Agent for prevention or improvement of inflammatory skin disease in dogs or cats

Abstract

To provide different agents or methods associated with an agent for prevention or improvement of inflammatory skin disease in dogs and cats.SOLUTION: An agent for prevention or improvement of inflammatory skin disease in dogs or cats contains bentonite as an active ingredient, the bentonite serving to adsorb bacteria causing inflammatory skin disease in dogs or cats.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for preventing or ameliorating inflammatory skin diseases in dogs or cats. [Background technology]

[0002] Epidemiological surveys have revealed that approximately half of pet dogs suffer from inflammatory skin diseases, including atopic dermatitis and pyoderma, and the importance of preventing and improving (treating) inflammatory skin diseases is increasing. The causative bacteria of inflammatory skin diseases include Staphylococcus pseudintermedius ( Staphylococcus pseudintermedius ) and Malassezia pachydermatis, a type of yeast-like fungus ( Malassezia pachydermatis ) have been identified. These bacteria (fungi) are normal flora present on dog skin, so they do not cause inflammatory skin diseases on healthy skin. However, when the balance of normal flora is disrupted and these bacteria overgrow, inflammatory skin diseases develop. Typically, excessive proliferation of these causative bacteria is treated with antibiotics. However, the overuse of antibiotics leads to the emergence of drug-resistant bacteria and adverse effects on the normal skin flora, resulting in many long-term negative effects. In particular, antibiotic-resistant (methicillin-resistant) strains have already emerged in Staphylococcus pseudintermedius, a bacterium known to cause canine pyoderma, and the development of methods for inhibiting the proliferation of causative bacteria that do not rely on antibiotics is needed. One example of such an antibiotic-free method is a proposed method for treating dermatitis and other conditions by potently activating the cellular immunity of dogs and cats. For example, Patent Document 1 discloses a therapeutic drug for immune diseases in dogs and cats, which contains canine interleukin-12, formed by the heterodimerization of proteins consisting of specific amino acid sequences. It is described that administration of this therapeutic drug to dogs with atopic dermatitis completely cured the condition. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 11-106350 Summary of the Invention [Problem to be solved by the invention]

[0004] The immune disease therapeutic drug described in Patent Document 1 is basically administered by injection, so treatment requires a visit to a veterinary clinic, etc., and there are limitations to its daily administration for preventive purposes. Therefore, there is a need for the development of preventive and ameliorative agents as external medicines that can be used daily in each household.

[0005] An objective of the present invention is to provide various agents and methods for preventing or ameliorating inflammatory skin diseases in dogs and cats. [Means for solving the problem]

[0006] The present inventors have conducted extensive research in light of the above-mentioned problems, and as a result have found that bentonite, a clay with an extensive track record of use in living organisms as a product, has the ability to bind (adsorb) to bacteria that cause inflammatory skin diseases in dogs and cats (i.e., bentonite can adsorb the bacteria that cause inflammatory skin diseases and remove them from the skin of dogs or cats), and further that it can also suppress the production of inflammatory cytokines caused by the causative bacteria. The present invention has been completed based on these findings.

[0007] The above-mentioned problems of the present invention have been solved by the following means. [1] An agent for preventing or improving inflammatory skin diseases in dogs or cats, which contains bentonite as an active ingredient and adsorbs causative bacteria of inflammatory skin diseases in dogs or cats using the bentonite. [2] An adsorbent for bacteria that cause inflammatory skin diseases, which contains bentonite as an active ingredient and adsorbs bacteria that cause inflammatory skin diseases in dogs or cats. [3] An inhibitor of inflammatory cytokine production, containing bentonite as an active ingredient, that suppresses the production of inflammatory cytokines caused by bacteria that cause inflammatory skin diseases in dogs or cats. [4] The agent according to any one of [1] to [3] above, wherein the inflammatory skin disease is pyoderma and the causative bacterium is Staphylococcus aureus. [5] The agent according to [4] above, containing 0.1 to 20 mass% of bentonite. [6] The agent according to [5] above, which is applied to the skin for 1 to 15 minutes. [7] The agent according to [6] above, wherein the agent is a clay pack, shampoo, treatment, lotion, ointment, or cream. [8] A method for preventing or ameliorating inflammatory skin diseases, comprising applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb bacteria that cause inflammatory skin diseases. [9] A method for adsorbing the causative bacteria of inflammatory skin diseases, comprising applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb the causative bacteria.

[10] A method for suppressing the production of inflammatory cytokines caused by causative bacteria of inflammatory skin diseases, comprising applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb the causative bacteria.

[11] The method according to any one of [8] to

[10] above, wherein the inflammatory skin disease is pyoderma and the causative bacterium is Staphylococcus aureus.

[12] The method according to

[11] above, wherein the bentonite is allowed to act at a concentration of 0.1 to 20% by mass.

[13] The method according to

[12] above, wherein the bentonite is allowed to act on the skin for 1 to 15 minutes.

[14] The method according to

[13] above, wherein the bentonite is applied in the form of a clay pack, shampoo, treatment, lotion, ointment, or cream. [Effects of the Invention]

[0008] The various agents of the present invention contain highly biosafe bentonite as an active ingredient, and by applying this directly to the skin of dogs or cats, they can reduce the number of live bacteria on the skin that cause inflammatory skin diseases and suppress the production of inflammatory cytokines, thereby exerting the effect of preventing or improving inflammatory skin diseases.Furthermore, according to the various methods of the present invention, by applying highly biosafe bentonite directly to the skin of dogs or cats, they can reduce the number of live bacteria on the skin that cause inflammatory skin diseases and suppress the production of inflammatory cytokines, thereby exerting the effect of preventing or improving inflammatory skin diseases. [Brief explanation of the drawings]

[0009]

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[0010] As used herein, "prevention" refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of an individual developing a disease or symptom. Furthermore, as used herein, "amelioration" refers to improving or alleviating a disease, symptom, or condition, preventing or delaying the worsening of a disease, symptom, or condition, or reversing, preventing, or delaying the progression of a disease, symptom, or condition.

[0011] The agent for preventing or ameliorating inflammatory skin diseases of the present invention contains bentonite as an active ingredient. The agent for preventing or ameliorating inflammatory skin diseases of the present invention can be applied directly to the skin of pet animals such as dogs or cats (e.g., areas prone to inflammatory skin diseases or areas affected by inflammatory skin diseases) to bind (adsorb) to the causative bacteria of inflammatory skin diseases present on the skin, and by removing the agent while still bound, the causative bacteria can be removed from the skin. In other words, the agent for preventing or ameliorating inflammatory skin diseases of the present invention does not sterilize or act as an antibacterial agent against causative bacteria, but rather eradicates them. In this specification, the term "sterilization" refers to killing bacteria, the term "antibacterial" refers to inhibiting bacterial growth, and the term "eradication" refers to removing or reducing bacteria from a target object. In particular, in areas affected by inflammatory skin diseases, the balance of the normal flora is disrupted, causing excessive proliferation of causative bacteria, and the skin barrier function is thought to be impaired. By directly applying the agent for preventing or improving inflammatory skin diseases of the present invention to such affected areas, the number of excessively proliferated causative bacteria can be reduced and the normal balance of the normal flora can be restored. Furthermore, even in cases where inflammatory skin diseases have not developed, excessive proliferation of causative bacteria can be prevented, thereby preventing inflammatory skin diseases.

[0012] [Agent for preventing or improving inflammatory skin diseases] Preferred embodiments of the present invention will be specifically described below, but the present invention is not limited to these embodiments except as defined in the present invention.

[0013] A first embodiment of the present invention is an agent for preventing or ameliorating inflammatory skin diseases (hereinafter also referred to as "the agent for preventing or ameliorating the present invention"), which contains bentonite as an active ingredient and uses the bentonite to adsorb causative bacteria of inflammatory skin diseases in dogs or cats. For example, by applying the agent for preventing or ameliorating the present invention in the form of an external preparation directly to the skin of a dog or cat (for example, an area prone to inflammatory skin diseases or an area affected by an inflammatory skin disease), the active ingredient bentonite interacts with the causative bacteria of inflammatory skin diseases (hereinafter also simply referred to as "causative bacteria") present on the skin and suppresses their activity, or binds to the causative bacteria and is then removed from the skin (reducing the number of viable causative bacteria of inflammatory skin diseases present on the skin), thereby restoring the normal skin flora to a normal balance, and as a result, it is thought that inflammatory skin diseases can be prevented or ameliorated. Looking at the preventive or ameliorative agent of the present invention from another aspect, another embodiment of the present invention is an adsorbent for causative bacteria of inflammatory skin diseases, which contains bentonite as an active ingredient and adsorbs causative bacteria of inflammatory skin diseases in dogs or cats. Looking at the preventive or ameliorative agent of the present invention from another perspective, another embodiment of the present invention is an inflammatory cytokine production inhibitor that contains bentonite as an active ingredient and suppresses the production of inflammatory cytokines caused by causative bacteria of inflammatory skin diseases in dogs or cats. It is believed that the active ingredient bentonite acts on the causative bacteria, thereby suppressing the production of inflammatory cytokines in skin cells, which ultimately leads to the prevention of inflammation. Hereinafter, the above-mentioned various agents of the present invention will also be collectively referred to as "agents of the present invention."

[0014] (inflammatory skin disease) Inflammatory skin diseases in dogs or cats include atopic dermatitis, for which Staphylococcus aureus and the like are risk factors, such as pyoderma, Malassezia dermatitis, etc. Among these, the agent of the present invention is preferably an agent for preventing or ameliorating pyoderma, etc.

[0015] The causative bacteria of the inflammatory skin disease are not particularly limited as long as they are bacteria that cause inflammatory skin diseases. Examples of such causative bacteria include Staphylococcus sp. Staphylococcus ) bacteria (Staphylococcus aureus), Malassezia genus ( Malassezia ) Fungi, etc. The Staphylococcus sp. Staphylococcus A specific example of such bacteria is Staphylococcus pseudintermedius ( ), a major pathogen of inflammatory skin diseases in dogs and cats. Staphylococcus pseudintermedius ) and Staphylococcus schleiferi ( Staphylococcus schleiferi ), Staphylococcus aureus ( Staphylococcus aureus ) etc. The Malassezia genus ( Malassezia ) A specific example of a fungus is Malassezia pachydermatis ( Malassezia pachydermatis ) etc.

[0016] (bentonite) Bentonite, the active ingredient in the agent of the present invention, has the ability to bind (or adsorb) to the causative bacteria of the inflammatory skin diseases. Although the specific mechanism by which bentonite binds to the causative bacteria of the inflammatory skin diseases is not clear, it is thought that the causative bacteria bind (adsorb) to the montmorillonite through electrical interaction with the montmorillonite contained in the bentonite.

[0017] Bentonite, the active ingredient of the agent of the present invention, is a clay whose main component is montmorillonite, a type of layered silicate mineral primarily composed of silica and alumina, and generally contains quartz, cristobalite, zeolite, feldspar, and other impurities. In the present invention, the term "bentonite" is used in a broader sense than usual. That is, it may be naturally occurring bentonite, purified bentonite (preferably purified montmorillonite) obtained by purifying naturally occurring bentonite, or synthetic montmorillonite. It may also be a pulverized product of any of these bentonites. The synthetic montmorillonite can be synthesized by conventional methods. For example, starting materials are mixed in the desired composition ratio to prepare a starting gel, and this gel is then subjected to hydrothermal treatment to synthesize montmorillonite. The hydrothermal synthesis method can be carried out using a Moray-type reaction vessel or an autoclave. Furthermore, by appropriately adjusting the composition of the starting materials during synthesis using conventional methods, the cation exchange capacity (CEC) of the resulting synthetic montmorillonite can be adjusted to a desired level. The content of the montmorillonite in the bentonite is preferably 50% by mass or more, more preferably 60% by mass or more, and may also be 70% by mass or more, 80% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more.

[0018] Examples of commercially available bentonite include Kunibond (trade name, manufactured by Kunimine Industries Co., Ltd.), Kunipia F (trade name, manufactured by Kunimine Industries Co., Ltd.), and Neokunibond (trade name, manufactured by Kunimine Industries Co., Ltd.). Furthermore, examples of naturally occurring bentonite (unrefined product) include Na (sodium) bentonite extracted from Tenryu ore in Hosogoe, Niigata Prefecture.

[0019] Montmorillonite has a layered structure consisting of thin plate-like crystals approximately 1 nm thick stacked one on top of the other, and cations such as alkali metals and alkaline earth metals are generally present between the crystal layers. There are no particular limitations on the cations present between the montmorillonite crystal layers in the bentonite used in the present invention. For example, one or more types of bentonite selected from Na-type, Li-type, K-type, NH4-type, Ca-type, Mg-type, Ba-type, Al-type, Fe-type, Cu-type, and Zn-type bentonite can be used. Among these, from the perspective of administering to the skin of dogs or cats, it is more preferable that the bentonite be Na-type, K-type, Ca-type, or Mg-type. As used herein, "major interlayer cations" refers to cations that account for 50% (mol %) or more of the interlayer cations in the montmorillonite. The proportion of these cations may be 60% or more, 70% or more, 80% or more, or even 90% or more. Regarding the ion exchange method for the interlayer cations in bentonite, methods commonly used for ion exchange of bentonite can be applied. From the same viewpoint as above, it is preferable that the bentonite used in the present invention contains 50 ppm or less of heavy metals and 2 ppm or less of arsenic.

[0020] In the present invention, the cation exchange capacity of the bentonite is preferably 40 meq (milliequivalent) or more per 100 g, more preferably 80 meq or more per 100 g, and even more preferably 100 meq or more per 100 g. The cation exchange capacity of the bentonite used in the present invention is typically 250 meq or less per 100 g. The cation exchange capacity of montmorillonite can be measured by a method based on the Schollenberger method (Clay Handbook, Third Edition, edited by the Clay Science Society of Japan, May 2009, pp. 453-454). More specifically, it can be measured by the method described in JBAS-106-77, Standard Test Method of the Japan Bentonite Industry Association.

[0021] In the present invention, the term "bentonite" refers to fine particle bentonite. More specifically, the particle size (primary particle size) of the bentonite is preferably 5 to 500 nm, more preferably 10 to 250 nm, even more preferably 15 to 200 nm, and particularly preferably 20 to 120 nm. By adjusting the particle size of the bentonite to fall within the above-mentioned preferred range, the specific surface area of ​​the bentonite particles increases, and therefore, when the bentonite is directly applied to the skin of a dog or cat as an aqueous dispersion, it can more efficiently bind to pathogenic bacteria. In the present invention, the "particle size" of the bentonite in the aqueous dispersion refers to the volume-based median size, which can be determined, for example, by a laser diffraction / scattering particle size distribution analyzer.

[0022] The form of the agent of the present invention is not particularly limited, and it can be in various forms that can be used as an agent for preventing or improving inflammatory skin diseases, an adsorbent for causative bacteria of inflammatory skin diseases, or an inhibitor of inflammatory cytokine production, such as in animal medicines or skin care products (cosmetics). For example, it can be in the form of a clay pack, shampoo, treatment, lotion, ointment, or cream. The agent of the present invention may also be bentonite itself. Furthermore, other ingredients that can be used in veterinary medicines, cosmetics, etc. may be added to the agent of the present invention in addition to bentonite, as long as the effects of the present invention are not impaired. For example, binders, antioxidants, humectants, fragrances, nonionic surfactants, anionic surfactants, oils, abrasives, pH adjusters, disinfectants, anti-inflammatory agents, water-soluble vitamins, preservatives, plant extracts, and other medicinal ingredients may be added as long as the effects of the present invention are not impaired.

[0023] The agent of the present invention can be made into a solid, liquid (dispersion liquid), gel, or other form according to the usual method, depending on the mode of use. To prepare various dosage forms, additives may be added to bentonite and prepared according to conventional methods. Commonly used additives can be used. Examples of additives include pharmaceutically acceptable excipients, liquid carriers, oily carriers, stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, disintegrants, lubricants, bulking agents, surfactants, dispersants, suspending agents, diluents, osmotic pressure adjusters, pH adjusters, preservatives, antioxidants, colorants, UV absorbers, moisturizers, thickeners, glossing agents, buffers, preservatives, flavoring agents, fragrances, coating agents, odor masking agents, and bacterial inhibitors.

[0024] The content of bentonite, the active ingredient, in the agent of the present invention is not particularly limited and can be determined appropriately depending on the species of dog or cat, the amount used, the binding ability to the causative bacteria, the condition and extent of the affected area, the use schedule, the formulation, and other factors. For example, the content of bentonite in the agent of the present invention is preferably 0.001% by mass or more, more preferably 0.005% by mass or more, even more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. It may also be 0.2% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 10% by mass or more, 20% by mass or more, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, or 80% by mass or more. The upper limit of the bentonite content in the agent of the present invention is not particularly limited, but may be 99% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, or 0.5% by mass or less.

[0025] [Method for preventing or improving inflammatory skin diseases] A second embodiment of the present invention is a method for preventing or ameliorating inflammatory skin diseases (hereinafter also referred to as the "preventive or ameliorating method of the present invention"), which comprises applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb causative bacteria of inflammatory skin diseases. For example, by directly applying bentonite to the skin of a dog or cat (e.g., an area prone to inflammatory skin diseases or an area affected by an inflammatory skin disease), the bentonite interacts with the causative bacteria of inflammatory skin diseases present on the skin and suppresses their activity, or binds to the causative bacteria and is then removed from the skin, thereby restoring the normal skin flora to a normal balance, and as a result, it is thought that inflammatory skin diseases can be prevented or ameliorated. Looking at the prevention or improvement method of the present invention from another aspect, another embodiment of the present invention is a method for adsorbing the causative bacteria of inflammatory skin diseases, which comprises applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb the causative bacteria. Looking at the preventive or ameliorative agent of the present invention from another perspective, another embodiment of the present invention is a method for suppressing the production of inflammatory cytokines caused by causative bacteria of inflammatory skin diseases, which comprises applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb the causative bacteria. It is believed that the action of bentonite on the causative bacteria results in the suppression of the production of inflammatory cytokines in skin cells. Hereinafter, the above-mentioned various methods of the present invention will be collectively referred to as "the method of the present invention." In the method of the present invention, the method of applying bentonite to the skin of a dog or cat includes a method of applying the agent of the present invention to the skin of a dog or cat. Hereinafter, "applying bentonite to the skin of a dog or cat" includes the application of the agent of the present invention to the skin of a dog or cat.

[0026] In the method of the present invention, the interval at which bentonite is applied to the skin of dogs or cats varies depending on the severity of the inflammatory skin disease and the condition of the skin, and therefore cannot be generally defined. However, it can be applied to the skin once or several times a day, or at any desired interval and for any period of time. For example, when the purpose is to prevent inflammatory skin disease, it is preferable to apply it to the skin at least once a week, more preferably at least once every three days, more preferably at least once every two days, and even more preferably at least once a day. Furthermore, when the purpose is to improve the condition of inflammatory skin disease, it is preferable to apply it more frequently than the above. It may be applied to the skin (the area affected by the inflammatory skin disease) at least twice a day, or even three or more times a day. Furthermore, the period of use is not particularly limited, and it is preferably continued until the skin symptoms are alleviated or completely cured. Specifically, for example, 1 week to 6 years is preferable, and 1 month to 1 year is more preferable.

[0027] The time for which bentonite is applied to the skin of a dog or cat during a single use is not particularly limited, but is preferably at least 10 seconds, more preferably at least 1 minute. The time can also be at least 3 minutes, or at least 5 minutes, or even at least 10 minutes. The time is preferably at most 1 hour, more preferably at most 30 minutes, and even more preferably at most 15 minutes. The bentonite applied to the skin of the dog or cat is preferably subsequently removed. For example, when the agent of the present invention is in the form of a clay pack, shampoo, treatment, lotion, ointment, cream, or the like, the agent can be subsequently washed off to remove the causative bacteria bound to the agent of the present invention.

[0028] In the method of the present invention, the amount of bentonite used per application to the skin of a dog or cat is not particularly limited. For example, the amount of bentonite used per application to the skin of a dog or cat is not particularly limited. Staphylococcus pseudintermedius ) is approximately 1 × 10 in the condition that causes pyoderma. 5 ~1×10 8 CFU / cm2 Therefore, it is preferable to administer an amount of bentonite that is within a range that can adequately capture the increased causative bacteria. On the other hand, if an excessive amount of bentonite is administered, the binding ability to the causative bacteria will decrease. Therefore, when an excessive amount of bentonite is administered, the binding ability to the causative bacteria will decrease. 2 It is preferable to administer the amount of bentonite so that it is 0.001 to 1 g per 1 cm of skin of a dog or cat, but it can also be administered so that it is 0.005 to 0.5 g, or it can also be administered so that it is 0.01 to 0.1 g. 2 In this case, the agent of the present invention can be used as it is, and from the same viewpoint as above, bentonite may be administered at 0.01 to 5 mass %, 0.05 to 1 mass %, or 0.1 to 0.5 mass %. [Example]

[0029] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0030] Experimental Example 1-1. Staphylococcus adsorption test The strains and bentonite used in this experiment were as follows: Strain: Staphylococcus aureus ( Staphylococcus aureus 17) Bentonite: Crushed Hosogoe ore (Na-type bentonite)

[0031] Staphylococcus aureus was inoculated into Luria-Bertani (LB) liquid medium (Kanto Chemical Co., Ltd.) and cultured with shaking at 37°C under aerobic conditions for 12 hours. After that, the viable cell count was increased to 1 × 10 in LB liquid medium (Kanto Chemical Co., Ltd.). 6 The Staphylococcus aureus bacterial solution was adjusted to CFU / ml to obtain a Staphylococcus aureus bacterial solution. The viable cell count was calculated by applying the bacterial solution to an LB agar medium (LB medium powder supplemented with 1.5% STAR Agar L-grade 01 (manufactured by Rikaken Co., Ltd.)), culturing it at 37°C under aerobic conditions for 24 hours, and then counting the number of colonies. Bentonite was added to the resulting bacterial suspension at 1%, 5%, or 20% by mass, and the mixture was mixed for 5 minutes. A portion of the mixture was then collected. The mixture was then centrifuged at 12,000 rpm for 5 minutes using a small centrifuge (model number: CFM-100, manufactured by AGC Technoglass Co., Ltd.) to collect the supernatant. The viable cell counts in the collected mixture and the supernatant were measured in the same manner as above. As a control sample, a supernatant was used that was prepared by mixing the Staphylococcus aureus bacterial suspension without bentonite for 5 minutes in the same manner as above, followed by centrifugation and collection. The results of the above test, which was performed multiple times, are shown in Figure 1. In the figure, "ns" means no significant difference, "*" means p<0.05, "**" means p<0.01, "***" means p<0.001, and "****" means p<0.0001. The same applies to the following figures.

[0032] (Statistical Test) Each graph in Figure 1 was analyzed for statistical significance at the 5% and 1% levels. First, a Bartlett test for equality of variances was performed. If this test indicated that the variances across all groups were equal (p > 0.05), a one-way analysis of variance was used to determine whether there were significant differences between groups. If significant differences were found between groups (p ≤ 0.05), Dunnett's multiple comparison test was used to determine whether there were significant differences between groups. If the Bartlett test indicated that the variances across groups were unequal (p ≤ 0.05), a Kruskal-Wallis test was used to determine whether there were significant differences between groups. If significant differences were found between groups (p ≤ 0.05), a Dunnett-type nonparametric multiple comparison test was used to determine whether there were significant differences between groups. Similar statistical processing was also carried out in the following examples.

[0033] Figure 1 clearly shows that in the sample in which bentonite was mixed with the Staphylococcus aureus solution for 5 minutes, the viable cell count in the supernatant obtained after subsequent centrifugation was significantly reduced compared to the control. This is thought to be due to the adsorption of Staphylococcus aureus by bentonite, followed by sedimentation by centrifugation, resulting in a reduction in the viable cell count in the supernatant. Furthermore, the sample in which bentonite was added at 1% by mass showed a particularly reduced viable cell count in the supernatant compared to the samples in which bentonite was added at 20% by mass or 5% by mass.

[0034] Experimental Example 1-2. Staphylococcus adsorption test The mixture and the supernatant were collected and the viable cell count was measured in the same manner as in Experimental Example 1-1, except that the bentonite content was changed to 1% by mass and 0.5% by mass, respectively, in the Staphylococcus aureus bacterial solution. The results are shown in Figure 2. FIG. 2 shows that the sample to which bentonite was added so as to give 0.5% by mass had the greatest reduction in the number of viable bacteria in the supernatant.

[0035] Experimental Example 2: Measurement of anti-inflammatory cytokines The bacterial strains, bentonite, and cultured cells used in this experiment were as follows: Strain: Staphylococcus aureus ( Staphylococcus aureus 17) Bentonite: Crushed Hosogoe ore (Na-type bentonite) Cultured cells: Human epidermal keratinocytes (HaCaT cells)

[0036] The anti-inflammatory effect of bentonite was investigated by measuring the amount of inflammatory cytokines (IL-6 and IL-8) produced by human epidermal keratinocytes induced by Staphylococcus aureus treated with bentonite. Human epidermal keratinocytes (HaCaT cells) were subcultured in D-MEM medium supplemented with 10% fetal bovine serum, and 1 × 10 4 The cells were seeded onto a 96-well culture plate at 100 cells / well. Cells in a state of approximately 80% confluence on the 2nd or 3rd day of passage were used for measurement. Staphylococcus aureus bacterial solution (1 x 10 5 Bentonite was mixed with a 10% by mass concentration of bentonite in a 10% by mass D-MEM medium (CFU / ml, D-MEM medium) and mixed for 5 or 15 minutes. The mixture was then centrifuged at 12,000 rpm for 5 minutes using a small centrifuge (model number: CFM-100, manufactured by AGC Technoglass Co., Ltd.) to recover the supernatant. The supernatant and D-MEM medium were mixed at a 1:1 volume ratio and added to a 96-well culture plate seeded with HaCaT cells. After 24 hours of culture, the amounts of IL-6 and IL-8 protein in the culture medium were measured by ELISA. As controls, Staphylococcus aureus solution and D-MEM medium were used instead of the supernatant. The results of seven trials are shown in Figure 3.

[0037] As is clear from Figure 3, when Staphylococcus aureus solution was added to HaCaT cells, the amounts of inflammatory cytokines IL-6 and IL-8 increased. In contrast, when the Staphylococcus aureus solution was mixed with bentonite, centrifuged, and the resulting supernatant was added to HaCaT cells, the amounts of inflammatory cytokines IL-6 and IL-8 were barely detectable.

[0038] Experimental Example 3: Pathological examination of pyoderma The bacterial strains, bentonite, and specimens used in this experiment were as follows: Strain: Staphylococcus aureus ( Staphylococcus pseudintermedius ) Bentonite: Crushed Hosogoe ore (Na-type bentonite) Subject: Mouse (BALB / c), female

[0039] Staphylococcus aureus was inoculated into LB liquid medium (Kanto Chemical Co., Ltd.) and cultured with shaking at 37°C under aerobic conditions for 12 hours. After that, the viable cell count in LB liquid medium (Kanto Chemical Co., Ltd.) was increased to 1 x 10 6The concentration of staphylococcus was adjusted to CFU / ml to obtain a staphylococcus solution (Test Solution 1). The viable cell count was calculated by applying the solution to an LB agar medium (LB medium powder supplemented with 1.5% STAR Agar L-grade 01 (manufactured by Rikaken Co., Ltd.)), culturing it at 37°C under aerobic conditions for 24 hours, and then counting the number of colonies. Bentonite was added to the obtained bacterial solution to a concentration of 10% by mass, and the mixture was mixed for 5 minutes. The mixture was centrifuged at 12,000 rpm for 5 minutes using a small centrifuge (model number: CFM-100, manufactured by AGC Technoglass Co., Ltd.) to recover the supernatant (Test Solution 3). For comparison, the supernatant obtained by centrifuging the above bacterial solution without adding bentonite was also prepared (Test Solution 2). 100 μl of each of the above test solutions 1 to 3 was administered intradermally to 7-week-old mice to induce skin abscesses. Four days after intradermal administration, the length and area of ​​the lesions in the mice were measured. Because the shape of the lesions varied from individual to individual, the length of the lesions was measured along the longest axis using a dedicated micrometer, PK-1012APX (Mitutoyo). The area of ​​the lesions was also measured using a Dermocamera DZ-D100 (CASIO) and calculated using ImageJ. Skin samples from the lesions were then collected for staphylococcal counts and histopathological analysis. Staphylococcus counts were calculated by placing the collected skin samples in sterilized PBS liquid medium, vortexing for 30 seconds, and then applying the resulting suspension to LB agar medium (LB medium powder supplemented with 1.5% STAR Agar L-grade 01 (Rikaken Co., Ltd.)). After 24 hours of aerobic incubation, colony counts were counted. Histopathological analysis was performed according to the method described by Kaneki et al., International Immunopharmacology 124 (2023) 110920. Specifically, the collected skin samples were fixed in 10% formalin solution and embedded in paraffin. Sections prepared from the embedded skin tissue were stained with hematoxylin and eosin and Gram stained according to standard methods. A third party performed a semi-quantitative analysis using indicators such as epidermal hyperplasia, crusts, and ulcers, dermal abscesses, inflammatory cell infiltration, and necrosis, as well as muscular infiltration, degeneration, necrosis, and hemorrhage. The pathological results are shown in Figures 4 and 5.

[0040] As is clear from Figure 4, the number of live staphylococci bacteria in the lesions of mice that had been intradermally administered the supernatant sample (Test Solution 3) was significantly reduced compared to the lesions of mice that had been intradermally administered the staphylococcal bacterial solution (Test Solution 1). Furthermore, the degree of reduction was significantly reduced compared to mice that had been intradermally administered the supernatant (Test Solution 2) obtained by simply centrifuging the staphylococcal bacterial solution. Furthermore, as is clear from Figure 5, the pathology scores of mice administered test solution 3 intradermally were significantly lower (or tended to be lower) than those of mice administered test solutions 1 and 2 intradermally.

Claims

1. An agent for preventing or improving inflammatory skin diseases in dogs or cats, which contains bentonite as an active ingredient and adsorbs causative bacteria of inflammatory skin diseases in dogs or cats using the bentonite.

2. An adsorbent for bacteria that cause inflammatory skin diseases, which contains bentonite as an active ingredient and adsorbs bacteria that cause inflammatory skin diseases in dogs or cats.

3. An inhibitor of inflammatory cytokine production, containing bentonite as an active ingredient, that suppresses the production of inflammatory cytokines caused by bacteria that cause inflammatory skin diseases in dogs or cats.

4. The agent according to any one of claims 1 to 3, wherein the inflammatory skin disease is pyoderma and the causative bacterium is Staphylococcus aureus.

5. The agent according to claim 4, containing 0.1 to 20% by mass of bentonite.

6. The agent according to claim 5, which is applied to the skin for 1 to 15 minutes.

7. The agent according to claim 6, which is a clay pack, shampoo, treatment, lotion, ointment, or cream.

8. A method for preventing or ameliorating inflammatory skin diseases, comprising applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb bacteria that cause inflammatory skin diseases.

9. A method for adsorbing the causative bacteria of inflammatory skin diseases, comprising applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb the causative bacteria.

10. A method for suppressing the production of inflammatory cytokines caused by causative bacteria of inflammatory skin diseases, comprising applying bentonite to the skin of a dog or cat and allowing the bentonite to adsorb the causative bacteria.

11. The method according to any one of claims 8 to 10, wherein the inflammatory skin disease is pyoderma and the causative bacterium is Staphylococcus aureus.

12. The method according to claim 11, wherein the bentonite is applied at a concentration of 0.1 to 20% by weight.

13. 13. The method of claim 12, wherein the bentonite is applied to the skin for 1 to 15 minutes.

14. 14. The method of claim 13, wherein the bentonite is applied in the form of a clay pack, shampoo, treatment, lotion, ointment, or cream.

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