Use of compounds in the treatment or prevention of atopic dermatitis

Abstract

The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidine-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide (also referred to herein as Compound 1) or a pharmaceutically acceptable salt thereof in the treatment or prevention of atopic dermatitis.

Description

[Technical Field]

[0001] The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidine-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide (also referred to herein as Compound 1) or a pharmaceutically acceptable salt thereof in the treatment or prevention of atopic dermatitis. [Background technology]

[0002] Atopic dermatitis (AD) is a common chronic, recurrent inflammatory skin disease. The main symptoms are dry skin, chronic eczematous skin lesions, and severe itching. In moderate and severe cases, it can even cause other disorders such as insomnia, anxiety, and depression, severely impacting quality of life. This disease is often accompanied by allergic asthma and allergic rhinitis and is a systemic autoimmune disorder.

[0003] Treatment for Alzheimer's disease (AD) can be broadly divided into two main categories: topical therapy and systemic therapy. Topical glucocorticoids are the first-line treatment for AD, but they cannot be used for life. Long-term use of glucocorticoids can lead to skin atrophy, telangiectasia, hyperpigmentation, and secondary infections. In particular, long-term use of glucocorticoids over a large area when the skin is damaged can lead to inhibition of the hypothalamic-pituitary-adrenal (HPA) axis, which can manifest as pseudo-Cushing's syndrome, steroid-induced diabetes, osteoporosis, exacerbation of infections, induction or exacerbation of gastrointestinal ulcers, and induction of psychiatric symptoms. Topical glucocorticoids have strict time constraints. Systemic administration of glucocorticoids should be avoided or kept to a minimum whenever possible. While dupilumab and JAK inhibitors have changed the situation where there were no available treatments for severely ill patients, effective remission has not been achieved in nearly 50% of severely ill patients, and significant side effects have occurred. For example, dupilumab has a side effect of conjunctivitis, and the JAK inhibitor ruxolitinib has a black-bordered warning on its product label alerting users to serious side effects, including the risk of severe infection, development of non-melanoma skin cancer, thrombosis, thrombocytopenia, anemia, and neutropenia. There is still a significant unmet clinical need in the field of Alzheimer's disease, and there is an urgent need to develop safer and more effective drugs that can be used over the long term.

[0004] Currently, the two commercially available TRK inhibitors (larotrectinib and entrectinib) are both targeted at cancer patients with TRK gene mutations. There are no commercially available drugs indicated for atopic dermatitis. Currently, three drugs are in clinical trials for atopic dermatitis: pegcantratinib, a TRKA inhibitor originally studied by Cephalon in Phase 2 (completed); BEN-2293, a pan-TRK inhibitor (capable of inhibiting TRKA, TRKB, and TRKC) in Phase 2 by BenevolentAI; and PBI-100, a pan-TRK inhibitor in Phase 1 by Pyramid Biosciences. Clinical research on pegcantratinib for atopic dermatitis was completed in 2010, while BEN-2293 and PBI-100 were still in trials. It appears that TRKA-specific inhibitors carry a risk of failing to treat atopic dermatitis. Regarding DDR1 and DDR2 inhibitors, no drugs for atopic dermatitis have entered clinical trials, and there have been no preclinical studies on DDR for which these are indicated. Similarly, there are no CSF1R inhibitors currently in the preclinical or clinical stages for atopic dermatitis.

[0005] Short-term stimulation with the hapten OXA (oxazolone) can induce allergic contact dermatitis, primarily caused by Th1 cell-mediated immunity, in mice. However, continuous and repeated contact with the hapten can induce Th2-mediated skin inflammation in mice, similar to that seen in human atopic dermatitis. Locally, erythema, scaling, and skin thickening are clearly visible on the skin. Histopathology reveals epidermal thickening, edema, and massive infiltration of inflammatory cells such as CD4+ T lymphocytes and mast cells into the dermis. This is one of the most common models for studying the pathological mechanisms of atopic dermatitis and screening therapeutic agents. This invention uses this model to identify the therapeutic effects of the above-mentioned compounds on atopic dermatitis.

[0006] CN113831344B discloses N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidine-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Specification of Chinese Patent No. 113831344 [Overview of the Initiative]

[0008] The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidine-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethinyl)-2-methylbenzamide (also referred to herein as Compound 1) or a pharmaceutically acceptable salt thereof in the treatment or prevention of atopic dermatitis. In the present invention, atopic dermatitis includes, according to age characteristics, infantile atopic dermatitis, early childhood atopic dermatitis, adolescent / adult atopic dermatitis, mild atopic dermatitis, moderate atopic dermatitis, severe atopic dermatitis according to severity, Th2 type atopic dermatitis, Th2 / Th17 mixed type atopic dermatitis, and Th2 / Th22 mixed type atopic dermatitis according to the type of inflammation.

[0009] [ka] compound 1

[0010] In a first aspect, the present invention provides the use of compound 1 or a pharmaceutically acceptable salt thereof in the manufacture of a drug for treating or preventing atopic dermatitis.

[0011] In one embodiment of the first aspect, the drug is administered topically. In another embodiment of the first aspect, the drug is administered orally.

[0012] In a second aspect, the present invention provides a method for treating or preventing atopic dermatitis, comprising administering to a subject in need thereof a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof.

[0013] In one embodiment of the second aspect, Compound 1 or a pharmaceutically acceptable salt thereof is administered topically. In another embodiment of the second aspect, Compound 1 or a pharmaceutically acceptable salt thereof is administered orally.

[0014] In a third aspect, the present invention provides a pharmaceutical composition for treating or preventing atopic dermatitis, comprising Compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable additive.

[0015] In one embodiment of the third aspect, the pharmaceutical composition is administered topically. In another embodiment of the third aspect, the pharmaceutical composition is administered orally. [[ID=X]]

[0016] <s In a fourth aspect, the present invention provides Compound 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of atopic dermatitis.

[0017] In one embodiment of the fourth aspect, Compound 1 or a pharmaceutically acceptable salt thereof is administered topically. In another embodiment of the fourth aspect, Compound 1 or a pharmaceutically acceptable salt thereof is administered orally. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] [Figure 1] FIGURE 1 is a graph showing the trend of changes in the body weight of mice from day 0 to day 26 in the topical administration experiment. [Figure 2] FIGURE 2 is a graph showing the ear thickness of mice from day 0 to day 26 in the topical administration experiment. [Figure 3A] FIGURE 3A is a photograph at a magnification of 20 times of a histological section of the ear of a mouse on day 26 in the topical administration experiment, stained with HE. [Figure 3B] Note: There seems to be an incorrect tag <s in the original text which is preserved as is in the translation as per the instructions. If this is an error, it may need to be corrected in the source text for a more accurate translation.Figure 3B is a graph showing the statistical analysis of the thickness of the epidermal tissue of the auricle of mice on day 26. [Figure 4A] Figure 4 shows inflammatory cell infiltration into the auricular tissue of mice on day 26 of the topical administration experiment. Figure 4A is a histopathological image. [Figure 4B] Figure 4B is a graph showing the score for inflammatory cell infiltration. [Figure 5] Figure 5 is a graph showing the thickness of the dorsal skin of mice on day 26 of the topical administration experiment. [Figure 6] Figure 6 is a graph showing the clinical scores of the dorsal skin of mice from day 0 to day 26 of the topical administration experiment. [Figure 7] Figure 7 is a line graph showing the percentage change in body weight of mice from day 0 to day 21 in the oral administration experiment. [Figure 8] Figure 8 is a line graph showing the percentage change in the thickness of the auricle of mice from day 0 to day 21 of the oral administration experiment. [Figure 9] Figure 9 is a line graph showing the clinical scores on the back of mice from day 0 to day 21 of the oral administration experiment. [Figure 10] Figure 10 shows a typical chart of HE-stained right ear tissue from mice in an oral administration experiment. [Figure 11] Figure 11 is a statistical graph showing the thickness of the epidermal layer in the right ear of mice in an oral administration experiment. [Figure 12] Figure 12 is a statistical graph showing inflammatory cell infiltration into the right ear tissue of mice in oral administration experiments. [Figure 13] Figure 13 is a representative chart showing the dermis of mice subjected to Masson staining in an oral administration experiment. [Figure 14] Figure 14 is a statistical graph showing the thickness of the dermis layer in the right ear of mice in an oral administration experiment. [Modes for carrying out the invention]

[0019] The present invention is further illustrated by the following embodiments illustrating the present invention, but is not limited thereto. [Examples]

[0020] Example 1: Inhibitory effect of Compound 1 on the kinase activity of TRK and DDR. Assay principle of the HTRF method The kinase phosphorylates the substrate, and the Eu-cryptate-labeled antibody binds to the phosphorylation site in the substrate. Streptavidin-XL665 binds to biotin in the substrate. When Eu and XL665 are in close proximity, Eu, acting as a donor, emits light (620 nm) after being excited by a light source (320 nm), transferring energy resonance to the nearby XL665 receptor, which then emits light (665 nm) after being excited. This specific signal is proportional to the phosphorylated substrate. When an inhibitor is added, the phosphorylation level is inhibited, the 665 nm emission becomes undetectable, and only the 620 nm emission is detected, allowing us to assess the level of inhibition of kinase activity by the compound.

[0021] TRKA Enzyme Activity Assay Compound 1 was serially diluted in DMSO to an initial concentration of 1 μM, and after 4-fold dilution, a total concentration of 10 was obtained. Isomorphic wells were set up for the assay. 25 nL of compound was transferred to a 384-well reaction plate (Greiner, catalog no. 784075) using a liquid handler (Echo, 665). A 2-fold concentrated kinase solution was prepared using the original kinase reaction buffer (5-fold concentrated buffer, MgCl 25 mM, DTT 1 mM, H2O, and MnCl 21 mM). 2.5 μL of TRKA (1.5 nM, Carna, 08-186) solution was transferred to the 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A mixed solution of 2x concentrated biotin-labeled tyrosine kinase substrate (1 μM) (Cisbio-PerkinElmer, catalog no. 61TK0BLE) and ATP (25 μM) was prepared using kinase reaction buffer. 2.5 μM of the substrate-ATP mixture was added to a reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixed solution of Eu-cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (cryptate) and 665 nm (XL665) were read using a BMG microplate reader. The ratio of fluorescence signals representing the degree of kinase activity in each well, i.e., (665nm / 620nm) × 10000, was calculated.

[0022] TRKB Enzyme Activity Assay Compound 1 was serially diluted in DMSO to an initial concentration of 1 μM, and after 4-fold dilution, a total concentration of 10 was obtained. Isomorphic wells were set up for the assay. 25 nL of compound was transferred to a 384-well reaction plate using a liquid handler (Echo, 665). A 2-fold concentrated kinase solution was prepared using the original kinase reaction buffer (5-fold concentrated buffer, MgCl 25 mM, DTT 1 mM, H2O, and MnCl 21 mM). 2.5 μL of TRKB (1.4 nM, Carna, 08-187) solution was transferred to the 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A mixed solution of 2-fold concentrated biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. A 2.5 μM mixture of substrate and ATP was added to the reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixture of Eu-cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (cryptate) and 665 nm (XL665) were read using a BMG microplate reader. The ratio of fluorescence signals representing the degree of kinase activity in each well, i.e., (665 nm / 620 nm) × 10000, was calculated.

[0023] TRKC Enzyme Activity Assay Compound 1 was serially diluted in DMSO to an initial concentration of 1 μM, and after 4-fold dilution, a total concentration of 10 was obtained. Isomorphic wells were set up for the assay. A 2-fold concentrated kinase solution was prepared using the original kinase reaction buffer (5-fold concentrated buffer, MgCl 25 mM, DTT 1 mM, H2O, and MnCl 21 mM). 2.5 μL of TRKC (1.5 nM, Carna, 08-197) solution was transferred to a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A mixed solution of 2-fold concentrated biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. 2.5 μM of the substrate-ATP mixture was added to the reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixed solution of Eu-cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (cryptate) and 665 nm (XL665) were read using a BMG microplate reader. The ratio of fluorescence signals representing the degree of kinase activity in each well, i.e., (665 nm / 620 nm) × 10000, was calculated.

[0024] DDR1 Enzyme Activity Assay Compound 1 was serially diluted in DMSO to an initial concentration of 1 μM, and after 4-fold dilution, a total concentration of 10 was obtained. Isomorphic wells were set up for the assay. A 2-fold concentrated kinase solution was prepared using the original kinase reaction buffer (5-fold concentrated buffer, MgCl 25 mM, DTT 1 mM, H2O, and MnCl 21 mM). 2.5 μL of DDR1 (2.7 nM, Carna, 08-113) solution was transferred to a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A mixed solution of 2-fold concentrated biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. 2.5 μM of the substrate-ATP mixture was added to the reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixed solution of Eu-cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (cryptate) and 665 nm (XL665) were read using a BMG microplate reader. The ratio of fluorescence signals representing the degree of kinase activity in each well, i.e., (665 nm / 620 nm) × 10000, was calculated.

[0025] DDR2 enzyme activity assay Compound 1 was serially diluted in DMSO to an initial concentration of 1 μM, and after 4-fold dilution, a total concentration of 10 was obtained. Isomorphic wells were set up for the assay. A 2-fold concentrated kinase solution was prepared using the original kinase reaction buffer (5-fold concentrated buffer, MgCl 25 mM, DTT 1 mM, H2O, and MnCl 21 mM). 2.5 μL of DDR2 (1.3 nM, Carna, 08-114) solution was transferred to a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A mixed solution of 2-fold concentrated biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. 2.5 μM of the substrate-ATP mixture was added to the reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixed solution of Eu-cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (cryptate) and 665 nm (XL665) were read using a BMG microplate reader. The ratio of fluorescence signals representing the degree of kinase activity in each well, i.e., (665 nm / 620 nm) × 10000, was calculated.

[0026] CSF1R enzyme activity assay Compound 1 was serially diluted in DMSO to an initial concentration of 10 μM, and a total concentration of 10 was obtained after a 4-fold dilution. Isomorphic wells were set up for the assay. A 2-fold concentrated kinase solution was prepared using the original kinase reaction buffer (5-fold concentrated buffer, MgCl 25 mM, DTT 1 mM, H2O, and MnCl 21 mM). 2.5 μL of CSF1R (1.3 nM, Carna, 08-114) solution was transferred to a 384-well reaction plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A mixed solution of 2-fold concentrated biotin-labeled tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared using the kinase reaction buffer. 2.5 μM of the substrate-ATP mixture was added to the reaction plate to initiate the reaction, and the plate was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with plate sealing film and incubated at 25°C for 40 minutes. 5 μL of a mixed solution of Eu-cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 was added to each well of the reaction plate. The plate was centrifuged at 1000 rpm for 1 minute and incubated at 25°C for 60 minutes. Fluorescence signals at 620 nm (cryptate) and 665 nm (XL665) were read using a BMG microplate reader. The ratio of fluorescence signals representing the degree of kinase activity in each well, i.e., (665 nm / 620 nm) × 10000, was calculated.

[0027] IC of compound 1 against TRKA, TRKB, TRKC, DDR1, DDR2, and CSF1R obtained from the above assay. 50 These are listed in Table 1.

[0028] Table 1. Inhibitory effect of Compound 1 on the kinase activity of TRK and DDR.

[0029] [Table 1] [Examples]

[0030] Example 2: Preparation of ointment of compound 1 The components of the formulation, based on the total weight of the formulation, are compound 1, which contains 8 mg / g of free base; DMSO, which accounts for 5% of the additives; PEG4000, which accounts for 20% of the additives; and PEG400, which accounts for 75% of the additives. PEG4000 and a portion of PEG400 were placed in a container, mixed, and heated in an 80°C water bath (oil bath) to dissolve. The mixture was continuously stirred during heating, and after thorough mixing, it was cooled to below 60°C to obtain mixed solution I. The weights of compound 1 powder and DMSO were measured and placed in the same container. Compound I was dissolved by sonication, the remaining PEG400 was added, and the mixture was thoroughly mixed to obtain mixed solution II. Mixed solution II was added to mixed solution I, the mixture was thoroughly mixed, placed in a container, cooled, and sealed to obtain the ointment (hereinafter also referred to as the "test drug"). [Examples]

[0031] Example 3: Method for evaluating the therapeutic effect of compound 1 ointment on a mouse model of OXA-induced atopic dermatitis. C57BL / 6 mice (female mice, weighing approximately 18.0 g, 6-8 weeks old) were obtained from Shanghai Southern Model Animal Biotechnology Co., Ltd., and divided equally based on body weight into several groups to receive the following treatments. Group 1: 1% oxazolone (abbreviated as OXA) + carrier (abbreviated as Group 1) Group 2: 1% OXA + Dexamethasone ointment (abbreviated as DEX) (abbreviated as Group 2) Group 3: 1% OXA + investigational drug (abbreviated as Group 3)

[0032] On day 0, the backs of the mice in each group were depilated using depilatory cream. 1% OXA was applied to the backs and right ears of the mice in each group. The 1% OXA solution was prepared by weighing 0.1 g of OXA, adding 10 mL of acetone solution, and mixing thoroughly until dissolved. After induction, the mice in each group were reared normally for 7 days. On days 7, 10, 12, 14, 17, 19, 21, and 24, 1% OXA was applied to the backs and right ears of the mice in each group at a rate of 40 mg / kg. The mice in Group 1 were coated with a carrier daily after induction with 1% OXA. Group 2 mice were administered DEX cream (0.75 mg / g dexamethasone acetate cream, NMPA drug approval number H44034170, China Resources Sanjiu Pharmaceutical Co., Ltd.) twice daily after induction with 1% OXA, applied at a dose of 11.25 mg / kg to the back and right ear of the mice. Group 3 mice were treated with the test drug (compound 1 at 8 mg / g) twice daily after induction with 1% OXA, at a dose of 100 mg / kg. The weight of the mice was measured three times a week, the thickness of the right ear was measured, and a clinical score was calculated for the back, based on the sum of three items: erythema (0-3 points), skin thickening (0-3 points), and scabbing (0-3 points). The mice were killed on day 26. Samples of the right ear of the mice were fixed with 4% PFA, embedded in paraffin, tissue sections were prepared, and HE staining was performed. The thickness of the epidermal layer was measured using a microscope, and inflammatory cell infiltration was scored.

[0033] result Table 2 and Figure 1 show the weight changes of mice in each group. The weight increase in Group 2, the group receiving the active drug DEX, was significantly less than that of Group 1, the carrier group, from day 14 to day 17 (p<0.05). The weight increase in Group 3, the group receiving the test drug, was significantly greater than that of Group 1, the carrier group, from day 14 to day 21. Statistical details are shown in Table 3.

[0034] Table 2 Average body weight of mice

[0035] [Table 2]

[0036] Table 3 Two-way ANOVA of statistical data on mouse body weight over time between groups

[0037] [Table 3] ns: No significant difference *:p<0.05 **:p<0.01 ***:p<0.001

[0038] Table 4 and Figure 2 show the increase in the thickness of the right ear auricle of mice from day 0 to day 26. Statistical analysis was performed on each group using two-way ANOVA. As shown in Table 5, a statistical difference occurred between group 2 (the DEX group) and group 1 (the carrier group) from day 12 to day 26, and a statistical difference also occurred between group 3 (the test drug group) and group 1 (the carrier group) from day 12 to day 26. Group 3 (the test drug group) was not statistically different from group 2 (the DEX group), indicating that both groups had a similar inhibitory effect on the increase in auricle thickness.

[0039] Table 4. Thickness of the auricle of a mouse (mm)

[0040] [Table 4]

[0041] Table 5 Two-way ANOVA of statistical data on the thickness of the auricle of mice over time between groups.

[0042] [Table 5] ns: No significant difference *:p<0.05

[0043] On day 26, pathological analysis was performed on mouse ear sections (Figure 3A). As shown in Figure 3B, the epidermal thickness was 40.54 μm ± 2.73 μm for group 1 (the carrier group), 28.34 μm ± 5.66 μm for group 2 (the DEX group), and 31.56 μm ± 2.78 μm for group 3 (the test drug group). Statistical analysis using one-way ANOVA showed that the epidermal thickness of group 2 (the active drug group) was significantly reduced compared to group 1 (the carrier group) (p<0.01), and the epidermal thickness of group 3 (the test drug group) was also significantly reduced (p<0.05). The drug efficacy was significant. Inflammatory cell infiltration is shown in Figure 4A, and its score is shown in Figure 4B (the rate of inflammatory cell infiltration into the dermis was evaluated as follows: <5% = 0 points, <33% = 1 point (mild), <67% = 2 points (moderate), >67% = 3 points (severe)). The degree of inflammatory cell infiltration was inhibited by the group receiving the active drug and the group receiving the test drug compared to the group receiving the carrier.

[0044] On day 26 of the experiment, mice in each group were euthanized, and dorsal skin was collected for thickness measurement. The values ​​for groups 1-3 were 0.76±0.02 mm, 0.51±0.01 mm, and 0.51±0.01 mm, respectively (Table 5). Statistical analysis using one-way ANOVA showed that groups 2 and 3 showed statistical differences compared to group 1, i.e., the carrier group (p<0.0001). There was no statistical difference between group 2 and group 3, indicating that both groups had similar efficacy in inhibiting dorsal skin thickness.

[0045] The clinical scores of the dorsal skin of mice from day 0 to day 26 are shown in Table 6 and Figure 6. According to the statistical analysis using two-way ANOVA, as shown in Table 7, a statistical difference occurred between group 2 (the dexamethasone group) and the carrier group from day 7 to day 19, and a statistical difference also occurred between group 3 (the test drug group) and the carrier group from day 7 to day 19. There was no statistical difference between group 2 (the dexamethasone group) and group 3 (the test drug group), indicating that both groups had similar efficacy in inhibiting clinical symptoms of the dorsal skin of mice.

[0046] Table 6 Clinical score of the back of a mouse

[0047] [Table 6]

[0048] Table 7 Two-way ANOVA of Statistical Data on Clinical Scores of Dorsal Skin of Mice Between Groups over Time

[0049] [Table 7] ns: No significant difference *:p<0.05

[0050] conclusion Based on measurements of auricle thickness, auricle epidermal layer thickness, inflammatory cell infiltration in auricle tissue, dorsal skin thickness, and clinical scores of the dorsal skin, compound 1 exhibits superior efficacy against symptoms in a mouse model of atopic dermatitis induced by 1% OXA. Compound 1 has similar efficacy to the effective drug dexamethasone cream, but with better weight gain in mice and better safety than the dexamethasone cream group. Glucocorticoids can cause skin atrophy, telangiectasia, hyperpigmentation, and secondary infections with long-term use, making them clinically unsuitable for long-term use and imposing strict time constraints. Compound 1 offers a safer and more effective new option for the treatment of atopic dermatitis. [Examples]

[0051] Example 4: Method for evaluating the therapeutic effect of orally administered compound 1 on a mouse model of OXA-induced atopic dermatitis. (1) Screening and grouping. A total of 60 female BALB / c mice, aged 6-8 weeks, were obtained from Shanghai Southern Model Animal Biotechnology Co., Ltd. and used as experimental animals for this project. The first grouping was performed before the start of the experiment. Mice were randomly assigned to a control group based on body weight (Group 1, 10 mice), and the remainder were designated as the model group. Eight days after sensitization (set as Day 0), the model group mice underwent a second grouping, and were randomly divided into five groups of 10 mice each (Group 2, Group 3, Group 4, Group 5, and Group 6) based on body weight and the thickness of the right ear auricle.

[0052] (2) Modeling of animals. The day of the first group division was set as day (-7). The backs of the mice were depilated (in an area of ​​approximately 2 cm x 3 cm) using depilatory cream. The backs and right ears of the model group mice were sensitized with an acetone solution of 1% OXA at ​​a concentration of 40 mg / kg, and the backs and right ears of the mice in Group 1 were coated with acetone. The 1% OXA solution was prepared by weighing 0.1 g of OXA powder, adding 10 mL of acetone solution, mixing well, and dissolving the powder. The backs and right ears of the mice in Group 1 were coated with acetone on days 0, 3, 5, 7, 10, 12, 14, 17, 19, and 21, and the backs and right ears of the mice in Groups 2, 3, 4, 5, and 6 were coated with an acetone solution of 1% OXA.

[0053] (3) Method of administration of the test substance. For 22 consecutive days from day 0 to day 21, mice in group 2 were given a carrier, mice in groups 3 to 5 were administered compound 1 intragastricly once daily for treatment at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg (based on free base), respectively, and mice in group 6 were administered abrocitinib, the active drug, intragastricly once daily at a dose of 17 mg / kg.

[0054] (4) Detection indicators. For 22 consecutive days from day 0 to day 21, the mice were weighed three times a week, the thickness of the right ear auricle was measured three times a week before administration, and clinical scores were obtained for the dorsal surface based on three aspects: erythema (0-3 points), skin thickening (0-3 points), and scabbing (0-3 points). The mice were killed on day 21. Skin from the right ear of the mice was set aside for pathological examination. Epidermal thickness and inflammatory cell infiltration were measured by HE staining, and dermal thickness was measured by Masson staining.

[0055] result Figure 7 shows the percentage change in body weight of mice during administration. Using two-way ANOVA, the model group (Group 2) showed a significant decrease in body weight compared to the control group (Group 1). **** p<0.0001, Table 8). Compared to Group 2, a model group administered with a carrier, compound 1 significantly inhibited weight loss when administered intragastricly at a dose of 3 mg / kg once daily for 22 consecutive days. ** (p<0.01, Table 8). When administered intragastricly at a dose of 10 mg / kg once daily for 22 consecutive days, compound 1 had no significant effect on weight change (p>0.05, Table 8). When administered intragastricly at a dose of 30 mg / kg once daily for 22 consecutive days, compound 1 had no significant effect on weight change (p>0.05, Table 8). Compared to Group 2, the model group administered the carrier, abrocitinib, the effective control drug, had no significant effect on weight change when administered intragastricly at a dose of 17 mg / kg once daily for 22 consecutive days (p>0.05, Table 8).

[0056] Analysis of the change rate of the body weight of mice

[0057]

Table 8

[0058] The change rate of the auricle thickness of the right ear of the mice during administration is shown in Fig. 8. Using two-way ANOVA, the model group of Group 2 had a significantly increased auricle thickness of the right ear compared to the control group of Group 1 ( **** p < 0.0001, Table 9). When compared with Group 2 of the model group administered with the carrier, Compound 1 could significantly reduce the auricle thickness of the right ear when administered intragastrically at a dose of 3 mg / kg once a day for 22 consecutive days ( * p < 0.05, Table 9). When administered intragastrically at a dose of 10 mg / kg once a day for 22 consecutive days, Compound 1 could significantly inhibit the auricle hypertrophy of the right ear ( **** p < 0.0001, Table 9). When administered intragastrically at a dose of 30 mg / kg once a day for 22 consecutive days, Compound 1 could significantly reduce the auricle thickness of the right ear ( **** p < 0.0001, Table 9), and significant inhibition continued from the 4th day of administration ( * p < 0.05, Table 10). When compared with Group 2 of the model group administered with the carrier, the control active drug, abrocitinib, could significantly inhibit the auricle hypertrophy of the right ear when administered intragastrically at a dose of 17 mg / kg once a day for 22 consecutive days ( **** p < 0.0001, Table 9).

[0059] Analysis of the change rate of the auricle thickness of the right ear of the mice in each experimental group

[0060]

Table 9

[0061] Table 10 Analysis of the rate of change in the thickness of the right ear auricle of mice at each time point.

[0062] [Table 10] Note: The percentage change in the thickness of the right auricle at each time point was compared using two-way ANOVA, and the p-values ​​were all compared with the p-values ​​for Group 2.

[0063] Figure 9 shows the clinical scores of the dorsal skin of mice during administration. Using two-way ANOVA, the model group (Group 2) showed a significant increase in the clinical scores of the dorsal skin compared to the control group (Group 1). **** p<0.0001, Table 11). Compared to Group 2, a model group administered with a carrier, compound 1 did not significantly inhibit the increase in dorsal skin clinical score when administered intragastricly at a dose of 3 mg / kg once daily for 22 consecutive days (p>0.05, Table 11). When administered intragastricly at a dose of 10 mg / kg once daily for 22 consecutive days, compound 1 was able to significantly reduce the dorsal skin clinical score. * p<0.05, Table 11). When administered intragastricly at a dose of 30 mg / kg once daily for 22 consecutive days, compound 1 significantly inhibited the increase in clinical scores on the dorsal skin. *** p<0.001, Table 11). Compared to Group 2, a model group administered a carrier, abrocitinib, the effective control drug, significantly reduced the clinical score of the dorsal skin when administered intragastricly once daily at a dose of 17 mg / kg for 22 consecutive days. * p<0.05, Table 11).

[0064] Table 11 Analysis of clinical scores of the dorsal skin of mice in each experimental group

[0065] [Table 11] Note: Clinical scores of the dorsal skin in each experimental group were compared using two-way ANOVA, and the p-values ​​were all compared to the p-value of Group 2.

[0066] Figure 10 shows HE-stained tissue sections of the right ears of mice. The thickness of the epidermal layer of the right ear was measured for each group of mice. Using one-way ANOVA, the thickness of the epidermal layer of the right ear in group 2 of the model group was significantly increased compared to group 1 ( **** p<0.0001, Figure 11, Table 12), infiltration of inflammatory cells such as eosinophils and macrophages was significantly increased. *** p<0.001, Figure 10, Figure 12, Table 13).

[0067] Compared to Group 2, a model group administered with a carrier, compound 1, when administered intragastricly at a dose of 3 mg / kg once daily for 22 consecutive days, significantly inhibited thickening of the epidermal layer of the right ear (p<0.05, Figure 11, Table 12) and significantly inhibited inflammatory cell infiltration. *** p<0.001, Figure 10, Figure 12, Table 13). When administered intragastricly at a dose of 10 mg / kg once daily for 22 consecutive days, compound 1 significantly reduced the thickness of the epidermal layer of the right ear ( * p<0.05, Figure 11, Table 12), significantly reduced inflammatory cell infiltration. *** p<0.001, Figure 10, Figure 12, Table 13). When administered intragastricly at a dose of 30 mg / kg once daily for 22 consecutive days, compound 1 significantly reduced the thickness of the epidermal layer of the right ear ( * p<0.05, Figure 11, Table 12), significantly reduced the number of inflammatory cells. *** p<0.001, Figure 10, Figure 12, Table 13).

[0068] Compared to Group 2, a model group administered a carrier, abrocitinib, the effective control drug, significantly reduced inflammatory cell infiltration when administered intragastricly at a dose of 17 mg / kg once daily for 22 consecutive days. *(p<0.001, Figures 10, 12, Table 13), it did not inhibit the thickening of the epidermal layer in the right ear (p>0.05, Figure 11, Table 12).

[0069] Table 12 Analysis of epidermal layer thickness of the right ear of mice in each experimental group

[0070] [Table 12] Note: The thickness of the epidermal layer of the right ear in each experimental group was compared using one-way ANOVA, and the p-values ​​were all compared to the p-value of group 2.

[0071] Table 13 Analysis of the amount of inflammatory cells in the right ear tissue of mice in each experimental group

[0072] [Table 13] Note: Data are expressed as mean ± standard error of the mean, and all significance levels are compared to the significance level of Group 2.

[0073] Figure 13 shows Masson-stained tissue sections of the right ears of mice. The thickness of the dermis of the right ears of mice from each group was measured. Using one-way ANOVA, the dermis thickness of Group 2 in the model group was significantly increased compared to Group 1. **** p<0.0001, Figure 14, Table 14).

[0074] Compared to Group 2, a model group administered a carrier, compound 1 significantly inhibited dermal thickening in the right ear when administered intragastricly at a dose of 3 mg / kg once daily for 22 consecutive days. **** p<0.0001, Figure 14, Table 14). When administered intragastricly at a dose of 10 mg / kg once daily for 22 consecutive days, compound 1 significantly reduced the thickness of the dermis layer in the right ear. **** p<0.0001, Figure 14, Table 14). When administered intragastricly at a dose of 30 mg / kg once daily for 22 consecutive days, compound 1 significantly reduced the thickness of the dermis layer in the right ear. ****p<0.0001, Figure 14, Table 14).

[0075] Compared to Group 2, a model group administered a carrier, abrocitinib, the effective control drug, significantly inhibited the increase in dermal thickness of the right ear when administered intragastricly at a dose of 17 mg / kg once daily for 22 consecutive days. *** p<0.001, Figure 14, Table 14).

[0076] Table 14 Analysis of dermal thickness of the right ear of mice in each experimental group

[0077] [Table 14] Note: The thickness of the dermis layer in the right ear of each experimental group was compared using one-way ANOVA, and the p-values ​​were all compared to the p-value of group 2.

[0078] conclusion Under experimental conditions, compound 1 administered orally at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg once daily for 22 consecutive days significantly inhibited thickening of the right ear auricle, epidermal layer thickening, and dermal layer thickening in OXA-induced atopic dermatitis model mice, and significantly reduced inflammatory cell infiltration. Clinical scores of OXA-induced atopic dermatitis in mice were significantly inhibited at doses of 10 mg / kg and 30 mg / kg. The compound had significant anti-inflammatory and symptom-inhibiting effects on atopic dermatitis.

[0079] The above-described examples and some embodiments should be interpreted, rather than limited, the invention as defined by the claims. Many modifications and combinations of the above-described features can be adopted without departing from the invention as described in the claims, for ease of understanding. All such modifications are included within the scope of the invention. All cited references are incorporated herein by reference in their entirety.

Claims

[Claim 1] The following structure in the manufacture of drugs for treating or preventing atopic dermatitis 【Chemistry 1】 The use of compound 1 having, or a pharmaceutically acceptable salt thereof.

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