Composition for preventing or treating atopic dermatitis, comprising crenolanib or pharmaceutically acceptable salt thereof

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis, comprising crenolanib or a pharmaceutically acceptable salt thereof. The composition according to the present invention has an effect of alleviating symptoms such as skin inflammation and pruritus of atopic dermatitis by regulating secretion of inflammatory markers (NO, PGE2, TNF-α, IL6), allergic markers (histamine, TNF-α), and inflammatory markers of keratinocytes (RANTES, MDC, TARC). Therefore, the composition according to the present invention can be advantageously used for preventing and treating atopic dermatitis.

Description

Composition for preventing or treating atopic dermatitis comprising crenolanib or a pharmaceutically acceptable salt thereof

[0001] The present invention relates to a composition for preventing or treating atopic dermatitis, and more preferably, to a composition for preventing or treating atopic dermatitis comprising crenolanib or a pharmaceutically acceptable salt thereof.

[0002] Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease, a type of autoimmune disease, that affects the stratum corneum, the skin's protective barrier. Typical symptoms include a red rash, dry skin, inflammation, and itching. Severe cases can lead to significant psychological problems and a reduced quality of life.

[0003] Although the cause of atopic dermatitis remains unclear, it is known that the onset or exacerbation of symptoms in other autoimmune diseases, including atopic dermatitis, may be due to abnormal autophagy associated with ULK2. ULK2, a serine / threonine kinase, is known to regulate autophagy by acting upstream of PIK3C3.

[0004] Corticosteroids, antihistamines, and immunosuppressants are used to treat atopic dermatitis. However, these treatments often have side effects or provide only temporary and incomplete symptom relief. Therefore, the industry needs a treatment that is safe and effective for long-term use.

[0005] Meanwhile, Crenolanib is a drug currently undergoing clinical trials as a treatment for acute myeloid leukemia (AML) and gastrointestinal stromal tumors (GIST). It inhibits the activity of Class III receptor tyrosine kinases (RTKs), such as FLT3 (FMS-like Tyrosine Kinase 3), PDGFR α (Platelet-Derived Growth Factor Receptor), and PDGFR β. These kinases are known to be closely related to the PI3K-AKT-mTOR signaling pathway, a representative inflammation-inducing signal.

[0006] Against this backdrop, the present inventors have confirmed that crenolanib or a composition comprising it has inhibitory effects on immune response and autophagy activity based on its inhibitory activity against the PI3K-AKT-mTOR signal pathway, which is a primary inhibitory target, and the newly identified ULK2, and have also confirmed that it has an effect of improving symptoms such as skin inflammation and itching of atopic dermatitis, thereby completing the present invention. Accordingly, it is expected that the composition according to the present invention can be usefully utilized as a pharmaceutical composition for the prevention or treatment of atopic dermatitis.

[0007] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis comprising crenolanib or a pharmaceutically acceptable salt thereof.

[0008] An object of the present invention is to provide a method for preventing or treating atopic dermatitis, comprising administering a therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0009] An object of the present invention is to provide a use of the present invention's crenolanib or a pharmaceutically acceptable salt thereof for preparing a medicament for preventing or treating atopic dermatitis.

[0010] An object of the present invention is to provide a composition comprising crenolanib or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of atopic dermatitis.

[0011] The present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising crenolanib or a pharmaceutically acceptable salt thereof.

[0012] In the present invention, crenolanib has a structure represented by the following chemical formula 1.

[0013] [Chemical Formula 1]

[0014]

[0015] The above-mentioned crenolanib is an anticancer drug developed to target FLT3 and PDGFRα / β, and is known to have the effect of inhibiting the PI3K-AKT-mTOR signal pathway, a representative inflammation-inducing signal, by inhibiting the activity of RTKs corresponding to Class Ⅲ, such as FLT3, PDGFRα, and PDGFRβ.

[0016] In one embodiment of the present invention, it was confirmed that crenolanib has an inhibitory effect on ULK2. ULK2 is a serine / threonine kinase known to regulate autophagy by acting upstream of PIK3C3. Abnormal autophagy may be associated with the onset or worsening of symptoms of autoimmune diseases, including atopic dermatitis.

[0017] That is, the composition according to the present invention has the effect of inhibiting the PI3K-AKT-mTOR signaling pathway, which is a signal that induces inflammation. Furthermore, the composition according to the present invention has the effect of inhibiting the secretion of ULK2, an autophagy regulatory protein. This indicates that the composition according to the present invention has an inhibitory effect on immune response and autophagy activity, and may exhibit a therapeutic effect on autoimmune diseases, including atopic dermatitis.

[0018] In the present invention, "atopic dermatitis" is a type of autoimmune disease. It is a chronic, relapsing inflammatory skin disease that causes abnormalities in the stratum corneum, the skin's protective barrier. Representative symptoms include a red rash, dry skin, skin inflammation, and itching.

[0019] Specifically, symptoms include dry, eczematous skin and papules, and lesion samples from atopic patients show epidermal hyperplasia, epidermal proliferation, and accumulation of lymphocytes and mast cells. Patients with atopic dermatitis may experience severe pruritus, which in turn induces inflammation of the skin lesions and further worsens clinical symptoms.

[0020] The term "pruritus" in the present invention refers to a disease that includes itching caused by a decrease in antibacterial activity and deterioration of barrier function due to a decrease in the lipid content of the stratum corneum of the skin, or itching caused by external stimuli such as temperature changes, chemical substances, and electrical stimulation. The scope of diseases according to the present invention also includes itching.

[0021] The composition according to the present invention is effective in improving symptoms of atopic dermatitis, such as skin inflammation and itching.

[0022] According to one embodiment of the present invention, the composition of the present invention significantly reduced skin inflammation caused by atopic dermatitis as measured by visual inspection. In other words, the composition of the present invention is effective in improving skin inflammation caused by atopic dermatitis.

[0023] Additionally, the composition according to the present invention significantly reduced scratching behavior caused by atopic dermatitis. In other words, the composition according to the present invention has the effect of suppressing itching caused by atopic dermatitis.

[0024] In addition, the composition according to the present invention has the effect of inhibiting the secretion of any one or more selected from the group consisting of inflammatory markers NO, PGE2, TNF-α, and IL6. In addition, the composition according to the present invention has the effect of inhibiting the secretion of any one or more selected from the group consisting of allergic markers Histamine and TNF-α. In addition, the composition according to the present invention has the effect of inhibiting the secretion of any one or more selected from the group consisting of inflammatory markers of keratinocytes RANTES, MDC, and TARC. Through this, it can be seen that the composition according to the present invention has anti-inflammatory, anti-allergic, and anti-dermatitis properties, and is effective in the treatment of atopic dermatitis.

[0025] The term "prevention" as used herein refers to any action that suppresses or delays the onset of atopic dermatitis through the administration of a composition. In the present invention, "treatment" refers to any action that improves or beneficially alters the symptoms of the disease through the administration of a composition.

[0026] In the present invention, “pharmaceutically acceptable salt” means a salt commonly used in the pharmaceutical industry, and for example, inorganic ionic salts manufactured with calcium, potassium, sodium, and magnesium, etc.; inorganic acid salts manufactured with hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, iodic acid, perchloric acid, and sulfuric acid, etc.; organic acid salts manufactured with acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid, etc.; sulfonic acid salts manufactured with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid, etc. Amino acid salts made from glycine, arginine, lysine, etc.; and amine salts made from trimethylamine, triethylamine, ammonia, pyridine, picoline, etc.; however, the types of salts meant in the present invention are not limited by these listed salts.

[0027] In addition, the crenolanib of the present invention includes not only pharmaceutically acceptable salts but also all salts, hydrates and solvates that can be prepared by conventional methods.

[0028] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, and may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.

[0029] The pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the pharmaceutical composition of the present invention may include, but is not limited to, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, and other pharmaceutically acceptable additives.

[0030] When the pharmaceutical composition of the present invention is formulated as an oral solid preparation, it includes tablets, pills, powders, granules, capsules, etc., and such solid preparations may include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., and include, but are not limited to, lubricants such as magnesium stearate and talc.

[0031] When the pharmaceutical composition of the present invention is formulated as an oral liquid, it includes a suspension, a solution, an emulsion, a syrup, etc., and includes, but is not limited to, a diluent such as water or liquid paraffin, a wetting agent, a sweetener, a fragrance, a preservative, etc.

[0032] When the pharmaceutical composition of the present invention is formulated for parenteral use, it includes a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and a suppository. Non-aqueous solvents and suspensions include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for a suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, and the like can be used, but are not limited to these.

[0033] The composition may be administered in single or multiple doses in a pharmaceutically effective amount. The term “pharmaceutically effective amount” as used herein means an amount sufficient to prevent or treat a disease at a reasonable benefit-risk ratio applicable to medical prevention or treatment, and the effective dosage level may be determined according to factors including the severity of the disease, the activity of the drug, the patient’s age, weight, health, sex, the patient’s sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field. For example, crenolanib or a pharmaceutically acceptable salt thereof may be administered at 0.0001 to 100 mg / kg per day, and the administration may be administered once a day or in several divided doses.

[0034] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes, including, but not limited to, oral administration, intrathecal, intra-auricular, intraperitoneal, intravenous, intramuscular, subcutaneous, intrauterine, sublingual, or intracerebrovascular injection. In addition, it can be applied directly to the affected area as needed.

[0035] The pharmaceutical composition of the present invention may contain 0.01 to 95 wt%, preferably 1 to 80 wt%, of crenolanib or a pharmaceutically acceptable salt thereof based on the total weight of the composition.

[0036] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, the pharmaceutical composition of the present invention can be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer the amount that achieves maximum efficacy with the minimum amount possible without causing side effects, a determination readily made by those skilled in the art.

[0037] The term "subject" of the present invention includes an animal or human whose symptoms can be improved by administration of a pharmaceutical composition according to the present invention. By administering a therapeutic composition according to the present invention to a subject, atopic dermatitis can be effectively prevented and treated.

[0038] The term "administration" in the present invention refers to introducing a given substance into a human or animal by any suitable method. The therapeutic composition according to the present invention may be administered orally or parenterally via any conventional route, as long as it can reach the target tissue. Furthermore, the therapeutic composition according to the present invention may be administered by any device capable of transporting the active ingredient to target cells.

[0039] Another aspect of the present invention provides a method for preventing or treating atopic dermatitis, comprising administering a therapeutically effective amount of crenolanib or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0040] The term "subject" of the present invention refers to any animal that exhibits or may exhibit atopic dermatitis, and typically may be an animal that can exhibit a beneficial effect from treatment with the present invention's crenolanib or a pharmaceutically acceptable salt thereof, but includes without limitation any subject that has atopic dermatitis or is likely to have such a disease. As described above, by administering the pharmaceutical composition of the present invention to a subject, the atopic dermatitis can be effectively prevented or treated.

[0041] Another aspect of the present invention provides a use of crenolanib or a pharmaceutically acceptable salt thereof of the present invention for preparing a medicament for preventing or treating atopic dermatitis.

[0042] Another aspect of the present invention provides a use of crenolanib or a pharmaceutically acceptable salt thereof for the prevention or treatment of atopic dermatitis.

[0043] Another aspect of the present invention provides a composition comprising crenolanib or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of atopic dermatitis.

[0044] The above-mentioned crenolanib or a pharmaceutically acceptable salt thereof may suppress itching of atopic dermatitis. The above-mentioned crenolanib or a pharmaceutically acceptable salt thereof may a) suppress the PI3K-AKT-mTOR signaling pathway, b) suppress the activity of ULK2, c) suppress the secretion of any one or more selected from the group consisting of NO, PGE2, TNF-α, and IL6, d) suppress the secretion of histamine, TNF-α, or all of them, and / or e) suppress the secretion of any one or more selected from the group consisting of RANTES, MDC, and TARC.

[0045] The present invention provides a food composition for preventing or improving atopic dermatitis, comprising crenolanib or a food-related acceptable salt thereof.

[0046] The term “improvement” of the present invention means any action by which atopic dermatitis is improved or beneficially changed by administration of the composition of the present invention.

[0047] In the food composition of the present invention, the crenolanib and its salt are preferably included in an amount of 0.00001 to 0.01 wt% relative to the food composition. If the amount is less than 0.00001 wt%, the effect is minimal, and if the amount exceeds 0.01 wt%, the increase in effect relative to the amount used is minimal, making it uneconomical.

[0048] Food-grade acceptable salt may be applied in the same manner as pharmaceutically acceptable salt.

[0049] When the food composition of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.

[0050] In the present invention, the food composition includes a health functional food.

[0051] The above “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body as defined in Act No. 6727 on Health Functional Foods, and “functionality” refers to consumption for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.

[0052] The food composition and health functional food of the present invention may include additional ingredients. For example, they may include biotin, folate, pantothenic acid, vitamins A, C, D, E, B1, B2, B6, B12, niacin, etc. In addition, they may include minerals such as chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn), iron (Fe), and calcium (Ca). In addition, they may include amino acids such as cysteine, valine, lysine, and tryptophan. In addition, food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), bactericides (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (pasting agents), film-forming agents, antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), gum bases, antifoaming agents, solvents, and improvers can be added. The above additives can be selected depending on the type of food and used in an appropriate amount.

[0053] The composition according to the present invention has anti-inflammatory, anti-allergic, and anti-dermatitis properties through the regulation of the secretion of inflammatory markers (NO, PGE2, TNF-α, IL6), allergic markers (histamine, TNF-α), and inflammatory markers of keratinocytes (RANTES, MDC, TARC). Furthermore, it is effective in improving symptoms of atopic dermatitis, such as skin inflammation and itching. Therefore, the composition according to the present invention can be usefully used for the prevention and treatment of atopic dermatitis.

[0054] Figure 1 is a graph showing the inhibitory efficacy of crenolanib against ULK2.

[0055] Figure 2 is an image showing the schedule of animal experiments using an atopic dermatitis-induced mouse model.

[0056] Figure 3 is an image visually observing the skin inflammation improvement effect of crenolanib in an atopic dermatitis-induced mouse model.

[0057] Figure 4 is a graph showing the atopic dermatitis score (SCORAD) and pruritus suppression effect of crenolanib in an atopic dermatitis-induced mouse model.

[0058] Figure 5 is a graph showing the TNF-α expression inhibitory effect of crenolanib in an atopic dermatitis-induced mouse model.

[0059] Figure 6 is a graph showing the epidermal improvement effect of crenolanib in an atopic dermatitis-induced mouse model.

[0060] Figure 7 is an image visually observing the spleen transformation inhibitory effect of crenolanib in an atopic dermatitis-induced mouse model.

[0061] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention and the scope of the present invention is not limited to these examples.

[0062] <Example>

[0063] Example 1. Confirmation of the inhibitory effect of crenolanib on ULK2.

[0064] Kinase-tagged T7 phage strains were prepared from E. coli hosts derived from the BL21 strain. E. coli were cultured, infected with T7 phage, and cultured at 32°C with shaking until lysis. After culture, the lysate was centrifuged and the supernatant was separated. Streptavidin-coated magnetic particles were reacted with the separated supernatant for 30 minutes. Afterwards, unbound and non-specifically bound ligands were removed using locking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT). The samples were reacted by adding 1x binding buffer (20% SeaBlock, 0.17x PBS, 0.05% Tween 20, 6 mM DTT), and stock (x111) solutions were prepared. The sample was diluted to a final concentration of 0.9% DMSO in 0.02 mL and washed with wash buffer (1x PBS, 0.05% Tween 20). After resuspending in elution buffer (1x PBS, 0.05% Tween 20, 0.5 μM nonbiotinylated affinity ligand), the reaction was performed at room temperature for 30 minutes and measured by qPCR.

[0065] As a result, as shown in Fig. 1, it was confirmed that crenolanib had excellent inhibitory efficacy against ULK2.

[0066] Example 2. Cell-based efficacy evaluation of crenolanib

[0067] Example 2-1. Confirmation of the anti-inflammatory activity of crenolanib

[0068] Mouse macrophage cell line (RAW 264.7) cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% P&S (Penicillin-Streptomycin). Cultured RAW264.7 cells were seeded at 4 × 10 to determine cytotoxicity. 3 After seeding cells / well (96-well plate), overnight incubation was performed. Crenolanib was treated at various concentrations (from 50 μM) and reacted for 24 hours (final 100 μL / well). 10 μL / well of Cell Counting Kit-8 (CCK-8, Dojindo, Japan) solution was added and reacted for 4 hours. The absorbance was measured at 450 nm. The measured value was compared with the control well to calculate cell viability%. Among them, the highest concentration showing cell viability of 95% or more was selected as the highest non-toxic concentration (Max. Conc.) and anti-inflammatory experiments were performed.

[0069] To confirm the anti-inflammatory activity, RAW 264.7 cells were seeded in 48-well plates and incubated overnight, and then cultured for 24 hours (final 500 μL / well) under the conditions of creolanib treatment and (±)-LPS treatment (1 μg / mL). For the quantification of NO, PGE2, TNF-α, and IL-6, the culture medium was collected and NO was quantified using Griess reagent (Promega), and PGE2, TNF-α, and IL-6 were quantified using each ELISA kit. The inhibition rate of inflammatory mediators and cytokine production induced by LPS treatment was calculated and shown in Table 1.

[0070] As a result, as shown in Table 1 below, the composition of Example 1 was found to inhibit the secretion of NO (31%), PGE2 (23%), TNF-α (39%), and IL6 (51%) by 23% to 51% at the non-toxic maximum concentration of 100 nM. This confirmed that crenolanib has anti-inflammatory activity.

[0071] NoCodeMaximum Non-Atoxic Concentration (Max Conc.)SecondaryNOPGE2TNF-αIL-6IC 50 % inh. at Max Conc.IC 50 % inh. at Max Conc.IC 50 % inh. at Max Conc.IC 50 % inh. at Max Conc.1 Example 1 (Crenolanib) 100 nM - 30.71 - 22.71 - 39.37 62.56 nM 50.69

[0072] Example 2-2. Confirmation of anti-allergic activity using mast cell lines

[0073] Human mast cell line (HMC-1.2) cells were cultured in IMDM (Iscove's Modified Dulbecco's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% P&S (Penicillin-Streptomycin). Cultured HMC-1.2 cells were maintained at a density of 2X10 4 After seeding cells / well (96-well plate), overnight incubation was performed. Crenolanib was treated at various concentrations (from 50 μM) and reacted for 6 hours (final 100 μL / well). 10 μL / well of CCK-8 solution was added and reacted for 4 hours. The absorbance was measured at 450 nm. The measured value was compared with the control well to calculate cell viability %. Among them, the highest concentration showing cell viability of 95% or more was selected as the highest non-toxic concentration (Max. Comc.) and an anti-allergy experiment was conducted.

[0074] To confirm the anti-allergic activity, HMC-1.2 cells were seeded in 48-well plates and incubated overnight. Then, they were cultured for 6 hours (final 500 μL / well) under the conditions of creolanib treatment and PMA and A23187 treatment (20 nM + 1 μM each, PA). To quantify the released mediators, the culture medium was collected and histamine and TNF-α were quantified using each ELISA kit. The inhibition rate of mediator production induced by PA treatment was calculated and shown in Table 2.

[0075] As a result, as shown in Table 2 below, the composition of Example 1 was found to inhibit the secretion of histamine and TNF-α by 48% and 22%, respectively, at the non-toxic maximum concentration of 50 nM. This confirmed that crenolanib has anti-allergic activity.

[0076] NoCodeNon-toxic Highest Concentration (Max Conc.)Secondary HistamineTNF-αIC 50 % inh. at Max Conc.IC 50 % inh. at Max Conc.1 Example 1 (Crenolanib) 50 nM - 47.56 - 21.91

[0077] Example 2-3. Confirmation of anti-dermatitis activity using keratinocyte cell lines

[0078] Human keratinocyte cell line (HaCaT) cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (Fetal Bovine Serum) and 1% P&S (Penicillin-Streptomycin). Cultured HaCaT cells were maintained at a density of 3X10 3After seeding cells / well (96-well plate), overnight incubation was performed. Crenolanib was treated at various concentrations (from 50 μM) and reacted for 24 hours (final 100 μL / well). 10 μL / well of CCK-8 solution was added and reacted for 4 hours. The absorbance was measured at 450 nm. The measured value was compared with the control well to calculate cell viability%. Among them, the highest concentration showing cell viability of 95% or more was selected as the highest non-toxic concentration (Max. Conc.) and anti-dermatitis experiment was conducted.

[0079] To confirm the anti-dermatitis efficacy, HaCaT cells were seeded in 6-well plates and incubated overnight. Then, they were cultured for 24 hours (final 1000 μL / well) under conditions of creolanib treatment and TNF-α and IFN-γ treatment (each 10 ng / mL, TI). To quantify the released chemokines, the culture medium was collected and RANTES, TARC, and MDC were quantified using each ELISA kit. The inhibition rate of chemokine production induced by TI treatment was calculated and is shown in Table 3.

[0080] As a result, as shown in Table 3 below, crenolanib was found to inhibit the secretion of RANTES, MDC, and TARC, which are inflammatory markers of keratinocytes, by 52%, 46%, and 53%, respectively, at the non-toxic maximum concentration of 2 μM. This confirmed that crenolanib possesses anti-dermatitis activity.

[0081] NoCodeMaximum Non-Toxic Concentration (Max Conc.)2ndRANTESMDCTARCIC 50 % inh. at Max Conc.IC 50 % inh. at Max Conc.IC 50 % inh. at Max Conc.1Example 1 (Crenolanib)2 μM1.90 μM52.04-46.101.64 μM52.85

[0082] Experimental Example 1. Establishment of a mouse model for atopic dermatitis.

[0083] To conduct animal experiments using crenolanib, a mouse model of atopic dermatitis induced by 2,4-dinitrochlorobenzene (DNCB) was established.

[0084] The experimental groups for animal experiments were organized as follows.

[0085]

[0086] The experimental groups for the above animal experiments were prepared as follows. First, a 1% DNCB solution was prepared using DNCB (2,4-dinitrochlorobenzene), an atopic dermatitis-inducing substance, and AOO solution (acetone + olive oil, 3:1) as an excipient. The prepared atopic dermatitis-inducing substance was periodically applied to both ears of the animals using a pipette at 0.025 mL / head and to the entire back subcutaneously at 0.15 mL / head, thereby inducing atopic dermatitis. Thereafter, 100 μL of the test substance was appropriately applied to the affected area once a day for each group, and changes were observed, as shown in Fig. 2.

[0087] For the crenolanib treatment group, a solution prepared with a final concentration of crenolanib of 1% (10 mg / ml) in AOO (acetone + olive oil 3:1) solution was used as the test substance, and for the Elidel treatment group, a 1% Elidel (Pimecrolimus) (leo-pharma, Denmark) solution was used.

[0088] Example 3. Confirmation of the skin inflammation-improving effect of crenolanib in an atopic dermatitis-induced mouse model.

[0089] To confirm the efficacy of atopic dermatitis treatment, visual evaluation of atopic symptoms and measurement of ear thickness were performed during animal experiments, as shown in Fig. 2, and the measurements were performed a total of 9 times from the start of administration to the day of atopic dermatitis induction. Ear thickness measurements were made using a digital thickness gauge (547-301, Mitutoyo Corp., Japan) on both ears of each animal, and the average thickness of both ears was calculated.

[0090] The images observing the inflammatory status of each experimental group are shown in Figure 3. As a result of comparing the inflammatory status of each group, dermatitis progressed significantly in the negative control group treated with DNCB compared to the normal control group, and skin inflammation was significantly reduced in the creolanib-treated group and the Elidel-treated group. That is, skin inflammation was observed to have improved in the creolanib-treated group and the Elidel-treated group compared to the negative control group based on visual inspection, confirming that creolanib has therapeutic efficacy in treating atopic dermatitis caused by DNCB.

[0091] Example 4. Confirmation of the dermatitis score (SCORAD) and pruritus suppression effect of crenolanib in an atopic dermatitis-induced mouse model.

[0092] To determine the efficacy of atopic dermatitis treatment, body weight and the macroscopic dermatitis score (SCORAD, erythema, edema, and hematoma; itching and dry skin; and erosion) were assessed. Furthermore, to confirm the itch-reducing effect, after a 10-minute acclimation training session the day before autopsy, behavior was filmed for 10 minutes and scratching behavior was assessed.

[0093] The dermatitis score (SCORAD) and scratching behavior of each experimental group are summarized and shown in Figure 4. As a result, the dermatitis score and scratching behavior increased in the negative control group treated with DNCB compared to the normal control group, and the dermatitis score and scratching behavior significantly decreased in the creolanib-treated group and the Elidel-treated group compared to the negative control group. In other words, the creolanib-treated group and the Elidel-treated group were observed to have improved dermatitis and itching compared to the negative control group, confirming that creolanib has therapeutic efficacy in treating atopic dermatitis caused by DNCB.

[0094] Example 5. Confirmation of the TNF-α expression inhibitory effect of crenolanib in an atopic dermatitis-induced mouse model.

[0095] To confirm the effect of suppressing the expression of TNF-α, a marker of atopic dermatitis, total RNA was extracted from mouse skin, and total RNA was extracted using TRIzolTM Reagent (15596026, Invitrogen TM After lysis using a 10 μl oligonucleotide buffer (Sigma, Carlsbad, CA, USA), the total RNA was extracted according to the protocol of the AccuPrep® Universal RNA Extraction Kit (K-3613, Bioneer, Daejeon, Korea). In addition, genomic DNA in total RNA was removed using the RNase-Free-DNase Set (79256, Qiagen, Hilden, Germany) according to the protocol.

[0096] Total RNA QC was performed using Nanodrop 2000 (Thermo science, Waltham, MA) to measure concentration, OD260 / 280, and OD260 / 230 values, and RNA integrity was confirmed using 5200 Fragment Analyzer (Agilent, California, USA).

[0097] cDNA synthesis was performed using 1 μg of total RNA according to the protocol of AccuPower® RocketScript Cycle RT PreMix (K-2201, Bioneer, Daejeon, Korea). Synthesis conditions were as follows: 12 cycles of (37°C for 30 s, 48°C for 4 min, and 55°C for 30 s) and 95°C for 5 min using the AllInOneCycler™ PCR system (Bioneer, Daejeon, Korea). Quantitative PCR (qPCR) was performed using the Exicycler 384™ Real-time Quantitative Thermal Block (A-2061, Bioneer, Daejeon, Korea) with 40 cycles of (95°C for 5 s, 58°C for 25 s, and 72°C for 30 s). The composition used for qPCR was 7.5 μl of AccuPower® 2X GreenStar Master Mix (K-6253, Bioneer, Daejeon, Korea), 5 μl of deionized sterile water (Bioneer), 1.5 μl each of sense and antisense primers (final concentration of 0.3 μM each), and 1 μl of cDNA, for a total volume of 15 μl.

[0098] qPCR data were normalized to the Gapdh gene expression level and relative quantitative analysis was performed using the △△CT value method.

[0099] The expression of TNF-α in each experimental group is summarized and shown in Figure 5. In the negative control group treated with DNCB, TNF-α expression increased compared to the normal control group, and the TNF-α expression levels in the creolanib-treated group and Elidel-treated group were significantly reduced compared to the negative control group. In other words, the creolanib-treated group and Elidel-treated group were observed to have a decreased expression of TNF-α, an inflammatory and allergic marker, compared to the negative control group, confirming that creolanib has a therapeutic effect on atopic dermatitis caused by DNCB.

[0100] Example 6. Confirmation of the epidermal improvement effect of crenolanib in an atopic dermatitis-induced mouse model.

[0101] The improvement of atopic dermatitis epidermal skin was confirmed through tissue staining. Skin tissue fixed in 10% neutral buffered formalin was trimmed to a certain thickness, then processed as usual, embedded in paraffin, and cut into 4-μm-thick sections, followed by H&E staining. All remaining tissue was stored in 10% neutral buffered formalin. Detailed procedures followed the standard pathological laboratory procedure of Biotoxtech Co., Ltd.

[0102] Histopathological examinations were performed on the skin of all provided individuals. The degree of infiltration of each inflammatory cell was examined on H&E-stained tissue slides, and the thickness of the epidermis and dermis was measured using the ImageJ (National Institute of Health, USA) program. Furthermore, the degree of infiltration of each type of inflammatory cell (eosinophils, mast cells, monocytes, and neutrophils) infiltrating the skin tissue was evaluated individually. Among these inflammatory cells, eosinophils, mast cells, and monocytes, which are considered to be directly related to the pathogenesis of atopic dermatitis, were comprehensively evaluated by adding their individual scores. Conversely, neutrophils, which can be secondary to epidermal erosions and ulcers caused by scratching behavior due to pruritus, were classified and evaluated separately. The scoring system based on toxicological pathology interpretation standards was applied to evaluate the degree of inflammatory cell infiltration.

[0103] As a result, the epidermal thickness and inflammatory cell infiltration level of each experimental group were summarized and shown in Fig. 6. In the negative control group treated with DNCG, the epidermal thickness and inflammatory cell infiltration level increased compared to the normal control group, and the epidermal thickness and inflammatory cell infiltration level of the crenolanib-treated group and the Elidel-treated group significantly decreased compared to the negative control group. In other words, the epidermal condition was observed to have improved in the crenolanib-treated group and the Elidel-treated group compared to the negative control group, and this confirmed that crenolanib has a therapeutic effect on atopic dermatitis caused by DNCB.

[0104] Example 7. Confirmation of the inhibitory effect of crenolanib on spleen transformation in a mouse model of atopic dermatitis.

[0105] After the above animal experiment was completed, the spleen was removed from the mouse when it was sacrificed to determine whether the drug was toxic, and the degree of deformation, such as size and weight, was measured. The image is shown in Figure 7.

[0106]

[0107] Comparing the spleens of each group, the negative control groups G2, G5, and G6 were larger and longer and deformed compared to G1, while the experimental groups G3, G4, G7, and G8, which were treated with creolanib or Elidel, showed relatively less deformity. In other words, it was observed that the creolanib or Elidel treatment groups did not induce splenomegaly compared to the negative control group, confirming that creolanib has the effect of suppressing spleen deformation caused by DNCB.

Claims

1. A pharmaceutical composition for preventing or treating atopic dermatitis comprising crenolanib or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition according to claim 1, wherein the composition inhibits the PI3K-AKT-mTOR signal pathway.

3. A pharmaceutical composition according to claim 1, wherein the composition inhibits the activity of ULK2.

4. A pharmaceutical composition according to claim 1, wherein the composition inhibits the secretion of at least one agent selected from the group consisting of NO, PGE2, TNF-α, and IL6.

5. A pharmaceutical composition according to claim 1, wherein the composition inhibits the secretion of at least one agent selected from the group consisting of Histamine and TNF-α.

6. A pharmaceutical composition according to claim 1, wherein the composition inhibits secretion of at least one compound selected from the group consisting of RANTES, MDC, and TARC.

7. A pharmaceutical composition according to claim 1, wherein the composition suppresses itching of atopic dermatitis.

8. Use of creolanib or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of atopic dermatitis.

9. The use of creolanib or a pharmaceutically acceptable salt thereof in clause 8, which suppresses itching of atopic dermatitis.

10. In paragraph 8, the crenolanib or a pharmaceutically acceptable salt thereof a) Inhibiting the PI3K-AKT-mTOR signaling pathway, or b) inhibiting the activity of ULK2, or c) inhibiting the secretion of any one or more selected from the group consisting of NO, PGE2, TNF-α and IL6; d) inhibiting the secretion of histamine, TNF-α or both, and / or e) Use for inhibiting the secretion of any one or more selected from the group consisting of RANTES, MDC and TARC.

11. A method for preventing or treating atopic dermatitis, comprising administering a therapeutically effective amount of creolanib or a pharmaceutically acceptable salt thereof to a subject in need thereof.

12. A method according to claim 11, wherein crenolanib or a pharmaceutically acceptable salt thereof suppresses itching of atopic dermatitis.

13. In clause 11, the crenolanib or a pharmaceutically acceptable salt thereof a) Inhibiting the PI3K-AKT-mTOR signaling pathway, or b) inhibiting the activity of ULK2, or c) inhibiting the secretion of any one or more selected from the group consisting of NO, PGE2, TNF-α and IL6; d) inhibiting the secretion of histamine, TNF-α or both, and / or e) A method for inhibiting the secretion of any one or more selected from the group consisting of RANTES, MDC and TARC.

14. Use of creolanib or a pharmaceutically acceptable salt thereof for the prevention or treatment of atopic dermatitis.

15. A composition comprising crenolanib or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of atopic dermatitis.

16. A food composition for preventing or improving atopic dermatitis, comprising creolanib or a food-chemically acceptable salt thereof.

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