Composition for preventing or treating atopic dermatitis

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating atopic dermatitis, the composition comprising a compound represented by chemical formula 1 or chemical formula 2 or a pharmaceutically acceptable salt thereof. That is, the present invention relates to pharmaceutical use of a compound represented by chemical formula 1 or chemical formula 2 or a pharmaceutically acceptable salt thereof for preventing or treating atopic dermatitis.

Description

Composition for the prevention or treatment of atopic dermatitis

[0001] The present application claims priority based on Korean Patent Application No. 10-2025-0051015 filed on April 18, 2025, and all contents disclosed in the specification and drawings of said application are incorporated into the present application.

[0002] The present invention relates to a pharmaceutical composition for the prevention or treatment of atopic dermatitis. That is, the present invention relates to the medicinal use of a specific compound (active ingredient) for the treatment or prevention of atopic dermatitis.

[0003] Atopic dermatitis (AD) is a chronic, recurring skin disease that is treated differently from general inflammatory diseases. Since the etiology of atopic dermatitis has not yet been clearly identified, it is considered to be a disease caused by the complex interaction of various factors, such as abnormalities in the immune system, damage to the skin barrier function, and genetic or environmental factors.

[0004] The immunological characteristic of atopic dermatitis is the selective activation of Th2 cells and the subsequent overexpression of inflammatory cytokines such as IL-4, IL-13, and TSLP. The action of these cytokines leads to increased production of IgE antibodies, promotion of eosinophil and mast cell infiltration, and induction of chronic inflammation in skin tissues, which can act as a major mechanism causing the disease to become chronic and recur repeatedly.

[0005] Atopic dermatitis occurs across all age groups but is particularly common in infancy and childhood, and its prevalence has recently been increasing in adults as well. Atopic dermatitis causes various clinical symptoms, such as erythema, edema, erosion, and crust formation, along with severe itching; it can lead to a significant decline in quality of life due to extreme itching, worsening of symptoms at night, and repeated relapses and exacerbations.

[0006] While corticosteroids, antihistamines, and immunosuppressants can be used as treatments for atopic dermatitis, existing therapies have limitations, such as side effects, temporary therapeutic effects, or merely symptom relief. Furthermore, as previously explained, the factors and mechanisms causing atopic dermatitis are diverse and complex; therefore, to adequately control the pathological state of atopic dermatitis involving intricate immune pathways, it is necessary to develop new treatments capable of controlling the disease through pathways different from existing therapies, and there is a demand for alternative treatment strategies that can be selected as an additional option.

[0007] [Prior Art Literature]

[0008] [Patent Literature]

[0009] (Patent Document 1) Korean Published Patent Application No. 10-2017-0087813

[0010] (Patent Document 2) Korean Registered Patent Publication No. 10-2541577

[0011] Although atopic dermatitis is a disease that severely impairs patients' quality of life, developing treatments for it is challenging because it is not caused by a single mechanism. As a complex immune disease involving the interaction of various pathways—such as structural defects in the skin barrier, signal imbalances between immune cells, changes in the skin microbiome, and sensory hypersensitivity of the nervous system—the immune pathways involved vary depending on the timing of disease onset and phenotype, with multiple different immune axes being simultaneously activated during the early and chronic stages. Consequently, various immunomodulatory candidate substances, such as IL-33 antagonists and TSLP inhibitors, have been attempted in clinical trials; however, there are numerous cases where these trials were discontinued due to unsatisfactory efficacy or safety concerns.

[0012] Accordingly, the present invention aims to provide a novel use of a compound that can be used as an active ingredient in a new treatment for atopic dermatitis, based on results confirmed through various experiments that the compound has excellent improvement and therapeutic effects on atopic dermatitis, and a pharmaceutical composition for the prevention or treatment of atopic dermatitis containing said compound.

[0013] To achieve the above objective, one aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a compound represented by the following Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof.

[0014] [Chemical Formula 1]

[0015]

[0016] [Chemical Formula 2]

[0017]

[0018] The inventors of the present invention have identified novel uses and effects of the compound of the present invention by proving through various experiments that the compound of the present invention is effective in treating atopic dermatitis, and have revealed that the compound of the present invention is suitable for development as a new treatment for atopic dermatitis by verifying its therapeutic effect on atopic dermatitis from various angles through animal experiments.

[0019] It has been confirmed through experiments that the compound of the present invention and the composition containing it can reduce the expression of inflammatory cytokines and suppress IgE antibodies to regulate excessive immune responses and regulate the balance of Th cells and Treg cells, thereby having the effect of significantly alleviating and improving clinical symptoms of atopic dermatitis.

[0020] Specifically, the compound of the present invention exhibited a therapeutic effect on atopic dermatitis in a dose-dependent manner. Specifically, it can inhibit the increase in skin thickness in skin tissues induced by atopic dermatitis and reduce tissue infiltration of mast cells and eosinophils, and can improve atopic dermatitis through various mechanisms such as inhibition of Th2 cells, restoration of the ratio of Treg cells, inhibition of the expression of various inflammatory cytokines, and reduction of total IgE and HDM-specific IgE in the blood.

[0021] In particular, the compound of the present invention exhibits a therapeutic effect similar to that of baricitinib, which is known as a standard treatment for atopic dermatitis. Furthermore, in animal experiments on mice, the compound of the present invention did not affect the survival of mice and did not cause changes in mouse body weight even after 28 days of administration, thus confirming its safety within the administered dosage range. Additionally, since the compound of the present invention exhibits high selectivity for the target, binding to and action on other targets are inhibited, resulting in a low likelihood of side effects and excellent safety.

[0022] Accordingly, the compound of the present invention and the composition containing it have characteristics suitable for use as a new therapeutic agent for treating atopic dermatitis.

[0023] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0024] Figure 1 shows a comparison of back skin photographs by administration group of HDM-iAD mice that induced atopic dermatitis through hair removal and application of HDM AD ointment, showing the photographs on Day 0 and Day 28, the end day of the experiment.

[0025] Figure 2 is a graph showing the changes in the clinical indicator evaluation score (Dermatitis score) of the dorsal skin of HDM-iAD mice according to the administration groups of Comparative Examples 1 to 3 and Examples 1 to 3 as the experiment progressed.

[0026] Figure 3a is a figure showing dorsal skin tissue isolated from HDM-iAD mice by administration group of Comparative Examples 1 to 3 and Examples 1 to 3, stained with H&E (Hematoxylin and Eosin) and viewed at 200x magnification.

[0027] FIG. 3b is a graph analyzing the correlation between dermatitis scores and epidermal thickness measurements by administration group for Comparative Examples 1 to 3 and Examples 1 to 3 (top of FIG. 3b) and a graph analyzing the correlation for each individual (bottom of FIG. 3b).

[0028] Figure 4a is a figure showing dorsal skin tissue isolated from HDM-iAD mice by administration group of Comparative Examples 1 to 3 and Examples 1 to 3, stained with TB (Toluidine Blue) and viewed at 200x magnification.

[0029] FIG. 4b is a graph analyzing the correlation between dermatitis scores and mast cell count measurements by administration group for Comparative Examples 1 to 3 and Examples 1 to 3 (top of FIG. 4b) and a graph analyzing the correlation for each individual (bottom of FIG. 4b).

[0030] Figure 5a is a figure showing dorsal skin tissue isolated from HDM-iAD mice by administration group of Comparative Examples 1 to 3 and Examples 1 to 3, stained with CR (Congo Red) and viewed at 200x magnification.

[0031] FIG. 5b is a graph analyzing the correlation between dermatitis scores and eosinophil count measurements by administration group for Comparative Examples 1 to 3 and Examples 1 to 3 (top of FIG. 5b) and a graph analyzing the correlation for each individual (bottom of FIG. 5b).

[0032] Figure 6a is a micrograph showing the results of immunofluorescence staining for GATA3 and CD3 on dorsal skin tissue isolated from HDM-iAD mice according to the administration groups of Comparative Examples 1 to 3, and Figure 6b is a micrograph showing the results of immunofluorescence staining for GATA3 and CD3 on dorsal skin tissue isolated from HDM-iAD mice according to the administration groups of Examples 1 to 3.

[0033] FIGS. 7a to 7c show the dermatitis scores and CD3 by administration group of Comparative Examples 1 to 3 and Examples 1 to 3, respectively. + Cell number, CD3 + GATA3 + Cell number and CD3 + GATA3 + / CD3 + These are graphs analyzing the correlation between cell ratios by administration group (top of each figure) and graphs analyzing the correlation by individual (bottom of each figure).

[0034] Figure 8a is a micrograph showing the results of immunofluorescence staining for FOXP3 and CD3 on dorsal skin tissue isolated from HDM-iAD mice according to the administration groups of Comparative Examples 1 to 3, and Figure 8b is a micrograph showing the results of immunofluorescence staining for FOXP3 and CD3 on dorsal skin tissue isolated from HDM-iAD mice according to the administration groups of Examples 1 to 3.

[0035] FIGS. 9a to 9c show the dermatitis scores and CD3 by administration group of Comparative Examples 1 to 3 and Examples 1 to 3, respectively. + Cell number, CD3 + FoxP3 + Cell number and CD3 + FoxP3 + / CD3 + These are graphs analyzing the correlation between cell ratios by administration group (top of each figure) and graphs analyzing the correlation by individual (bottom of each figure).

[0036] The present invention will be described in detail below.

[0037]

[0038] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a compound represented by the following formula 1 or formula 2 or a pharmaceutically acceptable salt thereof.

[0039] [Chemical Formula 1]

[0040]

[0041] [Chemical Formula 2]

[0042]

[0043] In this specification, the term "prevention" refers to any act of administering or applying a composition to a subject to suppress atopic dermatitis or delay its occurrence, and the composition of the present invention may be used for the purpose of preparing for atopic dermatitis in advance.

[0044] In this specification, the term "treatment" refers to any act of administering or applying a composition to a subject to improve the symptoms of atopic dermatitis that has already developed or is present, or to produce a beneficial effect.

[0045] In this specification, the term "improvement" refers to any act including the improvement, alleviation, or delay of some or all symptoms of atopic dermatitis, and may be used interchangeably with prevention or treatment.

[0046] The compound represented by Chemical Formula 1 above can be converted into a metabolite of the same form as the compound represented by Chemical Formula 2 in the body upon administration, and can exhibit activity in the form of the compound represented by Chemical Formula 2 in the body. In the present invention, the compound represented by Chemical Formula 1 and the compound represented by Chemical Formula 2 can be understood to exhibit equivalent effects or activities.

[0047] Since the compound of the present invention has excellent effects in preventing, treating, or improving atopic dermatitis, it is suitable for use as a pharmaceutical composition for preventing or treating atopic dermatitis.

[0048] Various factors, such as abnormalities in the immune system, damage to the skin barrier function, and genetic or environmental factors, have been proposed as causes of atopic dermatitis; however, the etiology has not yet been clearly identified, and the condition may develop due to complex interactions among these factors. Due to the nature of these diverse causes and the intertwining of various complex immune pathways, there are difficulties in developing strategies and drugs to treat atopic dermatitis. Just because a substance has activity that suppresses inflammatory responses or modulates immune responses does not mean that all of them can significantly improve or treat atopic dermatitis. This can be confirmed through cases of failure in the development and clinical trials of various candidate therapeutic substances for atopic dermatitis. Furthermore, therapeutic efficacy for atopic dermatitis may differ even among substances exhibiting identical or similar activities, or two substances with opposite activities may exhibit therapeutic effects.

[0049] In a situation where it is difficult to easily predict whether a substance can exhibit therapeutic activity against atopic dermatitis, it has been proven through animal experiments that the compound according to the present invention exhibits clear improvement and therapeutic effects on atopic dermatitis.

[0050] According to specific experimental examples of the present invention, when the compounds of the present invention were administered at various dosages to a mouse model in which atopic dermatitis was induced by the application of a house dust mite (HDM) ointment, significant improvement and therapeutic effects on atopic dermatitis were observed in terms of clinical indicators. Furthermore, in various experiments including specific histopathological analysis, confirmation of inhibition of the production of inflammation-related cytokines or IgE antibodies, and analysis of the balance of Th cells and Treg cells, therapeutic and improvement effects were observed in a dose-dependent manner. In particular, it was confirmed that the therapeutic effect was equivalent to or significantly superior to that of baricitinib, which is known as the standard treatment for atopic dermatitis.

[0051] As such, the compound of the present invention exhibits excellent preventive, therapeutic, or corrective effects for atopic dermatitis; however, while the specific pharmacological mechanism or side effects associated with the activity of the compound of the present invention have not been fully elucidated, the S1P receptor is one of the various possible receptors on which the compound of the present invention is expected to act. There are several subtypes of the S1P receptor, and the S1P receptor subtype on which a drug acts differs, and the resulting outcomes may vary. For example, among the receptors, S1PR5 is primarily expressed in oligodendrocytes of the CNS and is associated with myelin formation; therefore, there is a concern that side effects related to the central nervous system or neurodegeneration may occur due to the unintended action of a drug on S1PR5.

[0052] The inventors of the present invention sought to find a substance that exhibits excellent therapeutic activity against atopic dermatitis while possessing high selectivity and a low likelihood of side effects. The compound of the present invention exhibits high selectivity for S1PR1 and S1PR4 among S1P receptors, whereas it shows almost no selectivity for S1PR5. In experiments evaluated with the compound represented by Chemical Formula 2, the EC for S1PR1 50 is approximately 4.3 nM, EC for S1PR4 50 While is approximately 8.1 nM, the EC for S1PR5 50 It was found that the compound of the present invention has very high selectivity for S1PR1 and S1PR4, with an amount greater than 10 μM. Consequently, the compound of the present invention is a highly suitable active substance in terms of safety, as there is a low risk of it acting in the body other than for the intended effect.

[0053] The above compound may be administered to humans at a dose of 0.6 mg / kg / day to 6.0 mg / kg / day, specifically, at a dose of 0.6 mg / kg / day to 5.5 mg / kg / day, 0.65 mg / kg / day to 5.0 mg / kg / day, 0.70 mg / kg / day to 4.5 mg / kg / day, 0.75 mg / kg / day to 4.0 mg / kg / day, or 0.80 mg / kg / day to 3.5 mg / kg / day; however, the dosage range exhibiting a therapeutic effect may be wider depending on the characteristics and condition of the subject. Furthermore, since the therapeutic effect in animal experiments using the compound of the present invention was found to be dose-dependent, there is a possibility that a larger amount than the above numerical range may be administered.

[0054] When administered to animals other than humans, the above compound may be administered at a dose of 4.0 mg / kg / day to 100.0 mg / kg / day; specifically, the dose may be 5.0 mg / kg / day to 90.0 mg / kg / day, 6.0 mg / kg / day to 80.0 mg / kg / day, 7.0 mg / kg / day to 70.0 mg / kg / day, 8.0 mg / kg / day to 60.0 mg / kg / day, 9.0 mg / kg / day to 50.0 mg / kg / day, or 10.0 mg / kg / day to 40.0 mg / kg / day. However, since this is based on the dosage confirmed through animal experiments using mice, the dosage range exhibiting a therapeutic effect may be wider depending on the type of animal being applied. Furthermore, since the therapeutic effect in animal experiments using the compound of the present invention was found to be dose-dependent, there is a possibility that a larger amount than the above numerical range may be administered.

[0055] The above compound may be administered once to three times a day, specifically twice a day, but the frequency of administration may vary depending on the subject, the subject's condition, body weight, severity of the disease, etc.

[0056] The above pharmaceutical composition may be administered orally or parenterally, and specifically, may be administered orally.

[0057] The compound of the present invention may have at least one activity selected from the group consisting of inhibition of the Th2 immune pathway, increase in the proportion of Treg cells, inhibition of mast cell and eosinophil infiltration, inhibition of inflammatory cytokines, inhibition of IgE antibody production, and inhibition of action on S1PR5. However, the specific mechanism by which the compound of the present invention exerts an effect on atopic dermatitis has not been fully elucidated.

[0058] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or additive according to a method that can be easily carried out by a person skilled in the art to which the present invention belongs.

[0059] The above pharmaceutical composition may be prepared in any form of formulation commonly used in the technical field to which the present invention belongs. For example, it may be formulated into an oral or parenteral administration formulation, and the oral administration formulation may be a granule, powder, liquid, tablet, capsule, syrup, suspension, emulsion, aerosol, gel, etc., and the parenteral administration formulation may be an injection, suppository, topical preparation, etc., but is not limited thereto.

[0060] Liquid formulations may include solutions, syrups, and suspensions, and these liquid compositions may be contained within soft or hard capsules. These formulations may include pharmaceutically acceptable carriers, e.g., water, ethanol, polyethylene glycol, cellulose, or oil. The formulations may also include one or more emulsifiers and / or suspending agents.

[0061] In a tablet formulation, the amount of the active ingredient drug may be present in an amount of about 0.05% by weight to about 95% by weight relative to the total weight of the tablet, more generally in an amount of about 2% by weight to about 50% by weight of the formulation. The tablet may additionally include additives such as excipients, disintegrants, lubricants, binders, and solubilizers as needed. For example, the tablet may contain a disintegrant comprising about 0.5% by weight to about 35% by weight, more generally in an amount of about 2% by weight to about 25% by weight of the formulation. Examples of disintegrants may include, but are not limited to, lactose, starch, sodium starch glycolate, crospovidone, croscarmellose sodium, maltodextrin, or mixtures thereof. Suitable lubricants included for manufacturing tablets may be present in an amount of about 0.1% by weight to about 5% by weight, and talc, silicon dioxide, stearic acid, calcium, zinc or magnesium stearate, sodium stearyl fumarate, etc. may be used as lubricants, but the present invention is not limited to these types of additives. Gelatin, polyethylene glycol, sugar, gum, starch, polyvinylpyrrolidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. may be used as binders for manufacturing tablets, and mannitol, xylitol, lactose, dextrose, sucrose, sorbitol, starch, microcrystalline cellulose, etc. may be used as diluents for manufacturing tablets, but the present invention is not limited to these types of additives.

[0062] A solubilizing agent that may optionally be included in the tablet may be used in an amount of about 0.1% by weight to about 3% by weight relative to the total weight of the tablet, and, for example, polysorbate, sodium lauryl sulfate, sodium dodecyl sulfate, propylene carbonate, diethylene glycol monoethyl ether, dimethyl isosorbide, polyoxyethylene glycolated natural or hydrogenated castor oil, HCOR TM (Nikkol), oleyl ester, Gelucire TM ), Caprylic / Caprylic acid mono / diglyceride, Sorbitan fatty acid ester, Solutol HS TM The above may be used in the pharmaceutical composition according to the present invention, but the present invention is not limited to specific types of such solubilizing agents.

[0063] In this specification, the term “pharmaceutically acceptable salt” means a salt of an active compound prepared with an acid or a base, depending on a specific substituent based on the compound described herein. If the compound according to the invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a neutral compound with a sufficient amount of the desired base in a solvent-free or suitable inert solvent.

[0064] Acceptable salts include, for example, alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; ammonium salts; aliphatic amine salts, e.g., trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine or procaine salts; aralkyl amine salts, e.g., N,N-dibenzylethylenediamine salts; heterocyclic aromatic amine salts, e.g., pyridine salts, picolino salts, quinoline salts or isoquinoline salts; quaternary ammonium salts, e.g., tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts, e.g., arginine salts or lysine salts. Acid salts are, for example, inorganic acid salts, for example, hydrochloride, sulfate salt, nitrate salt, phosphate salt, carbonate salt, hydrogen carbonate or perchlorate; organic acid salts, for example, hemifumarate, acetate, propionate, lactate, malate, fumarate, tartaric acid salt, malate, citrate salt, ascorbate, formic acid; sulfonates, for example, methanesulfonate, isothionate, benzenesulfonate or p-toluenesulfonate; and acidic amino acid salts, for example, aspartate or glutamate. Solvents of the compounds of the present invention include, but are not limited to, alcoholates and hydrates.

[0065] In a preferred embodiment of the present invention, the pharmaceutically acceptable salt according to the present invention is a hemifumarate salt.

[0066] Another aspect of the present invention provides a pharmaceutical use of the compound or a pharmaceutically acceptable salt thereof for the prevention, treatment, or improvement of atopic dermatitis.

[0067] Another aspect of the present invention provides a method for the prevention or treatment of atopic dermatitis comprising administering a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof to a subject requiring treatment, improvement, or prevention of atopic dermatitis.

[0068] Another aspect of the present invention provides the compound or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of atopic dermatitis.

[0069] Another aspect of the present invention provides the use of said compound or a pharmaceutically acceptable salt thereof for the manufacture of a drug for the prevention or treatment of atopic dermatitis.

[0070]

[0071] The present invention will be explained in detail below through examples and experimental examples.

[0072] However, the following examples and experimental examples are intended to specifically illustrate the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

[0073] The compound represented by Chemical Formula 1 (2-amino-2-(2-(1-decyl-1H-1,2,3-triazole-4-yl)ethyl)propane-1,3-diol) can be prepared through the method described in Korean Published Patent Application No. 10-2017-0087813.

[0074] In addition, the hemifumarate salt of the above compound was used in the following experiments and evaluations, and was prepared using the method described in Korean Published Patent Application No. 10-2023-0065185.

[0075]

[0076] [Example]

[0077] PLM-2401 formulation for oral administration

[0078] A formulation for oral administration was prepared using the compound of the present invention (hereinafter also referred to as PLM-2401) prepared according to the preparation example as an active ingredient. The mixed solvent (vehicle) for the formulation of PLM-2401 was prepared by mixing 5% DW, 5% Tween 20, and 90% 0.1N HCl.

[0079] First, PLM-2401 was dissolved in a mixed solvent (5% DW, 5% Tween 20, 90% 0.1N HCl) to prepare a PLM-2401 solution with a concentration of 200 mg / ml, which was then divided into smaller portions and refrigerated according to the administration date for the animal experiment. Additionally, formulations corresponding to the dosage were prepared and used for each administration during the experiment.

[0080] PLM-2401 formulations for 20 mg / kg, 10 mg / kg, and 5 mg / kg dose administration (Examples 3, 2, and 1, respectively) were prepared by further diluting the PLM-2401 solution at a concentration of 200 mg / ml with the mixed solvent, and 200 μl was administered in animal experiments.

[0081]

[0082] [Comparative Example]

[0083] Positive control baricitinib preparation

[0084] Baricitinib, known to have therapeutic or improving effects on atopic dermatitis, was used as a positive control (Comparative Example 3). Baricitinib is used as a standard therapeutic agent for treating atopic dermatitis.

[0085] The baricitinib preparation was prepared by dissolving baricitinib in a solvent (0.5% methylcellulose in DW) to produce a stock solution with a concentration of 200 mg / ml, which was then aliquoted and refrigerated according to the administration date for the animal experiment. During the experiment, for each administration, an additional 2,995 μl of the solvent (0.5% methylcellulose in DW) was added to 5 μl of the baricitinib stock solution and vortexed to produce the preparation, and 200 μl was administered in the animal experiment (5 mg / kg dose).

[0086]

[0087] [Experimental Example]

[0088] Design of animal experiments for atopic dermatitis

[0089] To confirm the improvement and therapeutic effects of the compound of the present invention on atopic dermatitis, administration was performed on a mouse model with induced atopic dermatitis.

[0090] Female NC / Nga mice aged 8 or 7 weeks were obtained, and experiments were conducted at 9 weeks of age. An HDM (house dust mite)-induced atopic dermatitis mouse model, specifically the HDM-iAD (house dust mite-induced atopic dermatitis) mouse model, was utilized, and an HDM ointment (Biostir® AD ointment) was applied to the skin to induce atopic dermatitis. Since HDM is the primary cause of atopic dermatitis in humans, the HDM-iAD model is primarily used in validation testing phases during the development of therapeutic agents; in addition to efficacy evaluation, MOA studies can also be conducted simultaneously. Mice exhibiting no clinical symptoms such as disease or injury and possessing an appropriate body weight between 20 and 25 g were selected for the experiment, and all groups were randomly assigned based on the most recently measured body weight.

[0091] The mouse groups for the experiment were classified as shown in Table 1 below.

[0092] No. Group Dosage Frequency Route of Administration Number of Individuals G1 General Mouse (Comparative Example 1) --- N=10 G2 HDM-iAD / Vehicle (Comparative Example 2, Negative Control) NABID Oral Administration N=10 G3 HDM-iAD / Baricitinib (Comparative Example 3, Positive Control) 5 mg / kg QD Oral Administration N=10 G4 HDM-iAD / PLM-2401 (Example 1) 5 mg / kg BID Oral Administration N=10 G5 HDM-iAD / PLM-2401 (Example 2) 10 mg / kg BID Oral Administration N=10 G6 HDM-iAD / PLM-2401 (Example 3) 20 mg / kg BID Oral Administration N=10

[0093] In Table 1 above, the vehicle administered as the negative control was a solvent of 5% DW, 5% Tween 20, and 90% 0.1N HCl. The administration frequency BID means administration twice a day, and QD means administration once a day; however, for the G3 group administered QD, the positive control formulation was administered in the morning, followed by the same solvent (vehicle) as the negative control in the afternoon. Mice in all groups had their entire backs depilated using depilatory cream two days prior to the start of the experiment, and fine hairs were removed using clippers if necessary. To induce atopic dermatitis in mice, 90 mg of HDM ointment (Biostir® AD ointment) was applied and allowed to dry on Day 0 of the experiment, followed by additional skin applications on Days 7, 10, 14, 17, 21, and 24, for a total of seven applications. The experiment was conducted until day 28, and termination was decided when it was determined that the clinical index following the application of HDM ointment in the negative control group had reached its maximum.

[0094] The formulations or solvents of Examples 1 to 3 and Comparative Examples 2 and 3 were orally administered daily for 3 weeks starting from day 7 at the administration frequency according to Table 1 above. For each example and comparative example, the formulation or solvent was placed in a 1 ml / CC (W / O) syringe with a metal gavage inserted to contain the component to be administered, and after restraining the mouse, the tip of the gavage was inserted into the mouse's oral cavity to check whether negative pressure was applied, and then the inner tube of the syringe was pushed to administer 200 μl at a time.

[0095]

[0096] [Experimental Example 1]

[0097] Safety evaluation following the administration of the compound of the present invention

[0098] To evaluate safety according to the examples and comparative examples, the survival of mice was checked daily from the start of the experiment to the end of the experiment, and whether abnormal changes in body weight occurred depending on the administered component was checked by measuring the body weight of the mice. The body weight of the mice was measured twice a week and compared, and the values ​​comparing the body weight on day 0 and day 28 are shown in Table 2.

[0099] As a result of the safety evaluation, no mice died in any group over a period of 28 days, confirming that the administered compound PLM-2401 did not significantly affect mouse survival. Body weight also showed no significant changes in any group, including the group administered PLM-2401. Through these experimental results, the safety of the compound PLM-2401 of the present invention was confirmed for administration at doses of 5, 10, and 20 mg / kg.

[0100] Group Body Weight (g, Day 0) Body Weight (g, Day 28) Change in Body Weight (%, relative to Day 0) Mean SEM Mean SEM Mean SEM Comparative Example 1 (General Mouse) 23.19 2.14 24.13 1.74 104.29 4.54 Comparative Example 2 (Negative Control) 22.62.12 22.84 1.02 101.89.77 Comparative Example 3 (Baricitinib, 5 mg / kg) 23.78 2.64 24.07 2.09 101.56 5.31 Example 1 (PLM-2401, 5 mg / kg) 22.33 1.61 23.15 1.93 103.79 6.84 Example 2 (PLM-2401, 10 mg / kg)22.85 1.75 23.21 1.32 101 1.77 3.98 Example 3 (PLM-2401, 20 mg / kg)23.06 1.69 23.48 110 2.24 7.64

[0101] [Experimental Example 2]

[0102] Evaluation of clinical indicators for atopic dermatitis

[0103] Photos of changes in the mouse's skin were taken before applying HDM AD ointment to the mouse's dorsal skin, and photos of the results at the end of day 0 and day 28 are shown in Figure 1.

[0104] After 28 days, the negative control group showed the most severe skin lesions, with redness, edema, and crust formation on the back skin, as well as secondary skin damage caused by itching. On the other hand, the positive control group and the PLM-2401 treatment group showed much better skin conditions compared to the negative control group.

[0105] In addition, erythema / hemorrhage, scarring / dryness, edema, and excoriation / erosion of the skin were observed before applying HDM AD ointment to the dorsal skin of mice, and scores ranging from 0 to 3 points were assigned according to criteria (0 points: None, 1 point: Mild, 2 points: Moderate, 3 points: Severe). The sum of these scores was used as the clinical activity index, and the changes in the dermatitis score and the AUC (area under curve) values ​​were compared and presented in Figure 2, Table 3, and Table 4.

[0106] Group Dermatitis Score (Day 28) Mean SEM Comparison Example 1 (General Mouse) 0.00 0.00 Comparison Example 2 (Negative Control) 11.25 0.42 Comparison Example 3 (Baricitinib, 5 mg / kg) 4.65 0.80 Example 1 (PLM-2401, 5 mg / kg) 6.75 0.68 Example 2 (PLM-2401, 10 mg / kg) 5.80 0.72 Example 3 (PLM-2401, 20 mg / kg) 5.30 0.81

[0107] Group Dermatitis Score AUC Mean SEM Comparison Example 1 (General Mouse) 0.00 0.00 Comparison Example 2 (Negative Control) 115.23 6.54 Comparison Example 3 (Baricitinib, 5 mg / kg) 50.48 7.14 Example 1 (PLM-2401, 5 mg / kg) 63.38 6.40 Example 2 (PLM-2401, 10 mg / kg) 54.20 6.23 Example 3 (PLM-2401, 20 mg / kg) 50.10 6.95

[0108] As a result of evaluating dermatitis scores based on changes in skin symptoms, although the dermatitis scores in the entire group gradually increased over time, it was confirmed that dermatitis symptoms were statistically significantly reduced in both the positive control group (baritisinib) and the group administered the compound of the present invention PLM-2401 when compared to the dermatitis scores of the negative control group (Vehicle) (Fig. 2 and Table 3).

[0109] As can be seen in Table 4, the cumulative intensity of the treatment effect was confirmed by comparing the values ​​calculated as the cumulative area of ​​changes in atopic dermatitis symptoms over time, and a significant improvement effect was observed in Comparative Example 3 (baricitinib). In the case of the PLM-2401 administration group, a significant improvement effect was observed in the 5 mg / kg administration group (Example 1), and the 10 mg / kg and 20 mg / kg administration groups (Examples 2 and 3) showed excellent improvement effects similar to baricitinib, and in particular, the 20 mg / kg administration group showed the best improvement effect for atopic dermatitis among all groups.

[0110] Furthermore, when changes in skin thickness were measured and compared using a method similar to the measurement of atopic dermatitis scores, as shown in Table 5, the skin thickness of the mice other than the normal mice gradually increased; however, it was confirmed that the increase in skin thickness was statistically significantly reduced in both the positive control group (baricitinib) and the group administered with the compound of the present invention PLM-2401 compared to the negative control group (Vehicle). Examples 1 and 2 showed excellent effects in reducing the increase in skin thickness, and in particular, Example 3 was superior to the group administered with baricitinib (Comparative Example 3), showing the most excellent effect in reducing the increase in skin thickness.

[0111] Group Skin Thickness Change (%) Average SEM Comparison Example 1 (General Mouse) 107.99 0.5 Comparison Example 2 (Negative Control) 249.88 9.59 Comparison Example 3 (Baricitinib, 5 mg / kg) 173.24 8.44 Example 1 (PLM-2401, 5 mg / kg) 188.04 5.23 Example 2 (PLM-2401, 10 mg / kg) 183.01 5.31 Example 3 (PLM-2401, 20 mg / kg) 171.17 7.79

[0112] When synthesizing the experimental results described above, it can be confirmed that the compound PLM-2401 of the present invention has an effect of improving and treating atopic dermatitis-related symptoms at a level similar to or superior to that of baricitinib, which is known to have a therapeutic effect on atopic dermatitis, and furthermore, a concentration-dependent therapeutic effect was confirmed.

[0113]

[0114] [Experimental Example 3]

[0115] Confirmation of the concentration of antibodies related to atopic dermatitis

[0116] Atopic dermatitis is characterized by elevated blood IgE antibody concentrations associated with allergic immune responses, so to confirm the MOA of PLM-2401, the effect of PLM administration on reducing total IgE and HDM-specific IgE in the serum was examined.

[0117] To determine blood total IgE levels, eight mice were selected from each group, and serum was isolated. Using the Mouse Total IgE ELISA Kit, the serum was diluted 1 / 4,000 and added to a plate containing immobilized antibodies. After incubating for 2 hours and washing with wash buffer, detection antibodies were added and incubated for 1 hour. This was washed again with wash buffer, followed by the addition of streptavidin-HRP solution and incubation for 1 hour. After washing with wash buffer and incubation with TMB substrate for 25 minutes, stop solution was added, and absorbance was measured at 450 nm using a plate reader. The measurement results are shown in Table 6.

[0118] Group Total IgE Concentration (ng / ml) Mean SEM Comparative Example 1 (General Mouse) 0.68 0.14 Comparative Example 2 (Negative Control) 13.18 0.14 Comparative Example 3 (Baricitinib, 5 mg / kg) 9.02 0.96 Example 1 (PLM-2401, 5 mg / kg) 10.87 0.98 Example 2 (PLM-2401, 10 mg / kg) 9.61 0.73 Example 3 (PLM-2401, 20 mg / kg) 7.45 0.66

[0119] To measure HDM-specific IgE, serum was isolated from eight selected mice from each group. Using the Mouse HDM-specific IgE ELISA Kit, the serum was diluted 1 / 2 and placed in a plate, then incubated for 2 hours. After washing with wash buffer, the secondary antibody was added and incubated for 2 hours. Subsequently, the plate was washed again with wash buffer, and streptavidin-HRP solution was added and incubated for 30 minutes. After washing with wash buffer and incubating with TMB substrate for 25 minutes, a stop solution was added, and the absorbance was measured at 450 nm using a plate reader. The measurement results are shown in Table 7.

[0120] Group HDM-specific IgE concentration (ng / ml) Mean SEM Comparative Example 1 (General mouse) 0.24 0.05 Comparative Example 2 (Negative control) 1.55 0.11 Comparative Example 3 (Baricitinib, 5 mg / kg) 1.02 0.17 Example 1 (PLM-2401, 5 mg / kg) 0.85 0.14 Example 2 (PLM-2401, 10 mg / kg) 0.8 0.08 Example 3 (PLM-2401, 20 mg / kg) 0.86 0.13

[0121] As a result of confirming these inflammatory mediators at the protein level, it was confirmed that the total blood IgE concentration decreased in both the positive control group and the PLM-2401 administration group compared to the negative control group (Vehicle). In particular, the measurement results of Examples 1 to 3 showed that the total IgE concentration decreased in dependence on the administration concentration of PLM-2401, and a statistically significant decrease was confirmed in the baricitinib administration group (Comparative Example 3), the 10 mg / kg administration group of PLM-2401 (Example 2), and the 20 mg / kg administration group (Example 3).

[0122] In addition, the results of HDM-specific IgE measurement also showed that HDM-specific IgE was statistically significantly reduced in both the positive control group and the PLM-2401 administration group compared to the negative control group (Vehicle), indicating an excellent reduction effect even in the 5 mg / kg administration group.

[0123] Based on the results above, it was confirmed that the administration of PLM-2401 of the present invention regulates the immune response associated with the development of atopic dermatitis, thereby reducing the excessive production of IgE antibodies, and consequently, an improvement and therapeutic effect on atopic dermatitis is observed.

[0124]

[0125] [Experimental Example 4]

[0126] Confirmation of gene expression related to inflammation mediators in skin tissue (qRT-PCR)

[0127] The overexpression of inflammation-related cytokines can play an important role in the onset and exacerbation of atopic dermatitis, and these cytokines include IL-4, IL-13, and TSLP. Accordingly, the effect of reducing the expression of the above cytokines following the administration of PLM-2401 was confirmed through qRT-PCR.

[0128] On day 28 of the experiment, after the administration was terminated, ear skin tissue was isolated from mice of each group, and total RNA was isolated. Total RNA was measured using a kit (Easy-spin TM RNA was extracted using a total RNA extraction kit, and reverse transcription kit (PrimeScript) was used. TMcDNA was synthesized using a 1st strand cDNA Synthesis Kit. Transcript amounts were quantitatively analyzed using qRT-PCR (quantitative RT-PCR) with target probe activators for the target mediators IL-4, IL-13, and TSLP. An ABI 7500 (Applied Biosystems) was used, and IL-4, IL-13, and TSLP transcripts were measured in real-time using a Raji standard curve plot with log total RNA on the X-axis and Ct values ​​on the Y-axis. For quantification, a Raji standard curve plot was used with the X-axis log target strand (range: 0.000004 to 4 ng / ml) and Y-axis Ct values ​​of the standard transcripts for each target factor, and each measurement was quantified as a normalized ratio to the quantitative value of GAPDH. The ratio of the experimental results of each administration group to the quantitative values ​​of normal mice was measured and is presented in Tables 8 to 10.

[0129] Group IL-4 mRNA expression level (fold change) Mean SEM Comparison Example 1 (General mouse) 10.24 Comparison Example 2 (Negative control) 35 19.4 27 23.48 Comparison Example 3 (Baricitinib, 5 mg / kg) 48.8 6 16.4 Example 1 (PLM-2401, 5 mg / kg) 598.4 23 72.39 Example 2 (PLM-2401, 10 mg / kg) 298.8 31 30.25 Example 3 (PLM-2401, 20 mg / kg) 155.5 6 32.81

[0130] Group IL-13 mRNA expression level (fold change) Mean SEM Comparison Example 1 (General mouse) 10.43 Comparison Example 2 (Negative control) 43.76 25.77 Comparison Example 3 (Baricitinib, 5 mg / kg) 13.8 56.85 Example 1 (PLM-2401, 5 mg / kg) 7.5 5.57 Example 2 (PLM-2401, 10 mg / kg) 6.19 2.11 Example 3 (PLM-2401, 20 mg / kg) 0.89 0.26

[0131] Group TSLP mRNA expression level (fold change) Mean SEM Comparison Example 1 (General mouse) 10.31 Comparison Example 2 (Negative control) 42.06 11.01 Comparison Example 3 (Baricitinib, 5 mg / kg) 9.07 3.86 Example 1 (PLM-2401, 5 mg / kg) 21.8 7.3 Example 2 (PLM-2401, 10 mg / kg) 35.5 6 5.74 Example 3 (PLM-2401, 20 mg / kg) 15.6 4.46

[0132] As a result of the experiment, the expression of IL-4, IL-13, and TSLP in the negative control group (Vehicle) was significantly increased compared to normal mice, whereas in the positive control group and the PLM-2401 administration group, the expression levels of the above cytokines tended to decrease compared to the negative control group. In the case of the PLM-2401 administration groups, it was confirmed that the expression of IL-4, IL-13, and TSLP was reduced to a similar level when compared to Comparative Example 3, which was used as a positive control.

[0133] Through the above results, it was confirmed that the administration of the compound PLM-2401 of the present invention simultaneously reduced inflammatory cytokines along with clinical improvement, thereby demonstrating an improvement and therapeutic effect on atopic dermatitis.

[0134]

[0135] [Experimental Example 5]

[0136] Confirmation of inhibitory effect on histomorphological changes in skin tissue of atopic dermatitis

[0137] Histomorphological changes were examined to determine whether PLM-2401 had an inhibitory effect on inflammation at the tissue level, in addition to improving clinical indicators of atopic dermatitis. Since lesions of atopic dermatitis are characterized by epidermal thickening and increased infiltration of inflammatory cells (mast cells, eosinophils, etc.) within the dermis, the extent to which PLM-2401 alleviates and improves these tissue lesions was quantitatively measured.

[0138] Histological evaluation of H&E (Hematoxylin and Eosin) stained tissue

[0139] Eight individuals were selected from each mouse group, and dorsal skin tissue was isolated. The tissues were then fixed using a fixation solution (10% neutral buffered formalin solution) and embedded in paraffin to produce 3 μm thick tissue sections. The tissue sections were H&E stained, mounted, and examined under a microscope; all stained slides were scanned using a digital pathology scanner for evaluation. For each tissue, four sites (200x magnification) were selected and captured, and epidermal thickness was measured independently by at least two researchers.

[0140] Upon examination, the negative control group (Vehicle) showed not only a significantly increased epidermal thickness compared to normal mice but also hyperkeratotic lesions. In the dermis, neovascularization was increased, and along with tissue edema, the infiltration of inflammatory cells and extracellular matrix were significantly increased. In comparison, in both the positive control group and the PLM-2401 administration group, the increase in the epidermal layer was found to be reduced in a concentration-dependent manner, and the thickness decreased along with a reduction in infiltrating cells and extracellular matrix in the dermis (Fig. 3a).

[0141] As shown in Table 11 below, the epidermal thickness measurement results showed that the negative control group (Vehicle) had an epidermal thickness approximately 8.6 times greater than that of normal mice (Normal). Furthermore, compared to the negative control group, both the positive control group and the PLM-2401 administration group showed a statistically significant reduction in the increase in epidermal thickness. In particular, the PLM-2401 administration group exhibited a concentration-dependent pattern depending on the dosage, and the 20 mg / kg administration group (Example 3) showed effects similar to or superior to those of the positive control group. When examining the correlation between the dermatitis scores for each group identified in Experimental Example 2 and the epidermal thickness measurements (top of Fig. 3b), the correlation between the average dermatitis score and the average epidermal thickness of each group was found to be very high. Similarly, the correlation between individual dermatitis scores and epidermal thickness also showed statistical significance (bottom of Fig. 3b).

[0142] Group Epidermal Thickness (μm) Average SEM Comparative Example 1 (General Mouse) 14.87 0.78 Comparative Example 2 (Negative Control) 128.47 6.61 Comparative Example 3 (Baricitinib, 5 mg / kg) 65.78 2.61 Example 1 (PLM-2401, 5 mg / kg) 88.58 0.99 Example 2 (PLM-2401, 10 mg / kg) 76.39 4.75 Example 3 (PLM-2401, 20 mg / kg) 69.26 6.62

[0143] Histological evaluation of TB (Toluidine Blue) stained tissue

[0144] Eight individuals were selected from each mouse group, and dorsal skin tissue was isolated. The tissue was then fixed using a fixation solution (10% neutral buffered formalin solution) and embedded in paraffin to produce 3 μm thick tissue sections. The tissue sections were stained with Toluidine Blue, mounted, and examined under a microscope; all stained slides were scanned using a digital pathology scanner for evaluation. For each tissue, four sites (200x magnification) were selected and captured, and the number of mast cells was independently measured by at least two researchers.

[0145] As a result of the examination, a large number of mast cells infiltrated the dermis in the negative control group (Vehicle) compared to normal mice, whereas this phenomenon was found to be reduced in both the positive control group and the PLM-2401 administration group (Fig. 4a).

[0146] As shown in Table 12, quantitative measurement of the number of mast cells revealed that the number of mast cells in the negative control group (Vehicle) increased by approximately 6.6 times compared to normal mice (Normal). Conversely, both the positive control group and the PLM-2401 administration group showed a statistically significant reduction in the number of mast cells compared to the negative control group. In particular, the PLM-2401 administration group exhibited a concentration-dependent pattern depending on the dosage of PLM-2401. When examining the correlation between the dermatitis scores for each group identified in Experimental Example 2 and the mast cell count measurements (top of Fig. 4b), the correlation between the average dermatitis score and the average mast cell count for each group was found to be very high. Similarly, the correlation between individual dermatitis scores and mast cell counts also showed statistical significance (bottom of Fig. 4b).

[0147] Average SEM count of mast cells per group Comparative Example 1 (General mouse) 11.66 1.8 Comparative Example 2 (Negative control) 76.56 5.72 Comparative Example 3 (Baricitinib, 5 mg / kg) 32.25 1.37 Example 1 (PLM-2401, 5 mg / kg) 44.38 1.96 Example 2 (PLM-2401, 10 mg / kg) 39.91 2.36 Example 3 (PLM-2401, 20 mg / kg) 36.75 3.23

[0148] Histological evaluation of CR (Congo Red) stained tissue

[0149] Eight individuals were selected from each mouse group, and dorsal skin tissue was isolated. The tissues were then fixed using a fixation solution (10% neutral buffered formalin solution) and embedded in paraffin to produce 3 μm thick tissue sections. The tissue sections were stained with Congo Red, mounted, and examined under a microscope; all stained slides were scanned using a digital pathology scanner for evaluation. For each tissue, four sites (200x magnification) were selected and captured, and the number of eosinophils was independently measured by at least two researchers.

[0150] As a result of the examination, a large amount of eosinophils infiltrated the dermis in the negative control group (Vehicle) compared to normal mice, whereas this phenomenon was found to be reduced in both the positive control group and the PLM-2401 administration group (Fig. 5a).

[0151] As shown in Table 13, quantitative measurement of eosinophil counts revealed that the number of eosinophils in the negative control group (Vehicle) increased approximately 16-fold compared to normal mice (Normal). Conversely, both the positive control group and the PLM-2401 administration group showed a statistically significant decrease in eosinophil counts compared to the negative control group. In particular, the PLM-2401 administration group exhibited a concentration-dependent pattern depending on the dosage of PLM-2401. When examining the correlation between the dermatitis scores for each group identified in Experimental Example 2 and the eosinophil count measurements (top of Fig. 5b), the correlation between the average dermatitis score and the average eosinophil count for each group was found to be very high. Similarly, the correlation between individual dermatitis scores and eosinophil counts also showed statistical significance (bottom of Fig. 5b).

[0152] Group Eosinophil Count Average SEM Comparison Example 1 (General Mouse) 3.44 0.24 Comparison Example 2 (Negative Control) 49.56 3.22 Comparison Example 3 (Baricitinib, 5 mg / kg) 18.47 1.52 Example 1 (PLM-2401, 5 mg / kg) 271.83 Example 2 (PLM-2401, 10 mg / kg) 24.66 0.89 Example 3 (PLM-2401, 20 mg / kg) 21.09 2.71

[0153] Based on the comprehensive results of the histopathological evaluation described above, it was confirmed that PLM-2401 demonstrates efficacy comparable to that of baricitinib, a JAK inhibitor used as a standard treatment for atopic dermatitis in clinical practice. Furthermore, it significantly reduced the infiltration of mast cells and eosinophils, which are crucial to the pathogenesis of atopic dermatitis, and exhibited a therapeutic effect that was dose-dependent. Additionally, it was confirmed that the effect related to the clinical symptom-based dermatitis score identified in Experimental Example 2 was well reflected by the morphological quantitative evaluation index according to the present experiment.

[0154]

[0155] [Experimental Example 6]

[0156] Confirmation of the effect of regulating the balance of Th2 / Treg immune cells in the skin (immunofluorescence staining)

[0157] Changes in the helper T cell subset were analyzed through immunofluorescence staining targeting GATA3 and FoxP3, transcription factors expressed in immune cells.

[0158] For each mouse group, five individuals exhibiting representative dermatitis scores according to Experimental Example 2 were selected, and dorsal skin tissue was isolated. The tissue was then fixed using a fixation solution (10% neutral buffered formalin solution) and embedded in paraffin to produce 3 μm thick tissue sections. The tissue sections were baked at 60°C for 1 hour, after which the xylene paraffin was removed, and the tissues were rehydrated with ethanol of various concentrations. For antigen recovery, the tissue sections were treated in a pressure cooker under low pressure conditions using Tris-EDTA (pH 9.0) solution for 10 minutes, followed by blocking with 5% Normal Swine Serum for 20 minutes. Subsequently, the tissues were reacted with primary antibodies against FoxP3 and GATA3 at room temperature for 1 hour, followed by reaction with HRP-conjugated secondary antibodies for 30 minutes, and then treated with Opal 690 TSA (Tyramide signal amplification) solution to amplify fluorescence. Then, to remove the antigen, the slides were treated again in a pressure cooker under low pressure conditions using Tris-EDTA (pH 9.0) solution for 10 minutes. Next, for the second target staining, anti-CD3 antibody was used as the primary antibody, and fluorescence was amplified by treatment with Opal 520 TSA solution. After the second staining, counterstaining was performed with DAPI (4',6-diamidino-2-phenylindole) at room temperature for 3 minutes, and the slides were mounted on Vectashield (Vector Laboratories, CA, USA). The stained slides were photographed using a confocal microscope, and the number of double-positive cells (Treg and Th2 cells) was analyzed by color-labeling the cells using a device (Image J cell counter tool) and calculated as the number of positive cells per HPF (high power field, X400).

[0159] Th2 cells (CD3 + GATA3 + ) Analysis results of subtypes, CD3+ Regarding cell count (Table 14), it was found to be reduced in both the positive control and the PLM-2401 administration groups compared to the negative control (Vehicle), and in the case of the PLM-2401 administration groups, a dose-dependent decrease was observed. In particular, the 10 mg / kg (Example 2) and 20 mg / kg (Example 3) PLM-2401 administration groups showed a statistically significant decrease. Th2 cells (CD3 + GATA3 + ) Even in the case of the number (Table 15), the above CD3 + A pattern similar to the results regarding cell count was observed, and when compared to the negative control group, a statistically significant decrease was found in both the positive control group and the PLM-2401 administration group. And Th2 cells (CD3 + GATA3 + ) / CD3 + In the case of cell ratio (Table 16), it was found to be statistically significantly reduced in both the positive control group and the PLM-2401 administration group compared to the negative control group (Vehicle).

[0160] Group T (CD3 + ) Average SEM cell number Comparative Example 1 (General mouse) 20.15 2.38 Comparative Example 2 (Negative control) 92.58 78 Comparative Example 3 (Baricitinib, 5 mg / kg) 50.33 66 Example 1 (PLM-2401, 5 mg / kg) 65.85 9.41 Example 2 (PLM-2401, 10 mg / kg) 63.25 7.61 Example 3 (PLM-2401, 20 mg / kg) 50.35 4.34

[0161] Group Th2 (CD3 + GATA3 +) Average SEM cell number Comparative Example 1 (General mouse) 0.65 0.2 Comparative Example 2 (Negative control) 15.45 2.23 Comparative Example 3 (Baricitinib, 5 mg / kg) 3.5 0.84 Example 1 (PLM-2401, 5 mg / kg) 5.6 0.42 Example 2 (PLM-2401, 10 mg / kg) 5.4 0.67 Example 3 (PLM-2401, 20 mg / kg) 3.6 0.55

[0162] Group Th2 (CD3 + GATA3 + ) Cells (%) Mean SEM Comparative Example 1 (General Mouse) 2.96 0.91 Comparative Example 2 (Negative Control) 16.43 1.44 Comparative Example 3 (Baricitinib, 5 mg / kg) 6.79 1.56 Example 1 (PLM-2401, 5 mg / kg) 9.2 1.29 Example 2 (PLM-2401, 10 mg / kg) 8.6 10.6 Example 3 (PLM-2401, 20 mg / kg) 6.99 0.65

[0163] The dermatitis scores for each group confirmed in Experimental Example 2 above and the CD3 + Cell number, CD3 + GATA3 + Cell number and CD3 + GATA3 + / CD3 + When the correlation between cell ratios was examined (top of Figs. 7a to 7c), it was found to have a very high correlation with the average dermatitis score of each group. Similarly, the individual dermatitis score and the aforementioned CD3 + Cell number, CD3 + GATA3 + Cell number and CD3 + GATA3 + / CD3 + The correlation between cell ratios also showed statistical significance (bottom of Figs. 7a to 7c).

[0164] Treg cells (CD3 + FoxP3 + ) Analysis results of subtypes, CD3 +Regarding cell count (Table 17), a statistically significant decrease was observed in both the positive control and the PLM-2401 administration groups compared to the negative control (Vehicle). Treg cells (CD3 + FoxP3 + In the case of the number (Table 18), compared to the negative control (Vehicle), it increased in the positive control baricitinib (Comparative Example 3), the PLM-2401 10 mg / kg administration group (Example 2), and the 20 mg / kg administration group (Example 3), while it showed a decreasing trend in the PLM-2401 5 mg / kg administration group (Example 1), but no statistically significant results could be confirmed. Treg cells (CD3 + FoxP3 + ) / CD3 + In the case of cell ratio (Table 19), a distinct increase was observed in both the positive control group and the PLM-2401 administration group compared to the negative control group (Vehicle), and in particular, a statistically significant increase was observed in the positive control group baricitinib (Comparative Example 3), the 10 mg / kg administration group of PLM-2401 (Example 2), and the 20 mg / kg administration group (Example 3).

[0165] Group T (CD3 + ) Average SEM Cell Number Comparative Example 1 (General Mouse) 16.9 5 1.9 Comparative Example 2 (Negative Control) 88.2 5 5.5 Comparative Example 3 (Baricitinib, 5 mg / kg) 40.2 5 3.62 Example 1 (PLM-2401, 5 mg / kg) 52.2 5 10.65 Example 2 (PLM-2401, 10 mg / kg) 48.1 5 5.44 Example 3 (PLM-2401, 20 mg / kg) 44.7 4.09

[0166] GroupTreg(CD3 + FoxP3 +) Average SEM Cell Number Comparative Example 1 (General Mouse) 0.05 0.05 Comparative Example 2 (Negative Control) 6.05 2.1 Comparative Example 3 (Baricitinib, 5 mg / kg) 7.85 1.35 Example 1 (PLM-2401, 5 mg / kg) 4.9 0.67 Example 2 (PLM-2401, 10 mg / kg) 6.7 0.7 Example 3 (PLM-2401, 20 mg / kg) 6.9 0.65

[0167] GroupTreg(CD3 + FoxP3 + ) Cells (%) Average SEM Comparative Example 1 (General Mouse) 0.28 0.28 Comparative Example 2 (Negative Control) 6.34 2.13 Comparative Example 3 (Baricitinib, 5 mg / kg) 20.22 3.82 Example 1 (PLM-2401, 5 mg / kg) 11.2 2.69 Example 2 (PLM-2401, 10 mg / kg) 14.6 2.17 Example 3 (PLM-2401, 20 mg / kg) 18.88 4.35

[0168] The dermatitis scores for each group confirmed in Experimental Example 2 above and the CD3 + Cell number, CD3 + FoxP3 + Cell number and CD3 + FoxP3 + / CD3 + When the correlation between cell ratios was examined (top of Figs. 9a to 9c), it was found to have a very high correlation with the average dermatitis score of each group. Similarly, the individual dermatitis score and the aforementioned CD3 + Cell number, CD3 + FoxP3 + Cell number and CD3 + FoxP3 + / CD3 + The correlation between cell ratios also showed statistical significance (bottom of Figs. 9a to 9c).

[0169] Analysis of the Th2 / Treg cell ratio (Table 20) showed a distinct decrease in both the positive control group and the PLM-2401 administration group compared to the negative control group (Vehicle), and in particular, both the positive control group baricitinib (Comparative Example 3) and the PLM-2401 administration group (Examples 1 to 3) showed a statistically significant decrease.

[0170] Group Th2 / Treg Cells (%) Mean SEM Comparison Example 1 (General Mouse) 0.05 0.05 Comparison Example 2 (Negative Control) 5.93 1.51 Comparison Example 3 (Baricitinib, 5 mg / kg) 1.32 0.48 Example 1 (PLM-2401, 5 mg / kg) 1.74 0.45 Example 2 (PLM-2401, 10 mg / kg) 10.19 Example 3 (PLM-2401, 20 mg / kg) 0.65 0.18

[0171] Based on the above experimental results, it was anticipated that the compound PLM-2401 of the present invention exhibits an effect of improving and treating symptoms related to atopic dermatitis by reducing Th2 cells and increasing the proportion of Treg cells among helper T cell subtypes in skin tissue.

[0172]

[0173] Although the present invention has been described in detail above only with respect to the described embodiments and experimental examples, it is obvious to those skilled in the art that various modifications and variations are possible within the scope of the technical spirit of the invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Claims

A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 2] . A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 1, wherein the pharmaceutically acceptable salt is a hemifumarate salt. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 1, wherein the compound is administered to a human at a dose of 0.6 mg / kg / day to 6.0 mg / kg / day. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 1, wherein the compound is administered twice a day. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 1, wherein the pharmaceutical composition is administered orally. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 1, wherein the compound has at least one activity selected from the group consisting of inhibition of the Th2 immune pathway, increase in the proportion of Treg cells, inhibition of infiltration of mast cells and eosinophils, inhibition of inflammatory cytokines, inhibition of the production of IgE antibodies, and inhibition of action on S1PR5.

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