロタキサンの合成は、上記の特許文献１にも開示されており、その内容は参照により本明細書にも取り込まれる。好ましい直鎖状分子の分子数と環状分子の分子数との比率は１：４～１：５０であり、１：８～１：２０の比率がより好ましく、例えば、１：１０～１：１５の比率が用いられる。すなわち、好ましくは直鎖状分子１分子に４～５０個の環状分子が含まれ、より好ましくは８～２０個の環状分子が含まれ、例えば、１０～１５個の環状分子が含まれうる。 As a combination of a linear molecule and a cyclic molecule, a combination of β-cyclodextrin and poloxamer is preferable. The synthesis of polyrotaxane by a combination of β-cyclodextrin and poloxamer is also disclosed in Patent Document 1 described above, the contents of which are also incorporated herein by reference. The ratio of the number of molecules of linear molecules to the number of molecules of cyclic molecules is 1: 4 to 1:50, more preferably 1: 8 to 1:20, for example, 1:10 to 1:15. The ratio is used. That is, preferably 4 to 50 cyclic molecules are included in one molecule of the linear molecule, more preferably 8 to 20 cyclic molecules, for example, 10 to 15 cyclic molecules may be included.
　本発明において用いられる末端基（嵩高い置換基とも言う）としては、例えば、Ｏ－トリフェニルメチル（Ｏ－Ｔｒｔ）基、Ｓ－トリフェニルメチル（Ｓ－Ｔｒｔ）基、Ｎ－トリフェニルメチル（Ｎ－Ｔｒｔ）基が挙げられるが、限定はされない。末端基は、置換基を有するＯ－トリフェニルメチル基、Ｓ－トリフェニルメチル基、Ｎ－トリフェニルメチル基などであってもよい。好ましくは、Ｎ－トリフェニルメチル基が用いられる。Ｎ－Ｔｒｔ基は、弱酸性環境下において分解され、ポリロタキサン骨格が崩壊し、β－ＣＤなどの環状分子がリリースされる。末端基は、ペプチド結合、カーバメート結合、エステル結合、エーテル結合などを介して直鎖状分子に連結されうるが、好ましくはペプチド結合が用いられる。よって、本発明の一態様は、環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物に関する。β－シクロデキストリンが有する置換基は、好ましくはメチル基であるが、限定はされず、例えば、２－ヒドロキシエトキシエチル（ＨＥＥ）基、ヒドロキシエチル基、ヒドロキシプロピル基、ヒドロキシブチル基、ヒドロキシエトキシエチル基、Ｎ，Ｎ－ジメチルアミノエチル基（ＤＭＡＥ基と称することもある）、カルボキシル基、一級アミノ基、若しくはポリエチレングリコールなどの水溶性高分子や、トランスフェリンなどのタンパク質分子、オリゴアルギニンなどのペプチド分子などであってもよい。これらの基は、環状分子に直接結合していても、リンカーを介して結合していても良い。リンカーとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、カルバミン酸エステル結合（－Ｏ－ＣＯ－ＮＨ－）、エステル結合（－Ｏ－ＣＯ－）、カルボネート結合（－Ｏ－ＣＯ－Ｏ－）、エーテル結合（－Ｏ－）などが挙げられる。 Examples of terminal groups (also referred to as bulky substituents) used in the present invention include O-triphenylmethyl (O-Trt) group, S-triphenylmethyl (S-Trt) group, N-triphenylmethyl ( N-Trt) group, but not limited thereto. The terminal group may be a substituted O-triphenylmethyl group, S-triphenylmethyl group, N-triphenylmethyl group or the like. Preferably, an N-triphenylmethyl group is used. The N-Trt group is decomposed in a weakly acidic environment, the polyrotaxane skeleton is destroyed, and a cyclic molecule such as β-CD is released. The end group can be linked to the linear molecule via a peptide bond, a carbamate bond, an ester bond, an ether bond or the like, but preferably a peptide bond is used. Therefore, one embodiment of the present invention relates to an acid-decomposable polyrotaxane compound containing β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule. The substituent that β-cyclodextrin has is preferably a methyl group, but is not limited, and examples thereof include 2-hydroxyethoxyethyl (HEE) group, hydroxyethyl group, hydroxypropyl group, hydroxybutyl group, hydroxyethoxyethyl. Group, N, N-dimethylaminoethyl group (sometimes referred to as DMAE group), carboxyl group, primary amino group, or water-soluble polymer such as polyethylene glycol, protein molecule such as transferrin, peptide molecule such as oligoarginine It may be. These groups may be directly bonded to the cyclic molecule or may be bonded via a linker. The linker is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the linker may be a carbamate ester bond (—O—CO—NH—), an ester bond (—O—CO—), a carbonate bond (— O-CO-O-), ether bond (-O-) and the like.
　本発明に係るポリロタキサン化合物は、酸分解性の結合を介して末端基が直鎖状分子に連結された構造を有していても良い。酸分解性の結合としては、例えば、アセタール結合、ケタール結合、ジスルフィド結合、エステル結合、オルトエステル結合、ビニルエーテル結合、ヒドラジド結合、アミド結合などが挙げられるが、限定はされない。これらは、１種単独で使用してもよいし、２種以上を併用してもよい。末端基としては、例えば、Ｎ－トリチルグリシン、１以上のベンゼン環を有する基、１以上の第三ブチルを有する基等が使用されうるが、これらに限定はされない。１以上のベンゼン環を有する基としては、例えば、ベンジルオキシカルボニル（Ｚ）基、９－フレオレニルメチルオキシカルボニル（Ｆｍｏｃ）基、ベンジルエステル（ＯＢｚ）基等が挙げられ、また、１以上の第三ブチルを有する基としては、第三ブチルカルボニル（Ｂｏｃ）基、アミノ酸第三ブチルエステル（ＯＢｕ基）等が挙げられる。なお、酸分解性の結合と末端基とは、直接的に連結されている必要はなく、当業者に公知のリンカー部分を介して連結されていてもよい。 The polyrotaxane compound according to the present invention may have a structure in which a terminal group is linked to a linear molecule via an acid-decomposable bond. Examples of the acid-decomposable bond include, but are not limited to, an acetal bond, a ketal bond, a disulfide bond, an ester bond, an orthoester bond, a vinyl ether bond, a hydrazide bond, and an amide bond. These may be used individually by 1 type and may use 2 or more types together. As the terminal group, for example, N-tritylglycine, a group having one or more benzene rings, a group having one or more tert-butyls, and the like can be used, but are not limited thereto. Examples of the group having one or more benzene rings include a benzyloxycarbonyl (Z) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyl ester (OBz) group, and the like. Examples of the group having tertiary butyl include tertiary butylcarbonyl (Boc) group and amino acid tertiary butyl ester (OBu group). The acid-decomposable bond and the end group do not need to be directly linked, and may be linked via a linker moiety known to those skilled in the art.
　本発明に係るポリロタキサンの数平均分子量としては、特に制限はなく、目的に応じて適宜選択することができるが、１万～１０万程度とすることが好ましい。 The number average molecular weight of the polyrotaxane according to the present invention is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably about 10,000 to 100,000.
　本発明に係るポリロタキサン化合物としては、直鎖状分子がポリエチレングリコール（ＰＥＧ）およびポリプロピレングリコール（ＰＰＧ）を含むポロキサマーであり、環状分子がβ－シクロデキストリンであり、末端基がＮ－トリフェニルメチル基であり、末端基が直鎖状分子にペプチド結合を介して連結されていることが特に好ましい。この場合、ポロキサマーの分子量は４０００～７０００（例えば、約５０００）であり、直鎖状分子１分子あたりのβ－シクロデキストリンの数は１０～１５個（例えば、約１２個）でありうる。よって、本発明に係るポリロタキサン化合物は、以下に示す化学構造を有するポリロタキサンを含む：
Figure JPOXMLDOC01-appb-C000003
  

ここで、ｍは、ポロキサマー中のポリプロピレングリコールの繰返し単位の数を示す整数である（ここでは、括弧内にポリプロピレングリコールの繰返し単位を３つ記載しているため、「ｍ／３」と記載しているが、ｍが３の倍数である必要は無い）。ｎは、ポリエチレングリコールの繰返し単位の数を示す整数である。ｘは、β－シクロデキストリンの数を示す整数である。Ｒは、置換基を示しており化学結合Ｌを介してβ－シクロデキストリンに結合している。ここでは、β－シクロデキストリンが括弧内の環状構造として示されている。図示の都合上、β－シクロデキストリン上の置換基（例えば、メチル基）は１つのみが示されているが、置換基の数は複数であってもよい。場合によっては化学結合Ｌは介さなくてもよい。Ｚは、キャッピング分子（例えば、Ｎ－トリフェニルメチル基）を示しており化学結合Ｙ（例えば、ペプチド結合）を介して主軸高分子両末端に結合している。場合によっては化学結合Ｙは介さなくてもよい。 As the polyrotaxane compound according to the present invention, the linear molecule is a poloxamer including polyethylene glycol (PEG) and polypropylene glycol (PPG), the cyclic molecule is β-cyclodextrin, and the end group is an N-triphenylmethyl group. It is particularly preferred that the end group is linked to the linear molecule via a peptide bond. In this case, the molecular weight of the poloxamer can be 4000 to 7000 (eg, about 5000), and the number of β-cyclodextrin per linear molecule can be 10 to 15 (eg, about 12). Therefore, the polyrotaxane compound according to the present invention includes a polyrotaxane having the chemical structure shown below:
Figure JPOXMLDOC01-appb-C000003

Here, m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer (here, since 3 repeating units of polypropylene glycol are described in parentheses, “m / 3” is described). M need not be a multiple of 3). n is an integer indicating the number of polyethylene glycol repeating units. x is an integer indicating the number of β-cyclodextrin. R represents a substituent and is bonded to β-cyclodextrin through a chemical bond L. Here, β-cyclodextrin is shown as a cyclic structure in parentheses. For convenience of illustration, only one substituent (for example, a methyl group) on β-cyclodextrin is shown, but the number of substituents may be plural. In some cases, the chemical bond L may not be interposed. Z represents a capping molecule (for example, an N-triphenylmethyl group), and is bonded to both ends of the main polymer through a chemical bond Y (for example, a peptide bond). In some cases, the chemical bond Y may not be interposed.
細胞内環境
　本発明に係るポリロタキサン化合物は、酸性環境下で分解する酸分解性のポリロタキサン化合物であり、例えば、ｐＨ４．０～６．０の酸性環境下で分解する。上述のとおり、末端基としてＮ－Ｔｒｔ基を用いた場合、Ｎ－Ｔｒｔ基は弱酸性環境下において分解され、ポリロタキサン骨格が崩壊し、β－ＣＤなどの環状分子がリリースされる。 Intracellular environment The polyrotaxane compound according to the present invention is an acid-decomposable polyrotaxane compound that decomposes in an acidic environment. For example, it degrades in an acidic environment at pH 4.0 to 6.0. As described above, when an N-Trt group is used as a terminal group, the N-Trt group is decomposed in a weakly acidic environment, the polyrotaxane skeleton is destroyed, and a cyclic molecule such as β-CD is released.
　ヒトを含む真核生物の細胞内には、リソソームや後期エンドソームといった小胞が存在しており、これらの小胞の内腔は酸性化されていることが知られている。例えば、リソソームの内腔のｐＨは５前後である。よって、本発明に係るポリロタキサン化合物は、これらの小胞に取り込まれることにより分解されうる。本発明に係るポリロタキサン化合物は、分解に伴い、β－ＣＤなどの環状分子をリリースする。例えば、リソソーム内でβ－シクロデキストリンが放出された場合、リソソーム内に存在するコレステロールを包接することができ、それにより、リソソーム内のコレステロールの過剰蓄積を原因とするニーマン・ピック病Ｃ型などのライソゾーム病が治療もしくは予防されうる。 It is known that vesicles such as lysosomes and late endosomes exist in eukaryotic cells including humans, and the lumens of these vesicles are acidified. For example, the pH of the lysosomal lumen is around 5. Therefore, the polyrotaxane compound according to the present invention can be decomposed by being taken into these vesicles. The polyrotaxane compound according to the present invention releases a cyclic molecule such as β-CD upon decomposition. For example, when β-cyclodextrin is released in lysosomes, it can include cholesterol present in lysosomes, thereby causing Niemann-Pick disease type C, which is caused by excessive accumulation of cholesterol in lysosomes. Lysosomal disease can be treated or prevented.
　上述のとおり、メチル化されたβ－シクロデキストリンは、コレステロールの包接能が特に高いことが知られている。細胞内のコレステロールをメチル化されたβ－シクロデキストリンが包接することで、細胞内の過剰なコレステロールが引き起こす疾患の治療が可能であることが当業者には理解される。よって、本発明の一態様は、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞内のコレステロール蓄積に起因する疾患を治療するための医薬組成物に関する。また、別の観点からは、本発明の一態様は、対象に対して、好ましくはヒトに対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための方法に関する。さらに、本発明の一態様は、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用にも関する。細胞内のコレステロール蓄積に起因する疾患としては、ライソゾーム病、より具体的には、ゴーシェ病（Ｇａｕｃｈｅｒ病）、ニーマン・ピック病Ａ型（Ｎｉｅｍａｎｎ－Ｐｉｃｋ病Ａ型）、ニーマン・ピック病Ｂ型（Ｎｉｅｍａｎｎ－Ｐｉｃｋ病Ｂ型）、ニーマン・ピック病Ｃ型（Ｎｉｅｍａｎｎ－Ｐｉｃｋ病Ｃ型）、ＧＭ１ガングリオシドーシス、ＧＭ２ガングリオシドーシス（「Ｔａｙ－Ｓａｃｈｓ　Ｓａｎｄｈｏｆｆ　ＡＢ型」と称することもある。）、クラッベ病（Ｋｒａｂｂｅ病）、異染性白質変性症、マルチプルサルタファーゼ欠損症（Ｍｕｌｔｉｐｌｅ　ｓｕｌｆａｔｅｓｅ欠損症）、ファーバー病（Ｆａｒｂｅｒ病）、ムコ多糖症Ｉ型、ムコ多糖症ＩＩ型（「ハンター病」と称することもある。）、ムコ多糖症ＩＩＩ型（「サンフィリポ病」と称することもある。）、ムコ多糖症ＩＶ型、ムコ多糖症ＶＩ型（「マロトー・ラミー病」と称することもある。）、ムコ多糖症ＶＩＩ型（「スライ病」と称することもある。）、ムコ多糖症ＩＸ型（「Ｈｙａｌｕｒｏｎｉｄａｓｅ欠損症」と称することもある。）、シアリドーシス、ガラクトシアリドーシス、Ｉ－ｃｅｌｌ病／ムコリピドーシスＩＩＩ型、α－マンノシドーシス、β－マンノシドーシス、フコシドーシス、アスパルチルグルコサミン尿症、シンドラー／神崎病（Ｓｃｈｉｎｄｌｅｒ／神崎病）、ウォルマン病（Ｗｏｌｍａｎ病）、ダノン病（Ｄａｎｏｎ病）、遊離シアル酸蓄積症、セロイドリポフスチノーシス、ファブリー病が挙げられる。なお、前記ライソゾーム病は、オートファゴソームの蓄積を生じる、オートファジー機能異常に起因する疾患でもある。 As described above, methylated β-cyclodextrin is known to have particularly high cholesterol inclusion ability. It is understood by those skilled in the art that inclusion of methylated β-cyclodextrin in intracellular cholesterol enables treatment of diseases caused by excessive intracellular cholesterol. Thus, one embodiment of the present invention relates to a pharmaceutical composition for treating a disease caused by intracellular cholesterol accumulation, comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. From another viewpoint, one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human. The present invention relates to a method for treating or preventing Niemann-Pick disease type C (NPC). Furthermore, one aspect of the present invention is the use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing Neimanpick disease type C (NPC). Also related. Diseases caused by intracellular cholesterol accumulation include lysosomal disease, more specifically Gaucher disease (Gaucher disease), Niemann-Pick disease type A (Niemann-Pick disease type A), Niemann-Pick disease type B ( Niemann-Pick disease type B), Niemann-Pick disease type C (Niemann-Pick disease type C), GM1 gangliosidosis, GM2 gangliosidosis (sometimes referred to as “Tay-Sachs Sandhoff type AB”), Krabbe. Diseases (Krabe's disease), metachromatic leukodystrophy, multiple sulphase deficiency (Multisulfatese deficiency), Faber disease (Farber disease), mucopolysaccharidosis type I, mucopolysaccharidosis type II ), Mucopolysaccharidosis type III ( May be referred to as "Sanfiliposis"), mucopolysaccharidosis type IV, mucopolysaccharidosis type VI (sometimes referred to as "Maloto Lamy disease"), mucopolysaccharidosis type VII (referred to as "Sly disease") Mucopolysaccharidosis type IX (sometimes referred to as “Hyaluronidase deficiency”), sialidosis, galactosialidosis, I-cell disease / mucolipidosis type III, α-mannosidosis, β-mannosid Dorsis, fucosidosis, aspartylglucosamineuria, Schindler / Kanzaki disease (Schindler / Kanzaki disease), Wolman disease (Wolman disease), Danone disease (Danon disease), free sialic acid accumulation disease, ceroid lipofuscinosis, Fabry disease Is mentioned. The lysosomal disease is also a disease caused by autophagy dysfunction causing autophagosome accumulation.
　よって、本発明の一態様は、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物に関する。また、別の観点からは、本発明の一態様は、対象に対して、好ましくはヒトに対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための方法に関する。さらに、本発明の一態様は、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用にも関する。また、本発明の一態様は、上述のように、環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物にも関する。さらに、本発明の一態様は、環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物を含有する、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物にも関する。 Thus, one embodiment of the present invention relates to a pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC) comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. . From another viewpoint, one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human. The present invention relates to a method for treating or preventing Niemann-Pick disease type C (NPC). Furthermore, one aspect of the present invention is the use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing Neimanpick disease type C (NPC). Also related. Further, as described above, one embodiment of the present invention is an acid-decomposable product including β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at a terminal as a linear molecule. Also related to the polyrotaxane compound. Furthermore, one embodiment of the present invention contains an acid-decomposable polyrotaxane compound containing β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule. The present invention also relates to a pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC).
オートファジー
　本発明者らは、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を作用させることにより、細胞にオートファジーを誘導できることを見出した。シクロデキストリンは、好ましくはβ－シクロデキストリンである。よって、本発明の一態様は、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞においてオートファジーを誘導するための組成物に関する。また、別の観点からは、本発明の一態様は、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞においてオートファジーを誘導するための方法に関する。さらに、本発明の一態様は、細胞においてオートファジーを誘導するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用にも関する。 Autophagy The present inventors have found that autophagy can be induced in cells by acting an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. The cyclodextrin is preferably β-cyclodextrin. Accordingly, one aspect of the present invention relates to a composition for inducing autophagy in a cell, comprising an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules. From another point of view, one embodiment of the present invention relates to a method for inducing autophagy in a cell containing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. Furthermore, one aspect of the present invention also relates to the use of an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for inducing autophagy in a cell.
　また、本発明に係るメチル化ポリロタキサンは、細胞にオートファジー性細胞死を誘発しうる。オートファジー性細胞死を利用して、癌細胞に細胞死を誘導できることが当業者には知られている。よって、本発明の一態様は、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、癌を治療するための医薬組成物、好ましくはアポトーシス耐性の癌を治療するための医薬組成物に関する。また、別の観点からは、本発明の一態様は、対象に対して、好ましくはヒトに対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、癌を治療するため、特にアポトーシス耐性の癌を治療するための方法に関する。さらに、本発明の一態様は、癌を治療するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用にも関する。 The methylated polyrotaxane according to the present invention can induce autophagic cell death in cells. It is known to those skilled in the art that autophagic cell death can be used to induce cell death in cancer cells. Thus, one embodiment of the present invention is a pharmaceutical composition for treating cancer, preferably treating apoptosis-resistant cancer, comprising an acid-degradable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. The present invention relates to a pharmaceutical composition. From another viewpoint, one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human. It relates to a method for treating cancer, in particular for treating apoptosis-resistant cancer. Furthermore, one aspect of the present invention also relates to the use of an acid-degradable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating cancer.
　また、本発明に係るメチル化ポリロタキサンは、細胞にオートファジーを誘導することから、オートファジーの機能障害に起因する疾患を治療または予防するために使用されうることが当業者には理解される。よって、本発明の一態様は、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、オートファジーの機能障害に起因する疾患を治療または予防するための医薬組成物に関する。また、別の観点からは、本発明の一態様は、対象に対して、好ましくはヒトに対して、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を投与する工程を含む、オートファジーの機能障害に起因する疾患を治療または予防するための方法に関する。さらに、本発明の一態様は、オートファジーの機能障害に起因する疾患を治療または予防するための薬剤の製造における、メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物の使用にも関する。オートファジーの機能障害に起因する疾患としては、例えば、上述のライソゾーム病や、アルツハイマー病、パーキンソン病、ハンチントン病などの神経変性疾患が挙げられる。 Further, since the methylated polyrotaxane according to the present invention induces autophagy in cells, it will be understood by those skilled in the art that it can be used to treat or prevent diseases caused by autophagy dysfunction. Therefore, one embodiment of the present invention is a pharmaceutical composition for treating or preventing a disease caused by autophagy dysfunction, which comprises an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules. About. From another viewpoint, one embodiment of the present invention includes a step of administering an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules to a subject, preferably to a human. The present invention relates to a method for treating or preventing a disease caused by autophagy dysfunction. Furthermore, one aspect of the present invention is the use of an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclodextrin molecules in the manufacture of a medicament for treating or preventing a disease resulting from autophagy dysfunction. Also related. Examples of diseases caused by autophagy dysfunction include the above-mentioned lysosomal diseases, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.
製造方法
　メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物は、粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程を含む方法により、製造されうる。より具体的には、本発明に係る製造方法の一態様は、ポロキサマーの両末端に一級アミノ基を有するポロキサマーを得る工程（工程Ａ）、前記ポロキサマーとβ－シクロデキストリンとを反応させて擬ポリロタキサンを得る工程（工程Ｂ）、前記擬ポリロタキサンの両末端をＮ－トリチルグリシンでキャッピングする工程（工程Ｃ）、および粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程（工程Ｄ）を含む。 Production Method An acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules can be produced by a method comprising a step of reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide. . More specifically, one embodiment of the production method according to the present invention includes a step of obtaining a poloxamer having primary amino groups at both ends of the poloxamer (step A), and reacting the poloxamer with β-cyclodextrin to produce a pseudopolyrotaxane. (Step B), capping both ends of the pseudopolyrotaxane with N-tritylglycine (step C), and reacting acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide (Step D).
　本発明に係る方法において、粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる際には、脱水ジメチルスルホオキシドに溶解させたポリロタキサンが用いられうる。また、粉末化水酸化ナトリウムの存在下での酸分解性ポリロタキサンとヨウ化メチルとの反応は、３０分から３時間程度、好ましくは約１時間、撹拌することで行うことができる。撹拌は激しく行うことが好ましい。メチル基修飾数は、反応時のヨウ化メチルの当量により制御することが可能である。 In the method according to the present invention, when an acid-decomposable polyrotaxane and methyl iodide are reacted in the presence of powdered sodium hydroxide, a polyrotaxane dissolved in dehydrated dimethyl sulfoxide can be used. The reaction between the acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide can be carried out by stirring for about 30 minutes to 3 hours, preferably about 1 hour. Stirring is preferably performed vigorously. The number of methyl group modifications can be controlled by the equivalent of methyl iodide during the reaction.
医薬組成物
　本発明に係るポリロタキサン化合物は、上記のような疾患の治療または予防に用いる医薬組成物中の有効成分として利用することができる。よって、本発明の一つの態様は、疾患の治療または予防に用いる医薬組成物に関する。本発明に係る医薬組成物中のその他の成分としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、医薬的に許容され得る担体などが挙げられる。担体にも、特に制限はなく、例えば、剤形等に応じて適宜選択することができる。本発明に係る医薬組成物における含有量についても、特に制限はなく、目的に応じて適宜選択することができる。好ましくは、本発明に係る医薬組成物は、体温付近、例えば、３４℃～４２℃、より好ましくは３５℃～３８℃あるいは３７℃において水溶性である。 Pharmaceutical Composition The polyrotaxane compound according to the present invention can be used as an active ingredient in a pharmaceutical composition used for the treatment or prevention of the above-mentioned diseases. Therefore, one embodiment of the present invention relates to a pharmaceutical composition used for treatment or prevention of diseases. The other components in the pharmaceutical composition according to the present invention are not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include pharmaceutically acceptable carriers. There is no restriction | limiting in particular also in a support | carrier, For example, it can select suitably according to a dosage form etc. There is no restriction | limiting in particular also about content in the pharmaceutical composition based on this invention, According to the objective, it can select suitably. Preferably, the pharmaceutical composition according to the present invention is water-soluble at around body temperature, for example, 34 ° C to 42 ° C, more preferably 35 ° C to 38 ° C or 37 ° C.
　本発明に係る医薬組成物の剤形としては、特に制限はなく、所望の投与方法に応じて適宜選択することができ、例えば、注射剤（溶液、懸濁液、用事溶解用固形剤等）、吸入散剤などが挙げられる。注射剤としては、例えば、本発明に係るポリロタキサン化合物に、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤等を添加し、常法により皮下用、筋肉内用、静脈内用等の注射剤を製造することができる。ｐＨ調節剤及び前記緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどが挙げられる。安定化剤としては、例えば、ピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸などが挙げられる。等張化剤としては、例えば、塩化ナトリウム、ブドウ糖などが挙げられる。局所麻酔剤としては、例えば、塩酸プロカイン、塩酸リドカインなどが挙げられる。 There is no restriction | limiting in particular as a dosage form of the pharmaceutical composition based on this invention, According to the desired administration method, it can select suitably, For example, an injection (Solution, suspension, solid agent for use, etc.) And inhaled powders. As an injection, for example, a pH regulator, a buffer, a stabilizer, a tonicity agent, a local anesthetic, etc. are added to the polyrotaxane compound according to the present invention, and subcutaneous, intramuscular, intravenous, etc. are added by a conventional method. An injection for internal use can be produced. Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.
　本発明に係る医薬組成物の投与方法としては、特に制限はなく、例えば、医薬組成物の剤形、患者の状態等に応じて、局所投与、全身投与のいずれかを選択することができる。例えば、局所投与としては、脳室内投与などが挙げられる。 The administration method of the pharmaceutical composition according to the present invention is not particularly limited, and for example, either local administration or systemic administration can be selected according to the dosage form of the pharmaceutical composition, the patient's condition, and the like. For example, local administration includes intracerebroventricular administration.
　本発明に係る医薬組成物の投与対象としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、マウス、ラット、ウシ、ブタ、サル、イヌ、ネコなどが挙げられるが、好ましくはヒトである。 The subject of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys, dogs, cats and the like. However, it is preferably a human.
　本発明に係る医薬組成物の投与量としては、特に制限はなく、投与形態や、投与対象の年齢、体重、所望の効果の程度等に応じて適宜選択することができる。 The dosage of the pharmaceutical composition according to the present invention is not particularly limited, and can be appropriately selected depending on the dosage form, the age and weight of the administration subject, the degree of desired effect, and the like.
　本発明に係る医薬組成物の投与時期としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、上記疾患に感受性の患者に対して予防的に投与されてもよいし、症状を呈する患者に治療的に投与されてもよい。また、投与回数としても、特に制限はなく、投与対象の年齢、体重、所望の効果の程度等に応じて、適宜選択することができる。 The administration time of the pharmaceutical composition according to the present invention is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be administered prophylactically to a patient susceptible to the above-mentioned diseases, It may be administered therapeutically to patients presenting with symptoms. Moreover, there is no restriction | limiting in particular as frequency of administration, According to the age of an administration subject, a body weight, the grade of a desired effect, etc., it can select suitably.
　以下に、実施例を示して本発明を具体的に説明するが、これらにより本発明は何ら制限を受けるものではない。 Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.
＜例１：ポリロタキサンの製造＞
　線状高分子としてプルロニックＰ１０３（ＡＤＥＫＡ社製、商品名：アデカプルロニックＰ－１０３；ポリエチレングリコ－ル（以下、「ＰＥＧ」と称することがある。）とポリプロピレングリコ－ル（以下、「ＰＰＧ」と称することがある。）が、ＰＥＧ－ＰＰＧ－ＰＥＧの順に重合した共重合体；ＰＰＧ部分の数平均分子量は３２５０、ＰＥＧ部分の数平均分子量は８１０×２）を用い、以下のようにして、その両末端に一級アミノ基を結合させた。 <Example 1: Production of polyrotaxane>
Pluronic P103 (manufactured by ADEKA, trade name: Adekapluronic P-103; polyethylene glycol (hereinafter sometimes referred to as “PEG”) and polypropylene glycol (hereinafter referred to as “PPG”) as linear polymers. ) Is a copolymer polymerized in the order of PEG-PPG-PEG; the number average molecular weight of the PPG portion is 3250, the number average molecular weight of the PEG portion is 810 × 2), and is used as follows: A primary amino group was bonded to both ends.
　前記プルロニックＰ１０３を１０ｇ、１，１’－カルボニルジイミダゾ－ル（シグマ－アルドリッチ社製）を１２．８ｇ量り取り、広口瓶へ加えた。テトラヒドロフラン（関東化学社製）を１３５ｍＬ加えて溶解させ、室温で２４時間撹拌した。反応後、超純水を１ｍＬ加え１０分間撹拌した。エチレンジアミン（和光純薬社製）を２．４５ｇ量り取り、ナス型フラスコへ加え、テトラヒドロフランを１０ｍＬ加え溶解させた。プルロニックＰ１０３反応溶液をナス型フラスコへ滴下して加え、室温で２４時間撹拌した。反応後、分画分子量３５００の透析膜（スペクトラム社製）へ加え、メタノール（関東化学社製）に対し透析をすることで未反応物を除去した。ロータリーエバポレーターで濃縮することで両末端に一級アミノ基を有するプルロニックＰ１０３（以下、「Ｐ１０３－ＮＨ２」と称することがある。）を７．２２ｇ得た。 10 g of the above Pluronic P103 and 12.8 g of 1,1′-carbonyldiimidazole (manufactured by Sigma-Aldrich) were weighed and added to a wide-mouthed bottle. 135 mL of tetrahydrofuran (manufactured by Kanto Chemical Co., Inc.) was added and dissolved, and the mixture was stirred at room temperature for 24 hours. After the reaction, 1 mL of ultrapure water was added and stirred for 10 minutes. 2.45 g of ethylenediamine (manufactured by Wako Pure Chemical Industries, Ltd.) was weighed out and added to an eggplant type flask, and 10 mL of tetrahydrofuran was added and dissolved. The Pluronic P103 reaction solution was added dropwise to the eggplant-shaped flask and stirred at room temperature for 24 hours. After the reaction, an unreacted product was removed by dialysis against methanol (manufactured by Kanto Chemical Co., Inc.) in addition to a dialysis membrane (manufactured by Spectrum) having a molecular weight cut off of 3500. By concentrating with a rotary evaporator, 7.22 g of Pluronic P103 having a primary amino group at both ends (hereinafter sometimes referred to as “P103-NH 2 ”) was obtained.
　前記Ｐ１０３－ＮＨ２と、β－シクロデキストリン（以下、「β－ＣＤ」と称することがある。）を用い、以下のようにして、擬ポリロタキサンを調製した。 Using the P103-NH 2 and β-cyclodextrin (hereinafter sometimes referred to as “β-CD”), a pseudopolyrotaxane was prepared as follows.
　前記β－ＣＤを６．８１ｇ量り取り、広口瓶に加え、リン酸緩衝溶液３４０ｍＬに溶解させた。前記Ｐ１０３－ＮＨ２を１ｇ量り取り、少量のメタノールに溶解させた。Ｐ１０３－ＮＨ２溶液をβ－ＣＤ溶液に加え、室温で２４時間撹拌した。反応後、得られた沈殿物を遠心分離により回収した。回収した固体を凍結乾燥することで擬ポリロタキサンを３．８８ｇ得た。 6.81 g of the β-CD was weighed out, added to a wide-mouthed bottle, and dissolved in 340 mL of a phosphate buffer solution. 1 g of P103-NH 2 was weighed and dissolved in a small amount of methanol. P103-NH 2 solution was added to the beta-CD solution was stirred at room temperature for 24 hours. After the reaction, the resulting precipitate was collected by centrifugation. The recovered solid was freeze-dried to obtain 3.88 g of pseudopolyrotaxane.
　前記擬ポリロタキサンの両端部を、以下のようにしてＮ－トリチルグリシン（シグマ－アルドリッチ社製）でキャッピングすることにより、複数のβ－ＣＤを貫通した線状高分子の両端部に酸性ｐＨ環境下で脱離する嵩高い置換基を有するポリロタキサン（以下、「ＰＲＸ」と称することがある。）を得た。 By capping both ends of the pseudopolyrotaxane with N-tritylglycine (manufactured by Sigma-Aldrich) as follows, both ends of a linear polymer penetrating a plurality of β-CDs are subjected to an acidic pH environment. Thus, a polyrotaxane (hereinafter sometimes referred to as “PRX”) having a bulky substituent that is eliminated by the reaction was obtained.
　Ｎ－トリチルグリシンを７６０ｍｇ、Ｎ－Ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｅ（Ａｃｒｏｓ　Ｏｒｇａｎｉｃｓ社製）を２７６ｍｇ、Ｅｔｈｙｌ－３－（３－ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ）ｃａｒｂｏｄｉｉｍｉｄｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ（東京化成工業社製）を５３６ｍｇ量り取り、スクリュー管に加え、脱水Ｎ，Ｎ－ジメチルホルムアミド（和光純薬社製）を６．４６ｍＬ加え、室温で３時間撹拌した。前記擬ポリロタキサンに対し、超純水１７．２ｍＬ、メタノール８．６２ｍＬ、および前記Ｎ－トリチルグリシン反応溶液を加え、室温で２４時間撹拌した。反応後、得られた沈殿物を遠心分離により回収した。メタノール、Ｎ，Ｎ－ジメチルホルムアミド、超純水の順で得られた沈殿物を洗浄し、未反応物を除去した。回収した固体を凍結乾燥することで末端をＮ－トリチルグリシンでキャッピングされたポリロタキサンを１．７９ｇ得た。 760 mg of N-tritylglycine, 276 mg of N-Hydroxysuccinimide (Acros Organics), Ethyl-3- (3-dimethylaminopropyl) carbimide, Hydrochloride (Tokyo Chemical Industry Co., Ltd., N 6.46 mL of N-dimethylformamide (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 3 hours. To the pseudopolyrotaxane, 17.2 mL of ultrapure water, 8.62 mL of methanol, and the N-tritylglycine reaction solution were added and stirred at room temperature for 24 hours. After the reaction, the resulting precipitate was collected by centrifugation. The precipitate obtained in the order of methanol, N, N-dimethylformamide and ultrapure water was washed to remove unreacted substances. The recovered solid was freeze-dried to obtain 1.79 g of polyrotaxane having its end capped with N-tritylglycine.
　前記ポリロタキサンのβ－ＣＤにメチル（以下、「Ｍｅ」と称することがある。）基を以下のようにして導入し（図１）、メチル化ポリロタキサン（以下、「Ｍｅ－ＰＲＸ」と称することがある。）を得た（ポリロタキサン１分子当たりのβ－ＣＤ平均貫通数は１２．８；Ｍｅ基の修飾数は平均１２．２）。メチル化ＰＲＸの一般化学構造を以下に示す：
Figure JPOXMLDOC01-appb-C000004
  

ここで、ｍは、ポロキサマー中のポリプロピレングリコールの繰返し単位の数を示す整数である。ｎは、ポリエチレングリコールの繰返し単位の数を示す整数である。ｘは、β－シクロデキストリンの数を示す整数である。ここでは、β－シクロデキストリンが括弧内の環状構造として示されている。図示の都合上、β－シクロデキストリン上の置換基は１つのみが示されているが、置換基の数は複数であってもよい。 A methyl (hereinafter sometimes referred to as “Me”) group is introduced into β-CD of the polyrotaxane as follows (FIG. 1), and methylated polyrotaxane (hereinafter referred to as “Me-PRX”). (Average β-CD penetration number per molecule of polyrotaxane was 12.8; average number of Me group modifications was 12.2). The general chemical structure of methylated PRX is shown below:
Figure JPOXMLDOC01-appb-C000004

Here, m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer. n is an integer indicating the number of polyethylene glycol repeating units. x is an integer indicating the number of β-cyclodextrin. Here, β-cyclodextrin is shown as a cyclic structure in parentheses. For convenience of illustration, only one substituent on β-cyclodextrin is shown, but the number of substituents may be plural.
　前記ポリロタキサンを３００ｍｇ量り取り、脱水ジメチルスルホキシド（和光純薬社製）１５ｍＬに溶解させた。粉末化した水酸化ナトリウム（和光純薬社製）３２２ｍｇ、ヨウ化メチル（和光純薬社製）１６７μＬを続けて加え、１時間激しく撹拌した。反応後、ジエチルエーテル（昭和エーテル社製）を加え沈殿物を回収し、分画分子量３５００の透析膜へ加え、超純水に対し透析をすることで未反応物を除去した。回収した水溶液を凍結乾燥させることでメチル基を導入した、末端をＮ－トリチルグリシンでキャッピングされたポリロタキサン（Ｍｅ－ＰＲＸ）を２２７ｍｇ得た（図２）。 300 mg of the polyrotaxane was weighed and dissolved in 15 mL of dehydrated dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.). Powdered sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) 322 mg and methyl iodide (manufactured by Wako Pure Chemical Industries, Ltd.) 167 μL were continuously added, and the mixture was vigorously stirred for 1 hour. After the reaction, diethyl ether (manufactured by Showa Ether Co., Ltd.) was added to collect the precipitate, which was added to a dialysis membrane having a molecular weight cut off of 3500 and dialyzed against ultrapure water to remove unreacted substances. The recovered aqueous solution was lyophilized to obtain 227 mg of a polyrotaxane (Me-PRX) having a methyl group introduced and capped with N-tritylglycine (FIG. 2).
　前記Ｍｅ－ＰＲＸについて、溶離液を１０ｍＭ　ＬｉＢｒ含有ジメチルスルホオキシドとしたサイズ排除クロマトグラフィー測定、及び重水（関東化学社製）中で測定した４００ＭＨｚにおけるプロトン核磁気共鳴スペクトル測定を行った。 The Me-PRX was subjected to size exclusion chromatography measurement using 10 mM LiBr-containing dimethylsulfoxide as an eluent, and proton nuclear magnetic resonance spectrum measurement at 400 MHz measured in heavy water (manufactured by Kanto Chemical Co., Inc.).
　図２に示すサイズクロマトグラフィーの結果において、ＰＲＸ、及びＭｅ－ＰＲＸは溶出時間がβ－ＣＤよりも早く高分子量化していることから、合成が確認された。また、遊離β－ＣＤやＮ－トリチル基のピークが見られないことから、Ｍｅ－ＰＲＸは超分子構造を損なうことなく高純度で合成されたことが示された。図３に示すプロトン核磁気共鳴スペクトルの結果から、ポリロタキサンのβ－ＣＤに導入されたＭｅ基の修飾数がＰＲＸのβ－ＣＤ１分子あたり１２．２であることが明らかとなった。 In the result of size chromatography shown in FIG. 2, the synthesis of PRX and Me-PRX was confirmed because the elution time was higher than that of β-CD and the molecular weight was increased. In addition, since no peak of free β-CD or N-trityl group was observed, it was shown that Me-PRX was synthesized with high purity without impairing the supramolecular structure. From the result of the proton nuclear magnetic resonance spectrum shown in FIG. 3, it was revealed that the number of modifications of the Me group introduced into β-CD of polyrotaxane was 12.2 per β-CD molecule of PRX.
　また、プルロニックＰ１０３を主軸高分子としたＭｅ－ＰＲＸの他に、プルロニックＰ１２３（シグマ－アルドリッチ社製；平均分子量５８００）、プルロニックＰ１０５（第一工業製薬社製、商品名：エパンＵ－１０５；平均分子量６５００）、プルロニックＰ８４（ＡＤＥＫＡ社製、商品名：アデカプルロニックＰ－８４；平均分子量４２００）、プルロニックＬ１２１（シグマ－アルドリッチ社製；平均分子量４４００）を主軸高分子としたＭｅ－ＰＲＸを合成した。プロトン核磁気共鳴スペクトルの結果から、算出したβ－ＣＤ貫通数、Ｍｅ基修飾数、及び分子量を以下の表１に示す。
Figure JPOXMLDOC01-appb-T000005
  
In addition to Me-PRX using Pluronic P103 as the main axis polymer, Pluronic P123 (Sigma-Aldrich; average molecular weight 5800), Pluronic P105 (Daiichi Kogyo Seiyaku, trade name: Epan U-105; average) Me-PRX having a molecular weight of 6500), Pluronic P84 (manufactured by ADEKA, trade name: Adeka Pluronic P-84; average molecular weight 4200), Pluronic L121 (manufactured by Sigma-Aldrich; average molecular weight 4400) was synthesized as a main axis polymer. . The calculated β-CD penetration number, Me group modification number, and molecular weight from the results of proton nuclear magnetic resonance spectrum are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000005
　前記Ｎ－トリチルグリシンでキャッピングを行ったポリロタキサンについて、上記のように調製した軸高分子をプルロニックＰ１２３としたポリロタキサンのβ－ＣＤにヒドロキシエトキシエチル（以下「ＨＥＥ」）基を以下の様に導入し、水溶性のポリロタキサン（以下、「ＨＥＥ－ＰＲＸ」）を得た（ポリロタキサン当たりのβ－ＣＤの平均貫通数は１２．７；β－ＣＤ一つ当たりのＨＥＥ基の修飾数は６．５３）。ヒドロキシエトキシエチル化ＰＲＸの一般化学構造を以下に示す：
Figure JPOXMLDOC01-appb-C000006
  

ここで、ｍは、ポロキサマー中のポリプロピレングリコールの繰返し単位の数を示す整数である。ｎは、ポリエチレングリコールの繰返し単位の数を示す整数である。ｘは、β－シクロデキストリンの数を示す整数である。ここでは、β－シクロデキストリンが括弧内の環状構造として示されている。図示の都合上、β－シクロデキストリン上の置換基は１つのみが示されているが、置換基の数は複数であってもよい。 For the polyrotaxane capped with N-tritylglycine, a hydroxyethoxyethyl (hereinafter referred to as “HEE”) group was introduced into the β-CD of the polyrotaxane having the axial polymer prepared as described above as Pluronic P123 as follows. Thus, a water-soluble polyrotaxane (hereinafter referred to as “HEE-PRX”) was obtained (average number of penetrating β-CD per polyrotaxane was 12.7; the number of HEE group modifications per β-CD was 6.53) . The general chemical structure of hydroxyethoxyethylated PRX is shown below:
Figure JPOXMLDOC01-appb-C000006

Here, m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer. n is an integer indicating the number of polyethylene glycol repeating units. x is an integer indicating the number of β-cyclodextrin. Here, β-cyclodextrin is shown as a cyclic structure in parentheses. For convenience of illustration, only one substituent on β-cyclodextrin is shown, but the number of substituents may be plural.
　前記プルロニックＰ１２３を主軸高分子とした末端がＮ－トリチルグリシンでキャッピングされたポリロタキサンを２００ｍｇ量り取り、窒素雰囲気下でＮ，Ｎ－ジメチルホルムアミド１０ｍＬに溶解した。１，１’－カルボニルジイミダゾールを２８２ｍｇ加え、室温で２４時間撹拌した。その後、反応溶液に２－（２－アミノエトキシ）エタノール（東京化成工業社製）を９００μＬ加え、さらに室温で２４時間撹拌した。反応後、分画分子量３，５００の透析膜へ加え、超純水に対し透析をすることで未反応物を除去した。回収した水溶液を凍結乾燥することで、β－ＣＤにヒドロキシエトキシエチル基が導入され、プルロニックの末端がＮ－トリチルグリシンでキャッピングされたポリロタキサン（ＨＥＥ－ＰＲＸ）を得た。 200 mg of polyrotaxane having the terminal pluronic P123 as the main axis polymer and capped with N-tritylglycine was weighed and dissolved in 10 mL of N, N-dimethylformamide under a nitrogen atmosphere. 282 mg of 1,1'-carbonyldiimidazole was added and stirred at room temperature for 24 hours. Thereafter, 900 μL of 2- (2-aminoethoxy) ethanol (Tokyo Chemical Industry Co., Ltd.) was added to the reaction solution, and the mixture was further stirred at room temperature for 24 hours. After the reaction, it was added to a dialysis membrane having a molecular weight cut off of 3,500, and unreacted substances were removed by dialysis against ultrapure water. The recovered aqueous solution was lyophilized to obtain a polyrotaxane (HEE-PRX) in which hydroxyethoxyethyl groups were introduced into β-CD and the pluronic ends were capped with N-tritylglycine.
　前記ＨＥＥ－ＰＲＸについて、溶離液を１０ｍＭ　ＬｉＢｒ含有ジメチルスルホオキシドとしたサイズ排除クロマトグラフフィー測定、及び重水（関東化学社製）中で測定した、４００ＭＨｚにおけるプロトン核磁気共鳴スペクトル測定を行った。 The HEE-PRX was subjected to size exclusion chromatography measurement using 10 mM LiBr-containing dimethylsulfoxide as an eluent, and proton nuclear magnetic resonance spectrum measurement at 400 MHz measured in heavy water (manufactured by Kanto Chemical Co., Inc.).
　図４に示すサイズクロマトグラフィーの結果から、ＰＲＸ、及びＨＥＥ－ＰＲＸは溶出時間がβ－ＣＤよりも早く、高分子量化していることより合成が確認された。また、遊離β－ＣＤやＮ－トリチル基のピークが見られないことから、ＨＥＥ－ＰＲＸは超分子構造を損なうことなく高純度で合成されたことが示された。図５に示すプロトン核磁気共鳴スペクトルの結果から、ポリロタキサンのβ－ＣＤに導入されたＨＥＥ基の修飾数がＰＲＸのβ－ＣＤ１分子あたり６．５３であることが明らかとなった。 From the results of size chromatography shown in FIG. 4, the synthesis of PRX and HEE-PRX was confirmed because the elution time was faster than β-CD and the molecular weight was increased. In addition, since no peak of free β-CD or N-trityl group was observed, it was shown that HEE-PRX was synthesized with high purity without impairing the supramolecular structure. From the result of proton nuclear magnetic resonance spectrum shown in FIG. 5, it was revealed that the number of modifications of the HEE group introduced into β-CD of polyrotaxane was 6.53 per β-CD molecule of PRX.
＜例２：ｐＨ酸分解性＞
　１０ｍｇの１２．２Ｍｅ－Ｐ１０３を２ｍＬの１０ｍＭ酢酸緩衝食塩水（ｐＨ５．０）、または１０ｍＭリン酸緩衝食塩水（ｐＨ７．４）（以下、「ＰＢＳ」と称することがある。）に溶解し、３７℃で所定の時間（１、３、６、１２、２４、４８時間）静置した。時間毎に１００μＬ回収し、５０ｍＭ炭酸緩衝液（ｐＨ９．０）２００μＬ、アセトニトリル（関東化学社製）３００μＬと混合した。０．２２μｍのフィルタ－を通し、ガラスバイアルに加え、高速液体クロマトグラフィー（日本分光社製）測定を行った。カラムとしてＣｏｓｍｏｓｉｌ　５Ｃ１８－ＡＲ－ＩＩ　ｐａｃｋｅｄ　ｃｏｌｕｍｎ及びＣｏｓｍｏｓｉｌ　５Ｃ１８－ＡＲ－ＩＩ　ｇｕａｒｄ　ｃｏｌｕｍｎ（ナカライテスク社製）、溶離液として超純水：アセトニトリル＝２０：８０を使用し、カラム温度４０℃、流速０．８ｍＬ／ｍｉｎにて測定を行い、１２．２Ｍｅ－Ｐ１０３より脱離したＮ－トリチル基の２０５ｎｍにおける吸収強度を求めた。Ｎ－トリチル基が完全に脱離した際の吸収強度を１００％として、以下のようにしてＮ－トリチル基脱離率を算出した。結果を図６に示す。 <Example 2: pH acid decomposability>
10 mg of 12.2Me-P103 is dissolved in 2 mL of 10 mM acetate buffered saline (pH 5.0) or 10 mM phosphate buffered saline (pH 7.4) (hereinafter sometimes referred to as “PBS”). It was allowed to stand at 37 ° C. for a predetermined time (1, 3, 6, 12, 24, 48 hours). 100 μL was collected every hour and mixed with 200 μL of 50 mM carbonate buffer (pH 9.0) and 300 μL of acetonitrile (manufactured by Kanto Chemical Co., Inc.). Through a 0.22 μm filter, in addition to a glass vial, high performance liquid chromatography (manufactured by JASCO Corporation) was measured. Cosmosil 5C18-AR-II packed column and Cosmosil 5C18-AR-II guard column (manufactured by Nacalai Tesque) were used as the column, ultrapure water: acetonitrile = 20: 80 was used as the eluent, the column temperature was 40 ° C., and the flow rate was 0. The measurement was performed at 8 mL / min, and the absorption intensity at 205 nm of the N-trityl group eliminated from 12.2Me-P103 was determined. The absorption intensity when the N-trityl group was completely eliminated was defined as 100%, and the N-trityl group elimination rate was calculated as follows. The results are shown in FIG.
　また、各ｐＨ、時間毎の１２．２Ｍｅ－Ｐ１０３測定溶液を凍結乾燥し、溶離液を１０ｍＭ　ＬｉＢｒ含有ジメチルスルホオキシドとしたサイズ排除クロマトグラフィー分析（東ソー社製）により１２．２Ｍｅ－Ｐ１０３の分解とβ－ＣＤの放出を確認した。結果を図７に示す。 In addition, the 12.2Me-P103 measurement solution at each pH and time was freeze-dried, and size elution chromatography analysis (made by Tosoh Corp.) using 10 mM LiBr-containing dimethylsulfoxide as an eluent was performed to decompose 12.2Me-P103. Release of β-CD was confirmed. The results are shown in FIG.
［試料］
（１）１２．２Ｍｅ－Ｐ１０３（例１で作製）
（２）ＨＥＥ－ＰＲＸ [sample]
(1) 12.2Me-P103 (produced in Example 1)
(2) HEE-PRX
［Ｎ－トリチル基脱離率の算出方法］
脱離率（％）＝｛（各ｐＨ、時間におけるＭｅ－ＰＲＸ溶液の吸収強度）／（Ｎ－トリチル基が完全に脱離した際の吸収強度）｝×１００ [Calculation method of N-trityl group elimination rate]
Desorption rate (%) = {(Absorption intensity of Me-PRX solution at each pH and time) / (Absorption intensity when N-trityl group is completely eliminated)} × 100
　図６の結果から、１２．２Ｍｅ－Ｐ１０３はｐＨ５．０では３時間で６０．９％、２４時間で１００％のＮ－トリチル基が脱離したのに対し、ｐＨ７．４では４８時間後でも脱離率は１０．５％であった。また図７の結果より、Ｎ－トリチル基の脱離率に応じて１２．２Ｍｅ－Ｐ１０３に由来したピークが減少し、β－ＣＤ由来のピークが増加した。以上の結果より、生理ｐＨ条件下ではＭｅ－ＰＲＸは安定であるが、酸性ｐＨ環境下では末端のＮ－トリチルが脱離し、ポリロタキサン構造が崩壊することが明らかとなった。図８の結果から、ＨＥＥ－ＰＲＸはｐＨ５．０では３時間で６６．９％，２４時間で９８．８％のＮ－トリチル基が脱離したのに対し，ｐＨ７．４では４８時間後でも脱離率は７．３３％であった。また図９の結果より、Ｎ－トリチル基の脱離率に応じてＨＥＥ－ＰＲＸに由来したピークが減少し、β－ＣＤ由来のピークが増加した。以上より、ＨＥＥ－ＰＲＸはＭｅ－ＰＲＸと同様に酸性ｐＨ環境下で末端のＮ－トリチルが脱離し、ポリロタキサン構造が崩壊することが明らかとなった。 From the results of FIG. 6, 12.2Me-P103 had 60.9% of N-trityl group eliminated at 3 hours at pH 5.0 and 100% at 24 hours, while 48 hours at pH 7.4. The desorption rate was 10.5%. From the results shown in FIG. 7, the peak derived from 12.2Me-P103 decreased and the peak derived from β-CD increased according to the elimination rate of the N-trityl group. From the above results, it was found that Me-PRX is stable under physiological pH conditions, but terminal N-trityl is eliminated and the polyrotaxane structure is destroyed under an acidic pH environment. From the results shown in FIG. 8, HEE-PRX had 66.9% N-trityl groups eliminated at 3 hours at pH 5.0 and 98.8% at 24 hours, whereas 48 hours at pH 7.4. The desorption rate was 7.33%. From the results shown in FIG. 9, the peak derived from HEE-PRX decreased and the peak derived from β-CD increased according to the elimination rate of the N-trityl group. From the above, it has been clarified that HEE-PRX, like Me-PRX, loses terminal N-trityl under an acidic pH environment and collapses the polyrotaxane structure.
＜例３：温度応答性の評価＞
　各濃度のＭｅ－ＰＲＸ（表１）を１０ｍＭリン酸緩衝液（ｐＨ７．４）１ｍＬに石英セル中で溶解させた。ＵＶ／Ｖｉｓ分光光度計（日本分光社製）を用いて、６００ｎｍにおける透過率測定（昇温温度：１．０℃／ｍｉｎ）を行った。結果を図１０に示す。 <Example 3: Evaluation of temperature response>
Each concentration of Me-PRX (Table 1) was dissolved in 1 mL of 10 mM phosphate buffer (pH 7.4) in a quartz cell. Using a UV / Vis spectrophotometer (manufactured by JASCO Corporation), transmittance measurement at 600 nm (temperature increase temperature: 1.0 ° C./min) was performed. The results are shown in FIG.
　図１０の結果は、主軸高分子の異なる各Ｍｅ－ＰＲＸの透過率が２．５ｍｇ／ｍＬにおいて５０％となる温度（ＬＣＳＴ）は、１３．８Ｍｅ－Ｐ１０５（４６．９℃）＞１４．３Ｍｅ－Ｐ１２３（４０．０℃）＞１２．２Ｍｅ－Ｐ１０３（３９．１℃）＞１５．３Ｍｅ－Ｐ８４（３３．１℃）＞１５．２Ｍｅ－Ｌ１２１（３０．６℃）の順に低下することを示している。各プルロニック（ポロキサマー）は分子鎖に占めるＰＰＧ部分の重量分率が異なるが、ＰＰＧ重量分率が増加するにつれてＬＣＳＴが低下することが明らかとなった。また、メチル基修飾数が異なるプルロニックＰ１２３を軸高分子としたＭｅ－ＰＲＸでは、６．３Ｍｅ－Ｐ１２３では４３．０℃、１４．１Ｍｅ－Ｐ１２３では３６．５℃、２１．０Ｍｅ－Ｐ１２３では３５．９℃であり、メチル基導入数の増加に伴いＬＣＳＴが低下することが明らかとなった。Ｍｅ－ＰＲＸは構成成分により相転移温度を調整することが可能であることが示された。 The result of FIG. 10 shows that the temperature (LCST) at which the transmittance of each Me-PRX having a different main-axis polymer is 50% at 2.5 mg / mL is 13.8 Me-P105 (46.9 ° C.)> 14.3 Me. −P123 (40.0 ° C.)> 12.2 Me-P103 (39.1 ° C.)> 15.3 Me-P84 (33.1 ° C.)> 15.2 Me-L121 (30.6 ° C.) Show. Although each pluronic (poloxamer) has a different weight fraction of the PPG moiety in the molecular chain, it was revealed that the LCST decreases as the PPG weight fraction increases. Further, in Me-PRX using pluronic P123 having different methyl group modification number as the axial polymer, 6.3Me-P123 is 43.0 ° C, 14.1Me-P123 is 36.5 ° C, and 21.0Me-P123 is 35 It was found that the LCST decreased as the number of methyl group introduction increased. It has been shown that Me-PRX can adjust the phase transition temperature by the constituent components.
＜例４：オートファジーの誘導評価＞
　ＧＦＰ－ＬＣ３を安定発現するＨｅＬａ細胞を３５ｍｍガラスボトムディッシュに１．０×１０４ｃｅｌｌｓ／ｄｉｓｈの濃度で播種し、１日インキュベ－ションした。下記試料をβ－シクロデキストリン濃度換算で１ｍＭとなるように培地中に加えた。２４時間後に共焦点顕微鏡観察を行い、細胞中のＧＦＰ陽性のオートファゴソーム数を計測した。また、オートファジー阻害剤である３－メチルアデニン（メルク社製）を１０ｍＭの濃度で培地に添加した状態で前記と同様の実験を行った。結果を図１１に示す。 <Example 4: Induction evaluation of autophagy>
HeLa cells stably expressing GFP-LC3 were seeded in a 35 mm glass bottom dish at a concentration of 1.0 × 10 4 cells / dish and incubated for 1 day. The following sample was added to the medium so as to be 1 mM in terms of β-cyclodextrin concentration. After 24 hours, confocal microscopy was performed, and the number of GFP-positive autophagosomes in the cells was counted. Further, the same experiment as described above was performed in a state where 3-methyladenine (manufactured by Merck), an autophagy inhibitor, was added to the medium at a concentration of 10 mM. The results are shown in FIG.
［試料］
（１）１２．２Ｍｅ－Ｐ１０３（例１で作製）
（２）メチル－β－シクロデキストリン（以下、「Ｍｅ－β－ＣＤ」と称する。；下記構造式（１）で表される化合物；シグマ－アルドリッチ社製；製品番号３３２６１５；構造式（１）では、前記メチル基の修飾がｘ個（ｘ＝０～２１）の場合を示している。）
Figure JPOXMLDOC01-appb-C000007
  

構造式（１） [sample]
(1) 12.2Me-P103 (produced in Example 1)
(2) Methyl-β-cyclodextrin (hereinafter referred to as “Me-β-CD”; a compound represented by the following structural formula (1); manufactured by Sigma-Aldrich; product number 332615; structural formula (1) Shows the case of x modification of the methyl group (x = 0 to 21).
Figure JPOXMLDOC01-appb-C000007

Structural formula (1)
　各試料を添加した細胞中のＧＦＰ－ＬＣ３陽性のオートファゴソーム数の計測を行った結果、１２．２Ｍｅ－Ｐ１０３を作用させた細胞中には、未処理細胞、ＤＭ－β－ＣＤを作用させた細胞よりも有意に多いＧＦＰ－ＬＣ３陽性オートファゴソームが生成していることが確認された。これらの小胞は３－メチルアデニンにより生成数が減少したことより、オートファジーの誘導により生成したオートファゴソームであると考えられる。以上の結果より、Ｍｅ－ＰＲＸはオートファジーの誘導作用を示すことが明らかとなった。 As a result of measuring the number of GFP-LC3-positive autophagosomes in the cells to which each sample was added, untreated cells and DM-β-CD were allowed to act on the cells treated with 12.2Me-P103. It was confirmed that significantly more GFP-LC3-positive autophagosomes were generated than cells. These vesicles are considered to be autophagosomes produced by induction of autophagy because the number of produced vesicles was reduced by 3-methyladenine. From the above results, it was revealed that Me-PRX exhibits an autophagy-inducing action.
＜例５：細胞死の評価＞
　９６ｗｅｌｌプレートにＨｅＬａ細胞を０．５×１０４ｃｅｌｌｓ／ｗｅｌｌ播種し、一日インキュベーションした。下記試料をβ－シクロデキストリン濃度換算で、各濃度培地に加えた。４８時間後にＣｅｌｌＣｏｕｎｔｉｎｇＫｉｔ－８（同仁化学研究所社製）を添加し、３７℃で１．５時間インキュベーションした。マイクロプレートリーダー（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）で４５０ｎｍの吸光度を測定することで、細胞生存率を評価した。結果を図１２に示す。また、オートファジー阻害剤である３－メチルアデニンを１０ｍＭの濃度で培地に添加した状態で、下記試料をβ－シクロデキストリン濃度換算で１２．２Ｍｅ－ＰＲＸを１ｍＭ、Ｍｅ－β－ＣＤを２ｍＭとなるように培地中に加えた。前記と同様の実験を行い、細胞生存率を評価した。結果を図１３に示す。 <Example 5: Evaluation of cell death>
HeLa cells were seeded on a 96-well plate at 0.5 × 10 4 cells / well and incubated for one day. The following samples were added to each concentration medium in terms of β-cyclodextrin concentration. After 48 hours, CellCounting Kit-8 (manufactured by Dojindo Laboratories) was added and incubated at 37 ° C. for 1.5 hours. The cell viability was evaluated by measuring absorbance at 450 nm with a microplate reader (Thermo Fisher Scientific). The results are shown in FIG. In addition, with the autophagy inhibitor 3-methyladenine added to the medium at a concentration of 10 mM, the following samples were converted to β-cyclodextrin concentration of 12.2 Me-PRX at 1 mM and Me-β-CD at 2 mM. So that it was added to the medium. The same experiment as described above was performed to evaluate the cell viability. The results are shown in FIG.
［試料］
（１）１２．２Ｍｅ－Ｐ１０３（例１で作製）
（２）ＨＥＥ－ＰＲＸ（例１で作製）
（３）Ｍｅ－β－ＣＤ [sample]
(1) 12.2Me-P103 (produced in Example 1)
(2) HEE-PRX (prepared in Example 1)
(3) Me-β-CD
　各試料を添加した細胞の生存率を評価した結果、１２．２Ｍｅ－Ｐ１０３を作用させた細胞は、ＨＥＥ－ＰＲＸやＭｅ－β－ＣＤを作用させた細胞よりも有意に細胞生存率が低下、つまり細胞死を強く誘導していた。この結果は１２．２Ｍｅ－Ｐ１０３が、メチル化β－ＣＤを細胞内局所に放出し作用したためと考えられる。また３－メチルアデニンの存在下では、Ｍｅ－β－ＣＤを作用させた細胞は細胞生存率が低下したのに対し、１２．２Ｍｅ－Ｐ１０３を作用させた細胞では細胞生存率が増加した。つまり１２．２Ｍｅ－Ｐ１０３は、オートファジー阻害下では細胞死の誘導が抑制されたものと考えられる。以上の結果より、Ｍｅ－ＰＲＸはオートファジー細胞死の誘導作用を示すことが明らかとなった。 As a result of evaluating the viability of the cells to which each sample was added, the cells treated with 12.2Me-P103 had a significantly lower cell viability than the cells treated with HEE-PRX or Me-β-CD. In other words, cell death was strongly induced. This result is considered because 12.2Me-P103 acted by releasing methylated β-CD locally in the cell. In the presence of 3-methyladenine, cells treated with Me-β-CD decreased in cell viability, whereas cells treated with 12.2Me-P103 increased cell viability. In other words, 12.2Me-P103 is considered to suppress the induction of cell death under autophagy inhibition. From the above results, it became clear that Me-PRX has an effect of inducing autophagy cell death.
＜例６：アポトーシス抵抗性細胞に対する細胞死の誘導評価＞
　９６ｗｅｌｌプレートに、抗アポトーシス細胞としてアポトーシス亢進性タンパク質Ｂａｘ／ＢａｋをノックアウトしたＢａｘ／Ｂａｋ　ＤＫＯ　ＭＥＦ細胞を０．２×１０４ｃｅｌｌｓ／ｗｅｌｌ播種し、一日インキュベーションした。Ｃａｓｐａｓｅ阻害剤であるｚＶＡＤ－ＦＭＫを５０μＭの濃度で培地に添加した状態で、下記試料をβ－シクロデキストリン濃度換算で、１２．２Ｍｅ－ＰＲＸを０．７５ｍＭ、Ｍｅ－β－ＣＤを２ｍＭとなるよう培地に加えた。４８時間後にＣｅｌｌＣｏｕｎｔｉｎｇＫｉｔ－８（同仁化学研究所社製）を添加し、３７℃で１．５時間インキュベーションした。マイクロプレートリーダーで４５０ｎｍの吸光度を測定することで、細胞生存率を評価した。結果を図１４に示す。 <Example 6: Evaluation of cell death induction on apoptosis-resistant cells>
Bax / Bak DKO MEF cells in which a pro-apoptotic protein Bax / Bak was knocked out as anti-apoptotic cells were seeded on a 96-well plate at 0.2 × 10 4 cells / well and incubated for one day. In the state where zVAD-FMK, which is a caspase inhibitor, is added to the medium at a concentration of 50 μM, the following sample is converted to β-cyclodextrin concentration and 12.2 Me-PRX is 0.75 mM and Me-β-CD is 2 mM. To the culture medium. After 48 hours, CellCounting Kit-8 (manufactured by Dojindo Laboratories) was added and incubated at 37 ° C. for 1.5 hours. Cell viability was evaluated by measuring absorbance at 450 nm with a microplate reader. The results are shown in FIG.
［試料］
（１）１２．２Ｍｅ－Ｐ１０３（例１で作製）
（２）Ｍｅ－β－ＣＤ
（３）２，６－ジ－Ｏ－メチル－β－シクロデキストリン（以下、「ＤＭ－β－ＣＤ」と称することがある；下記構造式（２）で表される化合物；シグマ－アルドリッチ社製；製品番号Ｈ０５１３）
Figure JPOXMLDOC01-appb-C000008
  

構造式（２） [sample]
(1) 12.2Me-P103 (produced in Example 1)
(2) Me-β-CD
(3) 2,6-di-O-methyl-β-cyclodextrin (hereinafter sometimes referred to as “DM-β-CD”; compound represented by the following structural formula (2); manufactured by Sigma-Aldrich ; Product number H0513)
Figure JPOXMLDOC01-appb-C000008

Structural formula (2)
　各試料を添加した細胞の生存率を評価した結果、Ｍｅ－β－ＣＤ、ＤＭ－β－ＣＤを作用させた細胞は、正常細胞 (ＷＴ　ＭＥＦ)と比較してアポトーシス抵抗性細胞 (Ｂａｘ／Ｂａｋ　ＤＫＯ　ＭＥＦ + ｚＶＡＤ)において細胞生存率が有意に増加していた。一方１２．２Ｍｅ－Ｐ１０３を作用させた細胞では、ＷＴ　ＭＥＦ細胞の細胞生存率が７．５１％に対してＢａｘ／Ｂａｋ　ＤＫＯ　ＭＥＦ細胞 + ｚＶＡＤでは８．５０％であり，同等な殺細胞作用を示していた。この結果は、Ｍｅ－β－ＣＤやＤＭ－β－ＣＤがアポトーシスを誘導したのに対し、１２．２Ｍｅ－Ｐ１０３はアポトーシスとは異なる細胞死を誘導したと考えられる。Ｍｅ－ＰＲＸによるオートファジー細胞死は、アポトーシス抵抗性細胞種に対して有意に細胞死を誘導するものと期待される。 As a result of evaluating the viability of the cells to which each sample was added, cells treated with Me-β-CD and DM-β-CD were more resistant to apoptosis-resistant cells (Bax / Bak) than normal cells (WT MEF). The cell viability was significantly increased in DKO MEF 細胞 + 生存 zVAD). On the other hand, in the cells treated with 12.2Me-P103, the cell viability of WT MEF cells was 7.51%, while Bax / Bak DKO MEF cells + zVAD was 8.50%, which showed an equivalent cell killing effect. Was showing. This result suggests that Me-β-CD and DM-β-CD induced apoptosis, whereas 12.2Me-P103 induced cell death different from apoptosis. Autophagy cell death by Me-PRX is expected to induce cell death significantly for apoptosis-resistant cell types.
＜例７：ＮＰＣ病細胞中のコレステロール減少作用＞
　健常者由来皮膚線維芽細胞（以下、「ＮＨＦ」と称する。Ｃｏｒｅｉｌｌ　Ｉｎｓｔｉｔｕｔｅより入手）、ニーマン・ピック病Ｃ型患者由来皮膚繊維芽細胞（以下、「ＮＰＣ１」と称する。Ｃｏｒｅｉｌｌ　Ｉｎｓｔｉｔｕｔｅより入手）を用い、以下のようにして細胞内のコレステロールの集積を調べた前記ＮＰＣ１をガラスボトムディッシュに播種し（細胞数：１×１０４個／ディッシュ）、３７℃で２４時間培養後、下記試料をシクロデキストリン濃度に換算して１０μＭ～１０００μＭ添加し、さらに３７℃で２４時間培養した。前記培養後、４％パラホルムアルデヒド溶液で細胞を固定し、５０μｇ／ｍＬに調製したフィリピン（Ｐｏｌｙｓｃｉｅｎｃｅｓ社製）のＰＢＳ溶液を加え４５分間室温で静置した。ＰＢＳで３回洗浄した後、共焦点レーザー操作顕微鏡ＦｌｕｏＶｉｅｗ　ＦＶ１０ｉ（オリンパス社製）でコレステロールの局在を観察した。結果を図１５に示す。 <Example 7: Cholesterol-reducing action in NPC disease cells>
Using healthy human-derived skin fibroblasts (hereinafter referred to as “NHF”, obtained from Coreille Institute), Neiman-Pick disease type C patient-derived skin fibroblasts (hereinafter referred to as “NPC1”, obtained from Corell Institute) The NPC1, which was examined for intracellular cholesterol accumulation as follows, was seeded in a glass bottom dish (cell number: 1 × 10 4 cells / dish) and cultured at 37 ° C. for 24 hours. 10 μM to 1000 μM was added in terms of concentration, and further cultured at 37 ° C. for 24 hours. After the culture, the cells were fixed with a 4% paraformaldehyde solution, a PBS solution of Philippines (manufactured by Polysciences) prepared to 50 μg / mL was added, and the mixture was allowed to stand at room temperature for 45 minutes. After washing 3 times with PBS, the localization of cholesterol was observed with a confocal laser operating microscope FluoView FV10i (Olympus). The results are shown in FIG.
［試料］
（１）ヒドロキシプロピル－β－シクロデキストリン　１０００μＭ（以下、「ＨＰ－β－ＣＤ」と称することがある；下記構造式（３）で表される化合物；シグマ－アルドリッチ社製；製品番号３３２６０７；構造式（３）では、前記ヒドロキシプロピル基の修飾がｘ個（ｘ＝１～７）の場合を示している。）
（２）ＨＥＥ－ＰＲＸ（例１で作製）　１００μＭ（β－ＣＤ濃度換算）
（３）ＨＥＥ－ＰＲＸ　１０００μＭ（β－ＣＤ濃度換算）
（４）１２．２Ｍｅ－Ｐ１０３（例１で作製）　１００μＭ（β－ＣＤ濃度換算）
（５）１２．２Ｍｅ－Ｐ１０３　１０００μＭ（β－ＣＤ濃度換算）
Figure JPOXMLDOC01-appb-C000009
  

構造式（３） [sample]
(1) Hydroxypropyl-β-cyclodextrin 1000 μM (hereinafter sometimes referred to as “HP-β-CD”; compound represented by the following structural formula (3); manufactured by Sigma-Aldrich; product number 332607; structure (In Formula (3), the case where x modification (x = 1-7) of the said hydroxypropyl group is shown.)
(2) HEE-PRX (prepared in Example 1) 100 μM (converted to β-CD concentration)
(3) HEE-PRX 1000 μM (converted to β-CD concentration)
(4) 12.2Me-P103 (prepared in Example 1) 100 μM (converted to β-CD concentration)
(5) 12.2Me-P103 1000 μM (converted to β-CD concentration)
Figure JPOXMLDOC01-appb-C000009

Structural formula (3)
　図１５の結果から、前記ＮＨＦ細胞に比べ、前記ＮＰＣ１細胞では多量のコレステロールが細胞内に沈着している様子が認められた。前記ＮＰＣ１細胞に対し、前記ＨＰ－β－ＣＤを１０００μＭの濃度で作用させると蛍光強度が減少したことより、コレステロールの***が促進されたと予想される。一方、細胞内分解性結合を有するＨＥＥ－ＰＲＸでは１０００μＭの濃度で処理した場合には、前記ＮＨＦ細胞と同程度まで蛍光強度が減少し、前記ＨＰ－β－ＣＤよりも優れたコレステロール除去作用が示された。Ｍｅ－ＰＲＸ（１２．２Ｍｅ－ＰＲＸ）で処理したＮＰＣ１細胞も、前記ＮＨＦ細胞と同程度まで蛍光強度が減少し、前記ＨＰ－β－ＣＤよりも優れたコレステロール除去作用が示された。 From the results of FIG. 15, it was recognized that a larger amount of cholesterol was deposited in the NPC1 cells than in the NHF cells. When the HP-β-CD was allowed to act on the NPC1 cells at a concentration of 1000 μM, it was expected that the excretion of cholesterol was promoted because the fluorescence intensity decreased. On the other hand, in the case of HEE-PRX having intracellular degradable bonds, when treated at a concentration of 1000 μM, the fluorescence intensity decreases to the same level as that of the NHF cells, and the cholesterol removing action is superior to that of the HP-β-CD. Indicated. NPC1 cells treated with Me-PRX (12.2Me-PRX) also decreased in fluorescence intensity to the same extent as the NHF cells, indicating a cholesterol removal action superior to that of the HP-β-CD.
　本明細書には、本発明の好ましい実施態様を示してあるが、そのような実施態様が単に例示の目的で提供されていることは、当業者には明らかであり、当業者であれば、本発明から逸脱することなく、様々な変形、変更、置換を加えることが可能であろう。本明細書に記載されている発明の様々な代替的実施形態が、本発明を実施する際に使用されうることが理解されるべきである。また、本明細書中において参照している特許および特許出願書類を含む、全ての刊行物に記載の内容は、その引用によって、本明細書中に明記された内容と同様に取り込まれていると解釈すべきである。 While preferred embodiments of the invention are shown herein, it will be apparent to those skilled in the art that such embodiments are provided for purposes of illustration only, Various modifications, changes and substitutions may be made without departing from the invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the invention. In addition, the contents described in all publications, including patents and patent application documents referred to in this specification, are incorporated by reference in the same manner as the contents specified in this specification. Should be interpreted.
　上記のとおり、粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程を含む製造方法によって、メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物が製造された。特に、メチル化された複数のβ－シクロデキストリン分子を含む、酸分解性のポリロタキサン化合物は、リソソーム内のコレステロールの過剰蓄積を原因とするニーマン・ピック病Ｃ型などのライソゾーム病の治療もしくは予防、癌、特にアポトーシス耐性の癌の治療等において有用となりうる。 As described above, an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules is produced by a production method including a step of reacting an acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide. It was done. In particular, an acid-decomposable polyrotaxane compound containing a plurality of methylated β-cyclodextrin molecules is used to treat or prevent lysosomal diseases such as Niemann-Pick disease type C caused by excessive accumulation of cholesterol in lysosomes, It can be useful in the treatment of cancer, particularly cancer that is resistant to apoptosis.

Claims (23)

  1. 　メチル化された複数の環状分子と、末端基を有する直鎖状分子とを含む、酸分解性のポリロタキサン化合物。 An acid-decomposable polyrotaxane compound containing a plurality of methylated cyclic molecules and a linear molecule having a terminal group.
  2. 　細胞内の酸性環境下またはｐＨ４．０～６．０の酸性環境下で分解する、請求項１記載のポリロタキサン化合物。 2. The polyrotaxane compound according to claim 1, which decomposes in an acidic environment in a cell or an acidic environment having a pH of 4.0 to 6.0.
  3. 　環状分子がβ－シクロデキストリンである、請求項１または２のいずれか一項記載のポリロタキサン化合物。 The polyrotaxane compound according to any one of claims 1 and 2, wherein the cyclic molecule is β-cyclodextrin.
  4. 　β－シクロデキストリンの水酸基がメチル化されている、請求項３記載のポリロタキサン化合物。 The polyrotaxane compound according to claim 3, wherein the hydroxyl group of β-cyclodextrin is methylated.
  5. 　β－シクロデキストリンが１分子あたり６～２１個のメチル基を有する、請求項３または４記載のポリロタキサン化合物。 The polyrotaxane compound according to claim 3 or 4, wherein β-cyclodextrin has 6 to 21 methyl groups per molecule.
  6. 　直鎖状分子がポリエチレングリコール（ＰＥＧ）および／またはポリプロピレングリコール（ＰＰＧ）を含む、請求項１～５のいずれか一項記載のポリロタキサン化合物。 The polyrotaxane compound according to any one of claims 1 to 5, wherein the linear molecule contains polyethylene glycol (PEG) and / or polypropylene glycol (PPG).
  7. 　直鎖状分子がポロキサマーを含む、請求項１～６のいずれか一項記載のポリロタキサン化合物。 The polyrotaxane compound according to any one of claims 1 to 6, wherein the linear molecule comprises a poloxamer.
  8. 　直鎖状分子の分子量が４０００～７０００である、請求項１～７のいずれか一項記載のポリロタキサン化合物。 The polyrotaxane compound according to any one of claims 1 to 7, wherein the molecular weight of the linear molecule is from 4000 to 7000.
  9. 　直鎖状分子と環状分子との分子数の比率が１：１０～１：１５であることを特徴とする、請求項１～８のいずれか一項記載のポリロタキサン化合物。 The polyrotaxane compound according to any one of claims 1 to 8, wherein the ratio of the number of molecules of the linear molecule to the cyclic molecule is 1:10 to 1:15.
  10. 　末端基が、置換基を有する又は有さないＯ－トリフェニルメチル基、置換基を有する又は有さないＳ－トリフェニルメチル基、および置換基を有する又は有さないＮ－トリフェニルメチル基から成る群より選択される、請求項１～９のいずれか一項記載のポリロタキサン化合物。 The terminal group is an O-triphenylmethyl group with or without a substituent, an S-triphenylmethyl group with or without a substituent, and an N-triphenylmethyl group with or without a substituent. The polyrotaxane compound according to any one of claims 1 to 9, which is selected from the group consisting of:
  11. 　末端基が直鎖状分子にペプチド結合、カーバメート結合、エステル結合、またはエーテル結合を介して連結されている、請求項１～１０のいずれか一項記載のポリロタキサン化合物。 The polyrotaxane compound according to any one of claims 1 to 10, wherein the terminal group is linked to the linear molecule via a peptide bond, a carbamate bond, an ester bond, or an ether bond.
  12. 　下記構造式を有する、メチル化されたβ－シクロデキストリンと直鎖状分子を含むポリロタキサン化合物であって、
    Figure JPOXMLDOC01-appb-C000001
      

    　ここで、ｍはポロキサマー中のポリプロピレングリコールの繰返し単位の数を示す整数であり、ｎはポリエチレングリコールの繰返し単位の数を示す整数であり、ｘはβ－シクロデキストリンの数を示す整数であり、
    　β－シクロデキストリンが１分子あたり６～２１個のメチル基を有し、直鎖状分子がポリエチレングリコール（ＰＥＧ）および／またはポリプロピレングリコール（ＰＰＧ）を含み、直鎖状分子の平均分子量が４０００～７０００であり、β－シクロデキストリンの数ｘが１０～１５であり、末端基がＮ－トリフェニルメチル基から成り、酸分解性であり、全体の平均分子量が１８，０００～２７，０００であるポリロタキサン化合物。     A polyrotaxane compound comprising a methylated β-cyclodextrin and a linear molecule having the following structural formula,
    Figure JPOXMLDOC01-appb-C000001

    Here, m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer, n is an integer indicating the number of repeating units of polyethylene glycol, x is an integer indicating the number of β-cyclodextrins,
    β-cyclodextrin has 6 to 21 methyl groups per molecule, the linear molecule contains polyethylene glycol (PEG) and / or polypropylene glycol (PPG), and the average molecular weight of the linear molecule is 4000 to 7000, the number x of β-cyclodextrin is 10 to 15, the terminal group is composed of N-triphenylmethyl group, is acid-decomposable, and the total average molecular weight is 18,000 to 27,000 Polyrotaxane compound.
  13. 　メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物の製造方法であって、粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程を含む、製造方法。 A method for producing an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclic molecules, comprising a step of reacting an acid-decomposable polyrotaxane and methyl iodide in the presence of powdered sodium hydroxide .
  14. 　メチル化された複数の環状分子を含む、酸分解性のポリロタキサン化合物の製造方法であって、ポロキサマーの両末端に一級アミノ基を有するポロキサマーを得る工程、前記ポロキサマーとβ－シクロデキストリンとを反応させて擬ポリロタキサンを得る工程、前記擬ポリロタキサンの両末端をＮ－トリチルグリシンでキャッピングする工程、および粉末状の水酸化ナトリウム存在下で酸分解性ポリロタキサンとヨウ化メチルを反応させる工程を含む、製造方法。 A method for producing an acid-decomposable polyrotaxane compound comprising a plurality of methylated cyclic molecules, the step of obtaining a poloxamer having a primary amino group at both ends of the poloxamer, and reacting the poloxamer with β-cyclodextrin A pseudopolyrotaxane, a step of capping both ends of the pseudopolyrotaxane with N-tritylglycine, and a step of reacting an acid-decomposable polyrotaxane with methyl iodide in the presence of powdered sodium hydroxide. .
  15. 　メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞においてオートファジーを誘導するための組成物。 A composition for inducing autophagy in cells, containing an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  16. 　メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、癌を治療するための医薬組成物。 A pharmaceutical composition for treating cancer, comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  17. 　癌がアポトーシス耐性の癌である、請求項１５記載の医薬組成物。 The pharmaceutical composition according to claim 15, wherein the cancer is apoptosis-resistant cancer.
  18. 　メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、細胞内のコレステロール蓄積に起因する疾患またはオートファジーの機能障害に起因する疾患を治療または予防するための医薬組成物。 A pharmaceutical composition for treating or preventing a disease caused by intracellular cholesterol accumulation or autophagy dysfunction, comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules object.
  19. 　メチル化された複数のシクロデキストリン分子を含む、酸分解性のポリロタキサン化合物を含有する、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物。 A pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC) comprising an acid-decomposable polyrotaxane compound containing a plurality of methylated cyclodextrin molecules.
  20. 　環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物。 An acid-decomposable polyrotaxane compound containing β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having an N-triphenylmethyl group at the end as a linear molecule.
  21. 　環状分子として置換基を有するβ－シクロデキストリンと、直鎖状分子として末端にＮ－トリフェニルメチル基を有するポロキサマーとを含む、酸分解性のポリロタキサン化合物を含有する、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物。 Niemann-Pick disease type C (NPC) containing an acid-decomposable polyrotaxane compound containing β-cyclodextrin having a substituent as a cyclic molecule and a poloxamer having a terminal N-triphenylmethyl group as a linear molecule. Pharmaceutical composition for treating or preventing.
  22. 　下記構造式を有する、ヒドロキシエトキシエチル化されたβ－シクロデキストリンと直鎖状分子を含むポリロタキサン化合物であって、
    Figure JPOXMLDOC01-appb-C000002
      

    　ここで、ｍはポロキサマー中のポリプロピレングリコールの繰返し単位の数を示す整数であり、ｎはポリエチレングリコールの繰返し単位の数を示す整数であり、ｘはβ－シクロデキストリンの数を示す整数であり、
    　β－シクロデキストリンが１分子あたり６～２１個のヒドロキシエトキシエチル基を有し、直鎖状分子がポリエチレングリコール（ＰＥＧ）および／またはポリプロピレングリコール（ＰＰＧ）を含み、直鎖状分子の平均分子量が４０００～７０００であり、β－シクロデキストリンの数ｘが１０～１５であり、末端基がＮ－トリフェニルメチル基から成り、酸分解性であるポリロタキサン化合物。     A polyrotaxane compound comprising a hydroxyethoxyethylated β-cyclodextrin and a linear molecule having the following structural formula:
    Figure JPOXMLDOC01-appb-C000002

    Here, m is an integer indicating the number of repeating units of polypropylene glycol in the poloxamer, n is an integer indicating the number of repeating units of polyethylene glycol, x is an integer indicating the number of β-cyclodextrins,
    β-cyclodextrin has 6 to 21 hydroxyethoxyethyl groups per molecule, the linear molecule contains polyethylene glycol (PEG) and / or polypropylene glycol (PPG), and the average molecular weight of the linear molecule is A polyrotaxane compound having 4000 to 7000, the number x of β-cyclodextrin being 10 to 15, a terminal group consisting of an N-triphenylmethyl group, and being acid-decomposable.
  23. 　請求項２２に記載のポリロタキサン化合物を含む、細胞においてオートファジーを誘導するための組成物、アポトーシス耐性の癌を治療するための医薬組成物、細胞内のコレステロール蓄積に起因する疾患またはオートファジーの機能障害に起因する疾患を治療または予防するための医薬組成物、または、ニーマンピック病Ｃ型（ＮＰＣ）を治療または予防するための医薬組成物。 A composition for inducing autophagy in a cell, comprising a polyrotaxane compound according to claim 22, a pharmaceutical composition for treating apoptosis-resistant cancer, a disease caused by intracellular cholesterol accumulation, or a function of autophagy A pharmaceutical composition for treating or preventing a disease caused by a disorder, or a pharmaceutical composition for treating or preventing Niemann-Pick disease type C (NPC).
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CN114949253A (en) * 2022-06-01 2022-08-30 暨南大学附属第一医院（广州华侨医院） Double-drug combined-loading poly-rotaxane nano drug delivery system and preparation method and application thereof
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