Application of C-176 in the preparation of drugs for treating vitiligo

Abstract

This invention relates to the use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating vitiligo. The chemical structural formula of C-176 is as follows: This invention provides the use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating vitiligo. Experiments have demonstrated that C-176 is effective in treating vitiligo, providing a new and effective treatment option for vitiligo.

Description

Technical Field

[0001] This invention relates to novel uses of C-176, and more particularly to the use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating vitiligo. Background Technology

[0002] Vitiligo is an acquired depigmentation disorder with extremely complex etiologies, characterized primarily by the destruction and loss of function of epidermal melanocytes. Epidemiological surveys show that the incidence of vitiligo in the global population is approximately 1%. In recent years, the incidence of vitiligo has been increasing year by year, showing a trend towards younger ages, and the white patches often appear on areas such as the face and scalp, seriously affecting the physical and mental health of patients. Numerous pieces of evidence suggest that autoimmunity plays an important role in the progression of the disease, but the underlying mechanisms that induce vitiligo have not been fully elucidated. Currently, clinical treatments mainly involve non-targeted immunosuppressants, such as corticosteroids and calcineurin inhibitors. These drugs are only effective for a subset of patients and have significant side effects. Oxidative stress is generally considered to be the initiating factor in the pathogenesis of vitiligo. Under stress conditions, melanocytes produce and release damage-associated pattern molecules (inflammatory factors such as IFN-γ and IL-6), activating an autoimmune response that destroys melanocytes.

[0003] C-176 (CAS: 314054-00-7), chemically named N-(4-iodophenyl)-5-nitro-2-furancarboxamide, has the molecular formula C 11 H7IN2O4, with a molecular weight of 358.09, is a pale yellow solid; its chemical structural formula is as follows:

[0004]

[0005] C-176 is an inhibitor of the STING signaling pathway, exhibiting selectivity and blood-brain barrier permeability. C-176 can inhibit STING-mediated IFN-β production and possesses anti-inflammatory activity. However, currently, no research reports have been found regarding the use of C-176 in the treatment of vitiligo, either domestically or internationally. Summary of the Invention

[0006] The technical problem this invention aims to solve is that existing drugs for treating vitiligo are only effective for a subset of patients and have significant side effects. This invention provides the use of pharmaceutically acceptable salts of C-176 in the preparation of drugs for treating vitiligo and in the treatment of vitiligo complications. Furthermore, this invention also provides the use in the preparation of pharmaceutical compositions for treating vitiligo and vitiligo complications, wherein the pharmaceutical composition contains a therapeutically effective amount of C-176 or a pharmaceutically acceptable salt thereof.

[0007] Through extensive modern pharmacological research and in vivo pharmacodynamic experiments, this invention has demonstrated that C-176 can promote repigmentation in the skin lesions of vitiligo mice and inhibit the synthesis of inflammatory factors IFN-γ and IL-6 in the skin lesions of vitiligo mice.

[0008] Beneficial effects: This invention provides the use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a treatment for vitiligo. Experiments have demonstrated that C-176 is effective in treating vitiligo. This provides a new and effective treatment option for vitiligo. Attached Figure Description

[0009] Figure 1 This is an immunoblot image of the inflammatory factors IFN-γ and IL-6, and their upstream regulatory signaling pathway proteins, in human epidermal melanocytes. Among them, Figure 1 A is the Western blot image of the protein. Figure 1 B is a bar chart showing the relative expression levels of proteins.

[0010] Figure 1 A, Figure 1 B showed that compared with the blank control group, there were no significant changes in c-GAS, STING, IFN-γ, and IL-6 protein expression in the C-176 group, but the expression of c-GAS, STING, IFN-γ, and IL-6 protein was significantly increased in the H2O2 group. * 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 micrographs of skin tissue in a mouse model of vitiligo after ammoniacal silver staining with melanin. Left image: blank control group; middle image: vitiligo model group; right image: vitiligo model group treated with C-176.

[0012] The left image shows a large amount of melanin distribution in the skin tissue of the blank control group. The middle image shows a significant reduction in melanin in the skin tissue of the vitiligo model group. The right image shows an increase in melanin in the skin tissue after treatment with C-176 in addition to the vitiligo model. This indicates that C-176 can significantly promote repigmentation in the skin lesions of vitiligo mice.

[0013] Figure 3This is a bar chart showing the relative melanin content 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 significantly promoted repigmentation in the skin lesions of the vitiligo model mice. # P<0.05).

[0014] Figure 4 This is a bar chart showing the relative expression levels of inflammatory factors IFN-γ mRNA and IL-6 mRNA in the skin lesions of a vitiligo mouse model. Among them, Figure 4 A is a bar chart showing the relative expression levels of IFN-γ mRNA. Figure 4 B is a bar chart showing the relative expression levels of IL-6 mRNA.

[0015] Compared with the blank control group, the expression of IFN-γ mRNA and IL-6 mRNA in the dorsal 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 mRNA and IL-6 mRNA in the dorsal skin tissue of mice in the vitiligo model group. ## P<0.01).

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

[0017] Compared with the blank control group, the expression of proteins IFN-γ and IL-6 in the dorsal 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 proteins IFN-γ and IL-6 in the dorsal skin tissue of mice in the vitiligo model group. # P<0.05, ## P<0.01). Detailed Implementation

[0018] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention. They should not be construed as limiting the present invention.

[0019] The following are some of the pharmacodynamic tests and results of this invention:

[0020] Part 1: Effects of C-176 on the expression of inflammatory factors in a melanocyte oxidative stress model

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

[0022] Human epidermal melanocyte protein was then extracted. The cell supernatant culture medium in the six-well plate was discarded, and the cells were washed twice with pre-chilled PBS solution. The residual solution was discarded, and pre-prepared lysis buffer containing protease inhibitors was added. The cells were lysed on ice for 20 min. Cells were scraped off with a cell scraper and transferred to pre-chilled 1.5 mL centrifuge tubes. The tubes were centrifuged at 13,000 rpm for 15 min, and the supernatant was transferred to a new centrifuge tube. The supernatant was the extracted human epidermal primary melanocyte protein. After determining the protein concentration using the BCA method, the protein was mixed with 1 / 4 volume of 5× loading buffer and boiled in a constant temperature metal bath at 97°C for 7 min to denature the protein. The mixture was then cooled to room temperature.

[0023] The extracted proteins were subjected to Western blotting to determine the expression of the cGAS-STING signaling pathway and inflammatory factors IFN-γ and IL-6. SDS-PAGE electrophoresis was performed using 12% and 15% separating gels and a 4% stacking gel. After gel preparation, combs were inserted and the gels were allowed to solidify. 20 μg of protein sample was loaded onto the gels. The stacking gel was run at 80 V for 30 min, then increased to 120 V until the separating gel reached the bottom. NC membranes and transfer buffer were prepared. Transfer was performed in the following order: sponge, two layers of filter paper, and NC membrane. The membrane was cut into regions according to the molecular weight of the target protein, avoiding air bubbles. The transfer clamps were secured. Transfer conditions: 320 mA, 45 min, ice-water bath. TBST blocking buffer (5% skim milk powder) was prepared and blocked on a shaker at room temperature for 1.5 h. The membrane was then incubated with specific primary antibody overnight at 4°C. The primary antibody was recovered, and the membrane was washed with TBST for 7 min, 4 times. Goat anti-rabbit-HRP or goat anti-mouse-HRP was then added and incubated at room temperature for 1 h. The secondary antibody was recovered and washed four times with TBST for 7 min each time. The membrane was placed in an exposure apparatus, and chemiluminescent developing solution was added to develop the membrane. Images were taken using a gel imaging system. ImageJ and Graphpad Prism software were used for statistical analysis. All experiments were repeated at least three times. Data are expressed as mean ± standard deviation. Pairwise comparisons between groups were performed using t-tests, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. Experimental results are as follows: Figure 1 As shown.

[0024] Experimental results: Compared with the blank control group, there were no significant changes 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 suppressed 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 a melanocyte oxidative stress model.

[0026] Part Two: Effects of C-176 on repigmentation in the lesion area of ​​a mouse model of vitiligo

[0027] Experimental animals: C57 BL / 6J mice (5 weeks old) (20±2g), purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd. The experimental animals were housed separately in individual cages. Mice were randomly divided into 3 groups (n=6 per group), numbered sequentially as follows: (Group I) blank control group; (Group II) vitiligo model group (5% hydrogen peroxide); (Group III) vitiligo model group + C-176 (4mg / kg) treatment group. A 2cm×2cm area of ​​back hair was removed using an electric shaver. 24 hours later, 5% H2O2 was applied daily to the hairless area to initiate the modeling process, which lasted for four weeks. After successful modeling, 4mg / kg C-176 was applied to the affected skin area for three consecutive weeks. Skin tissue from the affected area was collected for Masson-Fontana silver staining of melanin. Experimental results are as follows: Figure 2 As shown.

[0028] Skin tissue (20 mg) was collected from the lesion area, aseptically cut, and then mixed with pre-cooled tissue lysis buffer (containing protease inhibitors). The tissue was thoroughly homogenized using a homogenizer. The centrifuged melanin precipitate was added to 200 μL of NaOH solution (1 mol / L, containing 10% DMSO) and lysed in an 80°C water bath for 2 hours to ensure complete melanin dissolution. The completely dissolved melanin solution was transferred to 96-well plates at a volume of 200 μL / well, and the absorbance was measured at 405 nm using a microplate reader. The experimental results are as follows: Figure 3 As 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 significantly promoted repigmentation in the skin lesions of mice in the vitiligo model group. #P<0.05).

[0030] Experiments have shown that C-176 can significantly promote repigmentation of pigment in the skin lesions of vitiligo mice.

[0031] Part 3: Effects of C-176 on the synthesis of inflammatory factors in the skin lesions of a mouse model of vitiligo

[0032] I. Effects of C-176 on the expression of inflammatory factors IFN-γ mRNA and IL-6 mRNA in the skin lesions of a vitiligo mouse model

[0033] Skin tissue from the lesion area of ​​the animal experiments described above was frozen and stored in liquid nitrogen. For the formal experiment, the frozen tissue (approximately 100 mg) was placed in a pre-cooled 1.5 mL centrifuge tube, minced, and placed in a mortar thoroughly cooled with liquid nitrogen. It was then ground with a pestle, with liquid nitrogen continuously added until it became a powder. 1 mL of RNAiso Plus (Total RNA extraction reagent) was added to completely cover the sample. The mixture was allowed to stand at room temperature for 10 minutes until it thawed. After that, it was ground again with the mortar until it became transparent. Transfer the homogenate to a 1.5 mL centrifuge tube, let stand at room temperature for 5 min, then centrifuge at 13000 rpm for 5 min at 4 °C. Transfer the supernatant to a new centrifuge tube, add 200 μL of chloroform, and shake vigorously for 15 s to thoroughly mix the components, forming an emulsion. Let stand at room temperature for 5 min, then centrifuge at 13000 rpm for 15 min at 4 °C. Carefully aspirate the upper clear aqueous phase to a new centrifuge tube (aspirate as little as possible to avoid aspirating the white film in the middle), then add an equal volume of pre-cooled isopropanol solution, and invert the tube once. Mix thoroughly once, let stand at room temperature for 10 min, centrifuge at 13000 rpm for 10 min at 4℃, discard the supernatant, add 1 mL of pre-cooled 75% ethanol (prepared with DEPC water), gently invert to wash the precipitate and allow it to float, centrifuge at 13000 rpm for 5 min at 4℃, discard the supernatant (as thoroughly as possible), open the centrifuge tube cap, dry for 5 min until no ethanol residue remains, add an appropriate amount of 0.1% DEPC water to dissolve according to the amount of RNA, and repeatedly pipette until the RNA is fully dissolved. RNA concentration and purity determination: pipette 2 μL of RNA for concentration determination, further dilute with DEPC water to a suitable concentration (usually between 200-1000 ng / μL), if OD 260 / OD 280 The ratio is between 1.8 and 2.2, indicating good purity, which 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] Gently pipette to mix, incubate at 42°C for 2 min, then add 4 μL of 5×HiScript III qRTSuperMix and centrifuge to the bottom of the tube.

[0038] Reaction conditions: reverse transcription was performed at 37℃ for 15 min; reverse transcriptase was inactivated at 85℃ for 5 s.

[0039] qRT-PCR:

[0040] Prepare the following reaction system on ice:

[0041]

[0042] The reaction program is set as follows:

[0043]

[0044] Relative quantitative analysis:

[0045] After qRT-PCR, Ct values ​​were obtained for each sample. Using GAPDH expression as an internal control, relative PCR quantification was employed. The formulas were: ΔCt = Cttest - Ctinternal, ΔΔCt = ΔCtexperimental group - ΔCtcontrol group, and mRNA relative expression level = 2^(-ΔΔCt). The relative levels of mRNA expression in the experimental group compared to the control group were calculated. The experimental results are as follows: Figure 4 As shown.

[0046] Experimental results: Compared with the blank control group, the expression of IFN-γ mRNA and IL-6 mRNA in the dorsal 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 mRNA and IL-6 mRNA in the dorsal skin tissue of mice in the vitiligo model group. ## P<0.01).

[0047] Experiments showed that C-176 could significantly inhibit the expression of inflammatory factors IFN-γ mRNA and IL-6 mRNA in the skin lesions of vitiligo mice.

[0048] II. Effects of C-176 on the expression of inflammatory proteins IFN-γ and IL-6 in the skin lesions of a mouse model of vitiligo.

[0049] Approximately 60 mg of skin tissue from the lesion area of ​​the animal experiment was taken, minced, and 1 mL of lysis buffer containing protease inhibitors was added. The tissue was homogenized at 12,000 rpm on ice. Twice the volume of extraction reagent was added to each 0.5 mL of tissue homogenate, and the mixture was thoroughly mixed by pipetting. The mixture was incubated on ice for 10 min, then centrifuged at 10,000 × g for 10 min at 4 °C. The solution separated into two phases, with a protein membrane in between. The upper and lower layers were discarded using a pipette, and the protein membrane adhered to the tube wall. The centrifuge tube cap was opened, and the tube was air-dried for 10 min. 200 μL of 2% SDS was added, and the tube was heated at 90 °C until the precipitate dissolved. After cooling to room temperature, the tube was centrifuged at 5,000 rpm for 5 min, and the supernatant was transferred to a new centrifuge tube. The supernatant was the extracted mouse skin tissue protein. After determining the protein concentration using the BCA method, the protein was mixed with 1 / 4 volume of loading buffer and boiled in a constant temperature metal bath at 97 °C for 10 min to denature the protein. The mixture was then cooled to room temperature. Perform Western blotting experiments following the same steps as described above. The experimental results are as follows: Figure 5 As shown.

[0050] Experimental results: Compared with the blank control group, the expression of proteins IFN-γ and IL-6 in the dorsal 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 proteins IFN-γ and IL-6 in the dorsal skin tissue of mice in the vitiligo model group. # P<0.05, ## P<0.01).

[0051] Experiments showed that C-176 could significantly inhibit the expression of inflammatory factor proteins IFN-γ and IL-6 in the skin lesions of vitiligo mice.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of C-176 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating vitiligo, wherein the chemical structural formula of C-176 is as follows:

2. The application as described in claim 1, characterized in that... The drug also contains pharmaceutically acceptable carriers or excipients.

3. The application as described in claim 1, characterized in that... The dosage form of the drug is a cream, ointment, lotion, gel, film, or spray.

Images