Topical application agent for the prevention or treatment of skin fibrosis, containing differentiation-inducing factors derived from cellular slime molds.

Abstract

This invention provides a pharmaceutical product that prevents or treats skin fibrosis diseases by inhibiting the conversion of adipocytes to myofibroblasts. [Solution] A preventive and / or therapeutic agent for skin fibrosis containing cellular slime mold-derived differentiation-inducing factor (DIF). This preventive and / or therapeutic agent prevents or treats skin fibrosis such as scleroderma by suppressing the conversion of adipocytes to myofibroblasts, and further, by topical application to the skin as a topical agent, DIF is efficiently delivered to adipocytes in the skin tissue, thereby suppressing adipocyte-myofibroblast conversion and effectively preventing or treating skin fibrosis.

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for skin fibrosis diseases, and more particularly to a topical preventive or therapeutic agent for said disease. [Background technology]

[0002] Myofibroblasts play a crucial role in the repair, regeneration, and maintenance of homeostasis of damaged tissue by producing diverse extracellular matrix and forming granulation tissue. Myofibroblasts emerge from various cell types, including fibroblasts, preadipocytes or adipocytes, epithelial cells (keratinocytes) or endothelial cells, pericytes, fibroblasts, and macrophages, often due to inflammatory responses. Since myofibroblasts highly express α-SMA (α-smooth muscle actin), the presence of myofibroblasts can be confirmed by detecting α-SMA-positive cells. As tissue regeneration progresses, myofibroblasts disappear, but their persistence leads to fibrosis in the tissue. Wound scarring (hypertrophic scars) and keloids are caused by myofibroblasts. The involvement of myofibroblasts converted from adipocytes in various diseases has been reported. In systemic sclerosis, the conversion of adipocytes to myofibroblasts, rather than dermal fibroblasts, is considered to be an important mechanism of fibrosis (Non-Patent Literature 1). Skin fibrosis, a characteristic clinical symptom of systemic sclerosis, is accompanied by the disappearance of dermal white adipose tissue (dWAT) and its replacement with a fibrous matrix, in which intradermal adipocytes lose the expression of their specific markers and acquire the expression of the myofibroblast marker (α-SMA) (Non-Patent Literature 2-4). In addition, adipocytes in the dermis differentiate into invasive myofibroblasts that release fatty acids to recruit macrophages and produce extracellular matrix, thereby controlling skin wound healing (Non-Patent Literature 5, 6).

[0003] Fibrosis is scarring and tissue hardening caused by excessive deposition of extracellular matrix (ECM) proteins by myofibroblasts in response to chronic inflammation. As the excessive accumulation of ECM progresses, the tissue hardens. The tension generated by tissue hardening is sensed by cells through mechanistic transmission via cell surface integrin receptors, activating YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). Activated YAP and TAZ migrate to the nucleus, contributing to increased activity of fibrosis genes such as CTGF (connective tissue growth factor) and PDGF (platelet-derived growth factor), which promote myofibroblast proliferation and activation. The YAP / TAZ pathway plays a major role in cutaneous fibrosis, where tension is a significant factor (Non-patent Literature 7, 8).

[0004] To date, several antifibrotic drugs have been developed. For example, pirfenidone, a TGF-β signaling inhibitor, and nintedanib, a triple kinase inhibitor that inhibits vascular endothelial growth factor receptors types 1-3, fibroblast growth factor receptors types 1-3, and platelet-derived growth factor receptor, have been approved for idiopathic pulmonary fibrosis. On the other hand, no drugs developed with the concept of inhibiting the conversion of adipocytes to myofibroblasts are known, and furthermore, there are currently no antifibrotic drugs that can be applied topically to the skin.

[0005] Cellular slime mold-derived differentiation-inducing factor (DIF) is a low-molecular-weight compound produced and secreted by a type of cellular slime mold, Dictyostelium discoideum. It is an active factor that induces differentiation into stalk cells when aggregated slime mold cells form a fruiting body. DIF has been reported to inhibit the inflammation-inducing prostaglandin synthase mPGES-1 (Patent Document 1), but currently, no anti-inflammatory drugs targeting mPGES-1 have been developed. Furthermore, it has been suggested that DIF-like factors accumulate in mitochondria and inhibit their function, thereby suppressing the proliferation of cancer cells (Non-Patent Literature 9). However, no fibrosis-inhibiting effects of DIF have been reported, either through systemic or local administration. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] W02011046200 [Non-patent literature]

[0007] [Non-Patent Document 1] Current Rheumatology Reports. 2020; 22(11): 79 [Non-Patent Document 2] Nat Rev Rheumatol. 2017; 13(2): 71-72 [Non-Patent Document 3] Curr Opin Rheumatol. 2017; 29(6): 585-90 [Non-Patent Document 4] Arthritis Rheum. 2015; 67(4): 1062-1073 [Non-Patent Document 5] Cell Stem Cell. 2020; 26(6): 801-803 [Non-Patent Document 6] Cell Stem Cell. 2020; 26(6): 880-895 [Non-Patent Document 7] Exp Dermatol. 2022; 31(10): 1477-1499 [Non-Patent Document 8] Front Pharmacol. 2022; 31(3): 320-329 [Non-Patent Document 9] PLOS ONE. 2013; 8(8):e72023

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide an agent for preventing or treating diseases caused by skin fibrosis by suppressing the Wnt / β-catenin pathway and the YAP / TAZ pathway, suppressing fibrosis, and maintaining subcutaneous adipose tissue.

Means for Solving the Problems

[0009] The present inventors found that by directly applying DIF to the skin of bleomycin-induced scleroderma model mice, skin fibrosis and thickening were suppressed. In addition, in the DIF administration group, the disappearance of subcutaneous adipose tissue was suppressed compared with the control group, suggesting that DIF suppresses the conversion of adipocytes to myofibroblasts, thereby suppressing skin fibrosis. Based on these findings, the present inventors conducted further research and as a result, completed the present invention.

[0010] That is, the present invention relates to the following. [1] An agent for preventing and / or treating skin fibrosis diseases containing a differentiation-inducing factor derived from cellular slime mold. [2] The differentiation-inducing factor derived from cellular slime mold is at least one compound selected from the formulas (I) to (IV):

[0011]

Chemical Formula

[0012] <able> The agent according to [1], which is at least one compound selected from the above or a pharmaceutically acceptable salt thereof. <able> [3] The agent according to [1], wherein the differentiation-inducing factor derived from cellular slime mold is a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof. [4] The agent according to any one of [1] to [3], wherein the skin fibrosis disease is at least one selected from the group consisting of scleroderma, hypertrophic scar, and keloid. [5] A drug described in any of [1] to [4] for local administration. [6] A topical skin preparation, as described in any of [1] to [4]. [7] An adipocyte-myofibroblast conversion inhibitor containing a differentiation-inducing factor derived from cellular slime mold. [Effects of the Invention]

[0013] According to the present invention, DIF inhibits the conversion of adipocytes to myofibroblasts, and therefore can prevent or treat diseases caused by skin fibrosis, such as scleroderma, hypertrophic scars, and keloids. According to the present invention, DIF can be used as a therapeutic agent that can be administered topically to the skin, is easy to administer, and minimizes the impact on other parts of the body by directly administering the drug to the lesion. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows that local administration of DIF-1 suppressed skin thickening in bleomycin-induced scleroderma model mice (administered concurrently with bleomycin). The top shows the administration schedule. The left image shows Masson Trichrome (MT) stained skin sections. The right image is a graph comparing skin thickness in the control group, vehicle group, and DIF-1 administered group. [Figure 2] The upper left of Figure 2 shows a photograph of skin tissue from a bleomycin-induced scleroderma model mouse, double-stained with osmium and hematoxylin-eosin (HE staining). The lower left of the same figure shows a CT image of skin tissue from an osmium-stained bleomycin-induced scleroderma model mouse. The upper section shows a coronal section, and the lower section shows a 3D reconstruction. The right side of the same figure shows a graph quantitatively evaluating the dWAT volume of a bleomycin-induced scleroderma model mouse using CT images. [Figure 3]Figure 3 shows that local administration of DIF-1 suppressed skin thickening in bleomycin-induced scleroderma model mice (DIF-1 administration started after completion of bleomycin administration). The upper left shows the administration schedule. The lower left photograph shows MT stained images of skin sections. The graph on the right compares skin thickness between the vehicle group (12 and 24 days) and the DIF-1 administration group. [Figure 4] Figure 4 shows the effect of DIF on adipocyte conversion. The top image shows a photograph of lipid droplets stained with Oil Red O in undifferentiated mesenchymal cells derived from rat subcutaneous fat. The bottom image shows the expression levels of adipocyte fatty acid-binding protein (A-FABP) and peroxisome proliferator-activated receptor gamma (PPARγ), which are adipocyte differentiation markers. [Figure 5] Figure 5 is a graph showing that DIF inhibits the YAP / TAZ inhibitory pathway related to fibrosis. [Figure 6] Figure 6 shows the effect of YAP / TAZ on adipocyte-myofibroblast conversion in mouse adipocyte progenitor cells 3T3L-1 cells stimulated with TGF-β1 (5 ng / ml) for 48 hours, using myofibroblasts expressing α-SMA as an indicator. [Figure 7] Figure 7 shows the effect of DIF on adipocyte-myofibroblast conversion in mouse adipocyte progenitor cells 3T3L-1 cells stimulated with TGF-β1 (5 ng / ml) for 60 hours, using myofibroblasts expressing α-SMA as an indicator. [Modes for carrying out the invention]

[0015] The present invention relates to a preventive and / or therapeutic agent for skin fibrosis disease containing a differentiation-inducing factor derived from cellular slime mold (also referred to as the pharmaceutical formulation of the present invention).

[0016] In this specification, the differentiation-inducing factor derived from cellular slime mold is defined by the following formula (A):

[0017] [ka]

[0018] (wherein, R 1 is selected from hydroxy and -O-C 1-3 alkyl; R 2 is selected from hydrogen, chloro, bromo, and iodo; R 3 is selected from -O-C 1-3 alkyl and hydroxy; R 4 is selected from hydrogen, chloro, bromo, and iodo; R 5 is selected from hydroxy, C 1-6 alkyl, and -O-C 1-3 alkyl; R 6 is selected from hydrogen, hydroxy, C 1-10 alkyl, and C 3-6 cycloalkyl; and, the -O-C 1-3 alkyl, C 1-6 alkyl, C 1-10 alkyl, and C 3-6 cycloalkyl may each independently be substituted with one or more substituents selected from hydroxy, amino, methyl, and methoxy on the carbon) refers to a compound represented by the formula or a pharmaceutically acceptable salt thereof.

[0019] More specifically, the differentiation-inducing factor derived from cellular slime mold (referred to as DIF) used in the present invention is represented by the following formulas (I) to (IV):

[0020] [Chemical formula]

[0021] refers to a compound represented by the formula or a pharmaceutically acceptable salt thereof.

[0022] In particular, DIF-1 and DIF-3 are preferred, with DIF-1 being more preferred, from the viewpoint of being highly effective in suppressing the conversion of adipocytes to myofibroblasts.

[0023] Examples of pharmaceutically acceptable salts of compounds of general formula (A) include inorganic acid addition salts (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.), organic carboxylic acid / sulfonic acid addition salts (e.g., formate, acetate, trifluoroacetate, maleate, tartrate, fumarate, citrate, lactate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), or metal salts such as sodium, potassium, magnesium, and calcium. Note that compounds of general formula (A) may also be in hydrate form.

[0024] Examples of salts in the basic group include salts with mineral acids such as hydrochloric acid, hydrobromic acid, and sulfuric acid; salts with organic carboxylic acids such as tartaric acid, formic acid, fumaric acid, maleic acid, malic acid, and citric acid; and salts with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, mesitylenesulfonic acid, and naphthalenesulfonic acid.

[0025] Examples of salts in acidic groups include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; and salts with amino acids such as lysine, arginine, and ornithine, and nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-efenamine, and N,N'-dibenzylethylenediamine.

[0026] DIF inhibits the conversion of adipocytes to myofibroblasts, making it useful as an adipocyte-to-myofibroblast conversion inhibitor. The present invention also provides an adipocyte-to-myofibroblast conversion inhibitor comprising DIF. In vivo or in vitro, contact of adipocytes with DIF inhibits the conversion of said adipocytes to myofibroblasts. Administration of an effective amount of DIF to a mammal (e.g., human) inhibits the conversion of adipocytes to myofibroblasts in said mammal. The mammal may be a patient with cutaneous fibrosis disease. Administration of an effective amount of DIF to a patient with cutaneous fibrosis disease inhibits the conversion of adipocytes to myofibroblasts in the skin tissue of said patient. In vitro, culturing adipocytes in the presence of DIF inhibits the conversion of said adipocytes to myofibroblasts.

[0027] Myofibroblasts converted from adipocytes produce diverse extracellular matrix, causing skin fibrosis; therefore, DIF is useful in the prevention or treatment of cutaneous fibrotic diseases. Cutaneous fibrotic diseases are a general term for diseases involving skin fibrosis. Examples of cutaneous fibrotic diseases include, but are not limited to, scleroderma (e.g., systemic scleroderma, localized scleroderma), hypertrophic scars, keloids, senile cutaneous atrophy, photolytic fibrosis, and dermatoporosis. Cutaneous fibrotic diseases can be treated in mammals that have developed them by administering an effective amount of DIF. Cutaneous fibrosis can be suppressed in mammals that have developed them by administering an effective amount of DIF. Cutaneous fibrotic diseases can be prevented in mammals at risk of developing them by administering an effective amount of DIF. By administering an effective dose of DIF to mammals at risk of developing cutaneous fibrosis, the risk of developing cutaneous fibrosis in those mammals can be reduced. For example, mammals that have undergone surgical procedures involving skin incisions, mammals that have undergone skin grafts, and mammals that have suffered wounds or burns are at risk of developing cutaneous fibrosis (particularly hypertrophic scarring). By administering an effective dose of DIF to these mammals, the development of cutaneous fibrosis (particularly hypertrophic scarring) in those mammals can be prevented, or the risk of developing cutaneous fibrosis (particularly hypertrophic scarring) in those mammals can be reduced.

[0028] Furthermore, by administering an effective amount of DIF to mammals that have developed cutaneous fibrosis diseases (such as scleroderma) characterized by a decrease in cutaneous adipose tissue due to enhanced adipocyte-myofibroblast conversion, it is possible to suppress atrophy of cutaneous adipose tissue and restore the amount of cutaneous adipose tissue. By administering an effective amount of DIF to mammals at risk of developing cutaneous fibrosis diseases, it is possible to prevent atrophy of cutaneous adipose tissue associated with the development of cutaneous fibrosis diseases in those mammals. By administering an effective amount of DIF to mammals at risk of developing cutaneous fibrosis diseases, it is possible to reduce the risk of atrophy of cutaneous adipose tissue associated with the development of cutaneous fibrosis diseases in those mammals.

[0029] Adipocyte-myofibroblast conversion in skin tissue occurs from the early stages of onset to the progression of cutaneous fibrotic diseases (e.g., scleroderma (e.g., systemic scleroderma, localized scleroderma), hypertrophic scars, keloids). Since DIF suppresses this adipocyte-myofibroblast conversion, it can exert excellent effects in preventing the onset of cutaneous fibrotic diseases (e.g., scleroderma (e.g., systemic scleroderma, localized scleroderma), hypertrophic scars, keloids), reducing the risk of onset, and delaying or suppressing the progression (exacerbation) of symptoms or conditions of cutaneous fibrotic diseases (e.g., skin fibrosis and thickening).

[0030] Furthermore, DIF inhibits skin fibrosis by suppressing adipocyte-myofibroblast conversion through downregulation of the YAP / TAZ signaling pathway, thus exhibiting excellent effects in preventing the onset of skin fibrotic diseases (e.g., scleroderma (e.g., systemic scleroderma, localized scleroderma), hypertrophic scars, keloids), reducing the risk of onset, and delaying or suppressing the progression (exacerbation) of symptoms or conditions of skin fibrotic diseases (e.g., skin fibrosis and thickening).

[0031] In this specification, "effective dose" means the amount that produces the desired effect (e.g., therapeutic effect) in a subject, for example, that in a subject administered this dose, the symptoms or condition of the disease are alleviated, reduced, or eliminated, or its progression is delayed or suppressed, compared to a subject who did not receive this dose. The effective dose can be determined by a physician as appropriate, depending on the subject's age, weight, sex, and the severity of their symptoms.

[0032] In one embodiment, cutaneous fibrosis is accompanied by an inflammatory response. Examples of cutaneous fibrosis accompanied by an inflammatory response include inflammatory scleroderma (inflammatory scleroderma); hypertrophic scars that occur during surgical procedures involving skin incision, skin grafting, wound or burn healing processes, etc. For such cutaneous fibrosis accompanied by an inflammatory response, DIF having an inhibitory effect on the inflammation-inducing prostaglandin synthase mPGES-1 is preferably applied. This is because, in addition to suppressing cutaneous fibrosis by inhibiting adipocyte-myofibroblast conversion, an anti-inflammatory effect due to mPGES-1 inhibition can be expected. It should be noted that even by applying DIF that does not have mPGES-1 inhibitory activity to cutaneous fibrosis accompanied by an inflammatory response, an inhibitory effect on cutaneous fibrosis by inhibiting adipocyte-myofibroblast conversion can be expected.

[0033] In one embodiment, cutaneous fibrosis is characterized by the absence of an inflammatory response. Examples of cutaneous fibrosis without an inflammatory response include inflammatory-negative scleroderma and keloids. Since the activity that inhibits adipocyte-myofibroblast conversion is independent of mPGES-1 inhibitory activity, applying DIF, which has mPGES-1 inhibitory activity, to cutaneous fibrosis without an inflammatory response can also be expected to have a preventive or therapeutic effect on cutaneous fibrosis.

[0034] The method for producing DIF may be a non-biological method, a biological method, or a combination thereof, but the non-biological method is simpler. Examples of non-biological methods include organic synthesis. Compounds, reagents, and equipment necessary for organic synthesis can be those that are generally available. The synthesis reaction may be carried out according to a method generally known to those skilled in the art, for example, the method described by Mark S. MASENTO et al. (Biochem. J. 1988; 256, 23-28). Alternatively, DIF may be prepared by a biological method, for example, by culturing microorganisms. Microorganisms suitable for preparing the target compound may be screened by a method known to those skilled in the art. Microorganisms suitable for preparing DIF are not limited, but are preferably fungi. Examples of fungal microorganisms include those belonging to the genera Dictyosterium (Dictyostelium) and Chaetomium. Examples of microorganisms include Dictyostelium discoideum, Dictyostelium mucoroides, Dictyostelium purpureum, and Polysphondylium violaceum. The culture of the microorganisms may be carried out under conditions known to those skilled in the art. The DIF thus produced may be used as is or purified, depending on the intended use. The purification method can be carried out by methods known to those skilled in the art. Compounds belonging to the DIF family are very stable and can be stored for long periods under normal conditions.

[0035] DIF can be formulated according to conventional methods to make therapeutic or prophylactic agents (also called pharmaceutical preparations) (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). Dosage forms of pharmaceutical preparations include, for example, liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. Pharmaceutical preparations may contain pharmaceutically acceptable carriers and / or additives in addition to DIF or a pharmaceutically acceptable salt thereof as the active ingredient. For example, the formulation may contain surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. However, the pharmaceutical formulation is not limited to these and may contain other commonly used carriers as appropriate. Specifically, examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. It may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, and amino acids.

[0036] The content of DIF or its pharmaceutically acceptable salts in a pharmaceutical preparation is not particularly limited, but for example, it may be approximately 0.00001 to 100% by weight or 0.0001 to 99.9% by weight of the total pharmaceutical preparation as DIF.

[0037] Pharmaceutical preparations can be administered orally or parenterally (e.g., transdermal, subcutaneous, nasal, pulmonary, intramuscular, or intravenous). Parenteral administration is preferred because it is relatively easy to administer and minimizes the impact on other parts of the body by directly administering the drug to the lesion site. Pharmaceutical preparations can also be administered systemically or locally, but local administration is preferred because it allows for direct administration to the lesion site and can be expected to have a rapid effect.

[0038] In one embodiment, the pharmaceutical formulation of the present invention is prepared as a topical skin preparation such as an ointment, plaster, patch, cream, lotion, or gel, together with pharmaceutically acceptable additives. By topically applying DIF to the skin as a topical skin preparation, DIF can be efficiently delivered to adipocytes in the skin tissue, thereby inhibiting adipocyte-myofibroblast conversion and effectively preventing or treating cutaneous fibrotic diseases. The content of DIF or its pharmaceutically acceptable salt in the topical skin preparation is usually 0.5 to 15% by weight, but is not limited thereto.

[0039] Additives used in ointments include bases, humectants, thickeners, and emulsifiers. Additives used in hard ointments or patches include thickeners, humectants, fillers, crosslinking agents, solvents, and emulsifiers. Additives used in creams include oil-soluble substances, water-soluble substances, and emulsifiers. Additives used in lotions include oil-soluble substances, water-soluble substances, and emulsifiers. Additives used in gels include bases, etc.

[0040] Examples of base materials include higher hydrocarbons, oils and fats, waxes, fatty acids, higher alcohols, lower alcohols, and esters. Examples of higher hydrocarbons include squalane, synthetic paraffin, liquid paraffin, white petrolatum, and microcrystalline wax; examples of waxes include beeswax, bleached beeswax, lanolin, and ceresin wax; examples of fatty acids include stearic acid and oleic acid; examples of higher alcohols include lanolin alcohol and cetostearyl alcohol; examples of lower alcohols include ethanol, isopropanol, and propylene glycol; and examples of esters include isopropyl myristate and stearyl myristate.

[0041] Examples of humectants include glycerin, propylene glycol, and 1,3-butylene glycol.

[0042] Examples of thickening agents include sodium alginate, gelatin, methylcellulose, carboxyvinyl polymer, and sodium polyacrylate.

[0043] Examples of emulsifiers include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants. Examples of cationic surfactants include cetyltrimethylammonium chloride, lauryldimethylbenzylammonium chloride, tetrabutylammonium chloride, and dioctadecyldimethylammonium chloride. Examples of anionic surfactants include sodium alkylbenzene sulfonate, sodium dodecyl sulfate, sodium coconut alcohol ethoxysulfate, sodium α-olefin sulfonate, and emulsified cetostearyl alcohol. Examples of amphoteric surfactants include N-alkyl-N,N-dimethylammonium betaine and imidazoline-type amphoteric surfactants. Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyoxyethylene hydrogenated castor oil, polyoxyl stearate, glycerin fatty acid esters, and diglycerin fatty acid esters. The above surfactants can be used individually or in combination.

[0044] Examples of fillers include kaolin, titanium dioxide, and zinc oxide.

[0045] Examples of crosslinking agents include acetaldehyde, dimethyl ketone, and aluminum sulfate.

[0046] Examples of solvents include alcohols such as ethanol and isopropanol.

[0047] Oil-soluble substances that can be used include higher hydrocarbons, waxes, fatty acids, higher alcohols, esters, etc. Examples of higher hydrocarbons include squalane, synthetic paraffin, liquid paraffin, white petrolatum, and microcrystalline wax; examples of waxes include beeswax, bleached beeswax, lanolin, and ceresin wax; examples of fatty acids include stearic acid and oleic acid; examples of higher alcohols include lanolin alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, cetostearyl alcohol, and cholesterol; and examples of esters include isopropyl myristate and stearyl myristate.

[0048] Water-soluble substances such as water, thickeners, and humectants can be used.

[0049] For topical skin preparations, pH adjusters, preservatives, macrogols, etc., may be added as needed. Examples of pH adjusters include diisopropanolamine, triisopropanolamine, triethanolamine, potassium hydroxide, sodium hydroxide, sodium citrate, phosphoric acid, tartaric acid, dl-malic acid, and glacial acetic acid. Examples of preservatives include thymol, dibutylhydroxytoluene, sodium edetate hydrate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, and propyl parahydroxybenzoate.

[0050] The dosage of DIF is adjusted as appropriate depending on the type of DIF, the type of disease, the severity of the disease, the dosage form, the age, sex, and weight of the recipient. For example, in the case of parenteral administration (e.g., transdermal administration), the dosage per dose can be 1 mg to 80 mg of DIF. The number of doses per day is one or more (e.g., two, three, or four times).

[0051] All references cited herein, including publications and patent documents, are incorporated herein by reference to the same extent as they are individually and specifically referred to and their entire contents are specifically described.

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. [Examples]

[0053] [Example 1] Effect of DIF-1 on bleomycin-induced scleroderma: Simultaneous administration Following the schedule shown in Figure 1, bleomycin (1.0 mg / mL, 50 μL) was locally injected subcutaneously into HoS:HR1 mice (8 weeks old) (black arrow). The vehicle group had 50 μl of acetone applied after local injection (white arrow). The DIF-1 group had a solution of 500 μg of DIF-1 dissolved in 50 μl of acetone applied after local injection (white arrow). The same procedure was performed a total of four times, every three days. 24 hours after the fourth procedure, the entire skin thickness was collected, stained with MT, and measured.

[0054] In a mouse model of bleomycin-induced scleroderma, topical administration of DIF-1 to the skin suppressed skin thickening (Figure 1).

[0055] [Example 2] Effect of DIF-1 on adipose tissue in a mouse model of bleomycin-induced scleroderma Osmium, a heavy metal, not only stains fat black and makes it visible, but it also makes it radiopaque, allowing for quantitative analysis using CT. Therefore, skin samples taken from bleomycin-induced scleroderma model mice prepared in the same manner as in Example 1 were stained with a 1% osmium(VIII) oxide solution for 24 hours, and the effect of DIF-1 on adipose tissue was evaluated by CT scanning and microscopic observation.

[0056] Adipose tissue was stained black by a combination of osmium staining and HE staining. In the vehicle group, white adipose tissue disappeared after bleomycin injection, whereas in the DIF-1 administration group, the disappearance of white adipose tissue was suppressed compared to the vehicle group. Regarding the retention of adipose tissue, DIF-1 was shown to significantly preserve adipose tissue through staining with the heavy metal osmium and quantitative evaluation of dWAT volume using CT images (Figure 2).

[0057] [Example 3] Effect of DIF-1 on bleomycin-induced scleroderma: Post-administration Following the schedule shown in Figure 3, HoS:HR1 mice (8 weeks old) were locally injected subcutaneously with bleomycin (1.0 mg / mL, 50 μL) on days 0, 3, 6, and 9, every three days for a total of four injections (black arrows). For the following two weeks, the vehicle group received acetone (50 μl) once daily, five times a week (from days 12 to 16 and from days 19 to 23) (white arrows). The DIF-1 group similarly received 500 μg of DIF-1 solution dissolved in 50 μl of acetone (white arrows). 24 hours after the last treatment, the entire skin layer was collected, stained with MT, and skin thickness was measured.

[0058] In a mouse model of bleomycin-induced scleroderma, topical administration of DIF-1 to the skin suppressed skin thickening (Figure 3).

[0059] [Example 4] Effect of DIF-1 on adipocyte conversion Undifferentiated mesenchymal cells derived from rat subcutaneous fat were isolated using a cell sorter. DIF-1 was added to adipocyte differentiation induction medium at the indicated concentration and cultured for 9 days. Lipid droplets were visualized by Oil Red O staining, and Oil Red O-positive areas were measured (Figure 4). In addition, the expression of A-FABP and PPARγ, adipocyte differentiation markers, was examined by Western blotting. It was revealed that DIF-1 promotes differentiation into adipocytes, demonstrating that DIF has an adipocyte differentiation-inducing effect in undifferentiated mesenchymal cells derived from rat subcutaneous fat.

[0060] [Example 5] YAP / TAZ suppression effect by DIF Human cervical cancer-derived HeLa cells were seeded and then treated with DIF-1 (30 μM) or Vehicle (ethanol) for 6 or 24 hours, respectively, and protein expression was analyzed by Western blotting. As shown in Figure 5, it was revealed that DIF-1 reduces the expression of YAP / TAZ proteins involved in fibrosis, thereby suppressing this pathway.

[0061] [Reference Example 1] Effects of YAP / TAZ on adipocyte-myofibroblast conversion Mouse adipocyte progenitor cells (3T3L-1) were stimulated with TGF-β1 (5 ng / ml) for 48 hours to induce conversion to myofibroblasts expressing α-SMA. The cells were stained with α-SMA and DAPI, and α-SMA-positive sites were measured. As shown in Figure 6, it was suggested that suppressing YAP / TAZ expression using siRNA against each mRNA suppresses adipocyte-myofibroblast conversion. Different siRNA sequence mixtures were used in #1 and #2. The siRNA sequences are shown below. #1 YAP:5′-UGGUACAUCAUCAGGGAUC-3′(Sequence ID 1) TAZ:5′-UAAACAGCUAAGGACGUCA-3′(Sequence ID 2) #2 YAP:5′-ACGUCCAAGAUUUCGGAAC-3′(Sequence ID 3) TAZ:5′-ACGGUGGCAACAUUGGUAC-3′(Sequence ID 4).

[0062] [Example 6] Effect of DIF on adipocyte-myofibroblast conversion Mouse adipocyte progenitor cells (3T3L-1) were stimulated with TGF-β1 (5 ng / ml) for 60 hours to induce conversion to myofibroblasts expressing α-SMA. DIF-1 was administered simultaneously with TGF-β1 at a concentration of 10 μM. Cells were stained for α-SMA and DAPI, and α-SMA-positive sites were measured. As shown in Figure 7, DIF-1 administration was suggested to suppress adipocyte-myofibroblast conversion.

[0063] Inhibiting YAP / TAZ expression in mouse adipocyte progenitor cells suppressed the conversion of adipocytes to myofibroblasts (Figure 6), and treating mouse adipocyte progenitor cells with DIF also suppressed the conversion of adipocytes to myofibroblasts (Figure 7). These findings suggest that DIF-induced YAP / TAZ suppression is the mechanism by which the conversion of adipocytes to myofibroblasts is inhibited.

[0064] The above findings suggest that DIF suppresses skin fibrosis by inhibiting adipocyte-myofibroblast conversion, and also suggests the possibility of promoting subcutaneous adipose tissue reconstruction. These results suggest that DIF-1 inhibits adipocyte-myofibroblast conversion and has preventive or therapeutic effects against diseases caused by skin fibrosis, such as scleroderma, hypertrophic scars, and keloids. [Industrial applicability]

[0065] DIF inhibits the conversion of adipocytes to myofibroblasts, thus potentially preventing or treating skin fibrosis associated with scleroderma, hypertrophic scars, keloids, etc. DIF has excellent transdermal absorption and can be administered topically to the skin as a topical agent.

Claims

1. A preventive and / or therapeutic agent for skin fibrosis containing a differentiation-inducing factor derived from cellular slime mold.

2. Cellular slime mold-derived differentiation-inducing factors are given by formulas (I) to (IV): The agent according to claim 1, which is at least one compound selected from or a pharmaceutically acceptable salt thereof.

3. Cellular slime mold-derived differentiation-inducing factor is given by formula (I): The agent according to claim 1, which is a compound represented by or a pharmaceutically acceptable salt thereof.

4. The agent according to claim 1, wherein the cutaneous fibrotic disease is at least one selected from the group consisting of scleroderma, hypertrophic scarring, and keloids.

5. The agent according to any one of claims 1 to 4, for local administration.

6. The agent according to any one of claims 1 to 4, which is a topical skin preparation.

7. An adipocyte-myofibroblast conversion inhibitor containing a differentiation-inducing factor derived from cellular slime mold.

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