Application of compound 1 in treating or preventing atopic dermatitis

Abstract

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

[0002] Atopic dermatitis (AD) is a common, chronic, recurring, inflammatory skin disease. Its primary symptoms are dry skin, chronic eczematous lesions, and intense itching. In moderate to severe cases, AD can even cause insomnia, anxiety, and depression, severely impacting quality of life. AD is a systemic autoimmune disease, often co-occurring with allergic asthma and allergic rhinitis.

[0003] AD treatment is mainly divided into two categories: topical medication and systemic treatment. Topical glucocorticoids are the first-line treatment for AD, but they should not be used long-term. Long-term use of glucocorticoids can cause local side effects such as skin atrophy, capillary dilation, pigmentation, and secondary infection. Long-term large-area application, especially when used on damaged skin, can also lead to hypothalamic-pituitary-adrenal (HPA) axis suppression, manifesting as Cushing's syndrome, steroid diabetes, osteoporosis, aggravated infection, induced or aggravated gastrointestinal ulcers, and induced psychiatric symptoms. Topical glucocorticoids have strict time limits. Systemic glucocorticoids should be avoided or used as little as possible. Although dupilumab and Jak inhibitors have changed the situation where some critically ill patients have no available treatment, nearly 50% of critically ill patients still do not achieve effective remission and have significant side effects. For example, dupilumab can cause conjunctivitis, and the Jak inhibitor ruxolitinib product label carries a black box warning, warning of serious side effects including: causing serious infections; developing non-melanoma skin cancer; forming blood clots; and developing thrombocytopenia, anemia, and neutropenia. There are still significant unmet clinical needs in the AD field, and there is an urgent need to develop safer and more effective drugs that can be used long-term.

[0004] Currently, the two marketed TRK inhibitors (larotrectinib and entrectinib) are both for patients with tumors harboring NTRK gene mutations, and there are no marketed drugs for atopic dermatitis. Three drugs are currently in clinical trials for atopic dermatitis: Cephalon's original TRKA inhibitor, Pegcantratinib, which is in Phase II clinical trials (discontinued), Benevolent AI's pan-TRK inhibitor BEN-2293 (which inhibits TRKA, TRKB, and TRKC), which is in Phase II clinical trials, and Pyrimad's pan-TRK inhibitor PBI-100, which is in Phase I clinical trials. Pegcantratinib's clinical studies for atopic dermatitis were terminated in 2010, while BEN-2293 and PBI-100 are still ongoing. This shows that inhibitors specifically targeting TRKA have a risk of failure in the treatment of atopic dermatitis. No DDR1 or DDR2 inhibitors have entered clinical trials for atopic dermatitis, and preclinical studies of DDRs for this indication are also blank. There are also no preclinical and clinical stage drugs for atopic dermatitis using CSF1R inhibitors.

[0005] Short-term exposure to the hapten OXA (oxazolone) induces contact allergic dermatitis in mice, primarily driven by Th1 cellular immunity. However, repeated exposure to the hapten OXA induces Th2-type skin inflammation, similar to human atopic dermatitis, characterized by localized erythema, scaling, and thickening. Histopathology reveals epidermal thickening and edema, along with extensive dermal infiltration of inflammatory cells such as CD4+ T lymphocytes and mast cells. This model is one of the most commonly used to study the pathogenesis of atopic dermatitis and screen for therapeutic drugs. The present invention utilizes this model to identify the therapeutic efficacy of compounds against atopic dermatitis.

[0006] CN113831344B discloses N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide. Summary of the Invention

[0007] The present invention relates to the use of N-(3-chloro-5-(trifluoromethyl)phenyl)-3-((6-(4-hydroxypiperidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)ethynyl)-2-methylbenzamide (also referred to as Compound 1 in the present invention) or a pharmaceutically acceptable salt thereof in the treatment or prevention of atopic dermatitis. In the present invention, atopic dermatitis includes infantile atopic dermatitis, childhood atopic dermatitis, and adolescent / adult atopic dermatitis classified by age characteristics; mild atopic dermatitis, moderate atopic dermatitis, and severe atopic dermatitis classified by severity; and Th2 type, Th2 / Th17 mixed type, and Th2 / Th22 mixed type atopic dermatitis based on inflammation type.

[0008]

[0009] In a first aspect, the present invention provides use of Compound 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing atopic dermatitis.

[0010] In one embodiment of the first aspect, the medicament is for external use. In another embodiment of the first aspect, the medicament is for oral use.

[0011] 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.

[0012] In one embodiment of the second aspect, the Compound 1 or a pharmaceutically acceptable salt thereof is administered externally. In another embodiment of the second aspect, the Compound 1 or a pharmaceutically acceptable salt thereof is administered orally.

[0013] 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 excipient.

[0014] In one embodiment of the third aspect, the pharmaceutical composition is for external use. In another embodiment of the third aspect, the pharmaceutical composition is for oral use.

[0015] In a fourth aspect, the present invention provides Compound 1 or a pharmaceutically acceptable salt thereof for use in treating or preventing atopic dermatitis.

[0016] In one embodiment of the fourth aspect, the Compound 1 or a pharmaceutically acceptable salt thereof is administered externally. In another embodiment of the fourth aspect, the Compound 1 or a pharmaceutically acceptable salt thereof is administered orally. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The graph shows the trend of changes in mouse body weight from day 0 to day 26 of the smear administration experiment.

[0018] Figure 2 The thickness of the mouse auricle on days 0 to 26 of the smear administration experiment is shown.

[0019] Figure 3A The HE staining picture of the mouse auricle tissue section on the 26th day of the smear administration experiment is shown, 20X magnification, Figure 3B Statistical analysis of mouse ear epidermal tissue thickness on day 26 is shown.

[0020] FIG4 shows the inflammatory cell infiltration in the auricle tissue of mice on the 26th day of the smear administration experiment. Figure 4A For tissue pathology pictures, Figure 4B Inflammatory cell infiltration was scored.

[0021] Figure 5 The thickness of the mouse back skin on day 26 of the topical administration experiment is shown.

[0022] Figure 6 The clinical scores of the mouse back skin on days 0-26 of the topical administration experiment are shown.

[0023] Figure 7 A line graph showing the body weight change rate of oral administration experimental mice from day 0 to day 21 is shown.

[0024] Figure 8 A line graph showing the change rate of auricle thickness in oral administration experimental mice from day 0 to day 21 is shown.

[0025] Figure 9 A line graph showing clinical scores on the back of mice given oral administration on days 0-21 is shown.

[0026] Figure 10 Shown are representative images of H&E staining of the right ear tissues of mice following oral administration.

[0027] Figure 11 Shown is a statistical graph of the epidermal thickness of the right ear of mice subjected to oral administration.

[0028] Figure 12 The graph shows the statistical graph of inflammatory cell infiltration in the right ear tissue of mice after oral administration.

[0029] Figure 12 Representative images of Masson staining of the dermis of mice following oral administration experiments are shown.

[0030] Figure 14 The graph shows the thickness of the dermis in the right ear of mice after oral administration. DETAILED DESCRIPTION

[0031] The present invention is further illustrated by the following examples which illustrate the invention but are not limited thereto.

[0032] Example 1: Inhibitory effect of compound 1 on TRK and DDR enzyme activities

[0033] HTRF method detection principle:

[0034] Kinases phosphorylate substrates, and antibodies labeled with Eu-cryptate (Eu-CryPtate) bind to the phosphorylation sites in the substrates. Streptavidin-XL665 binds to the substrate biotin. When Eu and XL665 are close to each other, Eu, as a donor, receives excitation from a light source (320 nm), emits emission light (620 nm) and resonantly transfers energy to the adjacent XL665 receptor, which then emits emission light (665 nm). The specific signal is proportional to the phosphorylated substrate. When an inhibitor is added, the phosphorylation level is suppressed, and the emission light at 620 nm, instead of 665 nm, can no longer be detected. This is used to evaluate the inhibitory effect of the compound on kinase activity.

[0035] TRKA enzyme activity assay:

[0036] Compound 1 was serially diluted in DMSO, starting at 1 μM. Fourfold dilutions were performed for a total of 10 dilutions, and assayed in duplicate. Using an Echo 665, 25 nL of compound was transferred to a 384-well plate (Cat. No. 784075, Greiner). A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl₂, 1 mM DTT, H₂O, 1 mM MnCl₂). 2.5 μL of TRKA (1.5 nM, 08-186, Carna) solution was transferred to the 384-well plate. The plate was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. Prepare a 2x mixture of biotinylated tyrosine kinase substrate (1 μM) (Cat. No. 61TK0BLE, Cisbio-PerkinElmer) and ATP (25 μM) in kinase reaction buffer. Add 2.5 μL of the substrate and ATP mixture to the reaction plate to initiate the reaction. Centrifuge at 1000 rpm for 60 seconds. Seal the plate with film and incubate at 25°C for 40 minutes. Add 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 to each well of the reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using a BMG microplate reader. Calculate the ratio of the fluorescence signals for each well (665 nm / 620 nm) x 10,000. The ratio indicates the degree of kinase activity.

[0037] TRKB enzyme activity assay:

[0038] Compound 1 was serially diluted in DMSO, starting at a 1 μM concentration. Fourfold dilutions were performed, with a total of 10 dilutions, and assayed in duplicate. 25 nL of compound was transferred to a 384-well plate using an Echo 665. A 2× kinase solution was prepared in 1× kinase reaction buffer (5× buffer, 5 mM MgCl₂, 1 mM DTT, H₂O, 1 mM MnCl₂). 2.5 μL of TRKB (1.4 nM, 08-187, Carna) solution was transferred to the 384-well plate. The solution was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotinylated tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared in kinase reaction buffer. 2.5 μL of the substrate and ATP mixture was added to the plate to initiate the reaction. The reaction was centrifuged at 1000 rpm for 60 seconds. Seal the plate with sealing film and incubate at 25°C for 40 minutes. Add 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 to each well of the reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using a BMG microplate reader. Calculate the ratio of the fluorescence signals for each well (665 nm / 620 nm) x 10,000. This ratio indicates the degree of kinase activity.

[0039] TRKC enzyme activity assay:

[0040] Compound 1 was serially diluted in DMSO, starting at a 1 μM concentration. Fourfold dilutions were performed, resulting in a total of 10 dilutions, and assayed in duplicate. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× buffer, 5 mM MgCl₂, 1 mM DTT, H₂O, 1 mM MnCl₂). 2.5 μL of TRKC (1.5 nM, 08-197, Carna) solution was transferred to a 384-well plate. The solution was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotinylated tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared in kinase reaction buffer. 2.5 μL of this mixture was added to the plate to initiate the reaction. The reaction was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with a film sealer and incubated at 25°C for 40 minutes. Add 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 to each well of the reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using a BMG microplate reader. Calculate the ratio of the fluorescence signals for each well (665 nm / 620 nm) x 10,000. This ratio indicates kinase activity.

[0041] DDR1 enzyme activity assay:

[0042] Compound 1 was serially diluted in DMSO, starting at a 1 μM concentration. Fourfold dilutions were performed, resulting in a total of 10 dilutions, and assayed in duplicate. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× buffer, 5 mM MgCl₂, 1 mM DTT, H₂O, 1 mM MnCl₂). 2.5 μL of DDR1 (2.7 nM, 08-113, Carna) solution was transferred to a 384-well plate. The solution was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotinylated tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared in kinase reaction buffer. 2.5 μL of this mixture was added to the plate to initiate the reaction. The reaction was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with a film sealer and incubated at 25°C for 40 minutes. Add 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 to each well of the reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using a BMG microplate reader. Calculate the ratio of the fluorescence signals for each well (665 nm / 620 nm) x 10,000. This ratio indicates kinase activity.

[0043] DDR2 enzyme activity assay:

[0044] Compound 1 was serially diluted in DMSO, starting at a 1 μM concentration. Fourfold dilutions were performed, resulting in a total of 10 dilutions, and assayed in duplicate. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× Buffer, 5 mM MgCl₂, 1 mM DTT, H₂O, 1 mM MnCl₂). 2.5 μL of DDR2 (1.3 nM, 08-114, Carna) solution was transferred to a 384-well plate. The solution was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotinylated tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared in kinase reaction buffer. 2.5 μL of this mixture was added to the plate to initiate the reaction. The reaction was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with a film sealer and incubated at 25°C for 40 minutes. Add 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 to each well of the reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using a BMG microplate reader. Calculate the ratio of the fluorescence signals for each well (665 nm / 620 nm) x 10,000. This ratio indicates kinase activity.

[0045] CSF1R enzyme activity assay:

[0046] Compound 1 was serially diluted in DMSO, starting at a 10 μM concentration. Fourfold dilutions were performed, resulting in a total of 10 dilutions, and assayed in duplicate. A 2× kinase solution was prepared using 1× kinase reaction buffer (5× buffer, 5 mM MgCl₂, 1 mM DTT, H₂O, 1 mM MnCl₂). 2.5 μL of CSF1R (1.3 nM, 08-114, Carna) solution was transferred to a 384-well plate. The reaction was centrifuged at 1000 rpm for 60 seconds and incubated at 25°C for 10 minutes. A 2× mixture of biotinylated tyrosine kinase substrate (1 μM) and ATP (25 μM) was prepared in kinase reaction buffer. 2.5 μL of this mixture was added to the plate to initiate the reaction. The reaction was centrifuged at 1000 rpm for 60 seconds. The plate was sealed with a film sealer and incubated at 25°C for 40 minutes. Add 5 μL of a mixture of Eu-Cryptate-labeled anti-tyrosine kinase antibody and streptavidin-XL665 to each well of the reaction plate. Centrifuge at 1000 rpm for 1 minute and incubate at 25°C for 60 minutes. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) using a BMG microplate reader. Calculate the ratio of the fluorescence signals for each well (665 nm / 620 nm) x 10,000. This ratio indicates kinase activity.

[0047] Table 1 lists the IC values of compound 1 for TRKA, TRKB, TRKC, DDR1, DDR2, and CSF1R obtained from the above assays. 50 .

[0048] Table 1: Inhibitory effect of compound 1 on TRK and DDR enzyme activities

[0049] <![CDATA[IC 50 (nM)]]> TRKA 4.397 TRKB 0.4325 TRKC 0.350 DDR1 0.7616 DDR2 37.93 CSF1R 14.71

[0050] Example 2: Preparation of Compound 1 Ointment

[0051] The formulation comprises 8 mg / g of free base Compound 1, 5% of the excipients by weight of DMSO, 20% of the excipients by weight of PEG4000, and 75% of the excipients by weight of PEG400. PEG4000 and a portion of PEG400 are mixed in a container and heated in an 80°C water bath (or oil bath) until melted. Stirring is continued during heating. After uniform stirring, the mixture is cooled to below 60°C to obtain a mixed solution I. Compound 1 powder and DMSO are weighed and placed in the same container. Compound 1 is dissolved by ultrasonication, and the remaining PEG400 is added and mixed to obtain a mixed solution II. Mixed solution II is added to mixed solution I, mixed thoroughly, filled, cooled, and sealed to obtain an ointment (hereinafter referred to as the "test drug").

[0052] Example 3: Evaluation of the therapeutic effect of compound 1 ointment on OXA-induced atopic dermatitis mouse model

[0053] method

[0054] C57BL / 6 mice (female, weighing approximately 18.0 g, 6-8 weeks old) were obtained from Shanghai Model Organisms Science Co., Ltd. and randomly divided into groups according to body weight to receive the following treatments:

[0055] Group 1: 1% oxazolone (abbreviated as OXA) + vehicle (referred to as Group 1);

[0056] Group 2: 1% OXA + dexamethasone ointment (abbreviated as DEX) (referred to as Group 2);

[0057] Group 3: 1% OXA + test drug (referred to as Group 3);

[0058] On day 0, the backs of mice in each group were depilated with depilatory cream, and the backs and right ears of mice in each group were smeared with 1% OXA. The 1% OXA solution was prepared by weighing 0.1 g of OXA powder, adding 10 mL of acetone solution, and mixing thoroughly to dissolve. After induction, the mice in each group were raised normally for 7 days, and then 1% OXA was applied to the back and right ear of each group of mice at 40 mg / kg on the 7th, 10th, 12th, 14th, 17th, 19th, 21st and 24th days. The mice in group 1 were induced with 1% OXA and then the vehicle was applied every day; the mice in group 2 were induced with 1% OXA and then DEX cream (compound dexamethasone acetate cream, 0.75 mg / g, national medicine standard H44034170, China Resources Sanjiu Pharmaceutical Co., Ltd.) was applied to the back and right ear of the treated mice at 11.25 mg / kg twice a day; the mice in group 3 were induced with 1% OXA and then the test drug (8 mg / g compound 1) was applied twice a day at 100 mg / kg. Mice were weighed and right ear thickness measured three times weekly. Back clinical scores were assessed for erythema (0-3 points), skin thickness (0-3 points), and scab formation (0-3 points). The clinical score was the sum of these three scores. Mice were sacrificed on day 26, and right ear specimens were fixed with 4% PFA, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Epidermal thickness was measured microscopically, and inflammatory cell infiltration was scored.

[0059] result

[0060] The body weight fluctuations of mice in each group are shown in Table 2 and Figure 1The weight gain of the positive drug DEX group in group 2 was significantly worse than that of the vehicle group in group 1 from day 14 to day 17 (p < 0.05). The weight gain of the test drug group in group 3 was significantly better than that of the vehicle group in group 1 from day 14 to day 21. The weight gain of the test drug group in group 3 was significantly better than that of the positive drug group DEX group from day 14 to day 21. The statistical details are shown in Table 3.

[0061] Table 2. Mean body weight of mice (g)

[0062]

[0063] Table 3. Statistical data of mouse weight among groups analyzed by Two-way Anova over time

[0064]

[0065] ns: no significant difference

[0066] *: p<0.05

[0067] **: p<0.01

[0068] ***: p < 0.001

[0069] The growth of the thickness of the right ear of mice from day 0 to day 26 is shown in Table 4 and Figure 2 Two-way ANOVA was used to analyze the groups. As shown in Table 5, from days 12 to 26, statistical differences were observed between the DEX group in group 2 and the vehicle group in group 1. From days 12 to 26, statistical differences were observed between the test drug group in group 3 and the vehicle group in group 1. No statistical differences were observed between the test drug group in group 3 and the DEX group in group 2, and both groups showed similar inhibitory effects on auricular thickness growth.

[0070] Table 4. Mouse auricle thickness (mm)

[0071]

[0072] Table 5. Statistical data of mouse auricle thickness among different groups using Two-way Anova analysis over time

[0073]

[0074] ns: no significant difference

[0075] *: p<0.05

[0076] On the 26th day, the mouse ear slices were sliced ( Figure 3A ) were used for pathological analysis. The values of epidermal thickness were as follows: 40.54 μm ± 2.73 μm in group 1 vehicle group, 28.34 ± 5.66 μm in group 2 DEX group, and 31.56 ± 2.78 μm in group 3 test drug group. Figure 3B As shown in Figure 2, according to One-way ANOVA statistical analysis, compared with the vehicle group in group 1, the epidermal thickness of the positive drug group in group 2 was significantly reduced (p<0.01), and the epidermal thickness of the test drug group in group 3 was also significantly reduced (p<0.05), indicating a significant therapeutic effect. Figure 4A As shown, the ratings are Figure 4B As shown in the figure (the infiltration of inflammatory cells in the dermis is less than 5% and is rated as 0 points, less than 33% is rated as 1 point for mild, less than 67% is rated as 2 points for moderate, and more than 67% is rated as 3 points for severe), the positive drug group and the test drug group inhibited the degree of infiltration of inflammatory cells relative to the vehicle group.

[0077] On the 26th day of the experiment, mice in each group were euthanized and the back skin thickness was measured. The values of groups 1 to 3 were 0.76±0.02mm, 0.51±0.01mm and 0.51±0.01mm, respectively. Figure 5 ), according to One-way ANOVA statistical analysis, compared with the vehicle group in group 1, groups 2 and 3 showed statistical differences (P<0.0001), and there was no statistical difference between groups 2 and 3, indicating that both groups had similar efficacy in inhibiting dorsal skin thickness.

[0078] The clinical scores of the back skin of mice from day 0 to day 26 are shown in Table 6 and Figure 6 Two-way ANOVA statistical analysis, as shown in Table 7, showed statistically significant differences between the dexamethasone group and the vehicle group in Group 2 from days 7 to 19, and between the test drug group and the vehicle group in Group 3 from days 7 to 19. There was no statistical difference between the dexamethasone group in Group 2 and the test drug group in Group 3, demonstrating similar efficacy in suppressing clinical symptoms on the back skin of mice.

[0079] Table 6. Clinical scores of mice back

[0080]

[0081] Table 7. Statistical data of the clinical scores of the mouse back skin among the groups analyzed by Two-way Anova over time

[0082]

[0083] ns: no significant difference

[0084] *: p<0.05

[0085] **: p<0.01

[0086] in conclusion

[0087] Based on the measurement of auricle thickness, auricle epidermal tissue thickness, auricle tissue inflammatory cell infiltration, back skin thickness and back skin clinical scores, compound 1 has excellent efficacy on the symptoms of 1% OXA-induced mouse atopic dermatitis model. Compound 1 has similar efficacy to the positive drug dexamethasone cream, and the weight gain of mice is better than that of the dexamethasone cream group, with better safety. Long-term use of glucocorticoids can cause side effects such as skin atrophy, capillary dilation, pigmentation, and secondary infection. They cannot be used long-term clinically and have strict restrictions on the duration of use. Compound 1 provides a safer and more effective new option for the treatment of atopic dermatitis.

[0088] Example 4: Method for evaluating the therapeutic effect of oral administration of Compound 1 on OXA-induced atopic dermatitis mouse model

[0089] (1) Screening and grouping: This project used a total of 60 female Balb / c mice aged 6 to 8 weeks, which were obtained from Shanghai South Model Organisms Technology Co., Ltd. The first grouping was performed before the start of the experiment. The mice were randomly divided into a control group (Group 1, 10 mice) according to their body weight, and the rest were model groups. On the 8th day after sensitization (denoted as Day 0), the model group mice were grouped for the second time and randomly divided into 5 groups (Group 2, Group 3, Group 4, Group 5, Group 6) according to their body weight and right ear auricle thickness, with 10 mice in each group.

[0090] (2) Animal modeling: The first day of grouping was designated as day (-7). The backs of mice were depilated with depilatory cream (area of approximately 2 cm × 3 cm). The backs and right ears of mice in the model group were sensitized with 1% OXA acetone solution at 40 mg / kg. Acetone was applied to the backs and right ears of mice in group 1. The 1% OXA solution was prepared by weighing 0.1 g of OXA powder, adding 10 mL of acetone solution, and mixing thoroughly to dissolve. On day 0, day 3, day 5, day 7, day 10, day 12, day 14, day 17, day 19, and day 21, acetone was applied to the backs and right ears of mice in group 1; 1% OXA acetone solution was applied to the backs and right ears of mice in groups 2, 3, 4, 5, and 6.

[0091] (3) Administration of the test substance: From day 0 to day 21, mice in group 2 were given the vehicle, and mice in groups 3 to 5 were given compound 1 at doses of 3 mpk, 10 mpk, and 30 mpk (calculated as free base), respectively, by gavage, once a day for treatment. Mice in group 6 were given the positive drug Abrocitinib at a dose of 17 mpk, by gavage, once a day for treatment.

[0092] (4) Detection indicators: From day 0 to day 21, mice were weighed three times a week for 22 consecutive days. The thickness of the right auricle of the mice was measured three times a week before administration. The back of the mice was scored for erythema (0-3 points), skin thickness (0-3 points), and scab (0-3 points). On day 21, the mice were sacrificed and the right ear skin was collected for pathological examination. The epidermal thickness and inflammatory cell infiltration were measured by H&E staining, and the dermal thickness was measured by Masson staining.

[0093] result

[0094] The weight change rate of mice during drug administration was as follows Figure 7 As shown in the results of a two-way ANOVA analysis, body weight in the model group 2 was significantly decreased compared to the control group 1 (****p<0.0001; Table 8). Compared with the vehicle-administered model group 2, compound 1 at a dose of 3 mpk administered orally once daily for 22 consecutive days significantly inhibited body weight loss (**p<0.01; Table 8). Compound 1 at a dose of 10 mpk administered orally once daily for 22 consecutive days had no significant effect on body weight change (p>0.05; Table 8). Compound 1 at a dose of 30 mpk administered orally once daily for 22 consecutive days had no effect on body weight change (p>0.05; Table 8). Compared with the vehicle-administered model group 2, the positive control drug abrocitinib at a dose of 17 mpk administered orally once daily for 22 consecutive days had no significant effect on body weight change (p>0.05; Table 8).

[0095] Table 8 Analysis of mouse body weight change rate

[0096]

[0097]

[0098] Note: Two-way ANOVA was used to compare the weight change rates of each experimental group, and the p values were compared with group 2.

[0099] The change rate of the thickness of the right ear of mice during drug administration was as follows Figure 8As shown in the results of the two-way ANOVA analysis, the thickness of the right auricle in the model group and group 2 was significantly increased compared with that in the control group (****p<0.0001; Table 9). Compared with the vehicle-administered model group 2, compound 1 at a dose of 3 mpk, administered orally once daily for 22 consecutive days, significantly reduced the thickness of the right auricle (*p<0.05; Table 9). Compound 1 at a dose of 10 mpk, administered orally once daily for 22 consecutive days, significantly inhibited the thickening of the right auricle (****p<0.0001; Table 9). Compound 1 at a dose of 30 mpk, administered orally once daily for 22 consecutive days, significantly reduced the thickness of the right auricle (****p<0.0001; Table 9), with the significant inhibition continuing from the fourth day of administration (*p<0.05; Table 10). Compared with the model group 2 administered with the vehicle, the positive control drug Abrocitinib at a dose of 17 mpk, administered orally once daily for 22 consecutive days, significantly inhibited the thickening of the right auricle (****p<0.0001; Table 9).

[0100] Table 9 Analysis of the change rate of the right ear auricle thickness of mice in each experimental group

[0101]

[0102] Note: Two-way ANOVA was used to compare the change rate of right ear auricle thickness among the experimental groups. The p values are compared with those in group 2.

[0103] Table 10 Analysis of the change rate of the auricle thickness of the right ear of mice at each time point

[0104]

[0105]

[0106] Note: Two-way ANOVA was used to compare the change rate of the right ear auricle thickness at each time point. The p values were compared with those in group 2.

[0107] Clinical scores of the back skin of mice during drug administration Figure 9As shown in the results of a two-way ANOVA analysis, the back skin clinical score in model group 2 was significantly increased compared to that in group 1 (****p<0.0001; Table 11). Compared with model group 2, which was given the vehicle, compound 1 at a dose of 3 mpk, administered orally once daily for 22 consecutive days, did not significantly inhibit the increase in back skin clinical score (p>0.05; Table 11). Compound 1 at a dose of 10 mpk, administered orally once daily for 22 consecutive days, significantly reduced the back skin clinical score (*p<0.05; Table 11). Compound 1 at a dose of 30 mpk, administered orally once daily for 22 consecutive days, significantly inhibited the increase in back skin clinical score (***p<0.001; Table 11). Compared with model group G2, which was given the vehicle, the positive control drug abrocitinib at a dose of 17 mpk, administered orally once daily for 22 consecutive days, significantly reduced the back skin clinical score (*p<0.05; Table 11).

[0108] Table 11 Clinical score analysis of the back skin of mice in each experimental group

[0109]

[0110] Note: Two-way ANOVA was used to compare the clinical scores of the back skin of each experimental group. The p values were compared with those of group 2.

[0111] The results of H&E staining of mouse right ear tissue sections are as follows Figure 10 As shown, the epidermal thickness of the right ear of mice in each group was measured, and one-way ANOVA analysis showed that the epidermal thickness of the right ear of the model group 2 was significantly increased compared with that of the group 1 (****p<0.0001; Figure 11 ; Table 12), the infiltration of inflammatory cells such as eosinophils and macrophages increased significantly (***p < 0.001; Figure 10 ; Figure 12 ; Table 13).

[0112] Compared with the model group 2 administered with the vehicle, compound 1 at a dose of 3 mpk, administered orally once daily for 22 consecutive days, significantly inhibited the thickening of the epidermis of the right ear (p < 0.05; Figure 11 ; Table 12 ) and inflammatory cell infiltration (***p < 0.001; Figure 10 ; Figure 12 ; Table 13); When compound 1 was administered orally once daily at a dose of 10 mpk for 22 consecutive days, the thickness of the epidermis of the right ear was significantly reduced (*p<0.05; Figure 11 ; Table 12), the accumulation of inflammatory cells was significantly reduced (***p<0.001; Figure 10 ; Figure 12; Table 13); Compound 1 at a dose of 30 mpk, once daily by gavage for 22 consecutive days, significantly reduced the thickness of the right ear epidermis (*p < 0.05; Figure 11 ; Table 12 ) and the number of inflammatory cells (***p < 0.001; Figure 10 ; Figure 12 ; Table 13).

[0113] Compared with the model group 2 administered with the vehicle, the positive control drug Abrocitinib at a dose of 17 mpk, administered orally once daily for 22 consecutive days, significantly reduced inflammatory cell infiltration (*p<0.001; Figure 10 ; Figure 12 ; Table 13 ), but failed to inhibit the thickening of the epidermal layer of the right ear (p>0.05; Figure 11 ; Table 12).

[0114] Table 12 Analysis of the thickness of the right ear epidermis of mice in each experimental group

[0115]

[0116] Note: One-way ANOVA was used to compare the thickness of the right ear epidermis of each experimental group. The p values were compared with those of group 2.

[0117] Table 13 Analysis of inflammatory cell content in the right ear tissue of mice in each experimental group

[0118]

[0119]

[0120] Note: Data are expressed as Mean ± SEM, and the significance levels are compared with G2.

[0121] The results of Masson staining of mouse right ear tissue sections are as follows Figure 13 As shown, the dermal thickness of the right ear of mice in each group was measured, and one-way ANOVA analysis showed that the dermal thickness of the right ear of the model group G2 was significantly increased compared with that of group 1 (****p<0.0001; Figure 14 ; Table 14).

[0122] Compared with the model group 2 administered with the vehicle, compound 1 at a dose of 3 mpk, administered orally once daily for 22 consecutive days, significantly inhibited the thickening of the right ear dermis (****p<0.0001; Figure 14 ; Table 14); When compound 1 was administered orally once daily at a dose of 10 mpk for 22 consecutive days, the thickness of the dermis of the right ear was significantly reduced (****p<0.0001; Figure 14; Table 14); Compound 1 at a dose of 30 mpk, once daily by gavage for 22 consecutive days, can significantly reduce the thickness of the dermis of the right ear (****p<0.0001; Figure 14 ; Table 14).

[0123] Compared with the model group 2 administered with the vehicle, the positive control drug Abrocitinib at a dose of 17 mpk, administered orally once daily for 22 consecutive days, significantly inhibited the increase in the thickness of the right ear dermis (***p<0.001; Figure 14 ; Table 14).

[0124] Table 14 Analysis of the thickness of the right ear dermis of mice in each experimental group

[0125]

[0126] Note: One-way ANOVA was used to compare the thickness of the right ear dermis in each experimental group. The p values were compared with those in group 2.

[0127] in conclusion

[0128] Under the conditions of this experiment, compound 1, administered orally once daily for 22 consecutive days, significantly inhibited auricular thickening, epidermal and dermal thickening, and reduced inflammatory cell infiltration in the right ear of mice in an OXA-induced atopic dermatitis model at doses of 3, 10, and 30 mpk. Doses of 10 and 30 mpk also significantly inhibited the clinical symptom score of OXA-induced atopic dermatitis in mice. This compound exhibits significant anti-inflammatory and atopic dermatitis symptom-suppressing effects.

[0129] The foregoing examples and description of certain embodiments should be considered as illustrative, rather than limiting, of the present invention as defined in the claims. As will be readily appreciated, many variations and combinations of the features described above may be employed without departing from the present invention as set forth in the claims. All such variations are intended to be within the scope of the present invention. All cited references are incorporated herein by reference in their entirety.

Claims

1. Use of Compound 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing atopic dermatitis, wherein Compound 1 has the following structure:

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