Medicine for treating atopic dermatitis

Abstract

The invention discloses an application of an STING inhibitor H-151 in preparation of a medicine for treating atopic dermatitis, the compound H-151 is applied to an atopic dermatitis model induced by oxazolone or 2, 4-dinitrochlorobenzene, and occurrence and development of atopic dermatitis can be effectively prevented. Therefore, the invention provides the medicine for preventing and treating atopic dermatitis, and the main active ingredients of the medicine comprise H-151 or pharmaceutically acceptable salts thereof. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a therapeutic drug for atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD) is a typical inflammatory skin disease, also known as atopic eczema. In addition to eczematous skin lesions, it is also accompanied by severe itching symptoms and is chronic and recurrent. Itching also affects people's quality of life by causing sleep disorders, anxiety and depression. AD is most common in children and occasionally affects adults. Its global incidence is increasing and is considered a public health problem. Moreover, children with AD are also at increased risk of developing food allergies, asthma, and allergic rhinitis. The pathogenesis of atopic dermatitis is not fully understood and is generally believed to be related to environmental factors, genetic susceptibility, skin barrier dysfunction, and immune abnormalities.

[0003] Topical glucocorticoids (hydrocortisone butyrate cream, betamethasone dipropionate cream, etc.) are first-line drugs for the treatment of AD, but long-term and large-area application of hormone drugs can easily lead to skin and systemic adverse reactions such as capillary dilation, wrinkles, skin atrophy, etc. Among topical non-hormonal drugs, calcineurin inhibitors tacrolimus and pimecrolimus are commonly used as first-line treatment drugs, but a burning sensation may occur when used. At present, new drugs for the treatment of atopic dermatitis mainly include phosphodiesterase 4 inhibitor crisaborole and JAK inhibitor ruxolitinib, but they have been observed to have local or systemic adverse reactions. Therefore, the market for atopic dermatitis treatment drugs is far from saturated, and there is an urgent need to find an active substance that can treat atopic dermatitis, is safe, stable and non-irritating.

[0004] H-151, also known as 1-(4-ethylphenyl)-3-(1H-indol-3-yl)urea (with the following structure), is an indole STING palmitoylation inhibitor (Nature 2018, 559, 269-273; EP3556362). It can inhibit the palmitoylation of STING to prevent the activation of STING, block the recruitment of downstream TANK binding kinase-1 (TBK1), the activation of interferon regulatory factor 3 (IRF3) and the release of type I interferon, inhibit the expression of inflammatory cytokines, and exert anti-inflammatory effects.

[0005]

[0006] Currently, H151 has been reported to be used to treat diseases such as psoriasis and acute kidney injury, but to date, there is no literature report on the therapeutic effect of compound H-151 on atopic dermatitis. Summary of the invention

[0007] Objective of the present invention: In order to solve the above technical problems, the present invention provides an application of a compound H-151 in the preparation of a drug for preventing and treating atopic dermatitis. The present invention proposes for the first time that compound H-151 can be used to prevent and treat atopic dermatitis with significant effects.

[0008] The present invention also provides a pharmaceutical composition with H-151 or a pharmaceutically acceptable salt thereof as a main active ingredient, and the pharmaceutical composition can be used for preventing or treating atopic dermatitis.

[0009] Technical solution: In order to achieve the above purpose, the structure of the compound H-151 described in the present invention is as follows:

[0010]

[0011] Among them, the compound H-151 has any one of the following medicinal effects: inhibiting the expression of inflammatory factors, reducing the epidermal thickness of skin lesions, and inhibiting the activation expression of proteins in the STING signaling pathway, and is used in the preparation of drugs for the treatment of atopic dermatitis.

[0012] Among them, the proteins of the STING signaling pathway include any one or more of IL23, IL6, IL10, IL1β, TNF-α, STING or ISG15.

[0013] The pharmaceutical composition for preventing or treating atopic dermatitis of the present invention comprises compound H-151 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

[0014] The invention relates to the use of the pharmaceutical composition for preventing or treating atopic dermatitis in the preparation of a drug for treating atopic dermatitis.

[0015] Among them, the drug dosage forms are ointments, creams, gels, external solutions, glues, plasters, rubber plasters, plasters, tinctures, lotions, paints, powders and freeze-dried powders.

[0016] Wherein, the drug administration method is skin application.

[0017] The skin includes tissue covering the surface of a human or animal body.

[0018] Use of the compound H-151 or the pharmaceutical composition of the present invention in the preparation of a drug for preventing or treating atopic dermatitis induced by oxazolone or 2,4-dinitrochlorobenzene.

[0019] Furthermore, the dosage of H-151 can vary according to the patient's condition or weight, disease severity, dosage form, administration route, and administration cycle, and can also be determined by a person skilled in the art. For example, the dosage of H151 can be 0.001 mg-100 mg / kg, and can be administered once or several times a day, or at intervals of multiple days.

[0020] Existing experiments have shown that H-151 has a significant inhibitory effect on STING in both humans and mice, showing high efficacy and selectivity as a small molecule antagonist. The above compound has not yet been used in the treatment of atopic dermatitis.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0022] The present invention proposes for the first time that H-151 can prevent and treat atopic dermatitis. In the present invention, H-151 can alleviate the atopic dermatitis phenotype after administration, reduce the epidermal thickness and mast cell count at the lesion site, and improve allergic reactions; at the same time, it can reduce the level of inflammatory factors, inhibit the expression of factors related to the STING signaling pathway, and has a significant therapeutic effect on atopic dermatitis.

[0023] The invention provides a new and effective technical means for preventing and treating atopic dermatitis and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow chart of the modeling of oxazolone-induced atopic dermatitis;

[0025] Figure 2 This is the appearance of the right ear of the mouse model of atopic dermatitis induced by oxazolone;

[0026] Figure 3 The swelling degree of the right ear of mice modeling atopic dermatitis induced by oxazolone (n=6 in each group);

[0027] Figure 4 The number of scratching in mice with atopic dermatitis induced by oxazolone (n=6 per group);

[0028] Figure 5 The weight of the 6 mm ear piece of the right ear of mice with oxazolone-induced atopic dermatitis (n=6 per group);

[0029] Figure 6 The weight of draining lymph nodes in mice with oxazolone-induced atopic dermatitis model (n=6 per group);

[0030] Figure 7The mRNA expression levels of genes related to the STING signaling pathway in the skin lesions of mice with atopic dermatitis induced by oxazolone (n=6), with GAPDH as the internal reference;

[0031] Figure 8 is the expression level of STING signaling pathway-related proteins in the skin lesions of the oxazolone-induced atopic dermatitis model (n=3 per group);

[0032] Fig. 9 Representative histological HE staining images of skin lesions in the oxazolone-induced atopic dermatitis model (n=6 in each group);

[0033] Fig.10 Toluidine blue staining images of skin lesion tissue sections of oxazolone-induced atopic dermatitis model (n=6 in each group);

[0034] Fig.11 The flowchart of the modeling of atopic dermatitis induced by 2,4-dinitrochlorobenzene;

[0035] Fig.12 This is the appearance of the right ear of a mouse model of atopic dermatitis induced by 2,4-dinitrochlorobenzene;

[0036] Fig.13 EASI score of the back of mice with atopic dermatitis induced by 2,4-dinitrochlorobenzene (n=6 in each group);

[0037] Fig.14 is the scratching frequency of mice with atopic dermatitis induced by 2,4-dinitrochlorobenzene (n=6 per group);

[0038] Fig.15 is the lymph node weight of mice with atopic dermatitis induced by 2,4-dinitrochlorobenzene (n=6 per group);

[0039] Fig.16 is the mRNA expression level of genes related to the STING signaling pathway in the skin lesions of mice with atopic dermatitis induced by 2,4-dinitrochlorobenzene (n=6), with GAPDH as the internal reference;

[0040] Fig.17 Representative histological HE staining images of skin lesions in the atopic dermatitis model induced by 2,4-dinitrochlorobenzene;

[0041] Fig.18 Toluidine blue staining images of skin lesion tissue sections in the atopic dermatitis model induced by 2,4-dinitrochlorobenzene. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0044] Example 1

[0045] Oxazolone (OXA)-induced atopic dermatitis model in mice

[0046] 1. Establishment of oxazolone-induced atopic dermatitis model in mice

[0047] 1) Experimental animals: Balb / C mice were purchased from Beijing Weitonglihua Company, female, 6-8 weeks old, with normal diet, drinking water and activities.

[0048] 2) Drug preparation: Acetone and olive oil were mixed in a volume ratio of 4:1 as solvent, and glucocorticoid betamethasone (CAS No.: 378-44-9) (purchased from Aladdin), phosphodiesterase 4 (PDE-4) inhibitor crisaborole (CAS No.: 906673-24-3) (purchased from Kangrui Pharmaceuticals) and JAK inhibitor ruxolitinib (CAS No.: 941678-49-5) (purchased from Borui Biopharmaceuticals) were selected as positive drugs, with doses of 0.025%, 2%, and 2% (W / V, g / ml), respectively. H-151 (prepared by referring to Nature, 2018, 559, 269-273) was set up with two dose groups of 2% and 4% (W / V, g / ml), all dissolved in the acetone olive oil mixed solution to prepare a working solution.

[0049] 3) Experimental groups:

[0050] The mice were randomly divided into the following 7 groups:

[0051] ①Blank control group (Control)

[0052] ②Model Group

[0053] ③H-151 low-dose group (2%)

[0054] ④H-151 high-dose group (4%)

[0055] ⑤ Betamethasone group (0.025%)

[0056] ⑥Ruxolitinib group (2%)

[0057] ⑦ Criborole group (2%)

[0058] 4) Animal modeling: The modeling process is as follows Figure 1As shown, on day -7, the skin barrier of the right ear of mice was destroyed by repeatedly peeling with medical tape except for the blank control group, and then 20 μL of 1% oxazolone solution was applied to both sides of the right ear of mice for initial sensitization. Starting from day 0, 20 μL of 0.2% oxazolone solution was applied to both sides of the right ear of mice every other day for stimulation, for a total of 10 times. At the same time, 20 μL of the corresponding group drug solution was applied to both sides of the right ear of each group once a day, and only 20 μL of acetone olive oil mixed solvent was applied to the blank control group, and the application time was the same as that of the other groups. All modeling and drug administration were performed after 1.5% isoflurane inhalation anesthesia.

[0059] 2. Photographing the right ear of mice and measuring ear thickness

[0060] On day -8 and 24 h after each application of 0.2% oxazolone, the thickness of the left and right ears of the mice were measured and photographed, and the swelling of the mice ears was calculated (ear swelling = (right ear thickness - left ear thickness) / left ear thickness).

[0061] like Figure 2 As shown in Figure 3, on the last day of modeling, the right ears of the mice in the model group had obvious redness, swelling and scabs, which were improved after the administration of H-151; the swelling of the right ears of the mice in the low-dose and high-dose H-151 administration groups was significantly reduced compared with the model group, and was equivalent to that of the crisaborole administration group.

[0062] 3. Mouse itch score measurement

[0063] Within 24 hours after the 10th stimulation with 0.2% oxazolone solution, the actions of the mice within 10 minutes were recorded with a video camera, and the number of times the mice scratched was recorded.

[0064] like Figure 4 As shown, the scratching frequency of mice in the low-dose and high-dose H-151 groups was significantly lower than that in the model group.

[0065] 4. Acquisition and preservation of right ear lesion tissue of mice

[0066] Within 24 hours after the 10th challenge with 0.2% oxazolone solution, the mice were killed by cervical dislocation, and the right ears of the mice were punched with a 6mm puncher and weighed. The right ear pieces were divided into two halves, one half was fixed with 4% paraformaldehyde for 24 hours and then embedded, and the other half was quickly frozen with liquid nitrogen and then stored in a -80℃ refrigerator.

[0067] like Figure 5 As shown, the right ear weights of the H-151 low-dose and high-dose groups were significantly lower than those of the model group and were comparable to those of the crisaborole-administered group.

[0068] 5. Acquisition and preservation of mouse lymphoid tissue

[0069] After the mice were killed by cervical dislocation, the draining lymph nodes were taken and weighed, then quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0070] like Figure 6 As shown, the lymphatic weight of mice in the low-dose and high-dose H-151 groups was slightly improved compared with the model group.

[0071] 6. Real-time fluorescence quantitative PCR detection of mRNA expression levels of genes related to the STING signaling pathway in skin lesions of atopic dermatitis model mice

[0072] 1) Collection of RNA from skin lesion tissue

[0073] (1) Take the skin lesion tissue out of the -80℃ freezer and place it in liquid nitrogen. Carefully and quickly cut 10 mg of tissue on an ice box and place it in a 2 mL QSP tube.

[0074] (2) Add 1 ml of Trizol, two small steel balls and one large steel ball, then place in a tissue homogenizer and grind for 30 seconds at 60 Hz for 3 times, and centrifuge at 13,000 r for 15 min at 4°C.

[0075] (3) Pipette the supernatant into a new 1.5 mL enzyme-free EP tube, add 1 / 5 volume of chloroform (i.e. 200 μL), shake up and down to mix, then place on ice for 10 min, and then centrifuge at 13,000 r for 15 min at 4°C.

[0076] (4) After centrifugation, the layered liquid is obtained. The upper layer is the aqueous phase containing RNA, the middle layer is the denatured protein, and the lower layer is the phenol solution. Pipette 200 μL of the aqueous layer into a new enzyme-free EP tube, add an equal amount of isopropanol, mix by inverting, place on ice for 10 minutes, and then centrifuge at 4°C for 10 minutes at 13,000 r.

[0077] (5) Pour off the supernatant and add 500 μL of 75% ethanol solution prepared with DEPC water to each tube. Gently flick the precipitate and centrifuge at 12,000 r / min at 4°C for 5 min.

[0078] (6) Pour off the supernatant, place the EP tube upside down on toilet paper, wait for the ethanol in the EP tube to evaporate completely at room temperature, add the corresponding volume of DEPC water according to the size of the precipitate to dissolve the precipitate, and then freeze it at -80℃.

[0079] 2) Reverse transcription to cDNA

[0080] (1) Take the RNA sample out of -80℃ and place it on ice to thaw, then centrifuge it at 12000r for 2min at 4℃. Use a pipette to mix, and then take 1μL of each RNA sample to detect the RNA concentration and OD260 / 280 ratio (absorbance ratio range: 1.8–2.0) using a Nano quantification instrument. Calculate the sample volume of 1000ng based on the measured concentration.

[0081] (2) Using a reverse transcription kit, according to the instructions, 1000 ng of RNA was taken for reverse transcription. The reaction system is shown in Tables 1 and 2 below:

[0082] Table 1 Reverse transcription step 1

[0083]

[0084] Program settings: 42°C, 2 minutes; 4°C, permanent

[0085] Table 2 Reverse transcription step 2

[0086]

[0087] Program settings: 37°C, 15 minutes; 85°C, 5 seconds; 4°C, permanent

[0088] (3) After the reaction, 20ul of cDNA was obtained. Add 80ul of RNase-free ddH2O, mix well, and store in a -20℃ refrigerator.

[0089] 3) qRT-PCR reaction system

[0090] The SYBR Green chimeric fluorescence method was used for qPCR reaction. The reaction system was configured according to Table 3.

[0091] The primer sequences are shown in Table 4.

[0092] Table 3 qRT-PCR reaction system

[0093]

[0094] The qRT-PCR instrument program settings were as follows: step one, 95°C, 30 s; step two, 95°C, 10 s, 60°C, 30 s, for a total of 40 cycles.

[0095] Table 4 Primer sequences

[0096]

[0097] Depend on Figure 7 It can be seen that H-151 administration can reduce the expression levels of STING and related inflammatory factors.

[0098] 7. Western blot was used to detect the expression levels of STING signaling pathway-related proteins in the skin lesions of atopic dermatitis model mice

[0099] 1) Extraction of skin lesion tissue protein

[0100] (1) Take RIPA protein lysis buffer, add 10 μL 100× phosphatase inhibitor and 10 μL 100× protease inhibitor per ml of protein lysis buffer and mix well.

[0101] (2) Take the skin lesion tissue out of the -80℃ freezer and place it in liquid nitrogen. Cut 10 mg of tissue on the ice box and place it in a 2 mL QSP tube. Add 2 small steel beads to each tube, then add 200 μL of RIPA lysis buffer, grind it in a tissue homogenizer at 60 Hz for 30 seconds for a total of 4 times, then centrifuge it at 13000 r for 15 min at 4℃, and aspirate the supernatant into a new 1.5 mL EP tube.

[0102] (3) After centrifugation, take out 2 μL of supernatant and add 38 μL of ddH2O, mix well and use for quantification. Transfer the remaining supernatant to another EP tube, add 5× loading buffer (1:4), mix well, place in a metal bath at 95°C for 10 min, centrifuge at room temperature for 2 min, and store the protein sample at -20°C.

[0103] Preparation of 5x loading buffer: 2g sodium dodecyl sulfate (SDS), 100mg bromophenol blue (BPB), 5ml 1M Tris-Hcl (PH=6.8), 10ml glycerol, 5ml β-mercaptoethanol (2-ME), mix well and use immediately.

[0104] (4) BCA protein quantification: Using the BCA protein concentration determination kit, according to the instructions, add the standard protein to each well of a 96-well plate in the gradient concentrations prepared as shown in Table 5, then add 200 μl of BCA reaction solution (solution A: solution B = 50:1). For the sample protein, take 20 μl / well and add 200 μl of BCA reaction solution. After incubation in a 37°C incubator for 30 minutes, use an enzyme reader to detect the absorbance of each well of the 96-well plate at 562 nm.

[0105] Table 5 BCA protein concentration test protein standard dilution scheme

[0106]

[0107] A standard curve is drawn based on the absorbance measured by the standard protein concentration, and then the concentration of each sample protein is calculated based on the standard curve, and the loading volume of each sample is calculated based on the protein concentration.

[0108] 2) Western blot detection

[0109] (1) Preparation of SDS-PAGE gel: prepare the lower gel according to the formula in Table 6, add it to the gel plate, use RO water or ethanol to seal the layer, wait for the lower gel to solidify, prepare the upper gel according to the formula in Table 7, pour out the RO water or ethanol liquid and turn it upside down, wait for the water / ethanol to evaporate completely, carefully add the upper gel (avoid bubbles), insert a comb, wait for the upper gel to solidify, soak the gel plate in 1X electrophoresis solution, and store it in a 4°C refrigerator.

[0110] Table 6 Recipes for separation gels of different concentrations

[0111]

[0112] Table 7 Concentrated gel formula

[0113]

[0114]

[0115] 10% APS is prepared for immediate use: 0.1g APS powder + 1ml ddH2O. After the powder is dissolved, mix well and place on ice for later use, keeping it away from light.

[0116] (2) Preparation of Western blot related solutions

[0117] ① Preparation of 10x electrophoresis buffer: Use a 1 / 10,000 precision electronic balance to weigh 72 g of glycine, 15.15 g of Tris Base, and 5 g of SDS, and dissolve them in 5000 mL of distilled water and store at room temperature.

[0118] ② Preparation of 1x running buffer: 900ml distilled water + 100ml 10x running buffer (9:1), mix well and use immediately.

[0119] ③ Preparation of 10x Transfer Buffer: Use a 1 / 10,000 precision electronic balance to weigh 14.4 g of glycine and 3.03 g of Tris, dissolve them in 1000 mL of distilled water, and store at room temperature.

[0120] ④ Preparation of 1x Transfer Buffer: 700 ml distilled water + 200 ml methanol + 100 ml 10x Transfer Buffer (7:2:1), mix well and pre-cool at 4°C.

[0121] ⑤ Preparation of 10x membrane washing solution (TBS): Use a 1 / 10,000 precision electronic balance to weigh 40.025g NaCl and 12.1g Tris Base and dissolve them in 5000mL distilled water. Place the mixture on an electric stirrer and heat to stir until dissolved. Use a pH meter to detect the pH value and use concentrated HCL to adjust the pH to 7.6.

[0122] ⑥ Preparation of 1x membrane washing solution (TBST): 900 ml distilled water + 100 ml 10x membrane washing solution + 1 ml Tween 20 (0.1%), store at room temperature.

[0123] ⑦ Preparation of 5% BSA blocking solution: Use a 1 / 10,000 precision electronic balance to weigh 2.5 g of BSA powder and dissolve it in 50 mL of ddH2O.

[0124] ⑧ Primary antibody and secondary antibody diluent: 5% BSA. Use a 1 / 10,000 precision electronic balance to weigh 2.5 g of BSA powder and dissolve it in 50 mL of ddH2O.

[0125] The primary and secondary antibodies used in the experiment are shown in Table 8.

[0126] Table 8 Primary and secondary antibodies used in the experiment

[0127]

[0128]

[0129] (3) Electrophoresis: Take out the gel from the 4℃ refrigerator, place the gel plate correctly in the electrophoresis tank, fill it with 1x electrophoresis solution, pull out the comb vertically and slowly, and then use a 200μl pipette to blow out the broken gel in the lane. Slowly add the protein sample to the lane in order, fill it with electrophoresis solution, adjust the voltage to 70V, change the voltage to 110V after 30min, and run for another 1.5h.

[0130] (4) Transfer to mold: Cut the PVDF membrane into 6cm x 9cm pieces and activate it in methanol for 1 minute. Transfer it to the pre-cooled transfer solution prepared in advance. Remove the rubber sheet and gently pry the thin sheet apart. Cut off the upper rubber layer and the rubber below the loading blue line. Place them in the order of black side of the transfer clip - sponge - filter paper - glue - PVDF membrane - filter paper - sponge - white side of the transfer clip. Be careful not to have bubbles between the glue and the PVDF membrane. Clamp the transfer clip and place it in the transfer tank. Finally, place the transfer tank in a container filled with crushed ice. Transfer the membrane at a constant current of 220mA for 60 minutes.

[0131] (5) Blocking: After the transfer is completed, the PVDF membrane is taken out and placed with the front side facing up in 5% BSA and placed in a shaker at room temperature for 1.5 h.

[0132] (6) Primary antibody incubation: Cut the PVDF membrane according to the target band position, place it in the primary antibody incubation box, pour in the primary antibody (1:1000, 5% BSA + 0.1% antibacterial preservative (Biyuntian, ST855)), and place it in a shaker at 4°C overnight.

[0133] (7) Secondary antibody incubation: The primary antibody was recovered, and the membrane was washed three times with TBST, each time for 8 min, and incubated with secondary antibody (1:10,000, prepared with 5% BSA + 0.1% antibacterial agent) for 1.5 h, and the membrane was washed three times with TBST, each time for 8 min.

[0134] (8) Exposure: Take out the membrane, gently press with a paper towel to absorb the moisture, add exposure solution (prepare ECL luminescent solution according to A solution: B solution = 1:1, store at room temperature away from light, and use it immediately after preparation), soak for 1 min, then take out and put it into the exposure instrument for exposure and development.

[0135] The experimental results are as follows Figure 8 It can be seen that the expression of STING protein in the lesions of atopic dermatitis mice increased, and H-151 administration mainly exerted its effect by affecting the NF-κB pathway

[0136] 8. HE staining of mouse skin lesion tissue to detect epidermal thickness

[0137] 1) Dewaxing of paraffin sections: sequentially place the sections into environmentally friendly dewaxing solution (purchased from Sewell Biotechnology, CR2207416) for 20 min - environmentally friendly dewaxing solution for 20 min - anhydrous ethanol for 5 min - anhydrous ethanol for 5 min - 75% alcohol for 5 min, and wash with tap water.

[0138] 2) Rewarming and fixation of frozen sections: Take the frozen sections out of the -20° refrigerator and restore them to room temperature. Fix them with tissue fixative for 15 minutes and then rinse with running water.

[0139] 3) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0140] 4) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin solution for 5 min.

[0141] 5) Dehydration and sealing: Dehydrate the slices in anhydrous ethanol for 3 times, 5 min each time - xylene for 5 min - xylene for 5 min to make them transparent, and seal the slices with neutral gum.

[0142] 6) Take pictures using a digital slide scanner.

[0143] like Fig. 9 As shown, both low-dose and high-dose administration of H-151 can significantly reduce the epidermal thickness of the lesion area in atopic dermatitis mice.

[0144] 9. Detection of mast cell count by toluidine blue staining of mouse skin lesions

[0145] 1) Dewax the paraffin sections to water: xylene 5 min - xylene 5 min - xylene 5 min - anhydrous ethanol 1 min - 95% ethanol 1 min - 75% ethanol 1 min - rinse with tap water for a few seconds.

[0146] 2) Toluidine blue staining: Add toluidine blue staining solution for 20-30 minutes, rinse with running water to wash away excess staining solution.

[0147] 3) Use 95% ethanol for color separation and control the color separation effect under the microscope.

[0148] 4) Dehydration and transparency: anhydrous ethanol for 1 min, xylene 3 times, 1-2 min each time, and neutral gum for sealing.

[0149] 5) Take pictures using a panoramic tissue slice scanner.

[0150] like Fig.10 As shown, both low-dose and high-dose administration of H-151 can reduce the number of mast cells in the lesions of atopic dermatitis mice and improve allergic reactions.

[0151] Example 2

[0152] 2,4-Dinitrochlorobenzene (DNCB)-induced atopic dermatitis model in mice

[0153] 1. Establishment of a mouse atopic dermatitis model induced by 2,4-dinitrochlorobenzene

[0154] 1) Experimental animals: Balb / C mice were purchased from Beijing Weitonglihua Company, female, 6-8 weeks old, with normal diet, drinking water and activities.

[0155] 2) Drug preparation: Acetone and olive oil were mixed in a ratio of 4:1 as solvent, and glucocorticoid betamethasone (purchased from Aladdin), phosphodiesterase 4 (PDE-4) inhibitor crisaborole (purchased from Kangrui Pharmaceuticals) and JAK inhibitor ruxolitinib (purchased from Borui Biopharmaceuticals) were selected as positive drugs, with doses of 0.25%, 2%, and 2%, respectively. H-151 (prepared by referring to Nature, 2018, 559, 269-273) was set up in two dose groups of 2% and 5%, and both were dissolved in the acetone olive oil mixed solution to prepare the working solution.

[0156] 3) Experimental groups:

[0157] The mice were randomly divided into the following 7 groups:

[0158] ⑧Blank control group (Control)

[0159] ⑨Model Group

[0160] ⑩H-151 low dose group (2%)

[0161] H-151 high-dose group (5%)

[0162] Betamethasone group (0.025%)

[0163] Ruxolitinib group (2%)

[0164] Criborole group (2%)

[0165] 4) Animal modeling: The modeling process is as follows Fig.11 As shown, on day -6, the mice were shaved on the back (first with a razor, then with a depilatory cream). On day -5, the mice were grouped according to their weight and hair removal status. On day -4, the skin on the back of the mice was repeatedly stripped with medical tape to destroy the skin barrier except for the blank control group, and then 200 μL of 1% DNCB solution was applied to the back of the mice for initial sensitization. Starting from day 0, 200 μL of 0.5% DNCB solution was applied to the back of the mice every other day for stimulation, for a total of 7 times. At the same time, 200ul of the corresponding drug solution was applied to the back of the mice every day, and the blank control group was only coated with 200 μL of acetone olive oil mixed solvent, and the application time was the same as that of the other groups. All modeling and drug administration were performed after 1.5% isoflurane inhalation anesthesia.

[0166] 2. Back photography of mice and EASI scoring

[0167] On day -5 and 24 h after each 0.5% DNCB application, the backs of the mice were scored by EASI and photographed. As shown in Table 9, the scores were as follows: 0 (none), 1 (mild), 2 (moderate) and 3 (severe) according to the severity. The total score is 15 points. Fig.12 As shown in Figure 13, on the last day of modeling, the model group mice had obvious redness, swelling and scabs on the back, which were improved after H-151 administration; the EASI scores of mice in the low-dose and high-dose H-151 administration groups were significantly lower than those in the model group and were comparable to those in the crisaborole administration group.

[0168] Table 9 Specific scoring requirements

[0169]

[0170]

[0171] The specific scoring principle for each item is: 0 means no such sign, 1 means the sign exists but needs careful observation, 2 means the sign can be seen immediately, and 3 means the sign is very obvious.

[0172] 3. Mouse itch score measurement

[0173] The number of scratching times of mice within 10 min was recorded within 24 hours after the seventh stimulation with 0.5% DNCB solution.

[0174] like Fig.14 As shown, the scratching frequency of mice in the low-dose and high-dose H-151 groups was slightly lower than that in the model group.

[0175] 4. Acquisition and preservation of mouse back skin lesion tissue

[0176] Within 24 hours after the seventh stimulation with 0.5% DNCB solution, the mice were killed by cervical dislocation, and the lesional skin on the back of the mice was cut off. Half of it was fixed with 4% paraformaldehyde for 24 hours and then embedded, and the other half was quickly frozen with liquid nitrogen and then stored in a -80℃ refrigerator.

[0177] 5. Acquisition and preservation of mouse lymphoid tissue

[0178] After the animals were killed by cervical dislocation, the draining lymph nodes were obtained and weighed, then quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0179] like Fig.15 As shown, compared with the model group, the lymph node weight of mice in the low-dose and high-dose H-151 groups was significantly improved.

[0180] 6. Real-time fluorescence quantitative PCR detection of mRNA expression levels of genes related to the STING signaling pathway in skin lesions of atopic dermatitis model mice

[0181] The specific steps are detailed in Example 1. Total RNA was extracted using Trizol reagent, and the obtained mRNA was reverse transcribed into cDNA using HiScript III RT SuperMix kit, and RT-PCR was used to detect the mRNA expression level of related genes.

[0182] Depend on Fig.16 It can be seen that H-151 administration can reduce the expression level of inflammatory factors related to the STING pathway.

[0183] 7. HE staining of mouse skin lesion tissue to detect epidermal thickness

[0184] The skin specimens from the back of the mice were fixed in 4% paraformaldehyde and embedded in paraffin. They were stained with hematoxylin and eosin (HE) and filmed with a digital slide scanner. The results were as follows: Fig.17 As shown, both low-dose and high-dose administration of H-151 can significantly reduce the epidermal thickness of the lesion area in atopic dermatitis mice.

[0185] 8. Detection of mast cell count by toluidine blue staining of mouse skin lesions

[0186] The skin specimens from the back of the mice were fixed in 4% paraformaldehyde, embedded in paraffin, stained with toluidine blue, and filmed with a digital slice scanner. Fig.18 As shown, both low-dose and high-dose administration of H-151 can reduce the number of mast cells in the lesions of atopic dermatitis mice and improve allergic reactions.

[0187] Example 3

[0188] Statistical analysis

[0189] All data in the present invention represent the mean ± standard error (mean + SEM) of at least three independent experiments. All data were plotted and statistically analyzed using GraphPad Prism and ImageJ software. One-way or two-way analysis of variance (ANOVA) or unpaired two-tailed Student's t test were used to statistically analyze the data. P < 0.05 was considered to be statistically significant. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

[0190] In summary, H-151 administration can alleviate the phenotype of atopic dermatitis, reduce the epidermal thickness and the number of mast cells in the lesions, and improve allergic reactions; it can reduce the level of inflammatory factors, inhibit the expression of factors related to the STING signaling pathway, and has a significant therapeutic effect on atopic dermatitis.

Claims

1. Use of STING inhibitor H-151 in the preparation of a drug for treating atopic dermatitis. The structure of the STING inhibitor H-151 is shown below:

2. The use according to claim 1, characterized in that: The compound H-151 has any one of the following medicinal effects: inhibiting the expression of inflammatory factors, reducing the epidermal thickness of skin lesions, and inhibiting the activation expression of proteins in the STING signaling pathway, and is used in the preparation of drugs for treating atopic dermatitis.

3. The application according to claim 2, characterized in that: The proteins of the STING signaling pathway include any one or more of IL23, IL6, IL10, IL1β, TNF-α, STING or ISG15.

4. A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that: The invention comprises compound H-151 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

5. Use of the pharmaceutical composition for preventing or treating atopic dermatitis according to claim 4 in the preparation of a drug for treating atopic dermatitis.

6. The use according to claim 5, characterized in that: The drug dosage forms are ointments, creams, gels, external solutions, glues, plasters, rubber plasters, plasters, tinctures, lotions, paints, powders and freeze-dried powders.

7. The use according to claim 1 or 5, characterized in that: The drug administration method is skin application.

8. The use according to claim 7, characterized in that: The skin includes tissue covering the surface of the human or animal body.

9. Use of H-151 according to claim 1 or the pharmaceutical composition according to claim 4 in the preparation of a drug for preventing or treating atopic dermatitis induced by oxazolone or 2,4-dinitrochlorobenzene.

10. The use according to claim 1 or 5 or 9, characterized in that: The dosage of H-151 is 0.001 mg-100 mg / kg, and the administration can be completed once or several times a day, or at intervals of multiple days.

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