WO2016121829A1 - Agent for treatment or prevention of dermatitis in which nanoparticles are used as active ingredient - Google Patents

Agent for treatment or prevention of dermatitis in which nanoparticles are used as active ingredient Download PDF

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WO2016121829A1
WO2016121829A1 PCT/JP2016/052379 JP2016052379W WO2016121829A1 WO 2016121829 A1 WO2016121829 A1 WO 2016121829A1 JP 2016052379 W JP2016052379 W JP 2016052379W WO 2016121829 A1 WO2016121829 A1 WO 2016121829A1
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dermatitis
amino acid
therapeutic
skin
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PCT/JP2016/052379
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French (fr)
Japanese (ja)
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昇一 城武
恵子 宇高
道之 笠井
栄紀 佐野
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昇一 城武
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Priority to JP2016572112A priority Critical patent/JP6847668B2/en
Publication of WO2016121829A1 publication Critical patent/WO2016121829A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin

Definitions

  • the present invention relates to a dermatitis treatment or prevention agent comprising nano-sized cyanoacrylate polymer particles as an active ingredient.
  • DDS drug delivery system
  • sustained release mainly for application to human medicine
  • drugs are conjugated to cyanoacrylate polymer particles.
  • DDS is known (Patent Documents 1 and 2 and Non-Patent Document 1).
  • Patent Documents 3 to 5 the present inventor has also disclosed a method for producing cyanoacrylate polymer particles, antibacterial agent-conjugated particles, and plasmid-conjugated particles with little variation in particle size.
  • Patent Documents 3 to 5 In the conventional polymer particle synthesis method, saccharides and polysorbate are allowed to coexist in the polymerization reaction system for the purpose of starting and stabilizing the anionic polymerization reaction of cyanoacrylate.
  • Patent Document 6 the cyanoacrylate polymer particles themselves have antibacterial activity against Gram-positive bacteria. Furthermore, in addition to the anticancer activity of amino acid-conjugated cyanoacrylate polymer particles (Patent Document 7), it was found that antibacterial activity can be exerted against various bacteria regardless of Gram stainability (Patent Documents 8 and 9). . Nano-sized polymer particles specifically adhere to the bacterial surface (cell wall), leading the bacteria to lysis.
  • Cyanoacrylate nanoparticles exhibit antibacterial activity with a mechanism of action completely different from antibiotics, and are also effective against multi-drug resistant bacteria such as MRSA (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin resistant enterococcus) .
  • MRSA Metal-resistant Staphylococcus aureus
  • VRE Vancomycin resistant enterococcus
  • Atopic dermatitis is a skin disease with a high prevalence in Japan. Increasing in recent years, patients are forced to suffer a lot of mental and physical pain.
  • Non-patent Document 2 The basics of pharmacotherapy for atopic dermatitis include the use of steroid topical drugs, calcineurin-inhibiting drugs, and anti-histamine / anti-allergic drugs as needed according to severity. Accordingly, oral steroids and oral calcineurin inhibitor are used (Non-patent Document 2). However, there are many patients who are afraid of side effects due to continuous use of steroids and avoid steroids, and there is a problem of infectivity due to immunosuppression, so new treatment means are required.
  • An object of the present invention is to provide a novel means capable of treating or preventing dermatitis such as atopic dermatitis.
  • the present invention contains at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient.
  • An agent for treating or preventing inflammation is provided.
  • the present invention contains at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient.
  • the present invention provides an inhibitor of type IV allergic reaction or type IV allergic reaction.
  • the present invention contains at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient.
  • a therapeutic or prophylactic agent for allergic symptoms involving type I allergic reaction or type IV allergic reaction is provided.
  • the present invention includes at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof in skin lesions to be treated or skin regions where prevention of the occurrence of dermatitis is desired.
  • Providing a method of treating or preventing dermatitis comprising administering an effective amount of cyanoacrylate polymer particles having an average particle size of less than 1000 nm.
  • the present invention provides a cyanoacrylate polymer having an average particle size of less than 1000 nm, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof for a subject in need thereof.
  • a method for suppressing a type I allergic reaction or a type IV allergic reaction comprising administering an effective amount of particles.
  • the present invention provides a cyanoacrylate polymer having an average particle size of less than 1000 nm, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof for a subject in need thereof.
  • a method of treating or preventing allergic symptoms involving type I allergic reactions or type IV allergic reactions comprising administering an effective amount of particles.
  • the present invention provides a novel means for treating or preventing dermatitis different from steroidal drugs.
  • the cyanoacrylate nanoparticles which are the active ingredients of the therapeutic or prophylactic agent of the present invention, are not used as a DDS for drugs, but the particles themselves exhibit a therapeutic and prophylactic effect for dermatitis.
  • the therapeutic or prophylactic agent of the present invention is preferably used even for patients who are anxious about steroidal drugs, patients who cannot use steroidal drugs due to side effects, and patients who are found to be susceptible to immunological decline. be able to.
  • the treatment schedule for NC / NgaSlc mice is shown (Study 1). It is the photograph of the skin lesion part of the NC / NgaSlc mouse
  • FIG. 1 It is the graph which scored the skin inflammation state of the lesioned part shown in FIG.
  • FIG. 1 is an HE-stained image of the dorsal skin tissue of NC / NgaSlc mice on the 8th day after applying DNFB after observing tape stripping-induced dermatitis according to the schedule shown in FIG. It is the result of having measured the blood IgE density
  • a comparison between the start of the experiment (blood collection 1) and the end of the experiment (blood collection 2) is shown.
  • the treatment schedule with respect to the BALB / c mouse which examined the effect of the nanoparticle water in the dermatitis prevention model is shown (examination 2).
  • FIG. 8 is an HE-stained image of dorsal skin tissue of BALB / c mice in which dermatitis was induced according to the schedule shown in FIG. 7. The dorsal skin 3 days after DNFB application was compared between the applied head side and the unapplied caudal side.
  • FIG. 14 is an HE-stained image of the dorsal skin tissue of BALB / c mice in which dermatitis was induced after spraying with nanoparticle water or water as a prevention model in the schedule shown in FIG. 13.
  • DNFB is applied to the back and tail side of the tissue on the third day.
  • the dorsal temporal skin applied to DNFB at the start of the experiment was also stained.
  • the dorsal temporal side of BALB / c mice in which dermatitis was induced by the schedule shown in FIG. 13 (DNFB was applied only at the start of the experiment as a treatment model, and then sprayed with nanoparticle water or water) and the caudal side (prevention model) As a result of spraying nanoparticle water or water, applying DNFB before the end of the experiment, and examining the expression levels of various cytokines on the third day).
  • the treatment schedule with respect to the BALB / c mouse made into the tape stripping induction dermatitis model is shown (examination 4).
  • FIG. 20 is an HE-stained image of the back skin tissue of BALB / c mice in which dermatitis was induced by repeated tape stripping according to the schedule shown in FIG. 19 (day 14). Two tissues randomly selected from each group of nanoparticle water or water spray are shown.
  • FIG. 20 shows the results of examining the expression levels of various cytokines in the back skin of BALB / c mice in which dermatitis was induced by repeated tape stripping according to the schedule shown in FIG. Measurements were made on cranial and caudal skin treated the same way, respectively.
  • the treatment schedule for hairless mice examined as a hapten-induced chronic dermatitis model by repeated DNFB application is shown (Study 5).
  • FIG. 25 shows the result of scoring skin inflammation for each group of hairless mice in which dermatitis was induced according to the schedule shown in FIG. 24 and examining changes in the score.
  • FIG. 25 is a photograph of a skin lesion of a hairless mouse in which dermatitis is induced according to the schedule shown in FIG. 24 (day 33).
  • a to D represent groups A to D, respectively.
  • the central A-1 to D-1 are the right auricular skin tissue, and the right A-2 to D-2 are the cranial dorsal skin tissue.
  • the bar in the tissue image indicates 100 ⁇ m.
  • 24 is the relative expression level of various cytokines at the site of inflammation induction in hairless mice in which dermatitis was induced by the schedule shown in FIG. 24 (day 34).
  • the particles used in the present invention are nano-sized polymer particles (average particle size of less than 1000 nm) obtained by anionic polymerization of a cyanoacrylate monomer, and amino acids, amino acid derivatives, oligomers and polymers thereof (hereinafter collectively referred to as “general names”). And at least one selected from “amino acid molecules”. Furthermore, at least 1 sort (s) selected from the group which consists of saccharide
  • amino acid molecules, sugars and polysorbates can be used as polymerization initiators / stabilizers for anionic polymerization of cyanoacrylate monomers.
  • a nanoparticle production method using sugar and / or polysorbate as a polymerization initiator / stabilizer is described in Patent Document 3, Patent Document 4 (antibacterial agent-conjugated particle), Patent Document 5 (plasmid-conjugated particle) and the like.
  • Nanoparticle production methods using amino acid molecules as polymerization initiators / stabilizers are described in Patent Documents 8 and 9 (use of amino acid molecules alone) and the like.
  • Patent Document 7 describes a production method in which an amino acid and a saccharide / polysorbate are used in combination. Any one of these polymerization initiators / stabilizers may be used, or two or more thereof may be used in combination.
  • an “amino acid” refers to a compound having an amino group and a carboxy group in the molecule. As defined in general amino acids, the amino group hydrogen is substituted for other parts in the molecule. Also included are imino acids, which are cyclic compounds that have been converted to secondary amines. Representative examples of amino acids that can be used in the present invention include 20 kinds of ⁇ -amino acids (arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, which constitute natural proteins.
  • Threonine phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, methionine
  • ⁇ -, ⁇ - and ⁇ -amino acid based molecules are also included.
  • arginine histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, methionine, ⁇ -Alanine, ⁇ -aminobutyric acid (GABA; neurotransmitter), carnitine, ⁇ -aminolevulinic acid, ⁇ -aminovaleric acid and the like.
  • GABA neurotransmitter
  • amino acids examples include arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, and methionine.
  • At least one selected from among the group consisting of glycine and aspartic acid can be mentioned, but not limited thereto.
  • amino acid derivative refers to a compound having a structure in which any group is modified or substituted in an amino acid as defined above.
  • amino acid derivatives that naturally exist as biological components can be preferably used in the present invention.
  • Specific examples of amino acid derivatives that can be used include creatine (arginine derivative with 1-methylguanidinoacetic acid), ornithine (arginine derivative with urea cycle product), thyroxine (aromatic amino acids triiodothyronine; T4), Desmosine (constituent component of keratin elastin and collagen; a structure in which three side chains of allicin and one side chain of lysine are combined), hydroxyproline and hydroxylysine (constituent components of gelatin and collagen), phosphoserine (serine and phosphate) Ester; casein component), theanine (tea component, glutamic acid derivative), kainic acid (component of seaweed insects), tricolominic acid (shime
  • amino acid “oligomer” refers to an oligopeptide in which 10 or less amino acid residues are bonded by peptide bonds
  • an amino acid “polymer” refers to a polypeptide in which 11 or more amino acid residues are bonded by peptide bonds. Say. Any of them may contain not only amino acids but also amino acid derivatives as residues.
  • the upper limit of the number of residues of the polypeptide is not particularly limited, but may be, for example, 500 residues or less.
  • those having 11 to 100 residues, 11 to 50 residues, 11 to 30 residues, 11 to 20 residues, or 11 to 15 residues can be preferably used.
  • Oligopeptides can be used more preferably than polypeptides. Among these, oligopeptides having 2 to 7 residues, 2 to 5 residues, or 2 or 3 residues can be more preferably used.
  • nano-sized (less than 1000 nm) cyanoacrylate polymer particles are used under the condition that any of the 20 ⁇ -amino acids constituting the natural protein does not use saccharides or polysorbate.
  • “Sugar” includes monosaccharides having a hydroxyl group (such as glucose, mannose, ribose and fructose), disaccharides having a hydroxyl group (such as maltose, trehalose, lactose and sucrose) and polysaccharides having a hydroxyl group (such as dextran and mannan). Etc.). These sugars may be either cyclic or chain-like, and when they are cyclic, they may be any one of pyranose type, furanose type and the like. In addition, there are various isomers of sugar, and any of them may be used.
  • Polysorbate includes various Tween-based surfactants such as polyoxyethylene sorbitan monolaurate (trade name “Tween 20”) and polyoxyethylene sorbitan monooleate (trade name “Tween 80”).
  • Monosaccharides, disaccharides and polysaccharides, and polysorbates can be used alone or in combination of two or more.
  • sugars and polysorbates described above glucose, dextran, and Tween 20 (trade name) are available at low cost, which is advantageous in terms of cost.
  • dextran dextran having a polymerization degree of about 50,000 or more in average molecular weight is preferable. There is no particular upper limit on the molecular weight of dextran, but it is usually about 500,000 or less.
  • Nanoparticles used in the present invention are particles synthesized using amino acid-based molecules as polymerization initiators / stabilizers and without using either sugar or polysorbate (that is, containing amino acid-based molecules, both sugar and polysorbate Particles that do not contain), as well as particles synthesized using amino acid molecules and sugars as polymerization initiators / stabilizers and without using polysorbates (that is, containing amino acid molecules and sugars, without polysorbates, such as SDS) Particles that do not contain other surfactants are preferred.
  • an alkyl cyanoacrylate monomer is preferable.
  • the alkyl group preferably has 1 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, still more preferably 3 to 5 carbon atoms, and may be linear or branched. Further, at least one of carbon atoms constituting the alkyl group may be replaced with a halogen atom (chlorine, bromine, iodine).
  • halogen atom chlorine, bromine, iodine.
  • preferable cyanoacrylate monomers include methyl-2-cyanoacrylate, ethyl-2-cyanoacrylate, n-propyl-2-cyanoacrylate, i-propyl-2-cyanoacrylate, and n-butyl-2-cyanoacrylate.
  • n-butyl-2-cyanoacrylate represented by the following formula, which is conventionally used as an adhesive for stitching wounds in the surgical field, can be preferably used.
  • At least one polymerization initiator / stabilizer is dissolved in a suitable solvent, and then at least one cyanoacrylate monomer is added under stirring, and the polymerization reaction proceeds by continuing appropriate stirring. You can do it.
  • at least one amino acid molecule is used as a polymerization initiator / stabilizer.
  • at least one polymerization initiator / stabilizer selected from the group consisting of sugar and polysorbate may be used in combination. Only one type of cyanoacrylate monomer may be used, or two or more types may be used.
  • the concentration of sugar and / or polysorbate in the polymerization reaction solution at the start of the reaction is not particularly limited. About 0.5% to 10%, preferably about 0.75% to 7.5%.
  • the concentration of sugar means w / v%, and the concentration of polysorbate means v / v%.
  • concentration ranges are “0.5 w / v% to 10 w / v%”. ”,“ 0.75 w / v% to 7.5 w / v% ”.
  • sugar when used in combination at 5 w / v% and polysorbate at 1 v / v%, the total concentration is 6%.
  • a monosaccharide eg glucose
  • it is preferably used at about 2.5 w / v% to 10 w / v%.
  • the concentration of amino acid molecules in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually 0.1 w / v % To 3w / v%.
  • the concentration of amino acid molecules used may be lower.
  • an aqueous solvent mainly composed of water for example, water, a lower alcohol aqueous solution, etc.
  • water is preferably used. Since anionic polymerization is initiated by hydroxide ions, the pH of the reaction solution affects the polymerization rate. When the pH of the reaction solution is high, the hydroxyl ion concentration is high, so that the polymerization is fast, and when the pH is low, the polymerization is slow.
  • an appropriate polymerization rate is usually obtained under acidic conditions with a pH of about 1.5 to 3.0.
  • the acid added to make the reaction solution acidic is not particularly limited, and either an inorganic acid or an organic acid can be used.
  • hydrochloric acid does not adversely affect the reaction and is volatilized after the reaction. Therefore, hydrochloric acid can be preferably used in the production of amino acid-based molecule-containing particles, but usable acid is not limited thereto.
  • the concentration of acid such as hydrochloric acid is not particularly limited, but can be appropriately selected within a range of about 0.0005N to 0.5N.
  • a method for synthesizing nano-sized polymer particles by polymerizing a cyanoacrylate monomer using an amino acid as a polymerization initiator a method called a mini-emulsion method is known (Weiss, CK et al., Preparatio, Macromolecules, 2007). , Vol. 40, p. 928-938; WO 2008/003706 A1), in this method, in a mini-emulsion composed of two phases of O phase containing lipophilic solvent such as hexadecane and W phase containing hydrochloric acid etc. Anionic polymerization is performed, and the use of a surfactant such as SDS is also essential.
  • a surfactant such as SDS
  • nanopolymer particles can be synthesized by a polymerization reaction in a single-phase aqueous solvent that does not contain an O phase. Therefore, it is necessary to use an organic solvent or a surfactant as a solvent. No.
  • nanopolymer particles can be produced using a polymerization initiator / stabilizer other than polysorbate. Therefore, including a Tween surfactant, an anionic surfactant, a cationic surfactant, Nanoparticles that do not contain any of an ionic surfactant, an amphoteric surfactant, and a nonionic surfactant can be prepared.
  • a Tween surfactant including a Tween surfactant, an anionic surfactant, a cationic surfactant, Nanoparticles that do not contain any of an ionic surfactant, an amphoteric surfactant, and a nonionic surfactant can be prepared.
  • the concentration of the cyanoacrylate monomer in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually about 0.5 v / v% to 2.0 v / v%, preferably about 0.8 v / v% to 1.2 v / v%. It is.
  • the reaction temperature is not particularly limited, but it is preferable because it is carried out at room temperature.
  • the reaction time varies depending on the pH of the reaction solution, the type of solvent, and the like, and therefore is appropriately selected depending on these factors. Although not particularly limited, the reaction time is usually about 10 minutes to 5 hours, preferably about 30 minutes to 4 hours. Since the obtained amino acid molecule-containing particles are usually used as neutral particles, it is preferable to neutralize the reaction solution by adding a base such as an aqueous sodium hydroxide solution after the reaction.
  • the reaction liquid after completion of the reaction may be filtered and washed with sterilized water as appropriate to collect the particles.
  • nano-sized cyanoacrylate polymer particles having an average particle diameter of less than 1000 nm can be easily produced.
  • the lower limit of the particle size is not particularly limited, but the particle size of the particles produced by the above polymerization reaction is usually about 7 nm or more.
  • the average particle size of the particles is 20 nm to 600 nm, more preferably 50 nm to 550 nm.
  • the size of the particles can be adjusted by adjusting the concentration and pH of the cyanoacrylate monomer in the reaction solution and the reaction time.
  • the particle size can also be adjusted by changing the concentration and type of the polymerization initiator / stabilizer (patent) References 3, 4 etc.).
  • concentration and type of the polymerization initiator / stabilizer (patent) References 3, 4 etc.).
  • the pH of the reaction solution is increased, when the reaction time is increased, and when the sugar concentration of the reaction solution is decreased, the particle size increases, and when polysorbate is used as a polymerization initiator / stabilizer, particles are increased. The size becomes smaller. By appropriately combining these reaction conditions, particles having a desired size can be produced.
  • the charge (zeta potential) of the nanoparticles is not particularly limited, but is usually about -50 mV to 0 mV.
  • the zeta potential indicates the charge on the particle surface and is an index of particle dispersibility.
  • the particle size and the zeta potential can be easily measured using, for example, a commercially available apparatus using a He / Ne laser (for example, a Zetasizer manufactured by Malvern Inst. UK).
  • Nanoparticles produced using amino acid-based molecules as polymerization initiators / stabilizers not only contain amino acid-based molecules attached to the particles, but also the -COO group in the amino acid structure of the ethylene end of cyanoacrylate. It is considered that it is bound to carbon and contained in the particle by a covalent bond.
  • the content of amino acid molecules in the particles obtained by the above method is usually about 20% to about 65%.
  • the content of amino acid-based molecules is determined by measuring the absorbance of the filter-passed solution when the filter is washed after polymerization at an appropriate wavelength, and the amount of amino-acid-based molecules in the filter-passed solution (ie, (Quantity) can be calculated by the following formula after obtaining by the absorbance method.
  • Amino acid molecule content (Amino acid molecule addition amount)-(Amount of amino acid molecule in the filter passage liquid)
  • Amino acid molecule content (%) Amino acid molecule content ⁇ Amino acid molecule addition x
  • the amino acid molecule-containing nanoparticles used in the present invention do not contain an antibacterial active ingredient against bacteria. It is known that cyanoacrylate polymer nanoparticles containing amino acids can exhibit antibacterial activity against both gram-positive and gram-negative bacteria, as described in Patent Documents 8 and 9, and are applied to the bacterial surface (cell wall). It exhibits antibacterial action by directing bacteria to lysis by specific adhesion. It has been confirmed that the cytotoxic activity of the nanoparticles is not exerted on normal mammalian cells, and the nanoparticles do not have in vivo toxicity. In addition, the amino acid molecule-containing nanoparticles used in the present invention do not contain an antibacterial active ingredient against fungi.
  • antibacterial active ingredient refers to a chemical substance component that can biochemically act on the metabolic pathway or physiological function of bacteria or fungi to inhibit the growth of the bacteria. Or it refers to antibiotics and other chemical components that can be used for fungal antibacterial activity. “No antibacterial active ingredient” means that it does not contain any antibacterial active ingredient, or even if it is contained in a very small amount, it cannot be used for antibacterial bacteria or fungi sensitive to the antibacterial active ingredient. This means that the antibacterial active ingredient is contained only in a trace amount.
  • the nanoparticles used in the present invention can also be said to be “substantially free” of antibacterial active ingredients against bacteria and fungi.
  • a trace amount that does not allow antibacterial activity means that the amount of antibacterial active ingredient contained in a particle per unit volume of the particle is defined as the concentration contained in the particle, and an antibacterial active ingredient having the same concentration as this content concentration It means an amount that does not prevent the growth of sensitive bacteria or fungi when it is allowed to act on sensitive bacteria or fungi alone without containing them.
  • the nanoparticles used in the present invention may be particles that do not contain any antibacterial active ingredient such as antibiotics.
  • the amino acid molecule-containing nanoparticles do not contain a chemical substance component having a therapeutic or preventive activity against dermatitis, such as a conventionally used dermatitis therapeutic agent.
  • a chemical substance component having a therapeutic or preventive activity against dermatitis such as a conventionally used dermatitis therapeutic agent.
  • the term “excluded” as used herein is the same as described above, and does not contain such a chemical substance component at all, or even if it contains such a chemical substance, it is extremely small, and can exert a therapeutic and preventive effect on skin lesions. It means that the chemical substance component is contained only in such a small amount as to be impossible.
  • Nanoparticles used in the present invention are not DDS as a dermatitis treatment drug, but the nanoparticles themselves control the skin inflammatory reaction through the improvement of skin barrier function and the immune response involved in controlling the number of superficial bacteria, Has the effect of treating and preventing inflammation.
  • the target dermatitis in the present invention is not particularly limited, but first, it may be dermatitis accompanied by pruritus (pruritic dermatitis). Specific examples of pruritic dermatitis include, but are not limited to, atopic dermatitis, contact dermatitis, infectious dermatitis, dermatitis due to psoriasis and autoimmune mechanism, drug eruption, and the like.
  • the target dermatitis may secondly be dermatitis related to skin injury.
  • dermatitis include, but are not limited to, radiation dermatitis, burns, acne and the like.
  • the amino acid molecule-containing nanoparticles have the action of suppressing type I and type IV allergic reactions. Therefore, the agent of the present invention comprising amino acid molecule-containing nanoparticles as an active ingredient is used as an inhibitor of type I allergic reaction or type IV allergic reaction, such as dermatitis involving type I allergic reaction or type IV allergic reaction. It is useful as an agent for treating or preventing allergic symptoms. It is known that the above pruritic dermatitis can involve type I allergic reaction or type IV allergic reaction.
  • the nano-sized cyanoacrylate polymer particles used in the present invention have antibacterial activity against bacteria (Patent Documents 8 and 9).
  • the antibacterial activity of the particles is expected to be effective against infected bacteria.
  • the agent of the present invention can be used, the dermatitis targeted in the present invention can be dermatitis other than dermatitis mainly caused by bacterial infection.
  • the agent of the present invention is used by locally administering to a skin lesion to be treated or a skin region where prevention of the occurrence of dermatitis is desired.
  • the local administration method include intracutaneous administration with injections, drops, etc., topical application with ointments, creams, patches, and the like.
  • the dosage is appropriately selected according to symptoms, age, body weight, administration method, etc., and is not particularly limited.
  • the amount of amino acid molecule-containing particles that are active ingredients for the target animal is about 0.01 ⁇ g to 10,000 mg per day. For example, it is about 1 ⁇ g to 100 mg, and is administered once or several times. Depending on the degree of symptom improvement, it may be administered once or several times daily for several days to several months, or once or several times every other day, or symptoms may occur Sometimes it may be administered.
  • the administration target of the agent of the present invention is a mammal, and examples thereof include humans, dogs, cats, rabbits, and hamsters.
  • the agent of the present invention may be used alone or in combination with other pruritic skin disease treatment or prevention agents and the like.
  • Standard treatments conventionally used for pruritic skin diseases are steroids, calcineurin inhibitors and antihistamines, but the agents of the present invention are considered to have a different mechanism of action from these standard treatments. Therefore, it is also possible to use the agent of the present invention in combination with a steroid agent or the like.
  • Nanosized polymer particles containing amino acids and dextran were produced according to the methods described in International Publication Nos. 2012/133648 and 2013/108871. The specific procedure is as follows.
  • the average particle diameter and zeta potential of the nanoparticles were measured using a commercially available zeta sizer (MalvernMInst. UK). Further, the amount of amino acid molecules in the filter passage liquid at the time of filter washing was determined by an absorbance method, and the amino acid content of the particles was calculated. The results are shown in Table 1.
  • Nanoparticles were suspended in sterilized water to prepare nanoparticle water (diluted to 0.3% (w / v), particle concentration 6 ⁇ g / ml) and used in the experiment. All of the mice used below were carried out in accordance with the experimental plan approved by the Kochi University Animal Experiment Committee, and were reared and experimented in an SPF environment.
  • NC / NgaSlc mice (Japan SLC, Shizuoka) are known to spontaneously develop dermatitis by tape stripping after shaving and depilation of the back, and are used as a model for atopic dermatitis (Matsuda , M., et.al. Int. Immunol. 9: 461, 1997).
  • 5 male 5-week-old NC / NgaSlc mice were used, and treatment was performed according to the schedule shown in FIG.
  • the back of the NC / NgaSlc mouse was shaved with a shaver and then removed with a hair removal cream (Epilat, Kracie Home Products, Tokyo).
  • the depilatory cream was wiped off, and tape stripping (Scotch Mending Tape, cat.no.810-3-24, 3M Japan, Tokyo) was performed 8 times with an adhesive tape the next day to induce dermatitis. The same operation was repeated when hair grew on the way. Three weeks later, in any mouse, the onset of dermatitis was confirmed over a wide area of the back (FIG. 2A).
  • mice that developed dermatitis 3 were the nanoparticle water spray group and 2 were the water spray group.
  • Nanoparticle water or sterilized water was sprayed every 2 days on the dorsal skin including the lesion.
  • a total liquid volume of 0.3 to 0.4 mL (1.8 to 2.4 ⁇ g as the amount of nanoparticles) was sprayed on 8 to 10 cm 2 of the back of one mouse.
  • the number of bacteria in the center of the back was measured on the 2nd, 6th and 8th days.
  • a mouse standard agar medium for viable cell count measured back central part of the (Petain check, Eiken) was pressed for 5 seconds, incubated for 24 hours at 37 ° C.
  • mice After leaving for 2 weeks from the 30th day in FIG. 1, all mice were again shaved and depilated for the purpose of inducing a hapten-induced type IV allergic reaction.
  • Photographs were taken on the second day after application of DNFB (Fig. 2D). On day 6 after application of DNFB, a serum sample was collected, then euthanized, and the skin on the back of the head with DNFB applied and the skin near the base of the tail without DNFB were collected and stained with hematoxylin-eosin (HE). Performed (FIGS. 5A-5D).
  • the degree of dermatitis of the mice A to D shown in FIG. 2 was scored according to the criteria shown in Table 2 below. Scoring was based on the literature reported by Fan et al. (Fan et al., Exp Biol Med, 226, 1045-1050, 2001). The degree of score was evaluated with a score of 0 to 3 for three skin lesions.
  • the number of colonies in the agar medium was 5, 7, and 8 for the nanoparticle water spray group, whereas that for the distilled water spray group was 48 and 85. It was confirmed that the antibacterial ability of nanoparticulate water was exhibited early after spraying.
  • Photographs were taken on days 0, 3, 7, 11, 13, 15, 22, 23, and 24, and dermatitis was scored according to the criteria shown in Table 2 above. The dorsal head side and caudal side were scored separately, and the total value was evaluated.
  • Blood sampling is performed before tape stripping (FIG. 7, blood sampling 1), 22 days after induction of tape stripping (FIG. 7, blood sampling 2), and 2 days after starting re-induction with DNFB application (FIG. 7, blood sampling 3).
  • the amount of IgE in the serum fraction was measured by sandwich ELISA.
  • the skin tissue on the dorsal head side and the caudal side of the mouse was collected. A part of the skin tissue was used for HE tissue staining evaluation. In addition, mRNA was extracted from the remaining part, and various cytokine expression levels (IL-1 ⁇ , TNF- ⁇ , IL-6, IL-17A, IL-4, IFN- ⁇ ) were examined by quantitative RT-PCR. . The expression level was normalized with respect to the HPRT gene and evaluated by relative expression level. Table 5 shows primer sequences for each mouse cytokine.
  • the degree of dermatitis induced when restimulated with DNFB application was milder in the nanoparticle water spray group than in the water spray group, suggesting a preventive effect.
  • the skin damage caused by shaving with a shaver and hair removal cream before DNFB application was lower in the nanoparticle water spray group.
  • HE skin histopathological image Fig. 11 shows an HE-stained image of skin tissue after reinduction of inflammation.
  • DNFB-applied dorsal epidermis thickening and dermal inflammation were milder in the nanoparticle water spray group than in the water spray group.
  • epidermal thickening was markedly suppressed in the nanoparticle water spray group, and no inflammatory cell infiltration was observed.
  • Cytokine expression level Fig. 12 shows the results of examining various cytokine expression levels by dividing the dermatitis induction part of the back into the dorsal head side (with DNFB application) and caudal side (without DNFB application).
  • cytokines associated with the disruption of barrier function such as IL-1 ⁇ , TNF- ⁇ , IL-6, etc., with or without nanoparticulate water spray, do not have DNFB applied to the dorsal temporal side and DNFB Expression was observed on the tail side of the painted area.
  • DNFB IL-17A, IL-4, and IFN- ⁇
  • cytokines produced by T cells were low in both cranial and caudal skin with and without nanoparticle water spray .
  • the nanoparticle water spray group is more effective than the distilled water spray group. It is concluded that Furthermore, compared with the distilled water spray group, the nanoparticle water spray group showed mild epidermal thickening and dermal inflammation, and the lesion was mild even after induction of dermatitis by DNFB application again. It is thought that spraying plays a role in controlling this and protecting the skin against repeated inflammation induction.
  • the blood IgE level at the end of the experiment was significantly higher than the blood IgE level at the start of the experiment and before reinducing the inflammation, but unlike the NC / NgaSlc mice, the BALB / c mice had nanoparticles. There was no significant difference between the water spray group and the water spray group.
  • Photographing was performed on days 0, 3, 7, 11, 13, 15, 22, 23, 24, and the therapeutic effect on skin inflammation was observed, and the degree of dermatitis was scored according to the criteria shown in Table 2 above. . It was scored separately for the dorsal head side (treatment model) and caudal side (prevention model) and evaluated.
  • Blood samples were collected on day 0 (blood collection 1), day 22 (blood collection 2) and day 24 (blood collection 3), and the IgE amount of the serum fraction was measured by sandwich ELISA.
  • mice skin tissues on the dorsal head side (treatment model) and caudal side (prevention model) of mice were collected, and HE tissue staining observation and cytokine expression level measurement were performed.
  • FIG. 14 shows a photograph of the back of the mouse
  • FIG. 15 shows score evaluation results of the inflammatory state.
  • the therapeutic effect on dermatitis induced by tape stripping and DNFB application was superior to nanoparticle water spray than water spray.
  • the degree of dermatitis induced when restimulated by DNFB application was lower in the nanoparticle water spray group than in the water spray group, and was superior in the preventive effect.
  • damage to the skin when shaving with a shaver and a hair removal cream before applying DNFB was lower in the nanoparticle water spray group.
  • FIG. 17 shows an HE stained image of the skin pathological tissue after induction of inflammation.
  • a preventive model of dermatitis the skin thickening on the dorsal and caudal side where nanoparticle water or water was sprayed first and dermatitis was induced before the end of the experiment was observed in the nanoparticle spray group than in the water spray group. It was mild.
  • the skin on the back of the head which was previously applied with DNFB and subsequently sprayed with nanoparticulate water or water, also showed that epidermal thickening was milder in the nanoparticle spray group than in the water spray group. there were.
  • FIG. 18 shows the results of examining the expression levels of various cytokines divided into models.
  • Cytokines associated with disruption of barrier function such as IL-1 ⁇ , TNF- ⁇ , and IL-6 are induced by applying DNFB before the end of the experiment as a model for preventing dermatitis with or without nanoparticle spraying.
  • DNFB was first applied as a treatment model for the caudal part and the existing dermatitis, and expression was observed in both the dorsal head side without DNFB application immediately before the end of the experiment. Furthermore, a high tendency was observed in the dorsal caudal side where DNFB was applied before skin collection.
  • cytokines produced by T cells IL-17A, IL-4, IFN Regardless of the presence or absence of nanoparticle spraying, - ⁇ increased in expression on the dorsal caudal side where DNFB was applied before the end of the experiment compared to the cranial side where DNFB was not applied before the end of the experiment.
  • the expression level of IFN- ⁇ tended to increase in the nanoparticle water spray group compared to the water spray group.
  • Tape stripping was repeatedly performed according to the schedule shown in FIG. After shaving and removing the back of 12-week-old BALB / c mice, measuring the amount of water transpiration (indicator of skin barrier function) and stripping the tape so that the value is in the range of 30-50 g / hm 2 ( 3-5 times).
  • the Multi Probe Adapter System (abbreviation MPA5) (software: CK-MPA-multi-probe version 1.5.1.4) of Courage + Khazaka electronic GmbH (Cologne, Germany)
  • a probe to which a Tewameter (registered trademark) probe (TM300MP) was connected was used. In the measurement, the probe was pressed against the left foot base so as not to be displaced.
  • the thickness of the epidermis is thicker in the water spray group than in the nanoparticle water spray group in the HE-stained skin sample collected on the 14th day, as shown in FIG.
  • cytokines associated with disruption of epidermal barrier function such as IL-1 ⁇ , TNF- ⁇ , IL-6 after repeated tape stripping was examined by RT-PCR method (results are shown in FIG. 23). Regardless of the presence or absence of nanoparticle water spray, the same level of expression was observed on both the dorsal caudal side and the cranial side.
  • the expression levels of cytokines produced by T cells were low for IL-4 and IFN- ⁇ , and there was no significant difference between the two groups, but IL- The expression level of 17A was higher in the water spray group than in the nanoparticle water spray group, suggesting that the skin barrier function was more impaired in the water spray group than in the nanoparticle water spray group.
  • the dermatitis score was defined as shown in Table 7 below, and the degree of dermatitis was scored. For each mouse, a score of 0 to 3 was assigned to 7 items of erythema, scales, wrinkles, protuberances, crusts, left auricle thickening / deformation, and right auricle thickening / deformation. did. The maximum score per animal is 21 points. The dermatitis score was measured on mice of each experimental group on day 5, day 12, day 19, day 26, day 33.
  • FIG. 25 shows the results of tracking the changes in the dermatitis scores on the back of the head and DNA after application of DNFB. Moreover, the image of the head side dorsal skin and both ears of each experimental group on day33 is shown in FIG.
  • the dermatitis score was clearly lower in group A sprayed with nanoparticle water together with DNFB application than group C (positive control group) and water spray B group that were not sprayed with nanoparticle water (FIG. 25).
  • Group D represents a negative control in which neither DNFB application nor spraying was performed.
  • dermatitis and auricular deformation were clearly observed in Group B and Group C, while the nanoparticle water spray group (Group A) was the same as the negative control (Group D), with marked symptoms. Suppressed.
  • Fig. 28 shows the results of measurement of skin moisture transpiration.
  • Group B and Group C the amount of transpiration increased significantly on day 12 after application of DNFB, and high transpiration was observed thereafter.
  • the nano-particle water spray group Group A
  • group A showed significantly lower values than groups B and C.
  • FIGS. 30A to 30D are typical pinna and cranial dorsal skin symptoms and HE-stained images of Groups A to D.
  • FIG. A-1, B-1, C-1, and D-1 are HE tissue staining photographs of the right auricular skin
  • A-2, B-2, C-2, and D-2 are HE on the dorsal skin. It is a tissue staining photograph.
  • the black bar indicates 100 ⁇ m.
  • Group C skin inflammation was strongly observed as in Group B, and changes in the auricle and back skin were observed more strongly than Group A.
  • results are shown in FIG.
  • the amount of anti-DNP antibody does not change much between the three groups: nanoparticle water spray group (A), water spray group (B), and non-spray group (C), and immune response to DNP (reaction between B cells and Th cells) was confirmed to be innocent. This suggests that it is unlikely that the hapten-induced immune response itself has decreased due to a decrease in the reactivity of DNFB to the skin due to the presence of nanoparticles associated with the skin.
  • Results are shown in FIG. In the nano-particle water spray group (Group A), the number of bacteria varies compared to the water spray group (Group B), the non-spray group (Group C), and the untreated group (Group D) that does not perform pretreatment or DNFB application. Less and lower value.
  • Results are shown in FIG.
  • the aggregation value of the nanoparticle water spray group (Group A) is significantly lower than that of the water spray group (Group B) and the group without spray spray (Group C).
  • the skin inflammation score value was lower than that in the water spray group and the non-spray group.
  • the skin inflammation score value was related not only to the thickening of the auricle, which is an index of immune responsiveness, but also to the back skin moisture transpiration amount and the water retention level, which is an index of skin barrier function.
  • the epithelial proliferation-related cytokines were expressed along with the Th17 cell line, whereas in the water spray group and the non-spray group, it was expressed through a skin damage-related signal.
  • the number of superficial bacteria in the nanoparticle water spray group was controlled to a certain low value compared with the other three groups.
  • nanoparticle water spray controls the skin inflammatory reaction through the improvement of the skin barrier function and the immune response involved in controlling the number of superficial bacteria.

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Abstract

Disclosed is a novel means that makes it possible to treat or prevent atopic dermatitis and other such types of dermatitis. The agent for the treatment or prevention of dermatitis according to the invention contains as an active ingredient cyanoacrylate polymer particles having an average particle size of less than 1000 nm (amino acid molecule-containing nanoparticles), said particles including at least one selected from the group comprising amino acids, amino acid derivatives, and oligomers and polymers of these. The amino acid molecule-containing nanoparticles have a suppressive effect on type I and type IV allergic reactions and are therefore also useful, for example, as an agent for the treatment or prevention of allergy symptoms such as dermatitis involving a type I allergic reaction or type IV allergic reaction.

Description

ナノ粒子を有効成分とする皮膚炎の治療又は予防剤A therapeutic or preventive agent for dermatitis comprising nanoparticles as an active ingredient
　本発明は、ナノサイズのシアノアクリレートポリマー粒子を有効成分とする皮膚炎の治療又は予防剤に関する。 The present invention relates to a dermatitis treatment or prevention agent comprising nano-sized cyanoacrylate polymer particles as an active ingredient.
　主としてヒトの医薬に応用すべく、薬物のデリバリーシステム（ＤＤＳ）や徐放化による薬物の効果向上を目的に、薬剤の微粒子化の研究が進んでおり、例えばシアノアクリレートポリマー粒子に薬剤を抱合させたＤＤＳが公知である（特許文献１、２及び非特許文献１）。本願発明者も、現在までに、粒径のばらつきが少ないシアノアクリレートポリマー粒子の製造方法、抗菌剤抱合粒子、及びプラスミド抱合粒子を開示している（特許文献３～５）。従来のポリマー粒子合成法では、シアノアクリレートのアニオン重合反応の開始及び安定化の目的で、重合反応系内に糖類やポリソルベートを共存させる。これらの過去の研究は、薬物のDDSと徐放化が目的であった。 In order to improve the effect of drugs by drug delivery system (DDS) and sustained release mainly for application to human medicine, research on finer drugs is progressing. For example, drugs are conjugated to cyanoacrylate polymer particles. DDS is known ( Patent Documents 1 and 2 and Non-Patent Document 1). To date, the present inventor has also disclosed a method for producing cyanoacrylate polymer particles, antibacterial agent-conjugated particles, and plasmid-conjugated particles with little variation in particle size (Patent Documents 3 to 5). In the conventional polymer particle synthesis method, saccharides and polysorbate are allowed to coexist in the polymerization reaction system for the purpose of starting and stabilizing the anionic polymerization reaction of cyanoacrylate. These past studies aimed at drug DDS and sustained release.
　その後、本願発明者は、シアノアクリレートポリマー粒子そのものにグラム陽性細菌に対する抗菌活性があることを見出した（特許文献６）。さらに、アミノ酸を抱合したシアノアクリレートポリマー粒子が抗がん活性を有するほか（特許文献７）、グラム染色性を問わず各種の細菌に対し抗菌力を発揮できることを見出した（特許文献８、９）。ナノサイズのポリマー粒子は、細菌表面（細胞壁）に特異的に接着し、細菌を溶菌に導く。シアノアクリレートナノ粒子は、抗生物質とは全く異なる作用機序で抗菌活性を発揮し、MRSA（Methicillin-resistant Staphylococcus aureus）やVRE（Vancomycin resistant enterococcus）等の多剤耐性菌に対しても有効である。 Subsequently, the present inventor found that the cyanoacrylate polymer particles themselves have antibacterial activity against Gram-positive bacteria (Patent Document 6). Furthermore, in addition to the anticancer activity of amino acid-conjugated cyanoacrylate polymer particles (Patent Document 7), it was found that antibacterial activity can be exerted against various bacteria regardless of Gram stainability (Patent Documents 8 and 9). . Nano-sized polymer particles specifically adhere to the bacterial surface (cell wall), leading the bacteria to lysis. Cyanoacrylate nanoparticles exhibit antibacterial activity with a mechanism of action completely different from antibiotics, and are also effective against multi-drug resistant bacteria such as MRSA (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin resistant enterococcus) .
　しかしながら、アトピー性皮膚炎等の皮膚炎に対するシアノアクリレートポリマー粒子の効果は知られていない。 However, the effect of cyanoacrylate polymer particles on dermatitis such as atopic dermatitis is not known.
　アトピー性皮膚炎は、日本国内でも有病率が高い皮膚疾患である。近年増加傾向にあり、患者は多くの精神的・身体的苦痛を強いられる。 Atopic dermatitis is a skin disease with a high prevalence in Japan. Increasing in recent years, patients are forced to suffer a lot of mental and physical pain.
　アトピー性皮膚炎の薬物療法の基本は、重症度に合わせた強度のステロイド外用薬、カルシニューリン阻害外用薬、必要に応じて抗ヒスタミン・抗アレルギー内服薬が併用され、最重症の場合にはさらに必要に応じてステロイド内服薬、カルシニューリン阻害内服薬が用いられる（非特許文献２）。しかしながら、ステロイド剤の大量連用による副作用を恐れてステロイド剤を忌避する患者が多く、また免疫抑制による易感染性の問題もあるため、新たな治療手段が求められている。 The basics of pharmacotherapy for atopic dermatitis include the use of steroid topical drugs, calcineurin-inhibiting drugs, and anti-histamine / anti-allergic drugs as needed according to severity. Accordingly, oral steroids and oral calcineurin inhibitor are used (Non-patent Document 2). However, there are many patients who are afraid of side effects due to continuous use of steroids and avoid steroids, and there is a problem of infectivity due to immunosuppression, so new treatment means are required.
特表平１１－５０３１４８号公報Japanese National Patent Publication No. 11-503148 特表２００２－５０４５２６号公報JP-T-2002-504526 特開２００８－１２７５３８号公報JP 2008-127538 A 国際公開第２００８／１２６８４６号公報International Publication No. 2008/126846 特開２００８－２０８０７０号公報JP 2008-208070 A 国際公開第２００９／０８４４９４号公報International Publication No. 2009/084494 国際公開第２０１０／１０１１７８号公報International Publication No. 2010/101178 国際公開第２０１２／１３３６４８号公報International Publication No. 2012/133648 国際公開第２０１３／１０８８７１号公報International Publication No. 2013/108871
　本発明は、アトピー性皮膚炎等の皮膚炎を治療又は予防できる新規な手段を提供することを目的とする。 An object of the present invention is to provide a novel means capable of treating or preventing dermatitis such as atopic dermatitis.
　本願発明者らは、皮膚炎モデルマウスを用いて鋭意研究した結果、抗菌活性が知られているナノサイズのシアノアクリレートポリマー粒子が皮膚炎に対しても有効であることを見出し、本願発明を完成した。 As a result of intensive research using dermatitis model mice, the present inventors have found that nano-sized cyanoacrylate polymer particles with known antibacterial activity are also effective against dermatitis, and completed the present invention. did.
　すなわち、本発明は、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子を有効成分として含有する、皮膚炎の治療又は予防剤を提供する。また、本発明は、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子を有効成分として含有する、I型アレルギー反応又はIV型アレルギー反応の抑制剤を提供する。さらに、本発明は、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子を有効成分として含有する、I型アレルギー反応又はIV型アレルギー反応が関与するアレルギー症状の治療又は予防剤を提供する。さらに、本発明は、治療すべき皮膚病変部、又は皮膚炎の発生の予防が望まれる皮膚領域に、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子の有効量を投与することを含む、皮膚炎の治療又は予防方法を提供する。さらに、本発明は、それを必要とする対象に対し、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子の有効量を投与することを含む、I型アレルギー反応又はIV型アレルギー反応の抑制方法を提供する。さらに、本発明は、それを必要とする対象に対し、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子の有効量を投与することを含む、I型アレルギー反応又はIV型アレルギー反応が関与するアレルギー症状の治療又は予防方法を提供する。 That is, the present invention contains at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient. An agent for treating or preventing inflammation is provided. Further, the present invention contains at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient. The present invention provides an inhibitor of type IV allergic reaction or type IV allergic reaction. Furthermore, the present invention contains at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient. Provided is a therapeutic or prophylactic agent for allergic symptoms involving type I allergic reaction or type IV allergic reaction. Furthermore, the present invention includes at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof in skin lesions to be treated or skin regions where prevention of the occurrence of dermatitis is desired. Providing a method of treating or preventing dermatitis comprising administering an effective amount of cyanoacrylate polymer particles having an average particle size of less than 1000 nm. Furthermore, the present invention provides a cyanoacrylate polymer having an average particle size of less than 1000 nm, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof for a subject in need thereof. Provided is a method for suppressing a type I allergic reaction or a type IV allergic reaction, comprising administering an effective amount of particles. Furthermore, the present invention provides a cyanoacrylate polymer having an average particle size of less than 1000 nm, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof for a subject in need thereof. A method of treating or preventing allergic symptoms involving type I allergic reactions or type IV allergic reactions, comprising administering an effective amount of particles.
　本発明により、ステロイド系薬剤とは異なる新規な皮膚炎の治療又は予防手段が提供される。本発明の治療又は予防剤の有効成分であるシアノアクリレートナノ粒子は、薬剤のDDSとして用いられるのではなく、粒子それ自体が皮膚炎の治療及び予防効果を現す。ステロイド系薬剤に不安を抱く患者や、副作用のためステロイド系薬剤を使用できない患者、さらには易感染性を示す免疫能低下を認める患者に対しても、本発明の治療又は予防剤を好ましく使用することができる。 The present invention provides a novel means for treating or preventing dermatitis different from steroidal drugs. The cyanoacrylate nanoparticles, which are the active ingredients of the therapeutic or prophylactic agent of the present invention, are not used as a DDS for drugs, but the particles themselves exhibit a therapeutic and prophylactic effect for dermatitis. The therapeutic or prophylactic agent of the present invention is preferably used even for patients who are anxious about steroidal drugs, patients who cannot use steroidal drugs due to side effects, and patients who are found to be susceptible to immunological decline. be able to.
NC/NgaSlcマウスに対する処置スケジュールを示す（検討１）。The treatment schedule for NC / NgaSlc mice is shown (Study 1). 図１に示すスケジュールで皮膚炎を誘発させたNC/NgaSlcマウスの皮膚病変部の写真である。Aは、初回テープストリッピングから３週間後の病変部。Bは、ナノ粒子水又は滅菌水の噴霧処置開始から２日目の病変部。Cは、ナノ粒子水又は滅菌水の噴霧処置開始から８日目の病変部。Dは、DNFB塗布開始から３日目の病変部。It is the photograph of the skin lesion part of the NC / NgaSlc mouse | mouth which induced dermatitis by the schedule shown in FIG. A is the lesion 3 weeks after the initial tape stripping. B is the lesion on the second day from the start of the spray treatment with nanoparticle water or sterilized water. C is a lesion on the eighth day from the start of the spray treatment with nanoparticle water or sterilized water. D is the lesion on the third day from the start of DNFB application. 皮膚炎誘導部における皮膚表面の細菌数を測定した結果である。It is the result of having measured the number of bacteria of the skin surface in a dermatitis induction part. 図２に示した病変部の皮膚炎症状をスコア化したグラフである。It is the graph which scored the skin inflammation state of the lesioned part shown in FIG. 図１に示すスケジュールで、テープストリッピング誘発性皮膚炎を観察した後に、DNFBを塗布して８日目のNC/NgaSlcマウスの背部皮膚組織のHE染色像である。FIG. 1 is an HE-stained image of the dorsal skin tissue of NC / NgaSlc mice on the 8th day after applying DNFB after observing tape stripping-induced dermatitis according to the schedule shown in FIG. 図１に示したスケジュールで皮膚炎を誘発させたNC/NgaSlcマウスの血中IgE濃度を測定した結果である。実験開始時（採血１）及び終了時（採血２）の比較を示す。It is the result of having measured the blood IgE density | concentration of the NC / NgaSlc mouse | mouth which induced dermatitis by the schedule shown in FIG. A comparison between the start of the experiment (blood collection 1) and the end of the experiment (blood collection 2) is shown. 皮膚炎予防モデルにおけるナノ粒子水の効果を検討した、BALB/cマウスに対する処置スケジュールを示す（検討２）。The treatment schedule with respect to the BALB / c mouse which examined the effect of the nanoparticle water in the dermatitis prevention model is shown (examination 2). 図７に示すスケジュールで皮膚炎を誘発させたBALB/cマウスの皮膚病変部の写真である。It is the photograph of the skin lesion part of the BALB / c mouse | mouth which induced dermatitis by the schedule shown in FIG. 図８に示した病変部の皮膚炎症状をスコア化したグラフである。It is the graph which scored the skin inflammation state of the lesioned part shown in FIG. 図７に示すスケジュールで皮膚炎を誘発させたBALB/cマウスの血中IgE濃度を測定した結果である（day0, day22, day24）。It is the result of having measured the blood IgE density | concentration of the BALB / c mouse which induced the dermatitis by the schedule shown in FIG. 7 (day0, day22, day24). 図７に示すスケジュールで皮膚炎を誘発させたBALB/cマウスの背部皮膚組織のHE染色像である。DNFB塗布後３日目の背部皮膚を、塗布した頭側と塗布していない尾側で比較した。FIG. 8 is an HE-stained image of dorsal skin tissue of BALB / c mice in which dermatitis was induced according to the schedule shown in FIG. 7. The dorsal skin 3 days after DNFB application was compared between the applied head side and the unapplied caudal side. 図７に示すスケジュールで皮膚炎を誘発させた皮膚炎予防モデル群のBALB/cマウスの背部頭側（DNFB塗布あり）及び尾側（DNFB塗布なし）の皮膚における各種サイトカインの発現量を調べた結果である。The expression levels of various cytokines in the dorsal head side (with DNFB application) and caudal side (without DNFB application) skin of BALB / c mice in the dermatitis prevention model group in which dermatitis was induced according to the schedule shown in FIG. It is a result. 皮膚炎治療モデルおよび予防モデルとして検討したBALB/cマウスに対する処置スケジュールを示す（検討３）。The treatment schedule for BALB / c mice examined as a dermatitis treatment model and prevention model is shown (Study 3). 図１３に示すスケジュールで皮膚炎を誘発させたBALB/cマウスの皮膚病変部の写真である。It is the photograph of the skin lesion part of the BALB / c mouse | mouth which induced dermatitis by the schedule shown in FIG. 図１４に示した病変部の皮膚炎の程度をスコア化したグラフである。It is the graph which scored the grade of the dermatitis of the lesioned part shown in FIG. 図１３に示すスケジュールで皮膚炎を誘発させたBALB/cマウスの血中IgE濃度を測定した結果である（day0, day22, day24）。It is the result of having measured the blood IgE density | concentration of the BALB / c mouse which induced the dermatitis by the schedule shown in FIG. 13 (day0, day22, day24). 図１３に示すスケジュールで、予防モデルとして、ナノ粒子水あるいは水を噴霧後、皮膚炎を誘発させたBALB/cマウスの背部皮膚組織のHE染色像である。予防モデルへの皮膚炎を誘発するため、背部尾側にDNFBを塗布して３日目の組織である。比較のため、治療モデルとして、実験開始時にDNFBに塗布した背部頭側の皮膚も染色した。FIG. 14 is an HE-stained image of the dorsal skin tissue of BALB / c mice in which dermatitis was induced after spraying with nanoparticle water or water as a prevention model in the schedule shown in FIG. 13. In order to induce dermatitis to the prevention model, DNFB is applied to the back and tail side of the tissue on the third day. For comparison, as a treatment model, the dorsal temporal skin applied to DNFB at the start of the experiment was also stained. 図１３に示すスケジュールで皮膚炎を誘発させたBALB/cマウスの背部頭側（治療モデルとして実験開始時にのみDNFBを塗布し、以後ナノ粒子水あるいは水を噴霧したもの）及び尾側（予防モデルとして、ナノ粒子水あるいは水を噴霧し、実験終了前にDNFBを塗布し、３日目）における各種サイトカインの発現量を調べた結果である。The dorsal temporal side of BALB / c mice in which dermatitis was induced by the schedule shown in FIG. 13 (DNFB was applied only at the start of the experiment as a treatment model, and then sprayed with nanoparticle water or water) and the caudal side (prevention model) As a result of spraying nanoparticle water or water, applying DNFB before the end of the experiment, and examining the expression levels of various cytokines on the third day). テープストリッピング誘発性皮膚炎モデルとしたBALB/cマウスに対する処置スケジュールを示す（検討４）。The treatment schedule with respect to the BALB / c mouse made into the tape stripping induction dermatitis model is shown (examination 4). 図１９に示すスケジュールに従い、テープストリッピングを繰り返して皮膚炎を誘発させたBALB/cマウスの皮膚炎の程度をスコア化したグラフである。It is the graph which scored the grade of the dermatitis of the BALB / c mouse | mouth which induced the dermatitis by repeating tape stripping according to the schedule shown in FIG. 図１９に示すスケジュールに従い、繰り返しテープストリッピングにより皮膚炎を誘発させたBALB/cマウスの皮膚炎誘導部における水分蒸散量を測定した結果である。It is the result of having measured the amount of water transpiration in the dermatitis induction part of the BALB / c mouse which induced dermatitis by repeated tape stripping according to the schedule shown in FIG. 図１９に示すスケジュールに従い、繰り返しテープストリッピングにより皮膚炎を誘発させたBALB/cマウスの背部皮膚組織のHE染色像である（day14）。ナノ粒子水あるいは水噴霧群それぞれから、無作為に選んだ2匹の組織を示す。FIG. 20 is an HE-stained image of the back skin tissue of BALB / c mice in which dermatitis was induced by repeated tape stripping according to the schedule shown in FIG. 19 (day 14). Two tissues randomly selected from each group of nanoparticle water or water spray are shown. 図１９に示すスケジュールで繰り返しテープストリッピングにより皮膚炎を誘発させたBALB/cマウスの背部皮膚における各種サイトカインの発現量を調べた結果である。同じ処置をした頭側および尾側の皮膚について、それぞれ測定した。FIG. 20 shows the results of examining the expression levels of various cytokines in the back skin of BALB / c mice in which dermatitis was induced by repeated tape stripping according to the schedule shown in FIG. Measurements were made on cranial and caudal skin treated the same way, respectively. 繰り返しDNFB塗布によるハプテン誘発性慢性皮膚炎モデルとして検討した、ヘアレスマウスに対する処置スケジュールを示す（検討５）。The treatment schedule for hairless mice examined as a hapten-induced chronic dermatitis model by repeated DNFB application is shown (Study 5). 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、皮膚炎症状をスコア化し、スコアの変化を調べた結果である。FIG. 25 shows the result of scoring skin inflammation for each group of hairless mice in which dermatitis was induced according to the schedule shown in FIG. 24 and examining changes in the score. 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウスの皮膚病変部の写真である（day33）。FIG. 25 is a photograph of a skin lesion of a hairless mouse in which dermatitis is induced according to the schedule shown in FIG. 24 (day 33). 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、耳介の厚さの変化を調べた結果である。It is the result of having investigated the change of the thickness of auricle about the individual of each group of hairless mice which induced dermatitis by the schedule shown in FIG. 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、頭側背部皮膚の水分蒸散量の変化を調べた結果である。It is the result of having investigated the change of the moisture transpiration | evaporation amount of the head side dorsal skin about the individual of each group of hairless mice which induced dermatitis by the schedule shown in FIG. 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、頭側背部皮膚の保水度の変化を調べた結果である。It is the result of having investigated the change of the water retention degree of the head side dorsal skin about the individual of each group of hairless mice which induced dermatitis by the schedule shown in FIG. 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウスの、day33における代表的な炎症状態及び炎症誘発部位のHE染色像である。A～DがそれぞれA群～D群を示す。中央のA-1～D-1が右耳介皮膚組織、右のA-2～D-2が頭側背部皮膚組織である。組織像中のバーは100μmを示す。[Fig. 25] Fig. 25 is a HE-stained image of a typical inflammatory state and a pro-inflammatory site on day 33 of a hairless mouse in which dermatitis was induced by the schedule shown in Fig. 24. A to D represent groups A to D, respectively. The central A-1 to D-1 are the right auricular skin tissue, and the right A-2 to D-2 are the cranial dorsal skin tissue. The bar in the tissue image indicates 100 μm. 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウスの、炎症誘発部位における各種サイトカインの、HPRTに対する相対的発現量である（day34）。24 is the relative expression level of various cytokines at the site of inflammation induction in hairless mice in which dermatitis was induced by the schedule shown in FIG. 24 (day 34). 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、血清中の抗DNP IgG抗体をELISAにより測定した結果である（day34）。It is the result of measuring the anti-DNP IgG antibody in serum by ELISA for individuals of each group of hairless mice in which dermatitis was induced by the schedule shown in FIG. 24 (day 34). 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、背部頭側の皮膚表在細菌数の変化を調べた結果である。It is the result of having investigated the change of the number of skin superficial bacteria of the back head side about the individual of each group of hairless mice which induced dermatitis by the schedule shown in FIG. 図２４に示すスケジュールで皮膚炎を誘発させたヘアレスマウス各群の個体について、還元血清中の抗S.aureus抗体（主としてIgG）の凝集価を調べた結果である。It is the result of having investigated the aggregation titer of the anti-S. Aureus antibody (mainly IgG) in a reduced serum about the individual of each group of hairless mice which induced dermatitis with the schedule shown in FIG.
　本発明で用いる粒子は、シアノアクリレートモノマーをアニオン重合して得られるナノサイズ（平均粒径1000 nm未満）のポリマー粒子であり、アミノ酸、アミノ酸誘導体、それらのオリゴマー及びポリマー（以下、これらを総称して「アミノ酸系分子」ということがある）から選択される少なくとも１種を含む。さらに、糖及びポリソルベートからなる群より選択される少なくとも１種を含んでいてよい。 The particles used in the present invention are nano-sized polymer particles (average particle size of less than 1000 nm) obtained by anionic polymerization of a cyanoacrylate monomer, and amino acids, amino acid derivatives, oligomers and polymers thereof (hereinafter collectively referred to as “general names”). And at least one selected from “amino acid molecules”. Furthermore, at least 1 sort (s) selected from the group which consists of saccharide | sugar and polysorbate may be included.
　アミノ酸系分子、糖及びポリソルベートは、シアノアクリレートモノマーのアニオン重合の重合開始・安定剤として使用できることが知られている。例えば、糖及び／又はポリソルベートを重合開始・安定剤として用いるナノ粒子製造法は、特許文献３、特許文献４（抗菌剤抱合粒子）、特許文献５（プラスミド抱合粒子）等に記載され公知である。アミノ酸系分子を重合開始・安定剤として用いるナノ粒子製造法は、特許文献８、９（アミノ酸系分子の単独使用）等に記載され公知である。また、特許文献７には、アミノ酸と糖類・ポリソルベートを併用する製造法が記載されている。これらの重合開始・安定剤は、いずれか１種のみを使用してもよいし、２種以上を組み合わせて使用してもよい。 It is known that amino acid molecules, sugars and polysorbates can be used as polymerization initiators / stabilizers for anionic polymerization of cyanoacrylate monomers. For example, a nanoparticle production method using sugar and / or polysorbate as a polymerization initiator / stabilizer is described in Patent Document 3, Patent Document 4 (antibacterial agent-conjugated particle), Patent Document 5 (plasmid-conjugated particle) and the like. . Nanoparticle production methods using amino acid molecules as polymerization initiators / stabilizers are described in Patent Documents 8 and 9 (use of amino acid molecules alone) and the like. Patent Document 7 describes a production method in which an amino acid and a saccharide / polysorbate are used in combination. Any one of these polymerization initiators / stabilizers may be used, or two or more thereof may be used in combination.
　本発明において、「アミノ酸」とは、分子内にアミノ基とカルボキシ基とを持つ化合物をいい、一般的なアミノ酸の定義の通り、アミノ基の水素が分子内の他の部分と置換して二級アミンとなった環状化合物であるイミノ酸も包含する。本発明で使用できるアミノ酸の代表的な例としては、天然のタンパク質を構成する２０種のα－アミノ酸（アルギニン、ヒスチジン、リジン、アスパラギン酸、グルタミン酸、アラニン、グリシン、ロイシン、バリン、イソロイシン、セリン、スレオニン、フェニルアラニン、トリプトファン、チロシン、シスチン又はシステイン、グルタミン、アスパラギン、プロリン、メチオニン）が挙げられるが、これらに限定されず、β－、γ－及びδ－アミノ酸系分子も包含される。具体例を挙げると、アルギニン、ヒスチジン、リジン、アスパラギン酸、グルタミン酸、アラニン、グリシン、ロイシン、バリン、イソロイシン、セリン、スレオニン、フェニルアラニン、トリプトファン、チロシン、シスチン又はシステイン、グルタミン、アスパラギン、プロリン、メチオニン、β－アラニン、γ－アミノ酪酸（ＧＡＢＡ；神経伝達物質）、カルニチン、γ－アミノレブリン酸、γ－アミノ吉草酸などが挙げられる。好ましいアミノ酸の例として、アルギニン、ヒスチジン、リジン、アスパラギン酸、グルタミン酸、アラニン、グリシン、ロイシン、バリン、イソロイシン、セリン、スレオニン、フェニルアラニン、トリプトファン、チロシン、シスチン又はシステイン、グルタミン、アスパラギン、プロリン、及びメチオニンから選択される少なくとも１種、中でも特に、グリシン及びアスパラギン酸からなる群より選択される少なくとも１種を挙げることができるが、これらに限定されない。 In the present invention, an “amino acid” refers to a compound having an amino group and a carboxy group in the molecule. As defined in general amino acids, the amino group hydrogen is substituted for other parts in the molecule. Also included are imino acids, which are cyclic compounds that have been converted to secondary amines. Representative examples of amino acids that can be used in the present invention include 20 kinds of α-amino acids (arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, which constitute natural proteins. Threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, methionine), but not limited to, β-, γ- and δ-amino acid based molecules are also included. Specific examples include arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, methionine, β -Alanine, γ-aminobutyric acid (GABA; neurotransmitter), carnitine, γ-aminolevulinic acid, γ-aminovaleric acid and the like. Examples of preferred amino acids include arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, and methionine. At least one selected from among the group consisting of glycine and aspartic acid can be mentioned, but not limited thereto.
　「アミノ酸誘導体」とは、上記定義によるアミノ酸においていずれかの基が修飾又は置換された構造を有する化合物をいう。生物体成分として天然に存在するアミノ酸誘導体は、通常、本発明で好ましく使用することができる。使用可能なアミノ酸誘導体の具体例を挙げると、クレアチン（アルギニン誘導体で1-メチルグアニジノ酢酸）、オルニチン（アルギニン誘導体で尿素サイクル産物）、サイロキシン（芳香族アミノ酸類であるトリヨウドサイロニン；Ｔ４）、デスモシン（角質エラスチンやコラーゲンの構成成分；3分子のアリシンの側鎖と1分子のリシンの側鎖が結合した構造）、ヒドロキシプロリン及びヒドロキシリジン（ゼラチンやコラーゲン構成成分）、ホスホセリン（セリンとリン酸のエステル；カゼイン構成成分）、テアニン（茶成分、グルタミン酸誘導体）、カイニン酸（海人草の虫下し成分）、トリコロミン酸（シメジの成分）やサルコシン（卵黄・ハム・豆類成分；Nメチルグリシン）等が挙げられるが、これらに限定されない。 “Amino acid derivative” refers to a compound having a structure in which any group is modified or substituted in an amino acid as defined above. In general, amino acid derivatives that naturally exist as biological components can be preferably used in the present invention. Specific examples of amino acid derivatives that can be used include creatine (arginine derivative with 1-methylguanidinoacetic acid), ornithine (arginine derivative with urea cycle product), thyroxine (aromatic amino acids triiodothyronine; T4), Desmosine (constituent component of keratin elastin and collagen; a structure in which three side chains of allicin and one side chain of lysine are combined), hydroxyproline and hydroxylysine (constituent components of gelatin and collagen), phosphoserine (serine and phosphate) Ester; casein component), theanine (tea component, glutamic acid derivative), kainic acid (component of seaweed insects), tricolominic acid (shimeji component), sarcosine (egg yolk / ham / bean component; N methylglycine), etc. However, it is not limited to these.
　アミノ酸の「オリゴマー」とは、１０個以下のアミノ酸残基がペプチド結合により結合したオリゴペプチドをいい、アミノ酸の「ポリマー」とは、１１個以上のアミノ酸残基がペプチド結合により結合したポリペプチドをいう。いずれも、アミノ酸だけではなくアミノ酸誘導体を残基として含んでいてよい。ポリペプチドの残基数の上限は特に限定されないが、例えば５００残基以下であり得る。ポリペプチドとしては、１１～１００残基、１１～５０残基、１１～３０残基、１１～２０残基、あるいは１１～１５残基のものが好ましく用いられ得る。 An amino acid “oligomer” refers to an oligopeptide in which 10 or less amino acid residues are bonded by peptide bonds, and an amino acid “polymer” refers to a polypeptide in which 11 or more amino acid residues are bonded by peptide bonds. Say. Any of them may contain not only amino acids but also amino acid derivatives as residues. The upper limit of the number of residues of the polypeptide is not particularly limited, but may be, for example, 500 residues or less. As the polypeptide, those having 11 to 100 residues, 11 to 50 residues, 11 to 30 residues, 11 to 20 residues, or 11 to 15 residues can be preferably used.
　オリゴペプチドはポリペプチドよりも好ましく用いられ得る。中でも、２～７残基、２～５残基、あるいは２又は３残基のオリゴペプチドがより好ましく用いられ得る。 Oligopeptides can be used more preferably than polypeptides. Among these, oligopeptides having 2 to 7 residues, 2 to 5 residues, or 2 or 3 residues can be more preferably used.
　上記した特許文献８～９に記載されている通り、天然のタンパク質を構成する２０種のα－アミノ酸のいずれでも、糖類やポリソルベートを使用しない条件でナノサイズ（1000nm未満）のシアノアクリレートポリマー粒子を合成できる。中性・酸性・塩基性アミノ酸のいずれでも、そして直鎖・芳香族・イミノ・含硫黄構造のいずれでも、糖類もポリソルベートも使用せずにナノ粒子を製造できることが示されている。従って、２０種のα－アミノ酸のみならず、上記したその他のアミノ酸及びアミノ酸誘導体もナノ粒子合成に使用することができるし、また、オリゴペプチドやポリペプチドも分子内にアミノ酸構造を有するので、やはりナノ粒子合成に使用することができる。 As described in the above-mentioned Patent Documents 8 to 9, nano-sized (less than 1000 nm) cyanoacrylate polymer particles are used under the condition that any of the 20 α-amino acids constituting the natural protein does not use saccharides or polysorbate. Can be synthesized. It has been shown that nanoparticles can be produced without using saccharides or polysorbates with any of neutral, acidic, basic amino acids, and linear, aromatic, imino, and sulfur-containing structures. Therefore, not only the 20 α-amino acids but also the other amino acids and amino acid derivatives described above can be used for nanoparticle synthesis, and since oligopeptides and polypeptides also have amino acid structures in the molecule, Can be used for nanoparticle synthesis.
　「糖」には、水酸基を有する単糖類（例えばグルコース、マンノース、リボース及びフルクトース等）、水酸基を有する二糖類（例えばマルトース、トレハロース、ラクトース及びスクロース等）及び水酸基を有する多糖類（例えばデキストランやマンナン等）が包含される。これらの糖は、環状、鎖状のいずれの形態であってもよく、また、環状の場合、ピラノース型やフラノース型等のいずれであってもよい。また、糖には種々の異性体が存在するがそれらのいずれでもよい。 “Sugar” includes monosaccharides having a hydroxyl group (such as glucose, mannose, ribose and fructose), disaccharides having a hydroxyl group (such as maltose, trehalose, lactose and sucrose) and polysaccharides having a hydroxyl group (such as dextran and mannan). Etc.). These sugars may be either cyclic or chain-like, and when they are cyclic, they may be any one of pyranose type, furanose type and the like. In addition, there are various isomers of sugar, and any of them may be used.
　「ポリソルベート」には、ポリオキシエチレンソルビタンモノラウレート（商品名 Tween 20）、ポリオキシエチレンソルビタンモノオレエート（商品名 Tween 80）等の各種のTween系界面活性剤が包含される。 “Polysorbate” includes various Tween-based surfactants such as polyoxyethylene sorbitan monolaurate (trade name “Tween 20”) and polyoxyethylene sorbitan monooleate (trade name “Tween 80”).
　単糖類、二糖類及び多糖類並びにポリソルベートは、単独で用いることもできるし、２種以上を組み合わせて用いることもできる。上記した糖及びポリソルベートのうち、グルコース、デキストラン、Tween 20（商品名）が安価に入手できコスト面で有利である。デキストランとしては、平均分子量５万程度以上の重合度であるデキストランが好ましい。デキストランの分子量の上限は特にないが、通常、分子量50万程度以下である。 Monosaccharides, disaccharides and polysaccharides, and polysorbates can be used alone or in combination of two or more. Among the sugars and polysorbates described above, glucose, dextran, and Tween 20 (trade name) are available at low cost, which is advantageous in terms of cost. As the dextran, dextran having a polymerization degree of about 50,000 or more in average molecular weight is preferable. There is no particular upper limit on the molecular weight of dextran, but it is usually about 500,000 or less.
　本発明で用いるナノ粒子としては、重合開始・安定剤としてアミノ酸系分子を使用し、糖及びポリソルベートのいずれも使用せずに合成した粒子（すなわち、アミノ酸系分子を含み、糖及びポリソルベートのいずれも含まない粒子）、並びに、重合開始・安定剤としてアミノ酸系分子及び糖を併用し、ポリソルベートを使用せずに合成した粒子（すなわち、アミノ酸系分子及び糖を含み、ポリソルベートを含まず、SDS等のその他の界面活性剤も含まない粒子）が好ましい。 Nanoparticles used in the present invention are particles synthesized using amino acid-based molecules as polymerization initiators / stabilizers and without using either sugar or polysorbate (that is, containing amino acid-based molecules, both sugar and polysorbate Particles that do not contain), as well as particles synthesized using amino acid molecules and sugars as polymerization initiators / stabilizers and without using polysorbates (that is, containing amino acid molecules and sugars, without polysorbates, such as SDS) Particles that do not contain other surfactants are preferred.
　シアノアクリレートモノマーとしては、アルキルシアノアクリレートモノマーが好ましい。アルキル基の炭素数は好ましくは１～８、より好ましくは２～６、さらに好ましくは３～５であり、直鎖でも分岐でもよい。またアルキル基を構成する炭素原子の少なくとも１つがハロゲン原子（塩素、臭素、ヨウ素）で置き換わっていてもよい。好ましいシアノアクリレートモノマーの具体例としては、メチル－２－シアノアクリレート、エチル－２－シアノアクリレート、ｎ－プロピル－２－シアノアクリレート、ｉ－プロピル－２－シアノアクリレート、ｎ－ブチル－２－シアノアクリレート、i-ブチル-2-シアノアクリレート、n-ペンチル-2-シアノアクリレート、ｎ－ヘキシル-2-シアノアクリレート、ｎ－ヘプチル-2-シアノアクリレート、n-オクチル-2-シアノアクリレート等を挙げることができる。これらの中でも特に、外科領域において傷口の縫合のための接着剤として従来用いられている、下記式で表されるn-ブチル-2-シアノアクリレート(nBCA)を好ましく用いることができる。 As the cyanoacrylate monomer, an alkyl cyanoacrylate monomer is preferable. The alkyl group preferably has 1 to 8 carbon atoms, more preferably 2 to 6 carbon atoms, still more preferably 3 to 5 carbon atoms, and may be linear or branched. Further, at least one of carbon atoms constituting the alkyl group may be replaced with a halogen atom (chlorine, bromine, iodine). Specific examples of preferable cyanoacrylate monomers include methyl-2-cyanoacrylate, ethyl-2-cyanoacrylate, n-propyl-2-cyanoacrylate, i-propyl-2-cyanoacrylate, and n-butyl-2-cyanoacrylate. I-butyl-2-cyanoacrylate, n-pentyl-2-cyanoacrylate, n-hexyl-2-cyanoacrylate, n-heptyl-2-cyanoacrylate, n-octyl-2-cyanoacrylate, etc. it can. Among these, in particular, n-butyl-2-cyanoacrylate (nBCA) represented by the following formula, which is conventionally used as an adhesive for stitching wounds in the surgical field, can be preferably used.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　ナノ粒子の製造工程では、適当な溶媒中に少なくとも１種の重合開始・安定剤を溶解させた後、撹拌下にて少なくとも１種のシアノアクリレートモノマーを加え、適宜撹拌を続けて重合反応を進行させればよい。本発明で用いるナノ粒子の製造においては、少なくとも１種のアミノ酸系分子を重合開始・安定剤として使用する。さらに糖及びポリソルベートからなる群より選択される少なくとも１種の重合開始・安定剤を組み合わせて用いてもよい。シアノアクリレートモノマーは、１種類のみ用いてもよいし、２種類以上を用いてもよい。 In the nanoparticle production process, at least one polymerization initiator / stabilizer is dissolved in a suitable solvent, and then at least one cyanoacrylate monomer is added under stirring, and the polymerization reaction proceeds by continuing appropriate stirring. You can do it. In the production of nanoparticles used in the present invention, at least one amino acid molecule is used as a polymerization initiator / stabilizer. Further, at least one polymerization initiator / stabilizer selected from the group consisting of sugar and polysorbate may be used in combination. Only one type of cyanoacrylate monomer may be used, or two or more types may be used.
　重合開始・安定剤として糖及び／又はポリソルベートを使用する場合、反応開始時の重合反応液中の糖及び／又はポリソルベートの濃度（複数種類用いる場合はその合計濃度）は、特に限定されないが、通常、0.5％～10％程度、好ましくは0.75％～7.5％程度である。なお、糖の濃度はw/v％、ポリソルベートの濃度はv/v％を意味し、例えば糖を単独で用いる場合には、上記した濃度範囲はそれぞれ「0.5w/v％～10w/v％」、「0.75w/v％～7.5w/v％」を意味する。また、糖を5w/v％、ポリソルベートを1v/v％で併せて用いる場合には、これらの合計濃度を6％というものとする。ただし、単糖類（例えばグルコース）のみを用いる場合には、2.5w/v％～10w/v％程度で用いることが好ましい。 When sugar and / or polysorbate is used as the polymerization initiator / stabilizer, the concentration of sugar and / or polysorbate in the polymerization reaction solution at the start of the reaction is not particularly limited. About 0.5% to 10%, preferably about 0.75% to 7.5%. The concentration of sugar means w / v%, and the concentration of polysorbate means v / v%. For example, when sugar is used alone, the above-mentioned concentration ranges are “0.5 w / v% to 10 w / v%”. ”,“ 0.75 w / v% to 7.5 w / v% ”. In addition, when sugar is used in combination at 5 w / v% and polysorbate at 1 v / v%, the total concentration is 6%. However, when only a monosaccharide (eg glucose) is used, it is preferably used at about 2.5 w / v% to 10 w / v%.
　重合開始・安定剤としてアミノ酸系分子を使用する場合、反応開始時の重合反応液中のアミノ酸系分子の濃度（複数種類用いる場合はその合計濃度）は、特に限定されないが、通常0.1w/v％～3w/v％程度である。糖及び／又はポリソルベートと併用する場合、アミノ酸系分子の使用濃度はこれより低い濃度であっても差し支えない。 When amino acid molecules are used as a polymerization initiator / stabilizer, the concentration of amino acid molecules in the polymerization reaction solution at the start of the reaction (the total concentration when multiple types are used) is not particularly limited, but is usually 0.1 w / v % To 3w / v%. When used in combination with sugar and / or polysorbate, the concentration of amino acid molecules used may be lower.
　重合反応の溶媒としては、水を主体とする水性溶媒（例えば水、低級アルコール水溶液など）を使用することができ、アミノ酸系分子含有粒子の製造の場合は、通常、水が好ましく用いられる。アニオン重合は水酸イオンにより開始されるので、反応液のpHは重合速度に影響する。反応液のpHが高い場合には、水酸イオンの濃度が高くなるので重合が速く、pHが低い場合には重合が遅くなる。アミノ酸系分子含有粒子を製造する場合には、通常、pHが1.5～3.0程度の酸性下で適度な重合速度が得られる。反応液を酸性にするために添加する酸は特に限定されず、無機酸及び有機酸のいずれでも使用することができる。例えば、塩酸は、反応に悪影響を与えず、反応後に揮散することから、アミノ酸系分子含有粒子の製造において好ましく用いることができるが、使用可能な酸はこれに限定されない。塩酸等の酸の濃度は、特に限定されないが、0.0005N～0.5N程度の範囲で適宜選択可能である。 As the solvent for the polymerization reaction, an aqueous solvent mainly composed of water (for example, water, a lower alcohol aqueous solution, etc.) can be used, and in the case of producing amino acid-based molecule-containing particles, usually water is preferably used. Since anionic polymerization is initiated by hydroxide ions, the pH of the reaction solution affects the polymerization rate. When the pH of the reaction solution is high, the hydroxyl ion concentration is high, so that the polymerization is fast, and when the pH is low, the polymerization is slow. When producing amino acid molecule-containing particles, an appropriate polymerization rate is usually obtained under acidic conditions with a pH of about 1.5 to 3.0. The acid added to make the reaction solution acidic is not particularly limited, and either an inorganic acid or an organic acid can be used. For example, hydrochloric acid does not adversely affect the reaction and is volatilized after the reaction. Therefore, hydrochloric acid can be preferably used in the production of amino acid-based molecule-containing particles, but usable acid is not limited thereto. The concentration of acid such as hydrochloric acid is not particularly limited, but can be appropriately selected within a range of about 0.0005N to 0.5N.
　アミノ酸を重合開始剤としてシアノクリレートモノマーを重合し、ナノサイズのポリマー粒子を合成する方法として、ミニエマルジョン法と呼ばれる手法が知られているが（Weiss, C.K. et al., Preparatio, Macromolecules, 2007, Vol. 40, p. 928-938; WO 2008/003706 A1）、この手法では、ヘキサデカン等の親油性溶媒を含むO相と塩酸等を含むW相の２相で構成されるミニエマルジョン中でアニオン重合が行われ、SDS等の界面活性剤の使用も必須である。本願発明者が開発した上記の方法によれば、O相を含まない単相の水性溶媒中での重合反応によりナノポリマー粒子を合成できるので、溶媒として有機溶媒や界面活性剤を使用する必要が無い。また、本願発明者の方法では、重合開始・安定剤としてポリソルベート以外のものを使用してナノポリマー粒子を製造することもできるので、Tween系の界面活性剤を含め、陰イオン界面活性剤、陽イオン界面活性剤、両性界面活性剤、及び非イオン界面活性剤のいずれの界面活性剤も含有しないナノ粒子を調製することができる。もっとも、ミニエマルジョン法により合成されたナノポリマー粒子の皮膚炎に対する効果は不明である。 As a method for synthesizing nano-sized polymer particles by polymerizing a cyanoacrylate monomer using an amino acid as a polymerization initiator, a method called a mini-emulsion method is known (Weiss, CK et al., Preparatio, Macromolecules, 2007). , Vol. 40, p. 928-938; WO 2008/003706 A1), in this method, in a mini-emulsion composed of two phases of O phase containing lipophilic solvent such as hexadecane and W phase containing hydrochloric acid etc. Anionic polymerization is performed, and the use of a surfactant such as SDS is also essential. According to the above method developed by the present inventor, nanopolymer particles can be synthesized by a polymerization reaction in a single-phase aqueous solvent that does not contain an O phase. Therefore, it is necessary to use an organic solvent or a surfactant as a solvent. No. In addition, in the method of the present inventor, nanopolymer particles can be produced using a polymerization initiator / stabilizer other than polysorbate. Therefore, including a Tween surfactant, an anionic surfactant, a cationic surfactant, Nanoparticles that do not contain any of an ionic surfactant, an amphoteric surfactant, and a nonionic surfactant can be prepared. However, the effect of nanopolymer particles synthesized by the miniemulsion method on dermatitis is unknown.
　反応開始時の重合反応液中のシアノアクリレートモノマーの濃度は、特に限定されないが、通常、0.5v/v％～2.0v/v％程度、好ましくは0.8v/v％～1.2v/v％程度である。 The concentration of the cyanoacrylate monomer in the polymerization reaction solution at the start of the reaction is not particularly limited, but is usually about 0.5 v / v% to 2.0 v / v%, preferably about 0.8 v / v% to 1.2 v / v%. It is.
　反応温度は、特に限定されないが、室温で行なうことが簡便で好ましい。反応時間は、反応液のpH、溶媒の種類等に応じて反応速度が異なるため、これらの要素に応じて適宜選択される。特に限定されないが、通常、反応時間は１０分～５時間程度、好ましくは３０分～４時間程度である。得られたアミノ酸系分子含有粒子は、通常、中性の粒子として用いられるので、反応終了後、水酸化ナトリウム水溶液等の塩基を反応液に添加して中和することが好ましい。反応終了後の反応液をフィルター濾過し、適宜滅菌水で洗浄して粒子を回収すればよい。 The reaction temperature is not particularly limited, but it is preferable because it is carried out at room temperature. The reaction time varies depending on the pH of the reaction solution, the type of solvent, and the like, and therefore is appropriately selected depending on these factors. Although not particularly limited, the reaction time is usually about 10 minutes to 5 hours, preferably about 30 minutes to 4 hours. Since the obtained amino acid molecule-containing particles are usually used as neutral particles, it is preferable to neutralize the reaction solution by adding a base such as an aqueous sodium hydroxide solution after the reaction. The reaction liquid after completion of the reaction may be filtered and washed with sterilized water as appropriate to collect the particles.
　上記の方法によれば、平均粒径が1000nm未満であるナノサイズのシアノアクリレートポリマー粒子を容易に製造することができる。粒子サイズの下限は特に限定されないが、上記の重合反応で製造される粒子の粒径は通常7nm程度以上となる。好ましくは、粒子の平均粒径は20nm～600nm、より好ましくは50nm～550nmである。粒子のサイズは、反応液中のシアノアクリレートモノマーの濃度やpH、反応時間を調節することによって調節することができる。また、重合開始・安定剤として糖及びポリソルベートから選択される少なくとも１種を用いる場合には、該重合開始・安定剤の濃度や種類を変えることによっても、粒子サイズを調節することができる（特許文献３、４等参照）。一般に、反応液のpHを高めた場合、反応時間を長くした場合、及び反応液の糖濃度を低くした場合には粒子サイズが大きくなり、重合開始・安定剤としてポリソルベートを用いた場合には粒子サイズが小さくなる。これらの反応条件を適宜組み合わせることで、所望のサイズの粒子を製造することができる。 According to the above method, nano-sized cyanoacrylate polymer particles having an average particle diameter of less than 1000 nm can be easily produced. The lower limit of the particle size is not particularly limited, but the particle size of the particles produced by the above polymerization reaction is usually about 7 nm or more. Preferably, the average particle size of the particles is 20 nm to 600 nm, more preferably 50 nm to 550 nm. The size of the particles can be adjusted by adjusting the concentration and pH of the cyanoacrylate monomer in the reaction solution and the reaction time. In addition, when at least one selected from sugar and polysorbate is used as the polymerization initiator / stabilizer, the particle size can also be adjusted by changing the concentration and type of the polymerization initiator / stabilizer (patent) References 3, 4 etc.). Generally, when the pH of the reaction solution is increased, when the reaction time is increased, and when the sugar concentration of the reaction solution is decreased, the particle size increases, and when polysorbate is used as a polymerization initiator / stabilizer, particles are increased. The size becomes smaller. By appropriately combining these reaction conditions, particles having a desired size can be produced.
　ナノ粒子の電荷（ゼータ電位）は、特に限定されないが、通常-50mV～0mV程度である。ゼータ電位とは、粒子表面の電荷を示すもので、粒子の分散性の指標となる。粒子サイズとゼータ電位は、例えばHe・Neレーザーを用いた市販の装置（例えばMalvern Inst.UK社製のゼータサイザー等）を用いて容易に測定することができる。 The charge (zeta potential) of the nanoparticles is not particularly limited, but is usually about -50 mV to 0 mV. The zeta potential indicates the charge on the particle surface and is an index of particle dispersibility. The particle size and the zeta potential can be easily measured using, for example, a commercially available apparatus using a He / Ne laser (for example, a Zetasizer manufactured by Malvern Inst. UK).
　アミノ酸系分子を重合開始・安定剤として用いて製造されたナノ粒子では、アミノ酸系分子が単に粒子に付着して含有されるのみならず、アミノ酸構造中の-COO基がシアノアクリレートのエチレン末端の炭素に結合し、共有結合により粒子に含有されていると考えられる。なお、上記方法で得られる粒子のアミノ酸系分子の含有率は、通常約２０％～約６５％程度である。アミノ酸系分子の含有率は、重合後にフィルター洗浄したときのフィルター通過液の吸光度を適当な波長で測定し、フィルター通過液中のアミノ酸系分子の量（すなわち粒子に結合しなかったアミノ酸系分子の量）を吸光度法により求めた後、下記の式によって算出することができる。
アミノ酸系分子含有量＝(アミノ酸系分子添加量)－(フィルター通過液中のアミノ酸系分子の量)
アミノ酸系分子含有率（％）＝アミノ酸系分子含有量÷アミノ酸系分子添加量×100 Nanoparticles produced using amino acid-based molecules as polymerization initiators / stabilizers not only contain amino acid-based molecules attached to the particles, but also the -COO group in the amino acid structure of the ethylene end of cyanoacrylate. It is considered that it is bound to carbon and contained in the particle by a covalent bond. The content of amino acid molecules in the particles obtained by the above method is usually about 20% to about 65%. The content of amino acid-based molecules is determined by measuring the absorbance of the filter-passed solution when the filter is washed after polymerization at an appropriate wavelength, and the amount of amino-acid-based molecules in the filter-passed solution (ie, (Quantity) can be calculated by the following formula after obtaining by the absorbance method.
Amino acid molecule content = (Amino acid molecule addition amount)-(Amount of amino acid molecule in the filter passage liquid)
Amino acid molecule content (%) = Amino acid molecule content ÷ Amino acid molecule addition x 100
　本発明で用いるアミノ酸系分子含有ナノ粒子は、細菌類に対する抗菌活性成分を含まない。アミノ酸を含有するシアノアクリレートポリマーナノ粒子がグラム陽性細菌及びグラム陰性細菌の両者に対して抗菌活性を発揮できることは、特許文献８、９に記載される通り公知であり、細菌表面（細胞壁）への特異的接着性により細菌を溶菌に導くことで抗菌作用を発揮する。ナノ粒子が有する細胞障害活性は、正常な哺乳動物細胞に対しては発揮されず、ナノ粒子にはin vivo毒性もないことが確認されている。また、本発明で用いるアミノ酸系分子含有ナノ粒子は、真菌に対する抗菌活性成分も含まない。 The amino acid molecule-containing nanoparticles used in the present invention do not contain an antibacterial active ingredient against bacteria. It is known that cyanoacrylate polymer nanoparticles containing amino acids can exhibit antibacterial activity against both gram-positive and gram-negative bacteria, as described in Patent Documents 8 and 9, and are applied to the bacterial surface (cell wall). It exhibits antibacterial action by directing bacteria to lysis by specific adhesion. It has been confirmed that the cytotoxic activity of the nanoparticles is not exerted on normal mammalian cells, and the nanoparticles do not have in vivo toxicity. In addition, the amino acid molecule-containing nanoparticles used in the present invention do not contain an antibacterial active ingredient against fungi.
　ここでいう「抗菌活性成分」とは、細菌又は真菌の代謝経路ないしは生理機能に生化学的に作用して該細菌の発育を阻止することができる化学物質成分をいい、具体的には、細菌又は真菌の抗菌に利用可能な抗生物質その他の化学物質成分を言う。「抗菌活性成分を含まない」とは、抗菌活性成分を全く含まないか、含んでいたとしてもごく微量であって、その抗菌活性成分に対し感受性である細菌又は真菌を抗菌することができない程度の微量にしか該抗菌活性成分を含んでいないことをいう。本発明で用いるナノ粒子は、細菌類及び真菌類に対する抗菌活性成分を「実質的に含まない」ということもできる。「抗菌することができない程度の微量」とは、粒子単位体積当たりに含まれる粒子中の抗菌活性成分量を粒子中の含有濃度と定義し、この含有濃度と同濃度の抗菌活性成分を粒子に含有させず単独で感受性細菌又は真菌に作用させた場合に、該感受性細菌又は真菌の発育を阻止できない量のことを意味する。本発明で用いるナノ粒子は、抗生物質等の抗菌活性成分を全く含まない粒子であり得る。 As used herein, the term “antibacterial active ingredient” refers to a chemical substance component that can biochemically act on the metabolic pathway or physiological function of bacteria or fungi to inhibit the growth of the bacteria. Or it refers to antibiotics and other chemical components that can be used for fungal antibacterial activity. “No antibacterial active ingredient” means that it does not contain any antibacterial active ingredient, or even if it is contained in a very small amount, it cannot be used for antibacterial bacteria or fungi sensitive to the antibacterial active ingredient. This means that the antibacterial active ingredient is contained only in a trace amount. The nanoparticles used in the present invention can also be said to be “substantially free” of antibacterial active ingredients against bacteria and fungi. “A trace amount that does not allow antibacterial activity” means that the amount of antibacterial active ingredient contained in a particle per unit volume of the particle is defined as the concentration contained in the particle, and an antibacterial active ingredient having the same concentration as this content concentration It means an amount that does not prevent the growth of sensitive bacteria or fungi when it is allowed to act on sensitive bacteria or fungi alone without containing them. The nanoparticles used in the present invention may be particles that do not contain any antibacterial active ingredient such as antibiotics.
　また、アミノ酸系分子含有ナノ粒子は、従来用いられている皮膚炎の治療薬のような、皮膚炎に対する治療又は予防活性を有する化学物質成分を含まない。ここでいう「含まない」も上記と同様であり、そのような化学物質成分を全く含まないか、含んでいたとしてもごく微量であって、皮膚病変部の治療及び予防効果を発揮することができない程度の微量にしか該化学物質成分を含んでいないことを意味する。本発明で用いるナノ粒子は、皮膚炎治療薬のDDSではなく、ナノ粒子それ自体が皮膚バリア機能の向上と表在細菌数制御の関与する免疫応答とを介して皮膚炎症反応を制御し、皮膚炎の治療及び予防効果を奏する。 In addition, the amino acid molecule-containing nanoparticles do not contain a chemical substance component having a therapeutic or preventive activity against dermatitis, such as a conventionally used dermatitis therapeutic agent. The term “excluded” as used herein is the same as described above, and does not contain such a chemical substance component at all, or even if it contains such a chemical substance, it is extremely small, and can exert a therapeutic and preventive effect on skin lesions. It means that the chemical substance component is contained only in such a small amount as to be impossible. Nanoparticles used in the present invention are not DDS as a dermatitis treatment drug, but the nanoparticles themselves control the skin inflammatory reaction through the improvement of skin barrier function and the immune response involved in controlling the number of superficial bacteria, Has the effect of treating and preventing inflammation.
　本発明において対象となる皮膚炎は、特に限定されないが、第一には掻痒を伴う皮膚炎（掻痒性皮膚炎）であり得る。掻痒性皮膚炎の具体例としては、アトピー性皮膚炎、接触皮膚炎、感染性皮膚炎、乾癬や自己免疫機序による皮膚炎、薬疹等を挙げることができるが、これらに限定されない。 The target dermatitis in the present invention is not particularly limited, but first, it may be dermatitis accompanied by pruritus (pruritic dermatitis). Specific examples of pruritic dermatitis include, but are not limited to, atopic dermatitis, contact dermatitis, infectious dermatitis, dermatitis due to psoriasis and autoimmune mechanism, drug eruption, and the like.
　また、対象となる皮膚炎は、第二には皮膚傷害に関連した皮膚炎であり得る。そのような皮膚炎の具体例としては、放射線性皮膚炎、熱傷、じょく瘡等を挙げることができるが、これらに限定されない。 Also, the target dermatitis may secondly be dermatitis related to skin injury. Specific examples of such dermatitis include, but are not limited to, radiation dermatitis, burns, acne and the like.
　下記実施例において確認されている通り、アミノ酸系分子含有ナノ粒子はI型とIV型のアレルギー反応を抑制する作用を有する。従って、アミノ酸系分子含有ナノ粒子を有効成分とする本発明の剤は、I型アレルギー反応又はIV型アレルギー反応の抑制剤として、例えばI型アレルギー反応又はIV型アレルギー反応が関与する皮膚炎等のアレルギー症状の治療又は予防剤として有用である。上記した掻痒性皮膚炎は、I型アレルギー反応又はIV型アレルギー反応が関与し得ることが知られている。 As confirmed in the Examples below, the amino acid molecule-containing nanoparticles have the action of suppressing type I and type IV allergic reactions. Therefore, the agent of the present invention comprising amino acid molecule-containing nanoparticles as an active ingredient is used as an inhibitor of type I allergic reaction or type IV allergic reaction, such as dermatitis involving type I allergic reaction or type IV allergic reaction. It is useful as an agent for treating or preventing allergic symptoms. It is known that the above pruritic dermatitis can involve type I allergic reaction or type IV allergic reaction.
　上述した通り、本発明で用いるナノサイズのシアノアクリレートポリマー粒子には細菌に対する抗菌活性があることが知られている（特許文献８、９）。感染性皮膚炎等の感染を主因とする皮膚炎に対しては、粒子の抗菌活性が感染した細菌に対しても効果を発揮すると期待されるため、感染を主因とする皮膚炎に対しても本発明の剤を使用することができるが、本発明で対象とする皮膚炎は、細菌感染を主因とする皮膚炎以外の皮膚炎であり得る。 As described above, it is known that the nano-sized cyanoacrylate polymer particles used in the present invention have antibacterial activity against bacteria (Patent Documents 8 and 9). For dermatitis mainly caused by infection such as infectious dermatitis, the antibacterial activity of the particles is expected to be effective against infected bacteria. Although the agent of the present invention can be used, the dermatitis targeted in the present invention can be dermatitis other than dermatitis mainly caused by bacterial infection.
　本発明の剤は、治療すべき皮膚病変部、又は皮膚炎の発生の予防が望まれる皮膚領域に局部投与して用いられる。局部投与方法としては、注射剤・点滴剤等による皮内投与、軟膏剤・クリーム剤・貼付剤等による局所適用等が挙げられる。投与量は、症状、年齢、体重、投与方法等に応じて適宜選択され、特に限定されないが、通常、対象動物に対し有効成分であるアミノ酸系分子含有粒子の量として１日0.01μg～10000mg程度、例えば1μg～100mg程度であり、１回ないし数回に分けて投与される。症状の改善の程度に応じ、数日ないし数ヶ月間にわたり、毎日１回若しくは数回、ないしは数日おきに１日若しくは数回、定期的に投与してもよいし、あるいは、症状が発生した時に投与してもよい。 The agent of the present invention is used by locally administering to a skin lesion to be treated or a skin region where prevention of the occurrence of dermatitis is desired. Examples of the local administration method include intracutaneous administration with injections, drops, etc., topical application with ointments, creams, patches, and the like. The dosage is appropriately selected according to symptoms, age, body weight, administration method, etc., and is not particularly limited. Usually, the amount of amino acid molecule-containing particles that are active ingredients for the target animal is about 0.01 μg to 10,000 mg per day. For example, it is about 1 μg to 100 mg, and is administered once or several times. Depending on the degree of symptom improvement, it may be administered once or several times daily for several days to several months, or once or several times every other day, or symptoms may occur Sometimes it may be administered.
　本発明の剤の投与対象は哺乳動物であり、例えばヒト、イヌ、ネコ、ウサギ、ハムスター等が挙げられる。 The administration target of the agent of the present invention is a mammal, and examples thereof include humans, dogs, cats, rabbits, and hamsters.
　本発明の剤は、単独で用いてもよいし、他の掻痒性皮膚疾患の治療又は予防剤等と併せて用いることもできる。掻痒性皮膚疾患に従来用いられている標準的治療薬はステロイド剤、カルシニューリン阻害薬や抗ヒスタミン薬であるが、本発明の剤はこれらの標準的治療薬とは作用機序が異なると考えられるので、本発明の剤とステロイド剤等とを組み合わせて用いることも可能である。 The agent of the present invention may be used alone or in combination with other pruritic skin disease treatment or prevention agents and the like. Standard treatments conventionally used for pruritic skin diseases are steroids, calcineurin inhibitors and antihistamines, but the agents of the present invention are considered to have a different mechanism of action from these standard treatments. Therefore, it is also possible to use the agent of the present invention in combination with a steroid agent or the like.
　以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
１．アミノ酸含有ナノ粒子の製造
　国際公開第２０１２／１３３６４８号公報および国際公開第２０１３／１０８８７１号公報に記載されている方法に準じて、アミノ酸及びデキストランを含有するナノサイズのポリマー粒子を製造した。具体的な手順は以下の通りである。 1. Production of amino acid-containing nanoparticles Nanosized polymer particles containing amino acids and dextran were produced according to the methods described in International Publication Nos. 2012/133648 and 2013/108871. The specific procedure is as follows.
　10 mLの0.01N HClに、100mgのデキストラン60K及び100mgのアミノ酸を溶解し、その液性pHを要時1N塩酸を用いてpH=3に調整した。アミノ酸としては、グリシン及びアスパラギン酸を使用した。 In 100 mL of 0.01N HCl, 100 mg of dextran 60K and 100 mg of amino acid were dissolved, and the liquid pH was adjusted to pH = 3 using 1N hydrochloric acid as necessary. As amino acids, glycine and aspartic acid were used.
　デキストラン及びアミノ酸を溶解させた上記の溶液を撹拌下、100μLのnBCAを加え、3時間撹拌し重合反応を実施した。1N NaOHを滴下して反応溶液を中和後(pH7.8)、さらに30分撹拌することで、アミノ酸及びデキストランを含有するナノサイズのポリマー粒子を得た。得られた粒子は、ミリポア社限外濾過器及びポール社製ワクチン製造向け透析限外濃縮装置にて2段階の限外濾過処理に付し、粒子精製品（粒子濃度2mg/mlのコロイド水溶液）を得た。 The above solution in which dextran and amino acids were dissolved was stirred, 100 μL of nBCA was added, and the mixture was stirred for 3 hours to carry out a polymerization reaction. After 1N NaOH was added dropwise to neutralize the reaction solution (pH 7.8), the mixture was further stirred for 30 minutes to obtain nano-sized polymer particles containing an amino acid and dextran. The obtained particles are subjected to a two-stage ultrafiltration treatment using a Millipore ultrafiltration device and a dialysis ultraconcentration device for the manufacture of vaccines manufactured by Pall, resulting in particle refined products (colloid aqueous solution with a particle concentration of 2 mg / ml). Got.
　市販のゼータサイザー（Malvern Inst.UK社製）を用いてナノ粒子の平均粒径及びゼータ電位を測定した。また、フィルター洗浄時のフィルター通過液中のアミノ酸分子の量を吸光度法により求め、粒子のアミノ酸含有率を算出した。結果を表１に示す。 The average particle diameter and zeta potential of the nanoparticles were measured using a commercially available zeta sizer (MalvernMInst. UK). Further, the amount of amino acid molecules in the filter passage liquid at the time of filter washing was determined by an absorbance method, and the amino acid content of the particles was calculated. The results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
２．マウス皮膚炎モデルにおけるナノ粒子の治療及び予防効果の検討１
　上記で製造したアスパラギン酸含有ナノ粒子を用いて実験を行なった。ナノ粒子は、滅菌水に懸濁してナノ粒子水（0.3％（w/v）に希釈、粒子濃度6μg/ml）を調製し、実験に用いた。以下に用いたマウスはすべて、高知大学動物実験委員会で承認された実験計画に沿って実施し、SPFの環境下で飼育、実験を行った。 2. Study of therapeutic and preventive effects of nanoparticles in a mouse dermatitis model 1
Experiments were performed using the aspartic acid-containing nanoparticles produced above. Nanoparticles were suspended in sterilized water to prepare nanoparticle water (diluted to 0.3% (w / v), particle concentration 6 μg / ml) and used in the experiment. All of the mice used below were carried out in accordance with the experimental plan approved by the Kochi University Animal Experiment Committee, and were reared and experimented in an SPF environment.
[方法]
　NC/NgaSlcマウス（日本SLC、静岡）は、背部の剃毛および除毛後、テープストリッピングにより皮膚炎を自然発症することが知られており、アトピー性皮膚炎のモデルとして使われている（Matsuda, M., et.al. Int. Immunol. 9:461, 1997）。本実験では、5週齢のNC/NgaSlcマウス雄5匹を使用し、図１に示すスケジュールで処置を行なった。NC/NgaSlcマウスの背部をシェーバーで剃毛した後、除毛クリーム（Epilat、クラシエホームプロダクツ株式会社、東京）にて除毛した。除毛クリームをふき取り、翌日に粘着テープによりテープストリッピング（Scotchメンディングテープ、cat.no.810-3-24、スリーエムジャパン株式会社、東京）を8回行い、皮膚炎を誘発した。途中毛が生えたら同様の操作を繰り返し行った。３週間後、いずれのマウスにおいても背部の広範囲にわたり皮膚炎の発症が確認された（図2A）。 [Method]
NC / NgaSlc mice (Japan SLC, Shizuoka) are known to spontaneously develop dermatitis by tape stripping after shaving and depilation of the back, and are used as a model for atopic dermatitis (Matsuda , M., et.al. Int. Immunol. 9: 461, 1997). In this experiment, 5 male 5-week-old NC / NgaSlc mice were used, and treatment was performed according to the schedule shown in FIG. The back of the NC / NgaSlc mouse was shaved with a shaver and then removed with a hair removal cream (Epilat, Kracie Home Products, Tokyo). The depilatory cream was wiped off, and tape stripping (Scotch Mending Tape, cat.no.810-3-24, 3M Japan, Tokyo) was performed 8 times with an adhesive tape the next day to induce dermatitis. The same operation was repeated when hair grew on the way. Three weeks later, in any mouse, the onset of dermatitis was confirmed over a wide area of the back (FIG. 2A).
　皮膚炎を発症したマウス５匹のうち、３匹をナノ粒子水噴霧群、２匹を水噴霧群とした。病変部を含む背部皮膚に、２日毎にナノ粒子水又は滅菌水を噴霧した。マウス１匹の背中8～10cm2に対して総液量0.3～0.4mL（ナノ粒子の量として1.8～2.4μg）を噴霧した。２日目、６日目および８日目に背部中央部の細菌数を測定した。具体的には、マウスの背部中央部に生菌数測定用標準寒天培地（ぺたんチェック、栄研化学）を5秒間押し当てた後、37℃フラン器で24時間培養し、6cm2の区画あたりのコロニー数をカウントした。寒天培地に触れた皮膚を水で湿らせたティシューペーパーで拭いた後、ナノ粒子水又は蒸留水を噴霧して１時間放置し、乾かした。皮膚病変については、２日目（図2B）及び８日目（図2C）に写真撮影を行なった。 Of the 5 mice that developed dermatitis, 3 were the nanoparticle water spray group and 2 were the water spray group. Nanoparticle water or sterilized water was sprayed every 2 days on the dorsal skin including the lesion. A total liquid volume of 0.3 to 0.4 mL (1.8 to 2.4 μg as the amount of nanoparticles) was sprayed on 8 to 10 cm 2 of the back of one mouse. The number of bacteria in the center of the back was measured on the 2nd, 6th and 8th days. Specifically, a mouse standard agar medium for viable cell count measured back central part of the (Petain check, Eiken) was pressed for 5 seconds, incubated for 24 hours at 37 ° C. incubator, partitions per 6 cm 2 The number of colonies was counted. The skin touched with the agar medium was wiped with tissue paper moistened with water, then sprayed with nanoparticle water or distilled water, left for 1 hour, and dried. The skin lesions were photographed on the second day (FIG. 2B) and the eighth day (FIG. 2C).
　図１の３０日目より２週間放置後、さらにハプテン誘発性IV型アレルギー反応を惹起させる目的で、全てのマウスについて再度背部の剃毛と除毛を行なった。翌日（1日目）と２日目に、マウスの背部頭側に0.5%ジニトロフルオロベンゼン（1-fluoro-2,4-dinitrobenzene、DNFB）（ナカライテスク株式会社、京都）を溶かしたオリーブ油－アセトン溶液（油：アセトン＝１：４）を20μL塗布した。ナノ粒子水噴霧および水噴霧は、除毛をした４４日目より毎日行った。DNFB塗布した場合はDNFBが乾燥した後に行った。DNFB塗布後２日目に写真撮影を行なった（図2D）。DNFB塗布後６日目に血清サンプルを採取後、安楽死させ、DNFBを塗布した頭側背部の皮膚とDNFBを塗布していない尾基部付近の皮膚を採取し、ヘマトキシリン－エオジン（HE）染色を行った（図5A-5D）。 After leaving for 2 weeks from the 30th day in FIG. 1, all mice were again shaved and depilated for the purpose of inducing a hapten-induced type IV allergic reaction. On the next day (1st day) and 2nd day, olive oil-acetone containing 0.5% dinitrofluorobenzene (1-fluoro-2,4-dinitrobenzene, DNFB) (Nacalai Tesque, Kyoto) on the back of the mouse 20 μL of the solution (oil: acetone = 1: 4) was applied. Nanoparticle water spray and water spray were performed every day from the 44th day after depilation. When DNFB was applied, it was done after DNFB had dried. Photographs were taken on the second day after application of DNFB (Fig. 2D). On day 6 after application of DNFB, a serum sample was collected, then euthanized, and the skin on the back of the head with DNFB applied and the skin near the base of the tail without DNFB were collected and stained with hematoxylin-eosin (HE). Performed (FIGS. 5A-5D).
　また、下記表2に示した基準で図2に示したA～Dのマウスの皮膚炎の程度をスコア化した。スコア化はFanらの報告した文献（Fan et al., Exp Biol Med, 226, 1045-1050, 2001）に基づき、３つの皮膚病変について、程度を0～3のスコアで評価した。 In addition, the degree of dermatitis of the mice A to D shown in FIG. 2 was scored according to the criteria shown in Table 2 below. Scoring was based on the literature reported by Fan et al. (Fan et al., Exp Biol Med, 226, 1045-1050, 2001). The degree of score was evaluated with a score of 0 to 3 for three skin lesions.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
[結果]
　皮膚炎発症マウスの背部中央部の細菌数をカウントした結果を表３及び図３に示す。ナノ粒子水噴霧群では表在細菌数がおよそ半分程度に減少した。 [result]
The results of counting the number of bacteria in the center of the back of mice with dermatitis are shown in Table 3 and FIG. In the nano-particle water spray group, the number of superficial bacteria decreased to about half.
　寒天培地のコロニー数は、ナノ粒子水噴霧群は5個、7個、8個であったのに対して蒸留水噴霧群は48個、85個であった。ナノ粒子水の抗菌能力は噴霧後の早い時間から発揮されることが確認された。 The number of colonies in the agar medium was 5, 7, and 8 for the nanoparticle water spray group, whereas that for the distilled water spray group was 48 and 85. It was confirmed that the antibacterial ability of nanoparticulate water was exhibited early after spraying.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　NC/NgaSlcマウスにテープストリッピングで惹起した皮膚炎の治癒の速度や程度には両群の間で大きな差は見られなかったが（図2A～2C、図4）、繰り返し除毛とテープストリッピングを行い、0.5%DNFB溶液を20μL塗布して惹起したハプテン誘導性皮膚炎においては、水噴霧群における皮膚炎の程度はナノ粒子水噴霧群皮膚炎よりも重症であった（図2D、図4）。 Although there was no significant difference between the two groups in the healing rate and extent of dermatitis caused by tape stripping in NC / NgaSlc mice (Figures 2A-2C, Figure 4), repeated hair removal and tape stripping were performed. In the hapten-induced dermatitis caused by applying 20 μL of 0.5% DNFB solution, the degree of dermatitis in the water spray group was more severe than that in the nanoparticle water spray group (Figure 2D, Figure 4) .
　HE染色皮膚切片を観察すると（図5）、ナノ粒子水噴霧群および水噴霧群いずれにおいても、DNFB塗布部位(図５A, C)では非塗布部位(図５B, D)に比べて、表皮肥厚と真皮内細胞浸潤、線維化が認められた。しかし、水噴霧群の方がナノ粒子水噴霧群よりもDNFB塗布による表皮肥厚、真皮の病変の程度が著明であった（図5Aと5C）。さらに、水噴霧群でみられたDNFB非塗布部位の軽度の表皮肥厚は、ナノ粒子水噴霧群では完全に抑制されていた（図5Bと5D）。 When the HE-stained skin section was observed (Fig. 5), in both the nanoparticle water spray group and the water spray group, the thickened epidermis at the DNFB application site (Fig. 5A, C) compared to the non-application site (Fig. 5B, D). Intradermal cell infiltration and fibrosis were observed. However, the water spray group showed a greater degree of epidermal thickening and dermal lesions due to DNFB application than the nanoparticle water spray group (FIGS. 5A and 5C). Furthermore, the mild epidermal thickening of the non-DNFB application site observed in the water spray group was completely suppressed in the nanoparticle water spray group (FIGS. 5B and 5D).
　また、I型アレルギー反応にナノ粒子が及ぼす影響を調べる目的で、DNFB塗布開始前（図１、４４日目）及びDNFB初回塗布から８日目（５２日目の実験終了時）のマウス血中のIgE濃度を固相化抗体（rat anti-mouse IgE, Southern Biotech, AL, USA）と酵素標識抗体（HRP-conjugated goat anti-mouse IgE antibody, BETHYL, TX, USA）で構成されたサンドイッチELISA法にて測定した。その結果、水噴霧群では、実験開始時のIgE量と比較して、実験終了時には5倍近くIgE量の上昇を示したのに対し、ナノ粒子水噴霧群ではIgE量の上昇は2-3倍程度にとどまっていた（表４、図６）。ナノ粒子水噴霧はIV型アレルギー反応にのみならずI型アレルギー反応の制御にも有効であることが確認された。 In addition, for the purpose of investigating the effects of nanoparticles on type I allergic reactions, blood in mice before the start of DNFB application (FIG. 1, day 44) and 8 days after the initial application of DNFB (at the end of the experiment on day 52) ELISA method consisting of immobilized antibodies (rat を anti-mouse IgE, Southern Biotech, AL, USA) and enzyme-labeled antibody (HRP-conjugated goat anti-mouse IgE antibody, BETHYL, TX, USA) Measured at As a result, the water spray group showed an increase in the IgE amount nearly 5 times at the end of the experiment compared to the IgE amount at the start of the experiment, whereas the nanoparticle water spray group showed an increase in the IgE amount of 2-3. It was only about twice (Table 4, Fig. 6). It was confirmed that nanoparticle water spray is effective not only for type IV allergic reaction but also for controlling type I allergic reaction.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
[結論]
　ナノ粒子水の有する殺菌作用は、テープストリッピングにより誘導したNC/NgaSlcマウスの皮膚炎に対する治療促進効果とDNFB塗布による皮膚炎誘導に対する予防効果を有することが示された。その効果は、殺菌作用によるバリア機能回復の促進と炎症反応の制御に基づくことが示唆された。 [Conclusion]
It was shown that the bactericidal action possessed by nanoparticulate water has a therapeutic promotion effect on dermatitis in NC / NgaSlc mice induced by tape stripping and a preventive effect on dermatitis induction by DNFB application. It was suggested that the effect was based on the promotion of barrier function recovery by bactericidal action and the control of inflammatory response.
３．マウス皮膚炎モデルにおけるナノ粒子の治療及び予防効果の検討２
　ナノ粒子の効果をさらに検証するため、MHC遺伝子型が知られているBALB/cマウスを用いて実験を行なった。まずは、ナノ粒子水による皮膚炎の予防効果をみる実験を行った。図７に示すスケジュールでテープストリッピングにより炎症を誘導し、一定期間後DNFB塗布により皮膚炎を再誘導した。この皮膚炎に対するナノ粒子水の治療効果について、皮膚炎症状態の経過観察、血中IgE濃度測定、およびHE組織染色標本観察を行い評価した。 3. Study of therapeutic and preventive effects of nanoparticles in mouse dermatitis model 2
In order to further verify the effects of nanoparticles, experiments were performed using BALB / c mice with known MHC genotypes. First, an experiment was conducted to examine the preventive effect of nanoparticle water on dermatitis. Inflammation was induced by tape stripping according to the schedule shown in FIG. 7, and dermatitis was re-induced by applying DNFB after a certain period. The therapeutic effect of nanoparticle water on this dermatitis was evaluated by observing the progress of skin inflammation, measuring blood IgE concentration, and observing HE tissue stained specimens.
[方法]
　BALB/cマウス（雌、8週齢）６匹の尾から採血した後、背部をシェーバーとクリームにより剃毛および除毛した。水でクリームをふき取り皮膚を乾燥した後、スコッチテープで８回ストリッピングし写真撮影をした（図８、day0）。 [Method]
After blood was collected from 6 tails of BALB / c mice (female, 8 weeks old), the back was shaved and removed with a shaver and cream. After wiping off the cream with water and drying the skin, stripping was performed 8 times with a scotch tape, and a photo was taken (FIG. 8, day 0).
　６匹のマウスはナノ粒子水噴霧群（3匹）と水噴霧群（3匹）の2群に分けて実験を行った。１日目に噴霧処理（ナノ粒子水噴霧は、マウス１匹の背中8～10cm2に対して総液量0.3～0.4mL、ナノ粒子量として1.8～2.4μgを噴霧）をした後、２日目以降は２０日目まで１日おきに噴霧処理を行なった。２２日目と２３日目にDNFB処理（マウス背部頭側に0.5%DNFBのオリーブ油－アセトン溶液（油：アセトン＝１：４）を20μL塗布）を行なった。 The six mice were divided into two groups, a nanoparticle water spray group (3 mice) and a water spray group (3 mice). After spraying on the first day (nanoparticle water spray is sprayed with a total liquid volume of 0.3-0.4 mL and a nanoparticle volume of 1.8-2.4 μg on the back of 8-10 cm 2 of one mouse), 2 days After the first, spraying was performed every other day until the 20th day. On the 22nd and 23rd days, DNFB treatment (20 μL of 0.5% DNFB olive oil-acetone solution (oil: acetone = 1: 4) was applied to the back of the mouse) was performed.
　写真撮影は０、３、７、１１、１３、１５、２２、２３、２４日目に行ない、上記表２に示した基準で皮膚炎をスコア化した。背部頭側と尾側を分けてスコア化し、その合計値によって評価した。 Photographs were taken on days 0, 3, 7, 11, 13, 15, 22, 23, and 24, and dermatitis was scored according to the criteria shown in Table 2 above. The dorsal head side and caudal side were scored separately, and the total value was evaluated.
　採血は、テープストリッピングする前（図７、採血1）、テープストリッピング誘導後22日目（図７、採血2）、DNFB塗布で再誘導開始後2日目（図７、採血3）に行ない、その血清分画のIgE量をサンドイッチELISAにより測定した。 Blood sampling is performed before tape stripping (FIG. 7, blood sampling 1), 22 days after induction of tape stripping (FIG. 7, blood sampling 2), and 2 days after starting re-induction with DNFB application (FIG. 7, blood sampling 3). The amount of IgE in the serum fraction was measured by sandwich ELISA.
　２４日目の実験終了時、マウスの背部頭側及び尾側の皮膚組織を採取した。皮膚組織の一部は、HE組織染色評価に用いた。また残りの一部は、mRNAを抽出し、定量的RT-PCRにより各種サイトカイン発現量（IL-1α、TNF-α、IL-6、IL-17A、IL-4、IFN-γ）を調べた。発現量は、HPRT遺伝子に対してノーマライズし、相対発現量で評価した。表５に各マウスサイトカインに対するプライマー配列を示す。 At the end of the experiment on the 24th day, the skin tissue on the dorsal head side and the caudal side of the mouse was collected. A part of the skin tissue was used for HE tissue staining evaluation. In addition, mRNA was extracted from the remaining part, and various cytokine expression levels (IL-1α, TNF-α, IL-6, IL-17A, IL-4, IFN-γ) were examined by quantitative RT-PCR. . The expression level was normalized with respect to the HPRT gene and evaluated by relative expression level. Table 5 shows primer sequences for each mouse cytokine.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
[結果]
1. 皮膚炎症状の判定結果
　テープストリッピング後、すべてのマウス背部に多数の糜爛を伴う紅斑を認めた（図８、day0）。テープストリッピングのみにより誘導された皮膚炎は、ナノ粒子水噴霧群では水噴霧群と比べて紅斑の消失が早く、糜爛も認められなかった（図８、day3）。両群の皮膚に明瞭な浮腫は認められなかった。一週間目以降はナノ粒子水噴霧群、水噴霧群ともに（番号8のマウス以外は）も炎症症状はほぼ同程度に回復した（図８、day7～day15）。経過日数に対する皮膚炎スコアの変化を図９に示す。ナノ粒子水噴霧群の方が水噴霧群のマウスと比べて、より早期から炎症程度が改善されることがスコアにおいて認められた。 [result]
1. Judgment result of skin inflammation symptoms After tape stripping, erythema with many wrinkles was observed on the back of all mice (FIG. 8, day 0). In the dermatitis induced only by tape stripping, the disappearance of erythema was faster in the nanoparticle water spray group than in the water spray group, and no wrinkles were observed (FIG. 8, day 3). There was no clear edema in the skin of both groups. From the 1st week onward, both the nanoparticle water spray group and the water spray group (except for mouse No. 8) recovered the inflammatory symptoms to almost the same level (FIG. 8, day 7 to day 15). The change in the dermatitis score with respect to the number of days elapsed is shown in FIG. It was observed in the score that the degree of inflammation was improved earlier in the nanoparticle water spray group than in the water spray group mice.
　DNFB塗布により再刺激した場合に誘導される皮膚炎の程度はナノ粒子水噴霧群の方が水噴霧群よりも軽度であり、予防効果が示唆された。特に、DNFB塗布前のシェーバーおよび除毛クリームによる剃毛による皮膚のダメージは、皮膚病変スコア（図９）に示したように、ナノ粒子水噴霧群の方が低かった。 The degree of dermatitis induced when restimulated with DNFB application was milder in the nanoparticle water spray group than in the water spray group, suggesting a preventive effect. In particular, as shown in the skin lesion score (FIG. 9), the skin damage caused by shaving with a shaver and hair removal cream before DNFB application was lower in the nanoparticle water spray group.
2. 血中IgE濃度
　実験開始時（0日目）、DNFBを塗布し皮膚炎を再誘導開始時（22日目）、および実験終了時（24日目）に採血した血中のIgE濃度を測定した結果を図１０に示す。ナノ粒子水噴霧群と水噴霧群との間で有意な差は認められなかった。 2. Blood IgE concentration The blood IgE concentration collected at the start of the experiment (day 0), at the start of reinduction of dermatitis by applying DNFB (day 22), and at the end of the experiment (day 24). The measurement results are shown in FIG. There was no significant difference between the nanoparticle water spray group and the water spray group.
3. HE皮膚病理組織像
　炎症再誘導後の皮膚組織のHE染色像を図１１に示す。DNFB塗布した背部頭側の表皮肥厚および真皮の炎症は、ナノ粒子水噴霧群では水噴霧群と比較して軽度であった。DNFB非塗布の背部尾側においても、表皮肥厚はナノ粒子水噴霧群では著明に抑制され、炎症細胞浸潤も認めなかった。 3. HE skin histopathological image Fig. 11 shows an HE-stained image of skin tissue after reinduction of inflammation. DNFB-applied dorsal epidermis thickening and dermal inflammation were milder in the nanoparticle water spray group than in the water spray group. Even on the dorsal caudal side where DNFB was not applied, epidermal thickening was markedly suppressed in the nanoparticle water spray group, and no inflammatory cell infiltration was observed.
4. サイトカイン発現量
　背中の皮膚炎誘導部を背部頭側（DNFB塗布あり）及び尾側（DNFB塗布なし）に分けて各種サイトカイン発現量を調べた結果を図１２に示す。DNFB塗布により再刺激した場合、IL-1α, TNF-α, IL-6などのバリア機能破壊と関連したサイトカインは、ナノ粒子水噴霧の有無を問わず、DNFB塗布された背部頭側およびDNFB非塗部の尾側共に発現が認められた。DNFB塗布により再刺激した場合、T細胞の産生するサイトカインであるIL-17A, IL-4, IFN-γは、ナノ粒子水噴霧の有無を問わず、頭側、尾側皮膚ともに発現が低かった。 4. Cytokine expression level Fig. 12 shows the results of examining various cytokine expression levels by dividing the dermatitis induction part of the back into the dorsal head side (with DNFB application) and caudal side (without DNFB application). When restimulated by DNFB application, cytokines associated with the disruption of barrier function such as IL-1α, TNF-α, IL-6, etc., with or without nanoparticulate water spray, do not have DNFB applied to the dorsal temporal side and DNFB Expression was observed on the tail side of the painted area. When re-stimulated with DNFB, IL-17A, IL-4, and IFN-γ, cytokines produced by T cells, were low in both cranial and caudal skin with and without nanoparticle water spray .
[結論]
　NC/NgaSlcマウスの場合と同様に、BALB/cマウスにおいても、マクロ的な評価ばかりでなく、ミクロ的な評価でもナノ粒子水噴霧群の方が蒸留水噴霧群よりも治療の効果は優れていると結論付けられる。さらに、ナノ粒子水噴霧群は蒸留水噴霧群と比較して表皮肥厚、真皮炎症は軽度であり、さらに、再度のDNFB塗布による皮膚炎誘導後においても病変は軽度であることから、ナノ粒子水噴霧が繰り返しの炎症誘導に対してこれを制御し皮膚を保護する役割を果たすと考えられる。 [Conclusion]
As in the case of NC / NgaSlc mice, in BALB / c mice, not only macroscopic evaluation but also microscopic evaluation, the nanoparticle water spray group is more effective than the distilled water spray group. It is concluded that Furthermore, compared with the distilled water spray group, the nanoparticle water spray group showed mild epidermal thickening and dermal inflammation, and the lesion was mild even after induction of dermatitis by DNFB application again. It is thought that spraying plays a role in controlling this and protecting the skin against repeated inflammation induction.
　実験終了時における血中のIgE量は実験開始時や再度の炎症誘導前の血中IgE量に比べて有意に上昇したが、NC/NgaSlcマウスの場合と異なり、BALB/cマウスにおいてはナノ粒子水噴霧群と水噴霧群との間には有意な差がみられなかった。 The blood IgE level at the end of the experiment was significantly higher than the blood IgE level at the start of the experiment and before reinducing the inflammation, but unlike the NC / NgaSlc mice, the BALB / c mice had nanoparticles. There was no significant difference between the water spray group and the water spray group.
４．マウス皮膚炎モデルにおけるナノ粒子の治療及び予防効果の検討３
　テープストリッピングやDNFB塗布による皮膚炎誘導後の表皮肥厚軽減および皮膚炎を再誘導した場合の炎症制御にナノ粒子水噴霧が有効であることが、先の皮膚炎の予防効果をみる実験で示された。この効果が、すでに皮膚炎が起こっているマウスに対しても見られるかどうか、また、それが皮膚バリア機能に関係するかどうかを明らかにする目的で、MHC遺伝子型が知られている純系のBALB/cマウスを用いて検証した。 4). Study on therapeutic and preventive effects of nanoparticles in mouse dermatitis model 3
The previous experiment showing the prevention effect of dermatitis shows that nanoparticle water spray is effective for reducing epidermal thickening after induction of dermatitis by tape stripping or DNFB application and for controlling inflammation when dermatitis is re-induced. It was. In order to clarify whether this effect is also seen in mice that have already had dermatitis and whether it is related to skin barrier function, a pure strain with known MHC genotype Verification was performed using BALB / c mice.
　BALB/cマウスにテープストリッピングとDNFB塗布で誘導した皮膚炎に対する治療効果とDNFB塗布による再誘導に対する予防効果を検討するため以下の項目を調べた。
(1)　皮膚炎の程度の記録とスコア化
(2)　DNFB塗布による皮膚炎再誘導後の血中IgE量測定
(3)　DNFB塗布による皮膚炎再誘導後の皮膚HE染色組織観察
(4)　DNFB塗布による皮膚炎再誘導後の皮膚炎関連炎症性サイトカイン発現量測定 The following items were investigated to examine the therapeutic effect on dermatitis induced by tape stripping and DNFB application in BALB / c mice and the preventive effect on re-induction by DNFB application.
(1) Recording and scoring the degree of dermatitis
(2) Measurement of blood IgE level after reinducing dermatitis by DNFB application
(3) Observation of skin HE stained tissue after reinduction of dermatitis by DNFB application
(4) Measurement of dermatitis-related inflammatory cytokine expression after reinduction of dermatitis by DNFB application
[方法]
　図１３に示すスケジュールでBALB/cマウス（雌、8週齢）を処置した。まず、マウスの背部をシェーバーとクリームにより剃毛および除毛し、8回のテープストリッピングを行い、皮膚のバリア破壊を行った。先にハプテン誘発性皮膚炎を誘発し、ナノ粒子水あるいは水噴霧による治療効果を検討する実験では、テープストリッピングの後、マウス背部頭側へDNFBを塗布（1日目及び2日目）して皮膚炎を誘導した。ナノ粒子水又は蒸留水の噴霧は、1日目及び2日目のDNFB塗布後と、それ以降は20日目まで1日おきに実施した。ナノ粒子水あるいは水噴霧の皮膚炎に対する予防効果を検討する目的で、２１日目に再度除毛処理をし、２２日目と２３日目にマウス背部尾側に0.5%DNFBのオリーブ油－アセトン溶液（油：アセトン＝１：４）を20μL塗布することで皮膚炎の誘導を行なった。 [Method]
BALB / c mice (female, 8 weeks old) were treated according to the schedule shown in FIG. First, the back of the mouse was shaved and removed with a shaver and cream, and tape stripping was performed 8 times to destroy the skin barrier. In an experiment to first induce hapten-induced dermatitis and examine the therapeutic effect of nanoparticle water or water spray, after tape stripping, DNFB was applied to the back of the mouse (Day 1 and Day 2). Dermatitis was induced. The spraying of nanoparticle water or distilled water was carried out after the application of DNFB on the 1st and 2nd days, and thereafter every other day until the 20th day. In order to examine the preventive effect of nanoparticle water or water spray against dermatitis, hair removal treatment was performed again on the 21st day, and on the 22nd and 23rd days, 0.5% DNFB olive oil-acetone solution was placed on the back of the mouse. Dermatitis was induced by applying 20 μL of (oil: acetone = 1: 4).
　写真撮影は０、３、７、１１、１３、１５、２２、２３、２４日目に行ない、皮膚炎症に対する治療効果を観察し、上記表２に示した基準で皮膚炎の程度をスコア化した。背部頭側（治療モデル）と尾側（予防モデル）とに分けてスコア化し、評価した。 Photographing was performed on days 0, 3, 7, 11, 13, 15, 22, 23, 24, and the therapeutic effect on skin inflammation was observed, and the degree of dermatitis was scored according to the criteria shown in Table 2 above. . It was scored separately for the dorsal head side (treatment model) and caudal side (prevention model) and evaluated.
　血液サンプルの採取は０日目（採血１）、２２日目（採血２）及び２４日目（採血３）に行ない、血清分画のIgE量をサンドイッチELISAにより測定した。 Blood samples were collected on day 0 (blood collection 1), day 22 (blood collection 2) and day 24 (blood collection 3), and the IgE amount of the serum fraction was measured by sandwich ELISA.
　２４日目の実験終了時、マウスの背部頭側（治療モデル）及び尾側（予防モデル）の皮膚組織を採取し、HE組織染色観察及びサイトカイン発現量測定を行なった。 At the end of the experiment on the 24th day, skin tissues on the dorsal head side (treatment model) and caudal side (prevention model) of mice were collected, and HE tissue staining observation and cytokine expression level measurement were performed.
[結果]
1. 皮膚炎症状の判定結果
　マウス背部の写真を図１４に、炎症状態のスコア評価結果を図１５に示す。テープストリッピングとDNFB塗布により誘導した皮膚炎に対する治療効果は、ナノ粒子水噴霧は水噴霧よりも優れていた。DNFB塗布により再刺激した場合に誘導される皮膚炎の程度はナノ粒子水噴霧群の方が水噴霧群よりも低く、予防効果に優れていた。特に、DNFB塗布前にシェーバーおよび除毛クリームによる剃毛を行なった際の皮膚のダメージは、皮膚炎スコア（図１５）に示したように、ナノ粒子水噴霧群の方が低かった。 [result]
1. Judgment result of skin inflammation symptoms FIG. 14 shows a photograph of the back of the mouse, and FIG. 15 shows score evaluation results of the inflammatory state. The therapeutic effect on dermatitis induced by tape stripping and DNFB application was superior to nanoparticle water spray than water spray. The degree of dermatitis induced when restimulated by DNFB application was lower in the nanoparticle water spray group than in the water spray group, and was superior in the preventive effect. In particular, as shown in the dermatitis score (FIG. 15), damage to the skin when shaving with a shaver and a hair removal cream before applying DNFB was lower in the nanoparticle water spray group.
2. 血中IgE濃度
　実験開始時（0日目）、背中後半部へのDNFB塗布による炎症再誘導開始時（22日目）、および実験終了時（24日目）に採血した血中のIgE濃度を測定した結果を図１６に示す。DNFB塗布により再刺激した場合の血中IgE濃度程度は、ナノ粒子水噴霧群の方が水噴霧群よりも低い傾向が認められた。 2. Blood IgE concentration IgE in blood collected at the start of the experiment (day 0), at the start of reinduction of inflammation by applying DNFB to the back half of the back (day 22), and at the end of the experiment (day 24) The results of measuring the concentration are shown in FIG. The blood IgE concentration when restimulated with DNFB application tended to be lower in the nanoparticle water spray group than in the water spray group.
3. HE組織染色像
　炎症誘導後の皮膚病理組織のHE染色像を図１７に示す。皮膚炎の予防モデルとして、先にナノ粒子水又は水の噴霧を行い、実験終了前に皮膚炎の誘導を行なった背部尾側の表皮肥厚は、ナノ粒子噴霧群の方が水噴霧群よりも軽度であった。皮膚炎の治療モデルとして、先にDNFB塗布を行い、追ってナノ粒子水又は水の噴霧を続けた背部頭側の皮膚についても、表皮肥厚はナノ粒子噴霧群の方が水噴霧群よりも軽度であった。 3. HE Tissue Stained Image FIG. 17 shows an HE stained image of the skin pathological tissue after induction of inflammation. As a preventive model of dermatitis, the skin thickening on the dorsal and caudal side where nanoparticle water or water was sprayed first and dermatitis was induced before the end of the experiment was observed in the nanoparticle spray group than in the water spray group. It was mild. As a treatment model for dermatitis, the skin on the back of the head, which was previously applied with DNFB and subsequently sprayed with nanoparticulate water or water, also showed that epidermal thickening was milder in the nanoparticle spray group than in the water spray group. there were.
4. サイトカイン発現量
　背部皮膚を頭側（先にDNFB塗布、続いてナノ粒子水又は水を噴霧する治療モデル）及び尾側（先にナノ粒子水又は水を噴霧、最後にDNFB塗布をする予防モデル）に分けて各種サイトカイン発現量を調べた結果を図１８に示す。 4. Cytokine expression level Prevention of dorsal skin on the head side (first treatment with DNFB followed by nanoparticle water or water spray) and caudal side (first with nanoparticle water or water spray, and finally DNFB application) FIG. 18 shows the results of examining the expression levels of various cytokines divided into models.
　IL-1α, TNF-α, IL-6などのバリア機能破壊と関連したサイトカインは、ナノ粒子噴霧の有無を問わず、皮膚炎の予防モデルとして、実験終了前にDNFBを塗布して誘導した背部尾側部、および先に存在する皮膚炎に対する治療モデルとして最初にDNFB塗布を行い、実験終了直前にはDNFB塗布をしていない背部頭側部のいずれにおいても発現が認められた。さらに、皮膚採取前にDNFB塗布をした背部尾側において、発現が高い傾向が認められた。 Cytokines associated with disruption of barrier function such as IL-1α, TNF-α, and IL-6 are induced by applying DNFB before the end of the experiment as a model for preventing dermatitis with or without nanoparticle spraying. DNFB was first applied as a treatment model for the caudal part and the existing dermatitis, and expression was observed in both the dorsal head side without DNFB application immediately before the end of the experiment. Furthermore, a high tendency was observed in the dorsal caudal side where DNFB was applied before skin collection.
　皮膚炎の予防モデルとして、最初にナノ粒子水又は水の噴霧を繰り返し、実験終了前にDNFBを塗布して皮膚炎を惹起した場合、T細胞の産生するサイトカインIL-17A, IL-4, IFN-γは、ナノ粒子噴霧の有無を問わず、DNFBを実験終了前に塗布した背部尾側は、実験終了前にはDNFB塗布をしていない頭側に比べて発現が上昇した。加えて、IFN-γの発現は、ナノ粒子水噴霧群の方が水噴霧群の方と比べて発現量が上昇する傾向が認められた。 As a preventive model of dermatitis, when the spray of nanoparticles or water is repeated first and DNFB is applied before the end of the experiment to induce dermatitis, cytokines produced by T cells IL-17A, IL-4, IFN Regardless of the presence or absence of nanoparticle spraying, -γ increased in expression on the dorsal caudal side where DNFB was applied before the end of the experiment compared to the cranial side where DNFB was not applied before the end of the experiment. In addition, the expression level of IFN-γ tended to increase in the nanoparticle water spray group compared to the water spray group.
[結論]
　NC/Ngaマウスの場合と同様にBALB/cにおいてもナノ粒子水の有する殺菌作用はテープストリッピング＋DNFB塗布により誘導した皮膚炎に対する治療効果とDNFB塗布による皮膚炎誘導に対する予防効果があることが示された。 [Conclusion]
As in the case of NC / Nga mice, the bactericidal action of nanoparticulate water in BALB / c is shown to have a therapeutic effect on dermatitis induced by tape stripping + DNFB application and a preventive effect on dermatitis induction by DNFB application. It was.
　NC/Ngaマウスの場合と同様にBALB/cにおいてもその効果は殺菌作用によるバリア機能回復の促進とそれに関連する炎症反応の制御に基づくことを示唆する結果が得られた。 As in the case of NC / Nga mice, results suggesting that the effect of BALB / c is based on the promotion of barrier function recovery by bactericidal action and the control of the inflammatory response related thereto.
５．マウス皮膚炎モデルにおけるナノ粒子水の治療及び予防効果の検討４
　ナノ粒子水噴霧がバリア機能の保持と炎症性応答制御に関与しているかどうかをさらに詳細に調べるために、12週齢BALB/cマウスに繰り返しテープストリッピングを行い、次の項目について調べた。
(1)　繰り返しテープストリッピングをする過程の皮膚炎症状態のスコア変化
(2)　繰り返しテープストリッピングをする過程の水分蒸散量変化
(3)　繰り返しテープストリッピング後の皮膚HE染色組織観察
(4)　繰り返しテープストリッピング後の皮膚炎関連炎症性サイトカイン発現量測定 5. Study of nanoparticle water treatment and prevention effect in mouse dermatitis model 4
In order to investigate in more detail whether nanoparticle water spray is involved in maintaining barrier function and controlling inflammatory response, 12-week-old BALB / c mice were subjected to repeated tape stripping to investigate the following items.
(1) Change in score of skin inflammatory condition during repeated tape stripping process
(2) Change in moisture transpiration during repeated tape stripping
(3) Observation of skin HE-stained tissue after repeated tape stripping
(4) Measurement of dermatitis-related inflammatory cytokine expression after repeated tape stripping
　図１９に示すスケジュールで繰り返しテープストリッピングを行った。12週齢BALB/cマウスの背部を剃毛および除毛した後、水分蒸散量（皮膚バリア機能の指標）を測定しながらその値が30から50g/hm2の範囲になるようにテープストリッピング（3～5回）をした。水分蒸散量の測定には、Courage + Khazaka electronic GmbH社（独国ケルン）のThe Multi Probe Adapter System（略称MPA5)（ソフトウェア：CK-MPA-multi-probe version 1.5.1.4）に蒸散量測定用のプローブTewameter(登録商標) probe(TM300MP)を接続したものを用いた。測定に際してはプローブを左足付け根部分にずれないように押し当てて行った。麻酔後に写真撮影と、その写真に基づいた皮膚炎症状態のスコア化、及び皮膚蒸散量の測定を行い、その後にナノ粒子水または蒸留水を噴霧した（各群4匹ずつ）。皮膚炎症の状態のスコア化は、上述の検討２、３と同様に、背部頭側部と尾側部とに分けてスコア化し、それぞれ、合計値によって評価した。ナノ粒子水噴霧群の水分蒸散量が10g/hm2前後の値を示した4日目、8日目および11日目に再び除毛とテープストリッピングを行った。14日目に写真撮影と水分蒸散量測定を行い、その後に採血と皮膚組織のサンプリング（凍結切片、固定標本およびRT-PCR用のRNA調製）を行った。 Tape stripping was repeatedly performed according to the schedule shown in FIG. After shaving and removing the back of 12-week-old BALB / c mice, measuring the amount of water transpiration (indicator of skin barrier function) and stripping the tape so that the value is in the range of 30-50 g / hm 2 ( 3-5 times). For measuring moisture transpiration, the Multi Probe Adapter System (abbreviation MPA5) (software: CK-MPA-multi-probe version 1.5.1.4) of Courage + Khazaka electronic GmbH (Cologne, Germany) A probe to which a Tewameter (registered trademark) probe (TM300MP) was connected was used. In the measurement, the probe was pressed against the left foot base so as not to be displaced. After anesthesia, photography, scoring of skin inflammation based on the photograph, and measurement of skin transpiration were performed, followed by spraying with nanoparticle water or distilled water (4 mice in each group). The scoring of the state of skin inflammation was scored separately for the dorsal head side and the caudal side in the same manner as in the examinations 2 and 3, and each was evaluated by the total value. Day 4 water loss of nanoparticles water spray group showed a value of about 10 g / hm 2, was performed again depilatory and tape stripping to 8 days and 11 days. On the 14th day, photography and measurement of water transpiration were performed, followed by blood sampling and skin tissue sampling (frozen sections, fixed specimens, and preparation of RNA for RT-PCR).
　皮膚炎状態をスコア評価した結果を図２０に示す。ナノ粒子噴霧群および水噴霧群共に、テープストリッピング毎にスコアは12程度まで上昇し、その翌日には2～3程度まで低下するという、バリア破壊と組織修飾の繰り返しが観察された。14日目の皮膚炎症状態は、ナノ粒子水噴霧群と水噴霧群との間に大きな差はなかった。 The results of score evaluation of the dermatitis state are shown in FIG. In both the nanoparticle spray group and the water spray group, repeated breaking of the barrier and tissue modification were observed, with the score rising to about 12 for each tape stripping and decreasing to about 2-3 on the following day. The skin inflammation state on day 14 was not significantly different between the nanoparticle water spray group and the water spray group.
　一方、皮膚の水分蒸散量を測定したところ（結果を図２１に示す）、テープストリッピングを繰り返していると、テープストリッピング毎に水噴霧群は60～70 g/hm2まで上昇するが、ナノ粒子水噴霧群のほうは最大50 g/hm2程度までしか上昇しなかった。テープストリッピング後、翌日には、ナノ粒子水噴霧群は10 g/hm2程度まで蒸散量が低下しているのに対し、テープストリッピングを繰り返していると、水噴霧群では、次第に蒸散量の回復が不十分となり、その値が徐々に上昇し、14日目には20 g/hm2以上を示した。このことは、繰り返しテープストリッピング後、皮膚バリア破壊後の修復機能は水噴霧群のほうがナノ粒子水噴霧群よりも劣っていることを示している。 On the other hand, when the amount of moisture transpiration in the skin was measured (results are shown in FIG. 21), when the tape stripping was repeated, the water spray group increased to 60-70 g / hm 2 for each tape stripping. more of the water spray group did not increase only up to a maximum of about 50 g / hm 2. After tape stripping, the next day, whereas the nanoparticles water spray group transpiration amount up to about 10 g / hm 2 is lowered and is repeated tape stripping, a water spray unit, gradually recovering transpiration Became insufficient, and its value gradually increased, and on the 14th day, it was over 20 g / hm 2 . This indicates that the water spray group is inferior to the nanoparticle water spray group in terms of the repair function after repeated tape stripping and skin barrier destruction.
　またこのことは、図２２に示すように、14日目に採取した皮膚のHE染色標本において、表皮の肥厚化程度がナノ粒子水噴霧群に比べて水噴霧群のほうが大きいことからも支持される。 This is also supported by the fact that the thickness of the epidermis is thicker in the water spray group than in the nanoparticle water spray group in the HE-stained skin sample collected on the 14th day, as shown in FIG. The
　さらに、繰り返しテープストリッピング後のIL-1α, TNF-α, IL-6などの表皮バリア機能破壊と関連したサイトカインの発現量をRT-PCR法で調べてみると（結果を図２３に示す）、ナノ粒子水噴霧の有無を問わず、背部尾側および頭側のいずれにおいても同程度の発現が認められた。一方、T細胞の産生するサイトカイン発現量はIL-4, IFN-γはその発現量は低く両者の群の間には大きな差がみられなかったが、表皮の破壊と関連性の高いIL-17Aは水噴霧群のほうがナノ粒子水噴霧群に比べて発現量が高く、皮膚バリア機能は水噴霧群のほうがナノ粒子水噴霧群よりも障害されていることを示唆していた。 Furthermore, when the expression level of cytokines associated with disruption of epidermal barrier function such as IL-1α, TNF-α, IL-6 after repeated tape stripping was examined by RT-PCR method (results are shown in FIG. 23), Regardless of the presence or absence of nanoparticle water spray, the same level of expression was observed on both the dorsal caudal side and the cranial side. On the other hand, the expression levels of cytokines produced by T cells were low for IL-4 and IFN-γ, and there was no significant difference between the two groups, but IL- The expression level of 17A was higher in the water spray group than in the nanoparticle water spray group, suggesting that the skin barrier function was more impaired in the water spray group than in the nanoparticle water spray group.
　ナノ粒子水の有する殺菌・保水作用は、繰り返しテープストリッピングによる皮膚損傷に対する治療と、該処理により誘導されうる皮膚炎症に対する予防に効果があることが示された。 It was shown that the sterilization / water retention action possessed by the nanoparticulate water is effective in treating skin damage by repeated tape stripping and preventing skin inflammation that can be induced by the treatment.
６．マウス皮膚炎モデルにおけるナノ粒子水の治療及び予防効果の検討５（ヘアレスマウスを用いた、ハプテン誘導性慢性皮膚炎モデルでの検討）
　剃毛、除毛に伴う物理的な皮膚バリア機能破壊の影響を避けるため、ヘアレスマウス（Hos:HR-1、日本SLC）を用い、また、アトピー性皮膚炎をはじめとする人の慢性皮膚炎に近い動物モデルを使ってナノ粒子水の抗菌、保水効果の検討を行うため、ハプテンを繰り返し塗布するマウス慢性皮膚炎モデルを作製し（Nakajima, S. et al., J. Invest. Dermatol, 134, 2122-2130, 2014らの方法をDNFBの抗原系に改変）、下記の８項目について調べた。背部皮膚にDNFBを塗布する実験群では、四肢による掻把の影響が少ないと思われる肩甲骨の位置の皮膚に塗布した。 6). Study of nanoparticle water treatment and prevention effect in mouse dermatitis model 5 (Study in hapten-induced chronic dermatitis model using hairless mouse)
Hairless mice (Hos: HR-1, Japan SLC) are used to avoid the effects of physical skin barrier function destruction associated with shaving and hair removal, and chronic dermatitis in humans including atopic dermatitis In order to investigate the antibacterial and water retention effects of nanoparticle water using an animal model close to the model, a mouse chronic dermatitis model with repeated application of hapten was prepared (Nakajima, S. et al., J. Invest. Dermatol, 134). , 2122-2130, 2014 et al. Were modified to DNFB antigen system), and the following 8 items were examined. In the experimental group in which DNFB was applied to the dorsal skin, it was applied to the skin at the location of the scapula where the effect of scratching by the extremities was considered to be small.
＜評価項目＞
(1)　皮膚炎症状態のスコア化：肉眼的な皮膚炎症程度の評価
(2)　両耳介の厚さ測定：耳介の炎症反応程度の評価
(3)　皮膚の水分蒸散量と保水度の測定：上背部皮膚表皮のバリア機能の破壊・回復程度の評価
(4)　HE組織染色標本観察：顕微鏡観察による皮膚炎症程度の評価
(5)　皮膚内に発現するサイトカインの種類と量の測定：背部頭側の皮膚と右耳介に発現するサイトカインの種類と発現量の評価
(6)　血清中の抗Hapten抗体量の測定：血清中の抗DNP IgG抗体量の評価
(7)　頭側背部皮膚の表在細菌数の測定：頭側背部皮膚の表在細菌数の評価
(8)　血清中の黄色ブドウ球菌に対するIgG抗体量の測定：ヘアレスマウスの背部皮膚から分離した黄色ブドウ球菌に対する血清IgG抗体量の評価 <Evaluation items>
(1) Scoring of skin inflammatory condition: Evaluation of macroscopic skin inflammation
(2) Measurement of thickness of both pinna: Evaluation of the degree of inflammatory reaction of pinna
(3) Measurement of skin moisture transpiration and water retention: Evaluation of the degree of destruction and recovery of the barrier function of the upper back skin epidermis
(4) HE tissue staining specimen observation: Evaluation of skin inflammation by microscopic observation
(5) Measurement of types and amounts of cytokines expressed in the skin: Evaluation of types and amounts of cytokines expressed in the dorsal temporal skin and right ear
(6) Measurement of serum anti-Hapten antibody level: Evaluation of serum anti-DNP IgG antibody level
(7) Measurement of the number of superficial bacteria in the cranial dorsal skin: Evaluation of the superficial bacteria count in the cranial dorsal skin
(8) Measurement of the amount of IgG antibody against S. aureus in serum: Evaluation of the amount of serum IgG antibody against S. aureus isolated from the back skin of hairless mice
＜マウス慢性皮膚炎モデルの作製方法＞
　ヘアレスマウス（Hos:HR-1）雌を一群５匹として、表６の通りに４つの実験群を作った。処置スケジュールを図２４に示す。 <Method for preparing mouse chronic dermatitis model>
Four experimental groups were made as shown in Table 6, with 5 female hairless mice (Hos: HR-1). The treatment schedule is shown in FIG.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　皮膚の写真撮影、常在細菌数、耳の厚さ、皮膚水分蒸散量、皮膚保水度、および皮膚炎スコアの測定は、マウスを麻酔してから行ない、これらの測定後にDNFB塗布を行なった。この間、DNFB塗布の翌日から１日おきにナノ粒子水の噴霧を行った（図２４）。day33に0.3%DNFBを塗布し、その１日後（day34）に両耳と背部の皮膚の採取と採血をおこなった。右耳介と頭側背部皮膚からはHE染色標本を作成し、また、RT-PCRによる各種サイトカインの測定を行った。血液からは血清中の抗DNP抗体及び抗S.aureus抗体の凝集価の測定を行った。 Measurement of skin photography, resident bacteria count, ear thickness, skin moisture transpiration, skin moisture retention, and dermatitis score was performed after anesthetizing the mice, and DNFB was applied after these measurements. During this time, spraying of nanoparticulate water was performed every other day from the day after the application of DNFB (FIG. 24). On day 33, 0.3% DNFB was applied, and one day later (day 34), skin of both ears and back was collected and blood was collected. HE stained specimens were prepared from the right auricle and cranial dorsal skin, and various cytokines were measured by RT-PCR. From blood, the aggregation titer of anti-DNP antibody and anti-S. Aureus antibody in serum was measured.
(1)　皮膚炎症のスコア化
　皮膚炎スコアは下記表７のように定義し、皮膚炎の程度をスコア化した。スコアは、マウス１匹について、紅斑、鱗屑、糜爛、***、痂皮、左耳介の肥厚・変形、右耳介の肥厚・変形の７項目について０～３の４段階のスコアを付け、合計した。１匹当たりの最大スコアは２１ポイントである。各実験群のマウスについて、day5, day12, day19, day26, day33に皮膚炎スコアを測定した。 (1) Scoring of skin inflammation The dermatitis score was defined as shown in Table 7 below, and the degree of dermatitis was scored. For each mouse, a score of 0 to 3 was assigned to 7 items of erythema, scales, wrinkles, protuberances, crusts, left auricle thickening / deformation, and right auricle thickening / deformation. did. The maximum score per animal is 21 points. The dermatitis score was measured on mice of each experimental group on day 5, day 12, day 19, day 26, day 33.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　DNFB塗布後の頭側背部及び両耳介の皮膚炎スコアの変化を追った結果を図２５に示す。またday33における各実験群の頭側背部皮膚および両耳介の画像を図２６に示す。DNFB塗布とともにナノ粒子水噴霧したA群では、ナノ粒子水噴霧をしなかったC群（陽性対照群）や水噴霧B群よりも明らかに皮膚炎スコアが低かった（図２５）。D群はDNFB塗布、噴霧いずれも施行しなかった陰性対照を示す。図２６に示す通り、B群とC群では皮膚炎、耳介変形が明らかに認められた一方、ナノ粒子水噴霧群（A群）は、陰性対照（D群）同等、著明に症状が抑制された。 FIG. 25 shows the results of tracking the changes in the dermatitis scores on the back of the head and DNA after application of DNFB. Moreover, the image of the head side dorsal skin and both ears of each experimental group on day33 is shown in FIG. The dermatitis score was clearly lower in group A sprayed with nanoparticle water together with DNFB application than group C (positive control group) and water spray B group that were not sprayed with nanoparticle water (FIG. 25). Group D represents a negative control in which neither DNFB application nor spraying was performed. As shown in FIG. 26, dermatitis and auricular deformation were clearly observed in Group B and Group C, while the nanoparticle water spray group (Group A) was the same as the negative control (Group D), with marked symptoms. Suppressed.
(2)　両耳介の厚さ測定
　各実験群のマウスについて、day5, day12, day19, day26, day33に左右耳介の厚さをそれぞれ厚みゲージで測定した。左右耳介の測定値の平均値をその個体の測定値としてグラフ化した。 (2) Measurement of thickness of both ear pinna For the mice of each experimental group, the thickness of the left and right pinna was measured with a thickness gauge on day 5, day 12, day 19, day 26, and day 33, respectively. The average value of the measured values of the left and right pinna was graphed as the measured value of the individual.
　結果を図２７に示す。水噴霧群（B群）及び陽性対照群（C群）では、DNFB塗布後のday12頃より耳介が肥厚し始めた一方、ナノ粒子水噴霧群（A群）は肥厚することなくday33には抑制効果が明らかであり、陰性対照群（D群）とほとんど変わらない結果を示した。 The results are shown in FIG. In the water spray group (Group B) and the positive control group (Group C), the auricles began to thicken around day12 after DNFB application, while the nanoparticle water spray group (Group A) did not thicken on day33. The inhibitory effect was clear and showed almost the same results as the negative control group (Group D).
(3)　皮膚の水分蒸散量と保水度測定
　各実験群のマウスについて、day5, day12, day19, day26, day33に上背部皮膚の水分蒸散量及び保水度を測定した。測定にはCourage + Khazaka electronic GmbH社（独国ケルン）のThe Multi Probe Adapter System（略称MPA5)（ソフトウェア：CK-MPA-multi-probe version 1.5.1.4）を使用した。蒸散量の測定では、MPA5に蒸散量測定用のプローブTewameter(登録商標) probe(TM300MP)を接続し、プローブを頭側背部皮膚に押し付けて測定を行なった。保水度の測定では、MPA5に保水度測定用プローブCorneometer (CM825MP)を接続し、頭側背部皮膚3か所にプローブを押し付けて保水度を測定し、3か所の測定値の平均値をその個体の測定値とした。 (3) Measurement of skin water transpiration and water retention The amount of water transpiration and water retention of the upper back skin was measured on day 5, day 12, day 19, day 26, and day 33 for mice in each experimental group. The Multi Probe Adapter System (abbreviation MPA5) (software: CK-MPA-multi-probe version 1.5.1.4) of Courage + Khazaka electronic GmbH (Cologne, Germany) was used for the measurement. In the measurement of the transpiration rate, a probe for measuring the transpiration amount Tewameter (registered trademark) probe (TM300MP) was connected to the MPA5, and the probe was pressed against the dorsal skin of the head and measured. To measure the water retention, connect a water retention measurement probe Corneometer (CM825MP) to the MPA5, and press the probe against 3 head and back skins to measure the water retention. The measured value of the individual was used.
　皮膚水分蒸散量の測定結果を図２８に示す。B群及びC群では、DNFB塗布後day12には蒸散量が明らかに上昇し、その後も高い蒸散量を示した。ナノ粒子水噴霧群（A群）では、一時的にわずかな蒸散量の上昇が認められたものの低い値を維持していた。day33には、A群はB群およびC群に比べ有意に低値を示した。 Fig. 28 shows the results of measurement of skin moisture transpiration. In Group B and Group C, the amount of transpiration increased significantly on day 12 after application of DNFB, and high transpiration was observed thereafter. In the nano-particle water spray group (Group A), although a slight increase in transpiration was observed, it remained low. On day 33, group A showed significantly lower values than groups B and C.
　保水度の測定結果を図２９に示す。B群及びC群ではDNFB塗布後、経時的に保水度が低下した一方、ナノ粒子水噴霧群（A群）では保水度の低下は全く見られず、無処置群（D群）と同様、保水能が維持された。day33には、ナノ粒子水噴霧群（A群）はB群、C群に比べて有意差を示した。 The measurement result of water retention is shown in FIG. In Group B and Group C, the water retention decreased over time after applying DNFB, while in the nanoparticle water spray group (Group A), no decrease in water retention was seen, as in the untreated group (Group D), Water retention capacity was maintained. On day 33, the nanoparticle water spray group (Group A) showed a significant difference compared to Group B and Group C.
(4)　HE組織染色標本観察
　各実験群のマウスより、day34（33日目にDNFB 0.3%の塗布後１日目）に右耳介と頭側背部皮膚を採取し、HE染色を行なった。 (4) Observation of HE tissue staining specimen The right auricle and cranial dorsal skin were collected from the mice of each experimental group on day 34 (1 day after application of DNFB 0.3% on day 33) and subjected to HE staining.
　図３０A～Dは、A群～D群の代表的な耳介および頭側背部皮膚の症状とHE染色像である。A-1、B-1、C-1、D-1は右耳介皮膚のHE組織染色写真であり、A-2、B-2、C-2、D-2は頭側背部皮膚のHE組織染色写真である。黒のバーは100μmを示す。 FIGS. 30A to 30D are typical pinna and cranial dorsal skin symptoms and HE-stained images of Groups A to D. FIG. A-1, B-1, C-1, and D-1 are HE tissue staining photographs of the right auricular skin, and A-2, B-2, C-2, and D-2 are HE on the dorsal skin. It is a tissue staining photograph. The black bar indicates 100 μm.
　水噴霧群（B群）では、肉眼的に炎症が強く、病理組織では耳介の表皮肥厚、および真皮の炎症、線維化も著明であった。一方、ナノ粒子水噴霧群（A群）は臨床症状、病理組織ともに明らかに抑制されていた。背部皮膚の病理組織では、差異は明かではないが、ナノ粒子水噴霧群（A群）では毛包周囲（ヘアレスマウスでは嚢胞状構造が形成される）の炎症細胞浸潤が軽度であった。 In the water spray group (Group B), inflammation was strong macroscopically, and in the pathological tissue, thickening of the epidermis, inflammation of the dermis, and fibrosis were also remarkable. On the other hand, in the nanoparticle water spray group (Group A), both clinical symptoms and histopathology were clearly suppressed. In the pathological tissue of the dorsal skin, the difference was not clear, but infiltration of inflammatory cells around the hair follicle (a cyst-like structure was formed in hairless mice) was mild in the nanoparticle water spray group (Group A).
　陽性対照群（C群）でもB群同様、強く皮膚炎症が認められ、A群に比べて耳介、背部皮膚の変化が強く認められた。 In the positive control group (Group C), skin inflammation was strongly observed as in Group B, and changes in the auricle and back skin were observed more strongly than Group A.
(5)　皮膚内に発現するサイトカインの種類と量の測定
　各実験群のマウスよりday34に採取した右耳介組織及び上背部皮膚組織におけるサイトカインの発現を定量的RT-PCRにより測定した。プライマーは上記表５に記載のものを用いた。発現量は、HPRT遺伝子に対してノーマライズし、相対発現量で評価した。 (5) Measurement of type and amount of cytokine expressed in skin Cytokine expression in right auricular tissue and upper back skin tissue collected on day 34 from mice of each experimental group was measured by quantitative RT-PCR. The primers described in Table 5 above were used. The expression level was normalized with respect to the HPRT gene and evaluated by relative expression level.
　結果を図３１に示す。耳介（図３１右）では、B群、C群の炎症性サイトカイン（IL-6,、IFN-γ）の発現がA群やD群よりも上昇し、B群、C群の耳介の炎症程度と関連性を示していた。背部皮膚（図３１左）では、A群以外にD群にもTNF-αやIL-17などのサイトカインの発現が認められた。このことは、ヘアレスマウスの場合はTh17細胞を介した皮膚恒常性の維持機構の存在を示唆していた。 The results are shown in FIG. In the auricle (FIG. 31 right), the expression of inflammatory cytokines (IL-6, IFN-γ) in groups B and C is higher than those in groups A and D. It was related to the degree of inflammation. In the back skin (FIG. 31 left), expression of cytokines such as TNF-α and IL-17 was observed in the D group as well as the A group. This suggested the existence of a maintenance mechanism of skin homeostasis via Th17 cells in the case of hairless mice.
(6)　血清中の抗hapten抗体量の測定
　各実験群のマウスよりday34に血液を採取し、血清中の抗DNP抗体量をDNP-BSAを抗原としたELISAにて測定した。5μg/mLのDNP-BSAでコートしたELISAプレートを使用し、マウス血清の希釈系列をサンプルとしてELISAを行なった。検出にはHRPO結合ヤギ抗マウスIgGを用いた。 (6) Measurement of serum anti-hapten antibody amount Blood was collected from mice of each experimental group on day 34, and the amount of anti-DNP antibody in serum was measured by ELISA using DNP-BSA as an antigen. An ELISA plate coated with 5 μg / mL DNP-BSA was used, and ELISA was performed using a dilution series of mouse serum as a sample. HRPO-conjugated goat anti-mouse IgG was used for detection.
　結果を図３２に示す。抗DNP抗体量は、ナノ粒子水噴霧群（A）、水噴霧群（B）、噴霧なし群（C）の3群間ではあまり変わらず、DNPに対する免疫応答（B細胞とTｈ細胞の反応）は遜色なく起こっていることが確認された。このことから、皮膚に会合したナノ粒子の存在によってDNFBの皮膚への反応性が低下するなどの理由により、ハプテン誘導性免疫応答自体が低下した、という可能性は低いことが示唆された。 Results are shown in FIG. The amount of anti-DNP antibody does not change much between the three groups: nanoparticle water spray group (A), water spray group (B), and non-spray group (C), and immune response to DNP (reaction between B cells and Th cells) Was confirmed to be innocent. This suggests that it is unlikely that the hapten-induced immune response itself has decreased due to a decrease in the reactivity of DNFB to the skin due to the presence of nanoparticles associated with the skin.
(7)　頭側背部皮膚の表在細菌数の測定
　各実験群のマウスについて、day5, day12, day19, day26, day33に上背部皮膚の表在細菌数を測定した。頭側背部皮膚に生菌数測定用標準寒天培地（ペタンチェック、栄研化学）を5秒間押し当てた後、37℃フラン器で24時間培養し、4cm2の区画あたりのコロニー数をカウントした。 (7) Measurement of the number of superficial bacteria on the cranial dorsal skin The number of superficial bacteria on the upper back skin was measured on mice from each experimental group on day 5, day 12, day 19, day 26, and day 33. After pressing the standard agar medium (Petancheck, Eiken Chemical Co., Ltd.) for viable count on the cranial dorsal skin for 5 seconds, the cells were cultured for 24 hours in a 37 ° C franc vessel, and the number of colonies per 4 cm 2 compartment was counted .
　結果を図３３に示す。ナノ粒子水噴霧群（A群）では、水噴霧群（B群）、噴霧なし群（C群）、前処理もDNFB塗布も行わない無処理群（D群）と比べて細菌数の変動が少なく、かつ低い値を示した。 Results are shown in FIG. In the nano-particle water spray group (Group A), the number of bacteria varies compared to the water spray group (Group B), the non-spray group (Group C), and the untreated group (Group D) that does not perform pretreatment or DNFB application. Less and lower value.
(8)　血清中の黄色ブドウ球菌に対するIgG抗体量の測定
　各実験群のマウスより、day34に採血し、血清を分離した。血清を非動化した後、血清と当量の0.2M 2-MEを加え37℃で１時間加温した。この処理により、腸管系で多くが産生されるIgM自然抗体がモノマー化し、凝集価への寄与が乏しくなる。その後、この血清を256倍まで倍々希釈し、これらヘアレスマウスの皮膚から分離したS.aureusを用いて作製した不活化S.aureus菌体液と混合して4℃で一晩反応させた。凝集が起こらなかった最大希釈倍率を凝集価とし、凝集価をグラフ化した。 (8) Measurement of the amount of IgG antibody against S. aureus in serum Blood was collected from the mice of each experimental group on day 34, and the serum was separated. After deactivating the serum, 0.2M 2-ME equivalent to the serum was added and heated at 37 ° C. for 1 hour. By this treatment, IgM natural antibodies that are produced in large amounts in the intestinal system become monomers, and the contribution to the aggregation value becomes poor. Then, this serum was diluted to 256 times, mixed with an inactivated S. aureus cell solution prepared using S. aureus isolated from the skin of these hairless mice, and reacted at 4 ° C. overnight. The maximum dilution ratio at which aggregation did not occur was defined as the aggregation value, and the aggregation value was graphed.
　結果を図３４に示す。ナノ粒子水噴霧群（A群）の凝集価は水噴霧群（B群）及び噴霧なし群（C群）よりも有意に低く、ナノ粒子水噴霧による血清中の抗S.aureus IgG抗体の減少が認められた。このことから、ナノ粒子水噴霧による皮膚バリア機能の速やかな修復の結果、IgG抗体産生を誘導するような皮膚表在細菌の真皮あるいはそれより深部への侵入が抑えられたことが示唆される。 Results are shown in FIG. The aggregation value of the nanoparticle water spray group (Group A) is significantly lower than that of the water spray group (Group B) and the group without spray spray (Group C). Was recognized. This suggests that as a result of rapid repair of the skin barrier function by nanoparticle water spraying, invasion of the skin surface bacteria that induce IgG antibody production into the dermis or deeper thereof was suppressed.
＜検討５まとめ＞
　ナノ粒子水を噴霧した群では、皮膚炎症スコア値は水噴霧群・非噴霧群のそれよりも低値であった。皮膚炎症スコア値は、免疫応答性の指標である両耳介の肥厚化のみならず、皮膚バリア機能の指標である背部皮膚水分蒸散量と保水度にも関連性が認められた。
　ナノ粒子水噴霧群の場合は、表皮増殖関連のサイトカインはTh17細胞系とともに発現し、一方、水噴霧群・噴霧なし群の場合は、皮膚ダメージ関連シグナルを介して発現することが示唆された。
　ナノ粒子水噴霧群の表在細菌数は他の3群と比べて一定の低値に制御されていた。ナノ粒子水噴霧による表在細菌数制御は、皮膚バリア性の向上との相乗効果により、血清中の抗S.aureus IgG抗体の減少をもたらした。
　以上の結果より、ナノ粒子水噴霧は皮膚バリア機能の向上と表在細菌数制御の関与する免疫応答とを介して皮膚炎症反応を制御することが示唆された。 <Summary of study 5>
In the group sprayed with nanoparticle water, the skin inflammation score value was lower than that in the water spray group and the non-spray group. The skin inflammation score value was related not only to the thickening of the auricle, which is an index of immune responsiveness, but also to the back skin moisture transpiration amount and the water retention level, which is an index of skin barrier function.
In the nanoparticle water spray group, it was suggested that the epithelial proliferation-related cytokines were expressed along with the Th17 cell line, whereas in the water spray group and the non-spray group, it was expressed through a skin damage-related signal.
The number of superficial bacteria in the nanoparticle water spray group was controlled to a certain low value compared with the other three groups. Control of the number of superficial bacteria by nanoparticle water spray resulted in a decrease in serum anti-S. Aureus IgG antibody due to a synergistic effect with improved skin barrier properties.
From the above results, it was suggested that nanoparticle water spray controls the skin inflammatory reaction through the improvement of the skin barrier function and the immune response involved in controlling the number of superficial bacteria.

Claims (19)

  1. 　アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子を有効成分として含有する、皮膚炎の治療又は予防剤。 A therapeutic or preventive agent for dermatitis comprising, as an active ingredient, cyanoacrylate polymer particles having an average particle size of less than 1000 nm, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof .
  2. 　前記粒子は、アミノ酸、アミノ酸誘導体、及びそれらのオリゴマーからなる群より選択される少なくとも１種を含む、請求項１記載の治療又は予防剤。 The therapeutic or prophylactic agent according to claim 1, wherein the particles include at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers thereof.
  3. 　前記粒子は、少なくとも１種のアミノ酸を含む、請求項２記載の治療又は予防剤。 The therapeutic or prophylactic agent according to claim 2, wherein the particles contain at least one amino acid.
  4. 　前記アミノ酸誘導体が、クレアチン、オルニチン、サイロキシン、デスモシン、ヒドロキシプロリン、ヒドロキシリジン、ホスホセリン、テアニン、カイニン酸、トリコロミン酸、及びサルコシンからなる群より選択される少なくとも１種である、請求項１又は２記載の治療又は予防剤。 The amino acid derivative is at least one selected from the group consisting of creatine, ornithine, thyroxine, desmosine, hydroxyproline, hydroxylysine, phosphoserine, theanine, kainic acid, tricolominic acid, and sarcosine. Treatment or prevention agent.
  5. 　前記アミノ酸が、アルギニン、ヒスチジン、リジン、アスパラギン酸、グルタミン酸、アラニン、グリシン、ロイシン、バリン、イソロイシン、セリン、スレオニン、フェニルアラニン、トリプトファン、チロシン、シスチン又はシステイン、グルタミン、アスパラギン、プロリン、メチオニン、β－アラニン、γ－アミノ酪酸、カルニチン、γ－アミノレブリン酸、及びγ－アミノ吉草酸からなる群より選択される少なくとも１種である、請求項１ないし４のいずれか１項に記載の治療又は予防剤。 The amino acid is arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, asparagine, proline, methionine, β-alanine The therapeutic or prophylactic agent according to any one of claims 1 to 4, which is at least one selected from the group consisting of γ-aminobutyric acid, carnitine, γ-aminolevulinic acid, and γ-aminovaleric acid.
  6. 　前記アミノ酸が、グリシン及びアスパラギン酸からなる群より選択される少なくとも１種である、請求項５記載の治療又は予防剤。 The therapeutic or prophylactic agent according to claim 5, wherein the amino acid is at least one selected from the group consisting of glycine and aspartic acid.
  7. 　前記粒子は、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種が共存する条件下において、シアノアクリレートモノマーをアニオン重合させることにより製造された粒子である、請求項１ないし６のいずれか１項に記載の治療又は予防剤。 The particle is a particle produced by anionic polymerization of a cyanoacrylate monomer under a condition in which at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof coexists. The therapeutic or prophylactic agent according to any one of 1 to 6.
  8. 　前記粒子は、糖及びポリソルベートからなる群より選択される少なくとも１種をさらに含む、請求項１ないし７のいずれか１項に記載の治療又は予防剤。 The therapeutic or prophylactic agent according to any one of claims 1 to 7, wherein the particles further contain at least one selected from the group consisting of sugar and polysorbate.
  9. 　前記粒子は、少なくとも１種の糖をさらに含む、請求項１ないし７のいずれか１項に記載の治療又は予防剤。 The therapeutic or prophylactic agent according to any one of claims 1 to 7, wherein the particles further contain at least one kind of sugar.
  10. 　前記糖が、水酸基を有する単糖類、水酸基を有する二糖類及び水酸基を有する多糖類からなる群より選択される少なくとも１種である、請求項８又は９記載の治療又は予防剤。 The therapeutic or prophylactic agent according to claim 8 or 9, wherein the sugar is at least one selected from the group consisting of a monosaccharide having a hydroxyl group, a disaccharide having a hydroxyl group, and a polysaccharide having a hydroxyl group.
  11. 　前記粒子は、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種と、糖及びポリソルベートからなる群より選択される少なくとも１種とが共存する条件下において、シアノアクリレートモノマーをアニオン重合させることにより製造された粒子である、請求項１ないし１０のいずれか１項に記載の治療又は予防剤。 The particles are cyanoacrylate under conditions where at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and at least one selected from the group consisting of sugars and polysorbates coexist. The therapeutic or prophylactic agent according to any one of claims 1 to 10, which is a particle produced by anionic polymerization of a monomer.
  12. 　前記シアノアクリレートがｎ－ブチルシアノアクリレートである請求項１ないし１１のいずれか１項に記載の治療又は予防剤。 The therapeutic or prophylactic agent according to any one of claims 1 to 11, wherein the cyanoacrylate is n-butyl cyanoacrylate.
  13. 　前記皮膚炎がアトピー性皮膚炎である請求項１ないし１２のいずれか１項に記載の治療又は予防剤。 The therapeutic or prophylactic agent according to any one of claims 1 to 12, wherein the dermatitis is atopic dermatitis.
  14. 　前記皮膚炎が、I型アレルギー反応又はIV型アレルギー反応が関与する皮膚炎である、請求項１ないし１２のいずれか１項に記載の治療又は予防剤。 The therapeutic or prophylactic agent according to any one of claims 1 to 12, wherein the dermatitis is dermatitis involving type I allergic reaction or type IV allergic reaction.
  15. 　アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子を有効成分として含有する、I型アレルギー反応又はIV型アレルギー反応の抑制剤。 Type I allergic reaction or type IV containing cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof An inhibitor of allergic reactions.
  16. 　アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子を有効成分として含有する、I型アレルギー反応又はIV型アレルギー反応が関与するアレルギー症状の治療又は予防剤。 Type I allergic reaction or type IV containing cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient, comprising at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof A therapeutic or prophylactic agent for allergic symptoms involving allergic reactions.
  17. 　治療すべき皮膚病変部、又は皮膚炎の発生の予防が望まれる皮膚領域に、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子の有効量を投与することを含む、皮膚炎の治療又は予防方法。 The skin lesion to be treated or the skin region where prevention of the occurrence of dermatitis is desired includes at least one selected from the group consisting of amino acids, amino acid derivatives, and oligomers and polymers thereof, and has an average particle size of 1000 nm. A method of treating or preventing dermatitis comprising administering an effective amount of cyanoacrylate polymer particles that is less than.
  18. 　それを必要とする対象に対し、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子の有効量を投与することを含む、I型アレルギー反応又はIV型アレルギー反応の抑制方法。 Administering to a subject in need thereof an effective amount of cyanoacrylate polymer particles comprising at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and having an average particle size of less than 1000 nm A method for suppressing a type I allergic reaction or a type IV allergic reaction.
  19. 　それを必要とする対象に対し、アミノ酸、アミノ酸誘導体、並びにそれらのオリゴマー及びポリマーからなる群より選択される少なくとも１種を含み、平均粒径が1000nm未満であるシアノアクリレートポリマー粒子の有効量を投与することを含む、I型アレルギー反応又はIV型アレルギー反応が関与するアレルギー症状の治療又は予防方法。 Administering to a subject in need thereof an effective amount of cyanoacrylate polymer particles comprising at least one selected from the group consisting of amino acids, amino acid derivatives, oligomers and polymers thereof, and having an average particle size of less than 1000 nm A method for treating or preventing allergic symptoms involving type I allergic reaction or type IV allergic reaction.
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