Application of VBIT-4 or pharmaceutically acceptable salt thereof in preparation of medicine for preventing or treating vitiligo and complications thereof

Abstract

The invention relates to application of VBIT-4 or pharmaceutically acceptable salt thereof in preparation of drugs for preventing or treating vitiligo and complications thereof. A large number of modern pharmacological scientific researches and in-vivo pharmacodynamic tests prove that VBIT-4 has a treatment effect on a leucoderma mouse model and inhibits synthesis of inflammatory factors and chemotactic factors in a leucoderma mouse skin lesion area. The mechanism of the invention is that VBIT-4 inhibits the expression of inflammatory factors and chemotactic factors in skin tissues so as to treat leucoderma, compared with the existing treatment means, VBIT-4 can promote the compound color of pigments in a skin lesion region on the back of a leucoderma mouse, has no obvious toxicity, and reduces the expression level of inflammatory factors (IL-1beta, IL-6 and IFN-alpha) and chemotactic factors (CXCL9 and CXCL10). In conclusion, the VBIT-4 is clear in action mechanism, remarkable in effect and free of obvious side effects, and has remarkable advantages and progress compared with existing drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of VBIT-4 or a pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating vitiligo, and preventing or treating complications of vitiligo. Background Art

[0002] Vitiligo is a common depigmentation disease that affects approximately 0.5% to 2% of the world's population. It is characterized by significant white spots caused by severe destruction and loss of function of melanocytes between the epidermis and dermis. Its external manifestations and corresponding symptoms cause patients to suffer from self-esteem and cause severe psychological distress, thus negatively affecting their lives. Multiple factors, including genetic variation, environmental triggers, autoimmunity, oxidative stress, and mitochondrial dysfunction, contribute to the occurrence and progression of vitiligo. Although the exact etiology of vitiligo is still unclear, oxidative stress is widely considered to be one of the key triggers of vitiligo. In vitiligo, stressed melanocytes may activate the innate immune system by producing and releasing damage-associated pattern molecules (DAMPs), providing the initial danger signal that triggers inflammation. This inflammation subsequently activates the adaptive immune system, promotes autoimmune destruction and promotes the progression of vitiligo. Notably, elevated levels of epidermal hydrogen peroxide (H2O2) have been observed in vivo in the skin lesions of patients with vitiligo. At millimolar levels, H2O2 can damage proteins, lipids, and DNA, leading to melanocyte apoptosis, which may be the cause of melanocyte loss in vitiligo lesions.

[0003] Although there are many treatment methods, the clinical efficacy is poor, which has a great impact on the patient's quality of life and psychology. The current main treatment method is to stimulate local abnormal melanocytes with drugs to promote the regeneration and growth of melanocytes, thereby preventing the development of the disease. However, the efficacy is not ideal, and the treatment methods are not generally effective for all patients.

[0004] VBIT-4 (CAS: 2086257-77-2), chemical name is N-(4-chlorophenyl)-4-hydroxy-3-(4-(4-(trifluoromethoxy)phenyl)piperazin-1-yl)butanamide. Molecular formula is C 21 H 23 ClF3N3O3, molecular weight 457.87, is a white solid. It dissolves in ethanol and DMSO. The chemical structure is as follows:

[0005]

[0006] VBIT-4 is used as an apoptosis inhibitor for the study of diseases related to apoptosis, such as neurodegenerative diseases and cardiovascular diseases. However, there are no reports on the use of VBIT-4 in the prevention or treatment of vitiligo in domestic and foreign research. Summary of the invention

[0007] The technical problem to be solved by the present invention is that the existing drugs for treating vitiligo have poor clinical efficacy, have a great impact on the quality of life and psychology of patients, and the treatment methods are not generally effective for all patients. Provided is the use of VBIT-4 or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing or treating vitiligo and its complications.

[0008] Furthermore, the present invention also provides a pharmaceutical composition for preventing or treating vitiligo and its complications, wherein the pharmaceutical composition contains a therapeutically effective amount of VBIT-4 or a pharmaceutically acceptable salt thereof.

[0009] The present invention uses a large number of modern pharmacological scientific studies and in vivo pharmacodynamic experiments to prove that VBIT-4 has a therapeutic effect on vitiligo mouse models and inhibits the synthesis of inflammatory factors and chemokines in the skin lesion area of ​​vitiligo mice.

[0010] The mechanism of the present invention is to inhibit the expression of inflammatory factors and chemokines in skin tissue through VBIT-4 to treat vitiligo. Compared with existing treatment methods, the present invention has the following advantages: At present, the main clinical vitiligo treatment drugs are glucocorticoids and immunosuppressants, and long-term use has significant side effects and is difficult to tolerate. The phototherapy treatment cycle is long, and patients need to visit the doctor many times, with poor compliance, and can cause symptoms such as skin photosensitivity and itching. Long-term use increases the risk of skin cancer. The present invention found through in vivo animal experiments that the results of the study using the vitiligo mouse model induced by hydrogen peroxide showed that VBIT-4 can promote the re-coloring of the pigment in the back lesion area of ​​vitiligo mice, without obvious toxicity, and reduce the expression levels of inflammatory factors (IL-1β, IL-6, IFN-α) and chemokines (CXCL9, CXCL10). In summary, VBIT-4 has a clear mechanism of action, significant effects, and no obvious side effects, and has significant advantages and progress compared to existing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1These are light microscopic photos of skin tissue in the lesion area of ​​the vitiligo mouse model after melanin ammonia silver staining. Among them, Figure A: blank control group; Figure B: vitiligo model group; Figure C: low-dose VBIT-4 (1 mg / kg) treatment based on the vitiligo model; Figure D: medium-dose VBIT-4 (5 mg / kg) treatment based on the vitiligo model; Figure E: high-dose VBIT-4 (10 mg / kg) treatment based on the vitiligo model.

[0012] Figure A shows that there is a large amount of melanin in the skin tissue of the blank control group. Figure B shows that the melanin in the skin tissue of the vitiligo model group is significantly reduced; Figure CE shows that after VBIT-4 treatment on the basis of the vitiligo model, the amount of melanin in the skin tissue increases. This shows that VBIT-4 can significantly promote the restoration of pigmentation in the skin lesions of vitiligo mice.

[0013] Figure 2 This is a bar graph of the relative expression of inflammatory factors IL-1βmRNA, IL-6mRNA, and IFN-αmRNA in the lesion area of ​​the vitiligo mouse model. Figure 2 A is a bar graph of the relative expression of IL-1β mRNA. Figure 2 B is a bar graph of the relative expression of IL-6 mRNA. Figure 2 C is a bar graph of the relative expression levels of IFN-α mRNA.

[0014] Compared with the blank control group, the expressions of IL-1βmRNA, IL-6mRNA, and IFN-αmRNA in the back skin tissue of mice in the vitiligo model group were significantly increased ( *** P<0.001). Compared with the vitiligo model group, the VBIT-4 group significantly inhibited the expression of IL-1βmRNA, IL-6mRNA, and IFN-αmRNA in the back skin tissue of mice in the vitiligo model group ( ## P<0.01, ### P<0.001).

[0015] Figure 3 This is a bar graph of the relative expression of chemokines CXCL9 mRNA and CXCL10 mRNA in the skin lesions of vitiligo mouse models. Figure 3 A is a bar graph of the relative expression of CXCL9 mRNA. Figure 3 B is a bar graph of the relative expression of CXCL10 mRNA.

[0016] Compared with the blank control group, the expression of CXCL9 mRNA and CXCL10 mRNA in the back skin tissue of mice in the vitiligo model group was significantly increased ( * P<0.05, ***P<0.001). Compared with the vitiligo model group, the VBIT-4 group significantly inhibited the expression of CXCL9 mRNA and CXCL10 mRNA in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ### P<0.001).

[0017] Figure 4 This is an immunoblot of inflammatory factors IL-1β, IL-6, and IFN-α in the skin lesions of a vitiligo mouse model. Figure 4 A is a protein immunoblot. Figure 4 B is a bar graph of relative protein expression.

[0018] Compared with the blank control group, the expression of IL-1β, IL-6, and IFN-α in the back skin tissue of mice in the vitiligo model group was significantly increased ( * P<0.05). Compared with the vitiligo model group, the VBIT-4 group significantly inhibited the expression of IL-1β, IL-6, and IFN-α in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ## P<0.01).

[0019] Figure 5 This is the immunoblot of chemokines CXCL9 and CXCL10 in the skin lesions of vitiligo mouse model. Figure 5 A is a protein immunoblot. Figure 5 B is a bar graph of relative protein expression.

[0020] Compared with the blank control group, the expression of proteins CXCL9 and CXCL10 in the back skin tissue of mice in the vitiligo model group was significantly increased ( ** P<0.01). Compared with the vitiligo model group, the VBIT-4 group significantly inhibited the expression of proteins CXCL9 and CXCL10 in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ## P<0.01).

[0021] Beneficial effects: The present invention provides the use of VBIT-4 or a pharmaceutically acceptable salt thereof in the preparation of a method for treating and / or preventing vitiligo. Experiments have shown that VBIT-4 or a pharmaceutically acceptable salt thereof has the efficacy of treating vitiligo, providing a new and effective treatment option for vitiligo. DETAILED DESCRIPTION

[0022] 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.

[0023] Below are some pharmacodynamic tests and results of the present invention:

[0024] Part I: Effect of VBIT-4 on the pigmentation of the skin lesions in a vitiligo mouse model

[0025] Experimental animals: Clean-grade female C57BL / 6J mice (five weeks old) (20±2g) were purchased from Shanghai Bikekeyi Biotechnology Co., Ltd. The experiment was carried out after acclimating to the environment for one week, and the mice were randomly divided into 5 groups (8 mice in each group): blank control group; vitiligo model group; vitiligo model group + VBIT-4 low-dose group (1mg / kg); vitiligo model group + VBIT-4 medium-dose group (5mg / kg); vitiligo model group + VBIT-4 (10mg / kg) high-dose 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, different doses of 1mg / kg, 5mg / kg, and 10mg / kg VBIT-4 were applied to the lesion area of ​​the mice 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 1 shown.

[0026] Experimental results: Compared with the blank control group, the epidermal melanin in the skin of mice in the vitiligo model group was significantly reduced. Compared with the vitiligo model group, the VBIT-4 medium-dose group and the VBIT-4 high-dose group could significantly promote the restoration of pigmentation in the skin lesion area of ​​mice in the vitiligo model group.

[0027] Experiments show that VBIT-4 can significantly promote the restoration of pigmentation in the lesion area of ​​the vitiligo mouse model.

[0028] Part II: Effects of VBIT-4 on the levels of inflammatory factors and chemokines in the skin lesions of vitiligo mouse models

[0029] 1. Effect of VBIT-4 on the expression of inflammatory factors IL-1β mRNA, IL-6 mRNA, IFN-α mRNA, chemokines CXCL9 mRNA, and CXCL10 mRNA in the skin lesions of vitiligo mouse models

[0030] Take the skin tissue of the lesion area of ​​the above animal experiment and freeze it in liquid nitrogen. In the formal experiment, take the frozen tissue (about 100 mg), put it into a pre-cooled 1.5 mL centrifuge tube, cut the tissue into pieces, scoop out the liquid nitrogen with a spoon and fully cool the mortar, and grind the tissue with a pestle. During this period, liquid nitrogen is continuously added until the tissue is ground into powder. Add 1 mL of RNAisoplus to each sample and completely cover the sample. Let it stand at room temperature for 10 minutes. After the sample is thawed, continue to grind it with a mortar until the lysate becomes transparent. The homogenate was transferred to a 1.5 mL centrifuge tube, allowed to stand at room temperature for 5 min, and 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 to each 1 mL of lysate, and the mixture 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 centrifuged at 4°C, 13,000 rpm for 15 min. The upper aqueous phase was carefully pipetted into a new centrifuge tube, and then an equal volume of precooled isopropanol solution was added, and the mixture was mixed by inversion. The mixture was allowed to stand at room temperature for 10 min, and centrifuged at 4°C, 13,000 rpm for 10 min. The supernatant was discarded, and 1 mL of precooled 75% ethanol (prepared with DEPC water) was added, and the mixture was gently inverted to wash the precipitate. The mixture was centrifuged at 4°C, 13,000 rpm for 5 min, and washed twice. The residual liquid was removed cleanly, the cap of the centrifuge tube was opened, and the mixture was dried for 5 min until no ethanol residue was left. According to the amount of RNA, an appropriate amount of 0.1% DEPC water was added to dissolve the RNA, and the RNA was repeatedly blown until it was fully dissolved. Take 2 μL RNA for concentration test and further dilute it with DEPC water to a suitable concentration (usually between 200-1000 ng / μL). 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.

[0031] To reverse transcribe RNA into cDNA, prepare the following reaction system on ice:

[0032]

[0033] Mix by gently pipetting, incubate at 42°C for 2 min, and then add 4 μL 5×HiScript III qRTSuperMix a , centrifuge to the bottom of the tube. Reverse transcription reaction conditions: 37℃, 15min for reverse transcription; 85℃, 5s to inactivate reverse transcriptase.

[0034] qRT-PCR: Prepare the following reaction system on ice:

[0035]

[0036] The reaction program was set up as follows:

[0037]

[0038]

[0039] Relative quantitative analysis: After qRT-PCR, the Ct value data of each sample was obtained. The expression of GAPDH was used as the internal reference. The PCR relative quantitative 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), and the relative level of gene expression of the experimental group mRNA compared with the control group was calculated. The experimental results are shown in Figure 2 , Figure 3 shown.

[0040] Experimental results: Compared with the blank control group, the expressions of IL-1βmRNA, IL-6mRNA, IFN-αmRNA, CXCL9 mRNA, and CXCL10 mRNA in the back skin tissue of mice in the vitiligo model group were significantly increased ( * P<0.05, *** P<0.001). Compared with the vitiligo model group, VBIT-4 significantly inhibited the expression of IL-1βmRNA, IL-6mRNA, IFN-αmRNA, CXCL9 mRNA, and CXCL10 mRNA in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ## P<0.01, ### P<0.001).

[0041] Experiments show that VBIT-4 can significantly inhibit the expression of mRNA of inflammatory factors and chemokines in the skin lesions of vitiligo mice.

[0042] Effect of VBIT-4 on the protein expression of inflammatory factors IL-1β, IL-6, IFN-α, CXCL9, and CXCL10 in the lesional area of ​​vitiligo mouse model

[0043] 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, and break the tissue with a high-speed mechanical homogenizer at 12000 rpm on ice. Add 2 times the volume of 1 mL of extraction reagent to each 0.5 mL of tissue homogenate, mix thoroughly, let stand at room temperature for 10 minutes, centrifuge at 4°C for 10 minutes, 10000×g, 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 liquid, pull the protein membrane apart with the gun head, and suck out the lower liquid. The protein membrane is attached to the tube wall. Open the centrifuge tube cap, air dry for 10 minutes, add an appropriate amount of 2% SDS to each sample, heat at 90°C until the precipitate dissolves, cool to room temperature, centrifuge at 5000rpm for 5 minutes, and the supernatant obtained by centrifugation is the extracted mouse skin tissue protein. After the protein concentration is determined by BCA method, mix with 1 / 4 volume of 5× loading buffer, and boil at 97°C in a constant temperature metal bath for 10 minutes to denature the protein. SDS-PAGE electrophoresis was performed, and separation gels with concentrations of 10%, 12%, and 15% and concentrated gels with concentrations of 4% were prepared. The gel was filled and the comb was inserted to wait for gelation. After the gel was prepared, 20-40 μg of protein samples were loaded and electrophoresed at 80V, 30min and 120V, 1h. After the electrophoresis was completed, PVDF membrane and transfer solution were prepared, and the corresponding area was cut according to the molecular weight of the target protein to avoid bubbles. The transfer clamp was clamped and transferred in an ice bath with a current of 0.32A. The transfer time was determined according to the molecular weight of the target protein. Blocked with TBST (0.05% Tween-20) containing 5% skim milk for 1.5h, and the blocking solution was washed. The primary antibody diluted according to the instructions was added and incubated at 4°C overnight. After recovering the primary antibody, TBST was washed for 7min, repeated 4 times, and then HRP-labeled secondary antibody was added to bind the primary antibody and incubated at room temperature for 1h. The secondary antibody was recovered and washed 4 times with TBST, each time for 7min. For chemiluminescence detection, the pre-mixed ECL chemical developer solution was added to the membrane, developed using a gel imaging system analyzer, and the image was collected. Band analysis was performed using Image J to count the grayscale of the bands, and GraphPad Prism 8.0.2 software was used to record and analyze the data. All data were expressed as mean ± standard deviation (SD), and one-way ANOVA was used for statistical analysis of multiple groups of data. The experimental results are shown in Figure 4 , Figure 5 shown.

[0044] Experimental results: Compared with the blank control group, the expression of proteins IL-1β, IL-6, IFN-α, CXCL9, and CXCL10 in the back skin tissue of mice in the vitiligo model group was significantly increased ( * P<0.05, **P<0.01). Compared with the vitiligo model group, VBIT-4 significantly inhibited the expression of IL-1β, IL-6, IFN-α, CXCL9, and CXCL10 in the back skin tissue of mice in the vitiligo model group ( # P<0.05, ## P<0.01).

[0045] Experiments show that VBIT-4 can significantly inhibit the expression of inflammatory factors and chemokine proteins in the skin lesions of vitiligo mice.

[0046] 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 VBIT-4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating vitiligo and its complications.

2. A pharmaceutical composition for preventing or treating vitiligo and its complications, characterized in that The pharmaceutical composition contains a therapeutically effective amount of VBIT-4 or a pharmaceutically acceptable salt thereof.

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