WO2017188257A1 - Polyrotaxane méthylé et procédé pour sa synthèse - Google Patents

Polyrotaxane méthylé et procédé pour sa synthèse 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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English (en)
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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Publication of WO2017188257A1 publication Critical patent/WO2017188257A1/fr

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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.

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Abstract

La présente invention concerne un composé de polyrotaxane clivable par un acide qui comprend : une pluralité de molécules annulaires méthylées ; et une molécule à chaîne linéaire qui présente un groupe terminal. Le composé de polyrotaxane clivable par un acide qui comprend une pluralité de molécules annulaires méthylées peut être produit au moyen d'un procédé de production qui comprend une étape consistant à faire réagir un polyrotaxane clivable par un acide avec de l'iodure de méthyle en présence d'hydroxyde de sodium en poudre. Le composé de polyrotaxane clivable par un acide qui comprend une pluralité de molécules annulaires méthylées peut être utilisé en tant qu'agent thérapeutique pour des maladies telles que des maladies lysosomales et le cancer.
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CN110527108A (zh) * 2019-08-29 2019-12-03 暨南大学 一种聚轮烷结构no供体材料及其制备方法与应用
CN110527108B (zh) * 2019-08-29 2021-07-27 暨南大学 一种聚轮烷结构no供体材料及其制备方法与应用
WO2022039206A1 (fr) * 2020-08-20 2022-02-24 国立大学法人 東京医科歯科大学 Composition pharmaceutique de traitement de la stéatohépatite non alcoolique
WO2023190243A1 (fr) * 2022-03-30 2023-10-05 デンカ株式会社 Procédé de préparation d'hydrogel supramoléculaire et application en tant que biomatériau
CN114949253A (zh) * 2022-06-01 2022-08-30 暨南大学附属第一医院（广州华侨医院） 一种双药联载的聚轮烷纳米递药系统及其制备方法与应用
CN114949253B (zh) * 2022-06-01 2022-12-16 暨南大学附属第一医院（广州华侨医院） 一种双药联载的聚轮烷纳米递药系统及其制备方法与应用

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