Application of C-176 in preparation of medicine for treating vitiligo

Abstract

The invention relates to an application of C-176 or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating leucoderma. The chemical structural formula of the C-176 is as follows: # imgabs0. The invention provides an application of the C-176 or the pharmaceutically acceptable salt thereof in preparation of medicines for treating leucoderma. Experiments prove that the C-176 has the effect of treating the vitiligo, and a new effective treatment choice is provided for the vitiligo.

Description

Technical Field

[0001] The present invention relates to a new use of C-176, and in particular to the use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating vitiligo. Background Art

[0002] Vitiligo is an acquired depigmentation disease with extremely complex etiology, characterized by the destruction and loss of function of epidermal melanocytes. Epidemiological surveys show that the incidence of vitiligo in the world's population is about 1%. In recent years, the incidence of vitiligo has increased year by year, and it is showing a trend of younger age, and white spots are more common in the head and face, seriously affecting the physical and mental health of patients. There is a lot of evidence that autoimmunity plays an important role in the progression of the disease, but the potential mechanism of vitiligo has not been fully elucidated. At present, clinical treatment drugs are mainly some non-targeted immunosuppressants, such as corticosteroids, calcineurin inhibitors, etc. These drugs are only effective for some patients and have large side effects. Oxidative stress is generally considered to be the initiating factor of vitiligo. Under stress conditions, melanocytes produce and release damage-related pattern molecules (inflammatory factors IFN-γ, IL-6, etc.), activating autoimmune responses to destroy melanocytes.

[0003] C-176 (CAS: 314054-00-7), chemical name N-(4-iodophenyl)-5-nitro-2-furancarboxamide, molecular formula C 11 H7IN2O4, molecular weight is 358.09, it is a light yellow solid; its chemical structure is as follows:

[0004]

[0005] C-176 is an inhibitor of the interferon gene stimulator pathway (STING signaling pathway) with selectivity and blood-brain barrier permeability. C-176 can inhibit the production of IFN-β mediated by STING and has anti-inflammatory activity. However, there are no reports on the use of C-176 in the treatment of vitiligo in domestic and foreign research. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the existing drugs for treating vitiligo are only effective for a part of patients and have large side effects. The present invention provides the use of a pharmaceutically acceptable salt of C-176 in the preparation of drugs for treating vitiligo and vitiligo complications. Furthermore, the present invention also provides the use of a pharmaceutical composition for the preparation of drugs for treating vitiligo and vitiligo complications, the pharmaceutical composition containing a therapeutically effective amount of C-176 or a pharmaceutically acceptable salt thereof.

[0007] The present invention has proved through a large number of modern pharmacological scientific studies and in vivo pharmacodynamic experiments that C-176 can promote the pigmentation restoration of the skin lesion area of ​​vitiligo mice and inhibit the synthesis of inflammatory factors IFN-γ and IL-6 in the skin lesion area of ​​vitiligo mice.

[0008] Beneficial effects: The present invention provides the use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a treatment for vitiligo. Experiments have shown that C-176 has the efficacy of treating vitiligo, providing a new and effective treatment option for vitiligo. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is an immunoblot of inflammatory factors IFN-γ, IL-6 and their upstream regulatory signaling pathway proteins in human epidermal melanocytes. Figure 1 A is a protein immunoblot. Figure 1 B is a bar graph of relative protein expression.

[0010] Figure 1 A. Figure 1 B shows that compared with the blank control group, there was no significant change in the c-GAS, STING, IFN-γ, and IL-6 proteins in the C-176 group, but the expression of c-GAS, STING, IFN-γ, and IL-6 proteins in the H2O2 group was significantly increased ( * P<0.05, ** P<0.01). Compared with the H2O2 group, the C-176+H2O2 group significantly inhibited the expression of c-GAS, STING, IFN-γ, and IL-6 proteins in melanocytes ( # P<0.05, ## P<0.01).

[0011] Figure 2 These are light microscopic photos of skin tissue in the lesion area of ​​the vitiligo mouse model after melanin ammonia silver staining. The left picture: blank control group; the middle picture: vitiligo model group; the right picture: C-176 treatment based on the vitiligo model.

[0012] The left picture shows that there is a large amount of melanin in the skin tissue of the blank control group. The middle picture shows that the melanin in the skin tissue of the vitiligo model group is significantly reduced; the right picture shows that after C-176 treatment based on the vitiligo model, the amount of melanin in the skin tissue increases. This shows that C-176 can significantly promote the restoration of pigmentation in the skin lesions of vitiligo mice.

[0013] Figure 3This is a bar graph of the relative content of melanin in the skin tissue of the vitiligo mouse model. Compared with the blank control group, the epidermal melanin in the skin of the vitiligo model mice was significantly reduced (**P<0.01). Compared with the vitiligo model group, the C-176 group can significantly promote the restoration of pigmentation in the skin lesions of the vitiligo model mice ( # P<0.05).

[0014] Figure 4 This is a bar graph of the relative expression of inflammatory factors IFN-γmRNA and IL-6mRNA in the skin lesions of vitiligo mouse models. Figure 4 A is a bar graph of the relative expression of IFN-γ mRNA. Figure 4 B is a bar graph of the relative expression of IL-6 mRNA.

[0015] Compared with the blank control group, the expressions of IFN-γmRNA and IL-6mRNA in the back skin tissue of mice in the vitiligo model group were significantly increased ( ** P<0.01). Compared with the vitiligo model group, C-176 significantly inhibited the expression of IFN-γmRNA and IL-6mRNA in the back skin tissue of mice in the vitiligo model group ( ## P<0.01).

[0016] Figure 5 This is an immunoblot of inflammatory factors IFN-γ and IL-6 in the skin lesions of a vitiligo mouse model. Figure 5 A is a protein immunoblot. Figure 5 B is a bar graph of relative protein expression.

[0017] Compared with the blank control group, the expression of IFN-γ and IL-6 in the back skin tissue of mice in the vitiligo model group was significantly increased ( ** P<0.01). Compared with the vitiligo model group, C-176 significantly inhibited the expression of IFN-γ and IL-6 in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ## P<0.01). DETAILED DESCRIPTION

[0018] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.

[0019] Below are some pharmacodynamic tests and results of the present invention:

[0020] Part I: Effects of C-176 on the expression of inflammatory factors in the melanocyte oxidative stress model

[0021] Human epidermal melanocytes were digested and counted, then seeded in six-well plates and placed in a constant temperature incubator at 37°C, 5% CO2 for 48 hours. A blank control group, C-176 (10 μM) group, H2O2 (100 μM) group, and C-176 (10 μM) + H2O2 (100 μM) group were set up and cultured for 24 hours.

[0022] After that, extract human epidermal melanocyte protein, discard the cell supernatant culture medium in the six-well plate, wash the cells twice with pre-cooled PBS solution, discard the residual solution, add the pre-prepared lysis solution containing protease inhibitors, and lyse on ice for 20 minutes. Scrape the cells with a cell scraper and transfer them to a pre-cooled 1.5mL centrifuge tube, centrifuge at 13000rpm for 15 minutes, and aspirate the supernatant into a new centrifuge tube. The supernatant is the extracted human epidermal primary melanocyte protein. After the protein concentration is determined by the BCA method, mix it with 1 / 4 volume of 5× loading buffer, boil it at 97℃ for 7 minutes in a constant temperature metal bath to denature the protein, and cool it at room temperature.

[0023] The extracted proteins were subjected to Western-Blot experiments to determine the expression of cGAS-STING signaling pathway and inflammatory factors IFN-γ and IL-6. SDS-PAGE electrophoresis was performed, and separation gels with concentrations of 12%, 15% and 4% concentrated gels were prepared. The gel was filled and the comb was inserted to wait for gelation. After the gel was made, 20μg of protein sample was loaded, and the concentrated gel voltage was 80V. After 30min, it was turned to 120V and ran to the bottom of the separation gel. Prepare NC membrane and transfer solution, transfer the membrane in the order of sponge, two layers of filter paper, and NC membrane. Cut the corresponding area according to the molecular weight of the target protein to avoid the generation of bubbles, clamp the transfer clip, and transfer the membrane conditions: 320mA, 45min, ice water bath. Prepare 5% skim milk powder in TBST blocking solution, block on a shaker at room temperature for 1.5h; incubate the membrane with specific primary antibody at 4℃ overnight. Recover the primary antibody, wash with TBST for 7min, 4 times; then add goat anti-rabbit-HRP or goat anti-mouse-HRP and incubate at room temperature for 1h. Recover the secondary antibody and wash with TBST 4 times, 7 minutes each time. Place in an exposure instrument, add chemiluminescent developer to the membrane for development, and take pictures with a gel imaging system. Image J and Graphpad Prism software were used to statistically analyze the data, and all experiments were repeated more than 3 times. Data are expressed as mean ± standard deviation. The t-test was used for pairwise comparison between groups, and one-way analysis of variance was used for comparison between multiple groups. P < 0.05 was considered statistically significant. The experimental results are shown in Figure 1 shown.

[0024] Experimental results: Compared with the blank control group, there was no significant change in the C-176 group, but the c-GAS-STING signaling pathway was activated in the H2O2 group, and the protein expression of inflammatory factors IFN-γ and IL-6 was significantly increased ( * P<0.05, ** P<0.01). Compared with the H2O2 group, the C-176+H2O2 group significantly inhibited the activation of the c-GAS-STING pathway in melanocytes and the expression of inflammatory factors IFN-γ and IL-6 ( # P<0.05, ## P<0.01).

[0025] Experimental conclusion: C-176 can significantly inhibit the expression of inflammatory factors IFN-γ and IL-6 in the melanocyte oxidative stress model.

[0026] Part II: Effect of C-176 on the pigmentation of the skin lesions in a vitiligo mouse model

[0027] Experimental animals: C57 BL / 6J mice (five weeks old) (20±2g) were purchased from Shanghai Bikekoyi Biotechnology Co., Ltd. The experimental animals were kept in individual cages. The mice were randomly divided into 3 groups (6 mice in each group) and numbered in sequence: (Group I) blank control group; (Group II) vitiligo model group (5% hydrogen peroxide); (Group III) vitiligo model group + C-176 (4mg / kg) treatment group. Use an electric shaver to remove the dorsal hair in a 2cm×2cm area. After 24h, 5% H2O2 was applied to the depilated area every day to start modeling, and the modeling period was four weeks. After the modeling was successful, 4mg / kg C-176 was applied to the lesion area of ​​the mouse for three consecutive weeks. The skin tissue of the lesion area was collected and Masson-Fontana melanin ammonia silver staining was performed. The experimental results are as follows Figure 2 shown.

[0028] Skin tissue (20 mg) from the lesion area was collected and cut aseptically, and then added to the pre-cooled tissue lysis solution (containing protease inhibitors). The melanin precipitate after centrifugation was added to 200 μL NaOH solution (1 mol / L, containing 10% DMSO) and lysed in an 80°C water bath for 2 hours to ensure that the melanin was fully dissolved. The completely dissolved melanin solution was transferred to a 96-well plate at a volume of 200 μL / well, and the absorbance value was measured at a wavelength of 405 nm using an enzyme reader. The experimental results are shown in Figure 2. Figure 3 shown.

[0029] Experimental results: Compared with the blank control group, the epidermal melanin in the skin of mice in the vitiligo model group was significantly reduced (**P<0.01). Compared with the vitiligo model group, the C-176 group could significantly promote the restoration of pigmentation in the skin lesions of mice in the vitiligo model group ( #P<0.05).

[0030] Experiments show that C-176 can significantly promote the restoration of pigmentation in the skin lesions of vitiligo mice.

[0031] Part III: Effects of C-176 on the synthesis of inflammatory factors in the lesional area of ​​vitiligo mouse model

[0032] 1. Effect of C-176 on the expression of inflammatory factors IFN-γmRNA and IL-6mRNA in the lesional area of ​​vitiligo mouse model

[0033] The skin tissue of the lesion area of ​​the above animal experiment was frozen in liquid nitrogen for preservation. In the formal experiment, the frozen tissue (about 100 mg) was taken and placed in a pre-cooled 1.5 mL centrifuge tube, the tissue was cut into pieces, placed in a mortar fully cooled with liquid nitrogen, and ground with a pestle. During the process, liquid nitrogen was continuously added until it became powdery. 1 mL of RNAiso Plus (Total RNA Extraction Reagent) was added to completely cover the sample, and it was left at room temperature for 10 minutes. After the sample was thawed, it was continued to be ground with a mortar until it became transparent. The homogenate was transferred to a 1.5 mL centrifuge tube, allowed to stand at room temperature for 5 min, and then centrifuged at 4°C, 13,000 rpm for 5 min. The supernatant was transferred to a new centrifuge tube, 200 μL of chloroform was added, and the tube was shaken vigorously for 15 s to fully mix the components to form an emulsion; allowed to stand at room temperature for 5 min, and then centrifuged at 4°C, 13,000 rpm for 15 min. The upper transparent aqueous phase was carefully pipetted into a new centrifuge tube (try to pipet as little as possible to avoid the white film in the middle), and then an equal volume of pre-cooled isopropanol solution was added, and the tube was inverted for 1 0 times, let stand at room temperature for 10 minutes, centrifuge at 4°C, 13000rpm for 10 minutes, discard the supernatant, add 1mL pre-cooled 75% ethanol (prepared with DEPC water), gently turn upside down, wash the precipitate and make it float, centrifuge at 4°C, 13000rpm for 5 minutes, discard the supernatant (as thoroughly as possible), open the centrifuge tube cap, dry for 5 minutes until there is no ethanol residue, add an appropriate amount of 0.1% DEPC water to dissolve according to the amount of RNA, and repeatedly blow until the RNA is fully dissolved. RNA concentration and purity determination: Pipette 2μL RNA for concentration detection, further dilute with DEPC water to an appropriate concentration (usually between 200-1000ng / μL), if OD 260 / OD 280 The ratio is between 1.8 and 2.2, which proves that the purity is good and can be used for subsequent experiments.

[0034] RNA was reverse transcribed into cDNA and the following reaction system was prepared on ice:

[0035]

[0036]

[0037] Mix by gently pipetting, incubate at 42°C for 2 min, add 4 μL 5×HiScript III qRTSuperMix, and centrifuge to the bottom of the tube.

[0038] Reaction conditions: 37°C, 15 min for reverse transcription; 85°C, 5 s to inactivate reverse transcriptase.

[0039] qRT-PCR:

[0040] Prepare the following reaction system on ice:

[0041]

[0042] The reaction program was set up as follows:

[0043]

[0044] Relative quantitative analysis:

[0045] After qRT-PCR was completed, the Ct value data of each sample was obtained. The expression level of GAPDH was used as the internal reference, and the PCR relative quantification method was used. According to the formula: ΔCt = Ct to be tested - Ct internal reference, ΔΔCt = ΔCt experimental group - ΔCt control group, mRNA relative expression = 2^(-ΔΔCt), the relative level of gene expression of mRNA in the experimental group compared with that in the control group was calculated. The experimental results are shown in Figure 4 shown.

[0046] Experimental results: Compared with the blank control group, the expression of IFN-γmRNA and IL-6mRNA in the back skin tissue of mice in the vitiligo model group was significantly increased ( ** P<0.01). Compared with the vitiligo model group, C-176 significantly inhibited the expression of IFN-γmRNA and IL-6mRNA in the back skin tissue of mice in the vitiligo model group ( ## P<0.01).

[0047] Experiments show that C-176 can significantly inhibit the expression of inflammatory factors IFN-γmRNA and IL-6mRNA in the lesion area of ​​vitiligo mice.

[0048] Effect of C-176 on the expression of inflammatory factor proteins IFN-γ and IL-6 in the skin lesions of vitiligo mouse models

[0049] Take about 60 mg of skin tissue from the lesion area of ​​the above animal experiment, cut it into pieces, add 1 mL of lysis solution containing protease inhibitors, and break the tissue with a high-speed mechanical homogenizer at 12000 rpm on ice. Add 2 times the volume of extraction reagent to each 0.5 mL of tissue homogenate, mix thoroughly, let stand on ice for 10 minutes, centrifuge at 4°C, 10000×g for 10 minutes, the solution is divided into upper and lower phases, the middle of the two phases is a protein membrane, use a pipette to discard the upper and lower liquids, and the protein membrane is attached to the tube wall. Open the centrifuge tube cap, air dry for 10 minutes, add 200 μL 2% SDS, heat at 90°C until the precipitate dissolves, cool at room temperature, centrifuge at 5000 rpm for 5 minutes, and aspirate the supernatant into a new centrifuge tube. The supernatant is the extracted mouse skin tissue protein. After the protein concentration is determined by BCA method, mix with 1 / 4 volume of loading buffer, boil at 97°C in a constant temperature metal bath for 10 minutes to denature the protein, and cool at room temperature. Perform the Western Blotting experiment in the same manner as above. Figure 5 shown.

[0050] Experimental results: Compared with the blank control group, the expression of IFN-γ and IL-6 in the back skin tissue of mice in the vitiligo model group was significantly increased ( ** P<0.01). Compared with the vitiligo model group, C-176 significantly inhibited the expression of IFN-γ and IL-6 in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ## P<0.01).

[0051] Experiments show that C-176 can significantly inhibit the expression of inflammatory factor proteins IFN-γ and IL-6 in the lesion areas of vitiligo mice.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating vitiligo.

2. The use according to claim 1, characterized in that The chemical structural formula of the C-176 is as follows:

3. A pharmaceutical composition for treating vitiligo, characterized in that The C-176 or a pharmaceutically acceptable salt thereof as described in claim 1 or 2 is used as the sole active ingredient.

4. The pharmaceutical composition according to claim 3, characterized in that Contains pharmaceutically acceptable carriers or excipients.

5. The pharmaceutical composition according to claim 3, characterized in that The dosage form of the drug is cream, ointment, cream preparation, gel, film or spray.

6. A pharmaceutical composition for treating vitiligo, characterized in that Contains C-176 or a pharmaceutically acceptable salt thereof as described in claim 1 or 2 and other active ingredients.

7. The pharmaceutical composition according to claim 5, characterized in that Contains the pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the drug is cream, ointment, cream preparation, gel, film or spray.

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