JP6847668B2 - A therapeutic or preventive agent for dermatitis containing nanoparticles as an active ingredient - Google Patents

Description

The present invention relates to a therapeutic or prophylactic agent for dermatitis containing nano-sized cyanoacrylate polymer particles as an active ingredient.

For the purpose of improving the effect of drugs by drug delivery system (DDS) and sustained release, mainly for application to human medicine, research on atomization of drugs is progressing, for example, cyanoacrylate polymer particles are bound to the drug. DDS is known (Patent Documents 1 and 2 and Non-Patent Document 1). To date, the inventor of the present application has also disclosed a method for producing cyanoacrylate polymer particles having little variation in particle size, antibacterial agent-conjugated particles, and plasmid-conjugated particles (Patent Documents 3 to 5). In the conventional polymer particle synthesis method, saccharides and polysorbates are allowed to coexist in the polymerization reaction system for the purpose of initiating and stabilizing the anionic polymerization reaction of cyanoacrylate. These past studies have aimed at DDS and sustained release of the drug.

After that, the inventor of the present application found that the cyanoacrylate polymer particles themselves have antibacterial activity against Gram-positive bacteria (Patent Document 6). Furthermore, it has been found that cyanoacrylate polymer particles conjugated with amino acids have anticancer activity (Patent Document 7) and can exert antibacterial activity against various bacteria regardless of Gram stainability (Patent Documents 8 and 9). .. The nano-sized polymer particles specifically adhere to the bacterial surface (cell wall) and lead the bacteria to lysis. Cyanoacrylate nanoparticles exert antibacterial activity by a completely different mechanism of action from antibiotics, and are also effective against multidrug-resistant bacteria such as MRSA (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin resistant enterococcus). ..

However, the effect of cyanoacrylate polymer particles on dermatitis such as atopic dermatitis is not known.

Atopic dermatitis is a skin disease with a high prevalence even in Japan. The number has been increasing in recent years, and patients are forced to suffer a lot of mental and physical distress.

The basics of drug therapy for atopic dermatitis are topical steroids of intensity according to the severity, topical calcineurin inhibitor, and antihistamine / antiallergic oral drugs as needed. Oral steroids and oral calcineurin inhibitors are used accordingly (Non-Patent Document 2). However, there are many patients who avoid steroids for fear of side effects due to continuous use of steroids, and there is also a problem of susceptibility to infection due to immunosuppression, so new therapeutic means are required.

Special Table No. 11-503148 Gazette Special Table 2002-504526 Gazette Japanese Unexamined Patent Publication No. 2008-127538 International Publication No. 2008/126846 Japanese Unexamined Patent Publication No. 2008-208070 International Publication No. 2009/0844494 International Publication No. 2010/10178 International Publication No. 2012/133648 International Publication No. 2013/108871

Christine Vauthier et al., Adv. Drug Deliv. Rev., 55, 519-548 (2003) Japan Dermatology Society Guideline "Atopic Dermatitis Practice Guideline", Japan Dermatology Society Atopic Dermatitis Practice Guideline Development Committee, Nikkikai Journal: 119 (8), 1515-1534, 2009 (Heisei 21)

An object of the present invention is to provide a novel means capable of treating or preventing dermatitis such as atopic dermatitis.

As a result of diligent research using a dermatitis model mouse, the inventors of the present application have found that nano-sized cyanoacrylate polymer particles known to have antibacterial activity are also effective against dermatitis, and completed the present invention. did.

That is, the present invention provides an agent for suppressing a type I allergic reaction or a type IV allergic reaction, which contains aspartic acid and dextran and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient. The present invention also comprises producing cyanoacrylate polymer particles having an average particle size of less than 1000 nm containing aspartic acid and dextran by anionic polymerization of a cyanoacrylate monomer under the condition where aspartic acid and dextran coexist. Provided is a method for producing an agent for suppressing a type I allergic reaction or a type IV allergic reaction.

INDUSTRIAL APPLICABILITY The present invention provides a novel therapeutic or preventive means for dermatitis different from steroidal agents. The cyanoacrylate nanoparticles, which are the active ingredients of the therapeutic or prophylactic agent of the present invention, are not used as DDS of the drug, but the particles themselves exhibit a therapeutic and preventive effect on dermatitis. The therapeutic or prophylactic agent of the present invention is preferably used for patients who are anxious about steroidal drugs, patients who cannot use steroidal drugs due to side effects, and patients with immunocompromised susceptibility. be able to.

The treatment schedule for NC / NgaSlc mice is shown (Examination 1). It is a photograph of the skin lesion part of NC / NgaSlc mouse which induced dermatitis by the schedule shown in FIG. A is the lesion 3 weeks after the first tape stripping. B is the lesion on the second day from the start of spraying with nanoparticle water or sterilized water. C is the lesion on the 8th day after the start of spraying with nanoparticle water or sterilized water. D is the lesion on the 3rd day after the start of DNFB application. This is the result of measuring the number of bacteria on the skin surface in the dermatitis induction part. It is a graph which scored the skin inflammation symptom of the lesion part shown in FIG. It is a HE-stained image of the back 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. This is the result of measuring the blood IgE concentration of NC / NgaSlc mice inducing dermatitis according to 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. The treatment schedule for BALB / c mice, which examined the effect of nanoparticle water in the dermatitis prevention model, is shown (Study 2). It is a photograph of the skin lesion part of BALB / c mouse which induced dermatitis by the schedule shown in FIG. 7. It is a graph which scored the skin inflammation symptom of the lesion part shown in FIG. These are the results of measuring the blood IgE concentration of BALB / c mice inducing dermatitis according to the schedule shown in FIG. 7 (day0, day22, day24). FIG. 7 is an HE-stained image of the back skin tissue of BALB / c mice in which dermatitis was induced according to the schedule shown in FIG. The dorsal skin 3 days after DNFB application was compared between the applied cranial side and the unapplied caudal side. The expression levels of various cytokines in the dorsal cranial (with DNFB application) and caudal (without DNFB application) skin of BALB / c mice in the dermatitis prevention model group that induced dermatitis according to the schedule shown in FIG. 7 were examined. The result. The treatment schedule for BALB / c mice examined as a dermatitis treatment model and a preventive model is shown (Examination 3). It is a photograph of the skin lesion part of BALB / c mouse which induced dermatitis by the schedule shown in FIG. It is a graph which scored the degree of dermatitis of the lesion part shown in FIG. These are the results of measuring the blood IgE concentration of BALB / c mice inducing dermatitis according to the schedule shown in FIG. 13 (day0, day22, day24). As a preventive model, the schedule shown in FIG. 13 is an HE-stained image of the back skin tissue of BALB / c mice in which dermatitis was induced after spraying nanoparticle water or water. Tissue 3 days after applying DNFB to the dorsal caudal side to induce dermatitis to the prophylactic model. For comparison, as a treatment model, the dorsal cranial skin applied to DNFB at the start of the experiment was also stained. The dorsal cranial side of BALB / c mice that induced dermatitis according to the schedule shown in FIG. 13 (DNFB was applied only at the beginning of the experiment as a treatment model, and then nanoparticle water or water was sprayed) and the caudal side (prevention model). As a result, nanoparticle water or water was sprayed, DNFB was applied before the end of the experiment, and the expression levels of various cytokines on the 3rd day) were examined. The treatment schedule for BALB / c mice used as a model of tape stripping-induced dermatitis is shown (Examination 4). It is a graph which scored the degree of dermatitis of BALB / c mouse which induced dermatitis by repeating tape stripping according to the schedule shown in FIG. It is a result of measuring the amount of water evaporation in the dermatitis-inducing part of BALB / c mice in which dermatitis was induced by repeated tape stripping according to the schedule shown in FIG. It 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 of the nanoparticle water or spray groups are shown. It is a result of examining the expression level 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. The cranial and caudal skins that underwent the same procedure were measured, respectively. The treatment schedule for hairless mice examined as a model of hapten-induced chronic dermatitis by repeated DNFB application is shown (Examination 5). It is a result of scoring the skin inflammation symptom of each group of hairless mice inducing dermatitis according to the schedule shown in FIG. 24 and examining the change in the score. It is a photograph of the skin lesion part of the hairless mouse which induced dermatitis by the schedule shown in FIG. 24 (day 33). It is a result of examining the change in the thickness of the auricle in each group of hairless mice in which dermatitis was induced by the schedule shown in FIG. 24. This is the result of examining the change in the amount of water evaporation of the cranial and back skin of each group of hairless mice in which dermatitis was induced by the schedule shown in FIG. 24. It is a result of examining the change of the water retention degree of the cranial back skin of each group of hairless mice which induced dermatitis by the schedule shown in FIG. 24. It is a HE-stained image of a typical inflammatory state and an inflammatory site on day 33 of a hairless mouse in which dermatitis was induced by the schedule shown in FIG. 24. A to D indicate 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 histology shows 100 μm. The relative expression levels of various cytokines at the inflammation-inducing site with respect to HPRT in hairless mice in which dermatitis was induced according to the schedule shown in FIG. 24 (day 34). It is the result of measuring the anti-DNP IgG antibody in serum by ELISA for the individual of each group of hairless mice which induced dermatitis by the schedule shown in FIG. 24 (day34). This is a result of examining changes in the number of superficial skin bacteria on the dorsal cranial side of individuals in each group of hairless mice in which dermatitis was induced according to the schedule shown in FIG. 24. It is a result of examining the agglutination value of the anti-S. aureus antibody (mainly IgG) in the reduced serum of the individuals of each group of hairless mice in which dermatitis was induced by the schedule shown in FIG. 24.

The particles used in the present invention are nano-sized (average particle size less than 1000 nm) polymer particles obtained by anionic polymerization of a cyanoacrylate monomer, and are amino acids, amino acid derivatives, their oligomers and polymers (hereinafter collectively referred to as these). Includes at least one selected from (sometimes referred to as "amino acid-based molecules"). Further, it may contain at least one selected from the group consisting of sugar and polysorbate.

It is known that amino acid-based molecules, sugars and polysorbates can be used as polymerization initiators and stabilizers for anionic polymerization of cyanoacrylate monomers. For example, a method for producing nanoparticles using sugar and / or polysorbate as a polymerization initiation / stabilizer is described and known in Patent Document 3, Patent Document 4 (antibacterial agent-conjugated particle), Patent Document 5 (plasmid-conjugated particle) and the like. .. Methods for producing nanoparticles using an amino acid-based molecule as a polymerization initiator / stabilizer are described in Patent Documents 8 and 9 (single use of an amino acid-based molecule) and are known. Further, Patent Document 7 describes a production method in which an amino acid and a saccharide / polysorbate are used in combination. Only one of these polymerization initiators / stabilizers may be used, or two or more thereof may be used in combination.

In the present invention, the "amino acid" refers to a compound having an amino group and a carboxy group in the molecule, and as defined by a general amino acid, the hydrogen of the amino group replaces another part in the molecule. It also includes imino acid, which is a cyclic compound that has become a secondary amine. Typical examples of amino acids that can be used in the present invention are 20 kinds of α-amino acids (arginine, histidine, lysine, aspartic acid, glutamic acid, alanine, glycine, leucine, valine, isoleucine, serine, etc. Examples include, but are not limited to, threonine, phenylalanine, tryptophan, tyrosine, cystine or cysteine, glutamine, aspartic acid, proline, methionine), but also include β-, γ- and δ-amino acid-based molecules. 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-aminobutamic acid and the like can be mentioned. Examples of preferred amino acids are from 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, especially at least one selected from the group consisting of glycine and aspartic acid, can be mentioned, but is not limited thereto.

The "amino acid derivative" refers to a compound having a structure in which any group is modified or substituted in the amino acid according to the above definition. Amino acid derivatives that naturally occur as biological components can usually be preferably used in the present invention. Specific examples of amino acid derivatives that can be used include creatin (1-methylguanidinoacetic acid for arginine derivatives), ornithine (urea cycle product for arginine derivatives), lysine (triiodosilonine, which is an aromatic amino acid; T4), and so on. Desmosin (constituents of keratinous elastin and collagen; a structure in which 3 molecules of arginine side chains and 1 molecule of lysine side chains are combined), hydroxyproline and hydroxylysine (gelatin and collagen components), phosphoserine (serine and phosphoric acid) Ester; casein component), theanin (tea component, glutamic acid derivative), arginine (seaman grass insect repellent component), tricholomic acid (shimeji component), sarcosine (egg yolk, ham, beans component; N-methylglycine), etc. However, it is not limited to these.

An amino acid "oligoform" is an oligopeptide in which 10 or less amino acid residues are bound by a peptide bond, and an amino acid "polymer" is a polypeptide in which 11 or more amino acid residues are bound by a peptide bond. Say. In each case, not only an amino acid but also an amino acid derivative may be contained as a residue. 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.

Oligopeptides can be used more preferably than polypeptides. Among them, oligopeptides having 2 to 7 residues, 2 to 5 residues, or 2 or 3 residues can be more preferably used.

As described in Patent Documents 8 to 9 described above, nano-sized (less than 1000 nm) cyanoacrylate polymer particles can be produced in any of the 20 α-amino acids constituting natural proteins under the condition that no saccharide or polysorbate is used. Can be synthesized. It has been shown that nanoparticles can be produced without the use of sugars or polysorbates in any of the neutral, acidic and basic amino acids, as well as in any of the linear, aromatic, imino and sulfur-containing structures. Therefore, not only 20 kinds of α-amino acids but also other amino acids and amino acid derivatives mentioned above can be used for nanoparticle synthesis, and oligopeptides and polypeptides also have an amino acid structure in the molecule. It can be used for nanoparticle synthesis.

"Sugars" include hydroxylated monosaccharides (eg glucose, mannose, ribose and fructose), hydroxylated disaccharides (eg maltose, trehalose, lactose and sucrose) and hydroxylated polysaccharides (eg dextran and mannan). Etc.) are included. These sugars may be in either a cyclic or chain form, and in the case of a cyclic form, they may be in either a pyranose type or a furanose type. Further, although various isomers are present in sugar, 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).

The monosaccharides, disaccharides and polysaccharides and the polysorbate can be used alone or in combination of two or more. Of the above-mentioned sugars and polysorbates, glucose, dextran, and Tween 20 (trade name) can be obtained at low cost, which is advantageous in terms of cost. As the dextran, a dextran having an average molecular weight of about 50,000 or more and a degree of polymerization is preferable. There is no particular upper limit to the molecular weight of dextran, but it is usually about 500,000 or less.

As the nanoparticles used in the present invention, particles synthesized by using an amino acid molecule as a polymerization initiator / stabilizer and without using either sugar or polysorbate (that is, containing amino acid molecule, both sugar and polysorbate). Particles that do not contain (that is, particles that do not contain) and particles that are synthesized without using polysorbate by using amino acid molecules and sugars as a polymerization initiator / stabilizer (that is, particles that contain amino acid molecules and sugars and do not contain polysorbate, such as SDS). Particles that do not contain other surfactants) are preferred.

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 the 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 and the like. it can. Among these, n-butyl-2-cyanoacrylate (nBCA) represented by the following formula, which is conventionally used as an adhesive for suturing wounds in the surgical field, can be preferably used.

In the nanoparticle production process, after at least one type of polymerization initiation / stabilizer is dissolved in an appropriate solvent, at least one type of cyanoacrylate monomer is added under stirring, and stirring is continued as appropriate to proceed with the polymerization reaction. Just let me do it. In the production of nanoparticles used in the present invention, at least one amino acid-based 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.

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 (the total concentration when a plurality of types are used) is not particularly limited, but is usually used. , 0.5% to 10%, preferably about 0.75% to 7.5%. The sugar concentration means w / v%, and the polysorbate concentration means v / v%. For example, when sugar is used alone, the above-mentioned concentration ranges are "0.5 w / v% to 10 w / v%", respectively. , "0.75w / v% ~ 7.5w / v%". When sugar is used at 5 w / v% and polysorbate is used at 1 v / v%, the total concentration of these is 6%. However, when only monosaccharides (for example, glucose) are used, it is preferable to use them at about 2.5 w / v% to 10 w / v%.

When an amino acid molecule is used as a polymerization initiator / stabilizer, the concentration of the amino acid molecule in the polymerization reaction solution at the start of the reaction (the total concentration when using a plurality of types) is not particularly limited, but is usually 0.1 w / v. It is about% to 3w / v%. When used in combination with sugar and / or polysorbate, the concentration of amino acid-based molecules used may be lower than this.

As the solvent for the polymerization reaction, an aqueous solvent mainly composed of water (for example, water, an aqueous solution of a lower alcohol, etc.) can be used, and in the case of producing amino acid-based molecule-containing particles, water is usually 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 concentration of hydroxide ions is high, so that the polymerization is fast, and when the pH is low, the polymerization is slow. When producing amino acid-based molecule-containing particles, an appropriate polymerization rate can usually be obtained under acidic pH of about 1.5 to 3.0. The acid added to acidify the reaction solution 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 volatilizes after the reaction, so that it can be preferably used in the production of amino acid-based molecule-containing particles, but the usable acid is not limited to this. The concentration of the acid such as hydrochloric acid is not particularly limited, but can be appropriately selected in the range of about 0.0005N to 0.5N.

A method called the mini-emulsion method is known as a method for synthesizing nano-sized polymer particles by polymerizing a cyanoacrylate monomer using an amino acid as a polymerization initiator (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, an O phase containing an oil-based solvent such as hexadecane and a W phase containing hydrochloric acid and the like. Anion polymerization is carried out, and the use of a surfactant such as SDS is also essential. According to the above method developed by the inventor of the present application, nanopolymer particles can be synthesized by a polymerization reaction in a single-phase aqueous solvent containing no O phase, so that it is necessary to use an organic solvent or a surfactant as the solvent. There is no. Further, in the method of the inventor of the present application, nanopolymer particles can be produced by using a compound other than polysorbate as a polymerization initiator / stabilizer, so that anionic surfactants including Tween-based surfactants and cations can be produced. It is possible to prepare nanoparticles that do not contain any of ionic surfactants, amphoteric surfactants, and nonionic surfactants. However, the effect of the nanopolymer particles synthesized by the mini-emulsion method on dermatitis is unknown.

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 0.5v / v% to 2.0v / v%, preferably about 0.8v / v% to 1.2v / v%. Is.

The reaction temperature is not particularly limited, but it is convenient and preferable to carry out the reaction at room temperature. Since the reaction rate varies depending on the pH of the reaction solution, the type of solvent, and the like, the reaction time is appropriately selected according to 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-based molecule-containing particles are usually used as neutral particles, it is preferable to add a base such as an aqueous sodium hydroxide solution to the reaction solution to neutralize the particles after the reaction is completed. After completion of the reaction, the reaction solution may be filtered and washed with sterilized water as appropriate to recover the particles.

According to the above method, nano-sized cyanoacrylate polymer particles having an average particle size 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, pH, and reaction time of the cyanoacrylate monomer in the reaction solution. Further, when at least one selected from sugar and polysorbate is used as the polymerization initiation / stabilizer, the particle size can be adjusted by changing the concentration and type of the polymerization initiation / stabilizer (Patent). Refer to References 3 and 4). Generally, when the pH of the reaction solution is increased, the reaction time is lengthened, or the sugar concentration of the reaction solution is decreased, the particle size becomes large, and when polysorbate is used as the polymerization initiation / stabilizer, the particles become large. The size becomes smaller. By appropriately combining these reaction conditions, particles of 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 surface of the particle and is an index of the dispersibility of the particle. The particle size and zeta potential can be easily measured using, for example, a commercially available device using a He / Ne laser (for example, a zeta sizer manufactured by Malvern Inst. UK).

In nanoparticles produced using amino acid-based molecules as a polymerization initiator / stabilizer, not only amino acid-based molecules are simply attached to the particles and contained, but also the -COO group in the amino acid structure is the ethylene terminal of cyanoacrylate. It is considered to be bound to carbon and contained in the particles by 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-passing solution when the filter is washed after polymerization at an appropriate wavelength, and measuring the amount of amino acid-based molecules in the filter-passing solution (that is, amino acid-based molecules that did not bind to particles). Amount) can be calculated by the following formula after being determined by the absorbance method.
Amino acid-based molecule content = (Amino acid-based molecule addition amount)-(Amino acid-based molecule content in the filter-passing solution)
Amino acid-based molecule content (%) = Amino acid-based molecule content ÷ Amino acid-based molecule addition amount x 100

The amino acid molecule-containing nanoparticles used in the present invention do not contain antibacterial active ingredients against bacteria. As described in Patent Documents 8 and 9, it is known that cyanoacrylate polymer nanoparticles containing amino acids can exert antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria, and can be applied to the bacterial surface (cell wall). It exerts an antibacterial effect by guiding bacteria to lysis by its specific adhesiveness. It has been confirmed that the cytotoxic activity of nanoparticles is not exerted on normal mammalian cells, and that nanoparticles have no in vivo toxicity. Further, the amino acid molecule-containing nanoparticles used in the present invention do not contain an antibacterial active ingredient against fungi.

The term "antibacterial active ingredient" as used herein refers to a chemical substance component capable of biochemically acting on the metabolic pathway or physiological function of a bacterium or fungus to inhibit the growth of the bacterium, and specifically, a bacterium. Alternatively, it refers to an antibiotic or other chemical component that can be used for antibacterial activity of fungi. "No antibacterial active ingredient" means that the antibacterial active ingredient is not contained at all, or even if it is contained, the amount is so small that bacteria or fungi that are sensitive to the antibacterial active ingredient cannot be antibacterial. It means that the antibacterial active ingredient is contained only in a very small 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 cannot be antibacterial" is defined as the amount of antibacterial active ingredient contained in the particles per particle unit volume as the content concentration in the particles, and the antibacterial active ingredient having the same concentration as this content concentration is used as the particles. It means an amount that cannot prevent the growth of the susceptible bacteria or fungi when it is allowed to act alone on the sensitive bacteria or fungi without being contained. The nanoparticles used in the present invention can be particles that do not contain any antibacterial active ingredients such as antibiotics.

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 therapeutic agent for dermatitis. The term "not contained" as used herein is the same as above, and it is possible that the chemical substance component is not contained at all, or even if it is contained, the amount is very small, and the effect of treating and preventing skin lesions can be exerted. It means that the chemical substance component is contained only in a trace amount that cannot be achieved. The nanoparticles used in the present invention are not the DDS of the dermatitis therapeutic agent, but the nanoparticles themselves control the skin inflammatory response through the improvement of the skin barrier function and the immune response involved in the control of the superficial bacterial count, and the skin. It has a therapeutic and preventive effect on inflammation.

The target dermatitis in the present invention is not particularly limited, but firstly, it may be pruritic dermatitis (pruritic dermatitis). Specific examples of pruritic dermatitis include, but are not limited to, atopic dermatitis, contact dermatitis, infectious dermatitis, psoriasis, dermatitis due to autoimmune mechanism, drug eruption and the like.

Also, the dermatitis of interest can secondly be a dermatitis associated with a skin injury. Specific examples of such dermatitis include, but are not limited to, radiation dermatitis, burns, and boil.

As confirmed in the examples below, amino acid molecule-containing nanoparticles have the effect of suppressing type I and type IV allergic reactions. Therefore, the agent of the present invention containing amino acid molecule-containing nanoparticles as an active ingredient can be used as an inhibitor of type I or type IV allergic reaction, such as dermatitis associated with type I or type IV allergic reaction. It is useful as a therapeutic or prophylactic agent for allergic symptoms. It is known that the above-mentioned pruritic dermatitis may involve a type I allergic reaction or a type IV allergic reaction.

As described above, the nano-sized cyanoacrylate polymer particles used in the present invention are known to have antibacterial activity against bacteria (Patent Documents 8 and 9). For dermatitis caused mainly by infection such as infectious dermatitis, the antibacterial activity of the particles is expected to be effective against infected bacteria, so it is also possible against dermatitis caused mainly by infection. Although the agent of the present invention can be used, the dermatitis targeted in the present invention can be a dermatitis other than a dermatitis mainly caused by a bacterial infection.

The agent of the present invention is locally administered 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 intradermal administration with an injection, a drip, etc., and topical application with an ointment, a cream, a patch, etc. The dose is appropriately selected according to the symptoms, age, body weight, administration method, etc., and is not particularly limited, but is usually about 0.01 μg to 10000 mg per day as the amount of amino acid molecule-containing particles which are active ingredients for the target animal. For example, it is about 1 μg to 100 mg, and is administered in one or several divided doses. Depending on the degree of improvement of the symptoms, it may be administered once or several times daily, or every few days once or several times for several days or months, or the symptoms occur. It may be administered from time to time.

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 therapeutic or preventive agents for pruritic skin diseases. Conventionally used standard therapeutic agents for pruritic skin diseases are steroids, carcinulin inhibitors and antihistamines, but the agents of the present invention are considered to have a different mechanism of action from these standard therapeutic agents. Therefore, it is also possible to use the agent of the present invention in combination with a steroid agent or the like.

Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.

1. 1. Production of Amino Acid-Containing Nanoparticles Nano-sized polymer particles containing amino acids and dextran were produced according to the methods described in International Publication No. 2012/133648 and International Publication No. 2013/108871. The specific procedure is as follows.

100 mg of dextran 60K and 100 mg of amino acid were dissolved in 10 mL of 0.01N HCl, and the liquid pH was adjusted to pH = 3 with 1N hydrochloric acid as needed. As amino acids, glycine and aspartic acid were used.

Under stirring, 100 μL of nBCA was added to the above solution in which dextran and amino acids were dissolved, and the mixture was stirred for 3 hours to carry out a polymerization reaction. After neutralizing the reaction solution by adding 1N NaOH (pH 7.8), the mixture was further stirred for 30 minutes to obtain nano-sized polymer particles containing amino acids and dextran. The obtained particles are subjected to two-step ultrafiltration treatment using a Millipore ultrafiltration membrane and a dialysis ultrafiltration device for vaccine production manufactured by Paul, and the particles are refined products (colloidal aqueous solution with a particle concentration of 2 mg / ml). Got

The average particle size and zeta potential of the nanoparticles were measured using a commercially available zeta sizer (manufactured by Malvern Inst. UK). In addition, the amount of amino acid molecules in the filter-passing liquid during filter cleaning was determined by the absorbance method, and the amino acid content of the particles was calculated. The results are shown in Table 1.

2. Examination of therapeutic and preventive effects of nanoparticles in a mouse dermatitis model 1
Experiments were carried out using the aspartic acid-containing nanoparticles produced above. The 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 the mice used below were bred and tested in an SPF environment according to the experimental plan approved by the Kochi University Animal Experiment Committee.

[Method]
NC / NgaSlc mice (Nippon SLC, Shizuoka) are known to spontaneously develop dermatitis by tape stripping after shaving and hair removal on the back, and are used as a model for atopic dermatitis (Matsuda). , M., et.al. Int. Immunol. 9: 461, 1997). In this experiment, five 5-week-old NC / NgaSlc mouse males were used and treated according to the schedule shown in FIG. The back of the NC / NgaSlc mouse was shaved with a shaver and then depilated with a depilatory cream (Epilat, Kracie Home Products Co., Ltd., Tokyo). The hair removal cream was wiped off, and the next day, tape stripping (Scotch mending tape, cat.no.810-3-24, 3M Japan Ltd., Tokyo) was performed 8 times with adhesive tape to induce dermatitis. When hair grew on the way, the same operation was repeated. After 3 weeks, the onset of dermatitis was confirmed over a wide area on the back in all mice (Fig. 2A).

Of the five mice that developed dermatitis, three were in the nanoparticle water spray group and two were in the water spray group. Nanoparticle water or sterile water was sprayed every two days on the back 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 the back of one mouse 8 ^{to 10 cm 2.} Bacterial counts in the central back were measured on days 2, 6, and 8. Specifically, a standard agar medium for measuring viable cell count (Petancheck, Eiken Chemical) was pressed against the center of the back of the mouse for 5 seconds, and then cultured in a 37 ° C. incubator for 24 hours, ^{per 6 cm 2} compartment. The number of colonies was counted. The skin that had come into contact with the agar medium was wiped with water-moistened tissue paper, then sprayed with nanoparticle water or distilled water and left for 1 hour to dry. For skin lesions, photographs were taken on the 2nd day (Fig. 2B) and the 8th day (Fig. 2C).

After being left for 2 weeks from the 30th day in FIG. 1, all the mice were shaved and depilated again for the purpose of further inducing a hapten-induced type IV allergic reaction. On the next day (1st day) and 2nd day, olive oil-acetone in which 0.5% dinitrofluorobenzene (1-fluoro-2,4-dinitrobenzene, DNFB) (Nacalai Tesque Co., Ltd., Kyoto) was dissolved on the dorsal cranial side of the mouse. 20 μL of the solution (oil: acetone = 1: 4) was applied. Nanoparticle water spraying and water spraying were performed daily from the 44th day after hair removal. When DNFB was applied, it was performed after the DNFB had dried. Photographs were taken 2 days after the application of DNFB (Fig. 2D). On the 6th day after applying DNFB, a serum sample was collected and then euthanized. The skin on the back of the head coated with DNFB and the skin near the base of the tail not coated with DNFB were collected and stained with hematoxylin-eosin (HE). I went (Fig. 5A-5D).

In addition, the degree of dermatitis in the mice A to D shown in FIG. 2 was scored according to the criteria shown in Table 2 below. The scoring was based on the literature reported by Fan et al. (Fan et al., Exp Biol Med, 226, 1045-1050, 2001), and the degree was evaluated with a score of 0 to 3 for three skin lesions.

[result]
The results of counting the number of bacteria in the central part of the back of mice with dermatitis are shown in Table 3 and FIG. In the nanoparticle spray group, the number of superficial bacteria decreased by about half.

The number of colonies on the agar medium was 5, 7, and 8 in the nanoparticle spray group, while it was 48, 85 in the distilled water spray group. It was confirmed that the antibacterial ability of nanoparticle water was exhibited from an early time after spraying.

Although there was no significant difference between the two groups in the rate or degree of healing of dermatitis caused by tape stripping in NC / NgaSlc mice (Figs. 2A-2C, Fig. 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 (Fig. 2D, Fig. 4). ..

When the HE-stained skin section was observed (Fig. 5), the epidermis thickened at the DNFB-applied site (Fig. 5A, C) compared to the non-applied site (Fig. 5B, D) in both the nanoparticle water spray group and the water spray group. Infiltration of cells in the epidermis and fibrosis were observed. However, the degree of epidermal thickening and dermis lesions due to DNFB application was more pronounced in the water spray group than in the nanoparticle water spray group (Figs. 5A and 5C). Furthermore, the mild epidermal thickening of the DNFB non-applied site observed in the water spray group was completely suppressed in the nanoparticle water spray group (Fig. 5B and 5D).

In addition, for the purpose of investigating the effect of nanoparticles on type I allergic reaction, in the blood of mice before the start of DNFB application (Figs. 1 and 44) and 8 days after the first application of DNFB (at the end of the experiment on day 52). IgE concentration is a sandwich ELISA method composed of a immobilized antibody (rat anti-mouse IgE, Southern Biotech, AL, USA) and an enzyme-labeled antibody (HRP-conjugated goat anti-mouse IgE antibody, BETHYL, TX, USA). Measured at. As a result, in the water spray group, the IgE amount increased by almost 5 times at the end of the experiment compared with the IgE amount at the start of the experiment, whereas in the nanoparticle water spray group, the IgE amount increased by 2-3. It stayed at about double (Table 4, Fig. 6). It was confirmed that nanoparticle spraying is effective not only for type IV allergic reaction but also for controlling type I allergic reaction.

[Conclusion]
It was shown that the bactericidal action of nanoparticle water has a therapeutic promoting 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 is based on the promotion of barrier function recovery by bactericidal action and the control of inflammatory response.

3. 3. Examination of therapeutic and preventive effects of nanoparticles in a mouse dermatitis model 2
In order to further verify the effect of nanoparticles, experiments were conducted using BALB / c mice with known MHC genotypes. First, we conducted an experiment to see 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 of time. The therapeutic effect of nanoparticle water on this dermatitis was evaluated by follow-up observation of the skin inflammatory state, measurement of blood IgE concentration, and observation of HE tissue-stained specimen.

[Method]
Blood was drawn from the tails of 6 BALB / c mice (female, 8 weeks old), and the back was shaved and depilated with a shaver and cream. After wiping the cream with water and drying the skin, the skin was stripped eight times with scotch tape and photographed (Fig. 8, day 0).

The six mice were divided into two groups, a nanoparticle water spray group (3 mice) and a water spray group (3 mice), and the experiment was conducted. Two days after spraying on the first day (nanoparticle water spray sprays a total liquid volume of 0.3 to 0.4 mL and a nanoparticle amount of 1.8 to 2.4 μg on ^{the back of one mouse 8 to 10 cm 2).} After the eyes, 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 cranial side of the back of the mouse).

Photography was performed 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 caudal and caudal sides of the back were scored separately and evaluated based on the total value.

Blood is collected before tape stripping (Fig. 7, blood collection 1), 22 days after tape stripping induction (Fig. 7, blood collection 2), and 2 days after re-induction by DNFB application (Fig. 7, blood collection 3). The amount of IgE in the serum fraction was measured by sandwich ELISA.

At the end of the experiment on the 24th day, the dorsal cranial and caudal skin tissues of the mice were collected. 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 to the HPRT gene and evaluated by the relative expression level. Table 5 shows the primer sequences for each mouse cytokine.

[result]
1. Results of judgment of dermatitis symptoms After tape stripping, erythema with a large number of erosions was observed on the backs of all mice (Fig. 8, day 0). In the dermatitis induced only by tape stripping, the erythema disappeared faster in the nanoparticle water spray group than in the water spray group, and no erosion was observed (Fig. 8, day 3). No clear edema was observed on the skin of both groups. After the first week, the inflammatory symptoms of both the nanoparticle water spray group and the water spray group (except for the number 8 mouse) recovered to almost the same level (Fig. 8, day7 to day15). The change in the dermatitis score with respect to the elapsed days is shown in FIG. The scores showed that the nanoparticle water spray group improved the degree of inflammation from an earlier stage than the mice in the water spray group.

The degree of dermatitis induced when re-stimulated by DNFB application was milder in the nanoparticle water spray group than in the water spray group, suggesting a preventive effect. In particular, the skin damage caused by shaving with the shaver and depilatory cream before applying DNFB was lower in the nanoparticle spray group, as shown in the skin lesion score (Fig. 9).

2. Blood IgE concentration The IgE concentration in the blood collected at the start of the experiment (day 0), at the start of re-induction of dermatitis by applying DNFB (day 22), and at the end of the experiment (day 24). The measurement results are shown in FIG. No significant difference was observed between the nanoparticle water spray group and the water spray group.

3. HE skin histopathological image The HE-stained image of the skin tissue after re-induction of inflammation is shown in FIG. Epidermal thickening and dermis inflammation on the dorsal cranial side to which DNFB was applied were milder in the nanoparticle water spray group than in the water spray group. Even on the dorsal caudal side without DNFB application, epidermal thickening was markedly suppressed in the nanoparticle spray group, and no inflammatory cell infiltration was observed.

4. Cytokine expression level Fig. 12 shows the results of examining the expression levels of various cytokines by dividing the dermatitis-inducing part of the back into the caudal side (with DNFB application) and the caudal side (without DNFB application). When re-stimulated by DNFB application, cytokines associated with barrier function disruption such as IL-1α, TNF-α, IL-6, with or without nanoparticle spraying, are DNFB-applied dorsal and non-DNFB Expression was observed on both the caudal side of the coated area. When re-stimulated by applying DNFB, the cytokines IL-17A, IL-4, and IFN-γ produced by T cells were low in expression on both the cranial and caudal skin with or without nanoparticle spraying. ..

[Conclusion]
As in the case of NC / NgaSlc mice, in BALB / c mice, the treatment effect of the nanoparticle spray group is superior to that of the distilled water spray group not only in the macro evaluation but also in the micro evaluation. It can be concluded that there is. Furthermore, the nanoparticle water spray group had milder epidermal thickening and dermis inflammation than the distilled water spray group, and the lesions were mild even after dermatitis was induced by reapplying DNFB. Spraying is thought to play a role in controlling repeated inflammation induction and protecting the skin.

The amount of IgE in blood at the end of the experiment was significantly higher than that at the start of the experiment and before the induction of inflammation again, but unlike the case of NC / NgaSlc mice, nanoparticles in BALB / c mice. No significant difference was found between the water spray group and the water spray group.

4. Examination of therapeutic and preventive effects of nanoparticles in a mouse dermatitis model 3
Experiments showing the preventive effect of dermatitis have shown that nanoparticle spraying is effective in reducing epidermal thickening after inducing dermatitis by tape stripping or applying DNFB and in controlling inflammation when re-inducing dermatitis. It was. For the purpose of clarifying whether this effect is also seen in mice that already have dermatitis and whether it is related to skin barrier function, the MHC genotype is known for pure strains. It was verified using BALB / c mice.

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 reinduction by DNFB application.
(1) Recording and scoring the degree of dermatitis
(2) Measurement of blood IgE level after reinduction of dermatitis by applying DNFB
(3) Observation of skin HE-stained tissue after reinduction of dermatitis by applying DNFB
(4) Measurement of dermatitis-related inflammatory cytokine expression level after reinduction of dermatitis by applying DNFB

[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 depilated with a shaver and cream, and tape stripping was performed 8 times to break the skin barrier. In an experiment in which hapten-induced dermatitis was first induced and the therapeutic effect of nanoparticle water or water spray was examined, DNFB was applied to the dorsal cranial side of the mouse after tape stripping (1st and 2nd days). Induced dermatitis. Spraying of nanoparticle water or distilled water was carried out every other day after the application of DNFB on the 1st and 2nd days and thereafter until the 20th day. For the purpose of examining the preventive effect of nanoparticle water or water spray on dermatitis, the hair was removed again on the 21st day, and on the 22nd and 23rd days, 0.5% DNFB olive oil-acetone solution was applied to the caudal side of the back of the mouse. Dermatitis was induced by applying 20 μL of (oil: acetone = 1: 4).

Photography was performed on days 0, 3, 7, 11, 13, 15, 22, 23, and 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. .. The dorsal cranial side (treatment model) and caudal side (prevention model) were scored and evaluated.

Blood samples were collected on the 0th day (blood collection 1), the 22nd day (blood collection 2), and the 24th day (blood collection 3), and the IgE amount of the serum fraction was measured by sandwich ELISA.

At the end of the experiment on the 24th day, skin tissues on the dorsal cranial side (treatment model) and caudal side (prevention model) of the mice were collected, and HE tissue staining was observed and cytokine expression level was measured.

[result]
1. Judgment result of dermatitis condition FIG. 14 shows a photograph of the back of a mouse, and FIG. 15 shows a score evaluation result of an inflammatory condition. The therapeutic effect on dermatitis induced by tape stripping and DNFB application was superior to that of nanoparticle spraying. The degree of dermatitis induced by re-stimulation by DNFB application was lower in the nanoparticle water spray group than in the water spray group, and the preventive effect was excellent. In particular, the damage to the skin when shaving with a shaver and a depilatory cream before applying DNFB was lower in the nanoparticle spray group as shown in the dermatitis score (FIG. 15).

2. Blood IgE concentration Blood IgE collected at the start of the experiment (day 0), at the start of reinduction of inflammation by applying DNFB to the latter half of the back (day 22), and at the end of the experiment (day 24). The result of measuring the concentration is shown in FIG. The blood IgE concentration when re-stimulated by applying DNFB tended to be lower in the nanoparticle water spray group than in the water spray group.

3. HE-stained image The HE-stained image of the skin pathological tissue after inflammation induction is shown in FIG. As a preventive model for dermatitis, the nanoparticle spray group was more likely than the water spray group to spray nanoparticle water or water first, and to induce dermatitis before the end of the experiment. It was mild. As a treatment model for dermatitis, the epidermal thickening of the nanoparticle spray group was milder than that of the water spray group for the skin on the dorsal head side where DNFB was applied first and then spraying of nanoparticle water or water was continued. there were.

4. Cytokine expression level Prevention of cranial skin (DNFB application first, then nanoparticle water or water spraying treatment model) and caudal side (nanoparticle water or water spray first, DNFB application last) The results of examining the expression levels of various cytokines by dividing them into models) are shown in FIG.

Cytokines associated with barrier function disruption such as IL-1α, TNF-α, and IL-6 were induced by applying DNFB before the end of the experiment as a preventive model for dermatitis with or without nanoparticle spraying. DNFB was first applied to the caudal region and as a treatment model for pre-existing dermatitis, and expression was observed in both the dorsal and cranial regions without DNFB application immediately before the end of the experiment. Furthermore, the expression tended to be high on the caudal side of the back where DNFB was applied before skin sampling.

As a preventive model for dermatitis, when nanoparticle water or water spray is repeated first and DNFB is applied before the end of the experiment to induce dermatitis, the cytokines IL-17A, IL-4, IFN produced by T cells. The expression of -γ was higher on the dorsal caudal side where DNFB was applied before the end of the experiment than on the cranial side where DNFB was not applied before the end of the experiment, regardless of the presence or absence of nanoparticle spraying. In addition, the expression level of IFN-γ tended to be higher in the nanoparticle water spray group than in the water spray group.

[Conclusion]
Similar to the case of NC / Nga mice, it was shown that the bactericidal action of nanoparticle water in BALB / c has a therapeutic effect on dermatitis induced by tape stripping + DNFB application and a preventive effect on dermatitis induction by DNFB application. It was.

Results suggest that the effect in BALB / c is based on the promotion of barrier function recovery by bactericidal action and the control of the associated inflammatory response, as in the case of NC / Nga mice.

5. Examination of therapeutic and preventive effects of nanoparticle water in a mouse dermatitis model 4
To further investigate whether nanoparticle spraying is involved in maintaining barrier function and controlling inflammatory response, 12-week-old BALB / c mice were repeatedly tape stripped to investigate the following:
(1) Changes in the score of skin inflammation during the process of repeated tape stripping
(2) Changes in the amount of water transpiration in the process of repeated tape stripping
(3) Observation of skin HE-stained tissue after repeated tape stripping
(4) Measurement of dermatitis-related inflammatory cytokine expression level after repeated tape stripping

Tape stripping was repeated according to the schedule shown in FIG. After shaving and removing the back of 12-week-old BALB / c mice, tape stripping ( ^{an index of skin barrier function) so that the value is in the range of 30 to 50 g / hm 2 while measuring the amount of water evaporation (index of skin barrier function).} 3-5 times). For measurement of evapotranspiration, use The Multi Probe Adapter System (abbreviated as MPA5) (software: CK-MPA-multi-probe version 1.5.1.4) of Cologne + Khazaka electronic GmbH (Cologne, Germany) for evapotranspiration measurement. A probe (registered trademark) probe (TM300MP) was connected. The measurement was performed by pressing the probe against the base of the left foot so as not to shift. After anesthesia, photographs were taken, the skin inflammation status was scored based on the photographs, and the amount of skin evapotranspiration was measured, and then nanoparticle water or distilled water was sprayed (4 animals in each group). The scoring of the state of skin inflammation was divided into the dorsal cranial part and the caudal side, and each was evaluated by the total value, as in the above examinations 2 and 3. Hair removal and tape stripping were performed again on the 4th, 8th, and 11th days when the water evaporation amount of the nanoparticle spray group ^{showed a value of around 10 g / hm 2.} On the 14th day, photography and water evapotranspiration measurement were performed, followed by blood sampling and skin tissue sampling (frozen sections, fixatives and RNA preparation for RT-PCR).

The result of score evaluation of the dermatitis state is shown in FIG. In both the nanoparticle spray group and the water spray group, repeated barrier destruction and tissue modification were observed, with the score increasing to about 12 for each tape stripping and decreasing to about 2 to 3 the next day. The skin inflammation state on the 14th day was not significantly different between the nanoparticle spray group and the spray group.

On the other hand, when the amount of water evaporation from the skin was measured (results are shown in FIG. 21), when tape stripping was repeated, the water spray group ^{increased to 60 to 70 g / hm 2 for} each tape stripping, but nanoparticles. In the water spray group, the increase was only up to about ^{50 g / hm 2.} The next day after tape stripping, the ^{amount of transpiration in the nanoparticle water spray group decreased to about 10 g / hm 2} , whereas when tape stripping was repeated, the amount of transpiration gradually recovered in the water spray group. Was insufficient, and the value gradually increased, ^{showing 20 g / hm 2} or more on the 14th day. This indicates that the repair function after repeated tape stripping and after skin barrier destruction is inferior in the water spray group to that in the nanoparticle water spray group.

This is also supported by the fact that, as shown in FIG. 22, in the HE-stained specimen of the skin collected on the 14th day, the degree of thickening of the epidermis was larger in the water spray group than in the nanoparticle water spray group. To.

Furthermore, when the expression levels of cytokines associated with the disruption of epidermal barrier function such as IL-1α, TNF-α, and IL-6 after repeated tape stripping were examined by RT-PCR (results are shown in FIG. 23), Similar expression was observed on both the dorsal and caudal sides with and without nanoparticle spraying. 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-, which is highly related to epidermal destruction. 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 impaired in the water spray group than in the nanoparticle water spray group.

It has been shown that the bactericidal and water-retaining action of nanoparticle water is effective in treating skin damage caused by repeated tape stripping and in preventing skin inflammation that can be induced by the treatment.

6. Examination of therapeutic and preventive effects of nanoparticle water in a mouse dermatitis model 5 (Examination in a hapten-induced chronic dermatitis model using a 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-retaining effects of nanoparticle water using an animal model close to that of the above, a mouse chronic dermatitis model in which hapten was repeatedly applied was created (Nakajima, S. et al., J. Invest. Dermatol, 134). , 2122-2130, 2014 et al. Modified to DNFB antigen system), and the following 8 items were investigated. In the experimental group in which DNFB was applied to the back skin, it was applied to the skin at the position of the scapula, which seems to be less affected by scratching by the limbs.

<Evaluation items>
(1) Scoring of skin inflammation status: Evaluation of the degree of macroscopic skin inflammation
(2) Measurement of thickness of both auricles: Evaluation of the degree of inflammatory reaction of the auricles
(3) Measurement of skin transpiration and water retention: Evaluation of the degree of destruction / recovery of the barrier function of the upper back skin epidermis
(4) HE tissue-stained specimen observation: Evaluation of the degree of skin inflammation by microscopic observation
(5) Measurement of the type and amount of cytokine expressed in the skin: Evaluation of the type and amount of cytokine expressed in the skin on the dorsal cranial side and the right pinna
(6) Measurement of anti-Hapten antibody level in serum: Evaluation of anti-DNP IgG antibody level in serum
(7) Measurement of the number of superficial bacteria on the cranial back skin: Evaluation of the number of superficial bacteria on the cranial back skin
(8) Measurement of IgG antibody level against Staphylococcus aureus in serum: Evaluation of serum IgG antibody level against Staphylococcus aureus isolated from the back skin of hairless mice

<How to make a mouse chronic dermatitis model>
Four experimental groups were prepared as shown in Table 6, with five female hairless mice (Hos: HR-1) in a group. The treatment schedule is shown in FIG.

Skin photography, resident bacterial counts, ear thickness, skin evapotranspiration, skin water retention, and dermatitis scores were measured after anesthetizing the mice, followed by DNFB application. During this period, nanoparticle water was sprayed every other day from the day after DNFB application (Fig. 24). 0.3% DNFB was applied on day 33, and one day later (day 34), skin was collected from both ears and back and blood was collected. HE-stained specimens were prepared from the right auricle and the cranial dorsal skin, and various cytokines were measured by RT-PCR. From blood, the agglutination titers of anti-DNP antibody and anti-S. aureus antibody in serum were measured.

(1) Scoring of dermatitis The dermatitis score was defined as shown in Table 7 below, and the degree of dermatitis was scored. The score is a total of 4 grades of 0 to 3 for 7 items of erythema, scales, erosion, scab, scab, left auricle thickening / deformation, and right auricle thickening / deformation for one mouse. did. The maximum score per animal is 21 points. Dermatitis scores were measured on day5, day12, day19, day26, and day33 for the mice in each experimental group.

FIG. 25 shows the results of tracking the changes in the dermatitis scores of the cranial back and both auricles after applying DNFB. Images of the cranial dorsal skin and binaural pinna of each experimental group on day 33 are shown in FIG. The dermatitis score was clearly lower in group A, which was sprayed with nanoparticles together with DNFB application, than in group C (positive control group) and group B, which was not sprayed with nanoparticles (Fig. 25). Group D shows a negative control that was neither treated with DNFB nor sprayed. As shown in FIG. 26, dermatitis and auricular deformity were clearly observed in groups B and C, while the nanoparticle spray group (group A) was equivalent to the negative control (group D) and had marked symptoms. It was suppressed.

(2) Measurement of thickness of both auricles The thickness of the left and right auricles was measured with a thickness gauge on day5, day12, day19, day26, and day33 for the mice in each experimental group. The average value of the measured values of the left and right auricles was graphed as the measured value of the individual.

The results are shown in FIG. In the water spray group (B group) and the positive control group (C group), the pinna began to thicken around day 12 after DNFB application, while the nanoparticle water spray group (A group) did not thicken on day 33. The inhibitory effect was clear, and the results were almost the same as those of the negative control group (Group D).

(3) Measurement of skin water evaporation and water retention The water evaporation and water retention of the upper back skin were measured on day5, day12, day19, day26, and day33 for the mice in each experimental group. The Multi Probe Adapter System (abbreviation MPA5) (software: CK-MPA-multi-probe version 1.5.1.4) of Cologne + Khazaka electronic GmbH (Cologne, Germany) was used for the measurement. In the measurement of the transpiration amount, a probe Tewameter (registered trademark) probe (TM300MP) for measuring the transpiration amount was connected to MPA5, and the probe was pressed against the cranial back skin for measurement. To measure the water retention, connect the probe Corneometer (CM825MP) for water retention measurement to MPA5, press the probe against the back skin on the cranial side to measure the water retention, and measure the average value of the measured values at the three points. It was used as an individual measurement value.

The measurement result of the amount of transpiration of skin water is shown in FIG. In groups B and C, the transpiration amount clearly increased on day 12 after the application of DNFB, and showed a high transpiration amount thereafter. In the nanoparticle spray group (Group A), a slight increase in the amount of transpiration was observed temporarily, but the value was maintained at a low value. On day33, group A showed significantly lower values than groups B and C.

The measurement result of the water retention degree is shown in FIG. In groups B and C, the water retention rate decreased over time after the application of DNFB, while in the nanoparticle spray group (group A), no decrease in water retention was observed, as in the untreated group (group D). Water retention capacity was maintained. On day33, the nanoparticle water spray group (A group) showed a significant difference compared to the B and C groups.

(4) Observation of HE tissue-stained specimens The right auricle and cranial dorsal skin were collected from the mice of each experimental group on day 34 (1st day after application of DNFB 0.3% on the 33rd day) and subjected to HE staining.

30A to 30D are typical auricular and cranial dorsal skin symptoms and HE-stained images of groups A to D. A-1, B-1, C-1, and D-1 are HE tissue-stained photographs of the right auricular skin, and A-2, B-2, C-2, and D-2 are HE tissues of the cranial back skin. It is a tissue-stained photograph. Black bars indicate 100 μm.

In the water spray group (Group B), the inflammation was macroscopically strong, and in the pathological tissue, the epidermal thickening of the auricle, and the inflammation and fibrosis of the dermis were also remarkable. On the other hand, the nanoparticle spray group (Group A) was clearly suppressed in both clinical symptoms and histopathology. In the histopathology of the dorsal skin, the infiltration of inflammatory cells around the hair follicles (a cystic structure was formed in hairless mice) was mild in the nanoparticle spray group (Group A), although the difference was not clear.

Similar to group B, the positive control group (group C) also showed strong skin inflammation, and changes in the auricle and back skin were stronger than in group A.

(5) Measurement of the type and amount of cytokines expressed in the skin The expression of cytokines in the right auricle tissue and upper back skin tissue collected on day 34 from the mice of each experimental group was measured by quantitative RT-PCR. The primers shown in Table 5 above were used. The expression level was normalized to the HPRT gene and evaluated by the relative expression level.

The results are shown in FIG. In the pinna (Fig. 31, right), the expression of inflammatory cytokines (IL-6 ,, IFN-γ) in groups B and C was higher than in groups A and D, and in the pinna of groups B and C It was associated with the degree of inflammation. In the back skin (Fig. 31, left), expression of cytokines such as TNF-α and IL-17 was observed in group D as well as group A. This suggested the existence of a Th17 cell-mediated maintenance mechanism of skin homeostasis in hairless mice.

(6) Measurement of anti-hapten antibody level in serum Blood was collected from the 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 diluted series of mouse serum as a sample. HRPO-bound goat anti-mouse IgG was used for detection.

The results are shown in FIG. The amount of anti-DNP antibody did not change much among the three groups, the nanoparticle spray group (A), the spray group (B), and the non-spray group (C), and the immune response to DNP (reaction between B cells and Th cells). Was confirmed to be happening as well. This suggests that it is unlikely that the hapten-induced immune response itself was reduced due to reasons such as the presence of nanoparticles associated with the skin reducing the reactivity of DNFB to the skin.

(7) Measurement of the number of superficial bacteria on the cranial back skin The number of superficial bacteria on the upper back skin was measured on day5, day12, day19, day26, and day33 for the mice in each experimental group. After pressing a standard agar medium (Petancheck, Eiken Chemical) for measuring viable cell count against the cranial back skin for 5 seconds, the cells were cultured in a 37 ° C. furan device for 24 hours, and the number of colonies per ^{4 cm 2 compartment was counted.} ..

The results are shown in FIG. In the nanoparticle water spray group (A group), the number of bacteria fluctuates compared to the water spray group (B group), the non-spray group (C group), and the untreated group (D group) without pretreatment or DNFB application. It was low and showed a low value.

(8) Measurement of IgG antibody level against Staphylococcus aureus in serum Blood was collected from the mice of each experimental group on day 34, and the serum was isolated. After immobilizing the serum, the serum and an equivalent amount of 0.2M 2-ME were added, and the mixture was heated at 37 ° C. for 1 hour. By this treatment, the IgM natural antibody, which is mostly produced in the intestinal system, is monomerized, and the contribution to the agglutination value is reduced. Then, the serum was diluted 2-fold to 256 times, mixed with an inactivated S. aureus cell fluid prepared using S. aureus separated from the skin of these hairless mice, and reacted at 4 ° C. overnight. The maximum dilution ratio at which agglutination did not occur was defined as the agglutination value, and the agglutination value was graphed.

The results are shown in FIG. The agglutination value of the nanoparticle water spray group (A group) was significantly lower than that of the water spray group (B group) and the non-spray group (C group), and the reduction of anti-S. aureus IgG antibody in serum by the nanoparticle water spray was reduced. Was recognized. This suggests that as a result of rapid repair of the skin barrier function by spraying nanoparticles, the invasion of superficial skin bacteria that induce IgG antibody production into the dermis or deeper than that was suppressed.

<Summary of Examination 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 found to be related not only to the thickening of both auricles, which is an index of immune responsiveness, but also to the amount of water evaporation from the back skin, which is an index of skin barrier function, and the degree of water retention.
It was suggested that in the nanoparticle water spray group, cytokines related to epidermal proliferation are expressed together with the Th17 cell line, while in the water spray group and the non-spray group, they are expressed via skin damage-related signals.
The number of superficial bacteria in the nanoparticle spray group was controlled to a certain low value compared to the other three groups. Controlling the number of superficial bacteria by spraying nanoparticles resulted in a decrease in anti-S. aureus IgG antibody in serum due to a synergistic effect with improved skin barrier properties.
These results suggest that nanoparticle spraying regulates the skin inflammatory response through improved skin barrier function and an immune response involving superficial bacterial count control.

Claims (3)

An inhibitor of type I or type IV allergic reaction, which comprises aspartic acid and dextran and contains cyanoacrylate polymer particles having an average particle size of less than 1000 nm as an active ingredient. Type I allergic reaction or IV, including the production of cyanoacrylate polymer particles with an average particle size of less than 1000 nm containing aspartic acid and dextran by anionic polymerization of cyanoacrylate monomers under conditions in which aspartic acid and dextran coexist. A method for producing an inhibitor of type allergic reaction . The method according to claim 2 , wherein the cyanoacrylate is n-butyl cyanoacrylate.

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