Application of Viseno in the preparation of drugs for treating vitiligo

Abstract

The present invention discloses the use of Viseno in the preparation of a drug for treating vitiligo. The present invention belongs to the technical field of vitiligo treatment. As an oral antagonist of CCR9, Viseno affects the role of CCR9 / CCL25 in the pathogenesis of vitiligo by blocking the biological activity of its natural ligand CCL25. Compared with the prior art, the present invention has the advantages of being specific and targeted, so the therapeutic effect will be more obvious.

Description

Technical Field

[0001] The present invention relates to the technical field of vitiligo treatment, and more particularly to the application of Viseno in the preparation of a medicine for treating vitiligo. Background Art

[0002] Vitiligo is a common chronic acquired autoimmune skin disease in clinical practice, characterized by obvious white spots caused by the destruction of melanocytes. In the revised classification proposed by the 2012 Global Consensus Conference on Vitiligo, vitiligo is divided into three categories: non-segmental vitiligo (NSV), segmental vitiligo (SV), and undetermined / unclassified group, of which segmental vitiligo is a relatively rare disease. According to the distribution of skin lesions, vitiligo is further divided into systemic (GV), acrofacial, mucosal, localized, universal, and mixed types, with systemic and acrofacial types being the most common. In recent years, the incidence of vitiligo has increased year by year, with a global incidence of 0.5% to 2.0%, and both adults and children are affected. Because this disease has a significant impact on the social and emotional aspects of patients' lives, it also imposes a huge psychological burden on patients. Studies have shown that this psychological pressure can aggravate the occurrence and development of inflammatory skin diseases, causing a vicious cycle that perpetuates the disease, and can also lead to severe depression.

[0003] The occurrence of vitiligo is complex and multifactorial, and its pathogenesis is the result of the interaction between genetic components, metabolic factors related to cellular oxidative stress, adhesion of melanocytes to epithelial cells, and immunity (innate and acquired), which ultimately leads to the attack on melanocytes. Given its autoimmune background, vitiligo is often associated with other autoimmune diseases or immune-mediated diseases. Current studies have also emphasized the central role of immune cells and their mediators in the immunopathogenesis of vitiligo. Among them, key immune cells include helper T cells 1 cells, cytotoxic T cells, regulatory and resident memory T cells, as well as dendritic cells, natural killer and ILC-1 cells. In addition, key mediators include interferon-γ (IFN-γ), CXC chemokine ligand 9 (CXCL9) and 10 (CXCL10), and interleukin-15 (IL-15).

[0004] To date, vitiligo can be treated through different forms of phototherapy, surgery, and topical treatments, such as glucocorticoids, immunosuppressants, calcineurin inhibitors, and vitamin D. However, current treatments are still not ideal, they are non-specific, non-targeted, have limited efficacy, and have the problem of recurrence after cessation of treatment. Targeted therapies, such as biologics targeting cytokines and small molecule inhibitors targeting intracellular signaling molecules, are recently emerging as promising treatments for autoimmune diseases.

[0005] Chemokines are structurally related proteins with relatively small molecular weights that can target cell chemotaxis and promote inflammatory responses. CC chemokine receptor 9 (CCR9) is mainly expressed on the surface of immune cells and intestinal cells, and is involved in the differentiation of immature T lymphocytes and certain inflammatory responses; chemokine ligand 25 (CCL25) is the only ligand of CCR9, which is mainly secreted by thymic epithelial cells and intestinal epithelial cells. It plays a key role in mobilizing CCR9-positive cells to migrate and home to target tissues. In addition, adhesion defects between epidermal cell components are also related to the pathogenesis of vitiligo. E-cadherin is a protein that helps keratinocytes anchor each other, and its expression is reduced in melanocytes of vitiligo patients. Melanocyte marker (Melan-A) is a melanoma-related antigen that can be recognized by autologous cytotoxic T lymphocytes. It is positive in melanocytes of normal skin, but negative or lowly expressed in melanocytes of vitiligo patients. Arachidonic acid-15-lipoxygenase (ALOX15) is a regulatory protein involved in the lipid peroxidation process of ferroptosis (an iron-dependent, new type of programmed cell death that is different from apoptosis, necrosis, and autophagy). Its expression is increased in patients with vitiligo, promoting the occurrence of melanocyte ferroptosis.

[0006] In summary, how to provide a targeted, effective, and non-recurring vitiligo treatment method is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides the use of Viseno in the preparation of drugs for treating vitiligo. Studies have found that CC chemokine receptor 9 (CCR9) and its ligand CC chemokine ligand 25 (CCL25) are involved in the occurrence and development of a variety of inflammatory diseases. The present invention's research found that it has a higher expression in the skin tissue of vitiligo patients. Viseno is an oral antagonist of CCR9 that can block the biological activity of its natural ligand CCL25. It is being developed and applied clinically to the treatment of inflammatory bowel diseases such as Crohn's disease. Since the research data of the present invention shows that CCR9 / CCL25 plays an important role in the onset of vitiligo, we consider applying Viseno to the treatment of vitiligo, and hope that it can improve the problems existing in the existing treatment methods for vitiligo and provide a new treatment method.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] Application of CCL25 activity inhibitors in the preparation of drugs for treating vitiligo.

[0010] Furthermore, the CCL25 activity inhibitor is Viseno.

[0011] Application of Viseno in the preparation of medicines for treating vitiligo.

[0012] A medication for vitiligo, including Viseno.

[0013] As an oral antagonist of CCR9, Viseno affects the role of CCR9 / CCL25 in the pathogenesis of vitiligo by blocking the biological activity of its natural ligand CCL25. Therefore, the present invention can be used as a new method for treating vitiligo.

[0014] It can be seen from the above technical solution that compared with the prior art, the beneficial effect achieved by the present invention is: the present invention blocks the biological activity of its exclusive ligand CCL25 by targeting CCR9, an important cytokine in the pathogenesis of vitiligo, thereby producing therapeutic effects. Compared with the prior art, the advantage of the present invention is that it has specificity and targeting, so the therapeutic effect will be more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0016] Figure 1 This is a schematic diagram of restraint stress in Example 1 of the present invention;

[0017] Figure 2 It is a representative image of the back of mice in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+Ver: monobenzone + restraint + Ver group;

[0018] Figure 3 is the skin lesion score of mice in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone+restraint group, Mon+RS+Ver: monobenzone+restraint+Ver group;

[0019] Figure 4 The immunofluorescence results of E-cadherin and Melan-A in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+Ver: monobenzone + restraint + Ver group, Mon+RS+tacrolimus: monobenzone + restraint + tacrolimus group;

[0020] Figure 5The ELISA test results of E-cadherin in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone+restraint group, Mon+RS+Ver: monobenzone+restraint+Ver group;

[0021] Figure 6 The immunofluorescence results of ALOX15 in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+Ver: monobenzone + restraint + Ver group, Mon+RS+tacrolimus: monobenzone + restraint + tacrolimus group;

[0022] Figure 7 The ELISA test results of CCL25 in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone+restraint group, Mon+RS+Ver: monobenzone+restraint+Ver group;

[0023] Figure 8 The immunofluorescence results of CCR9 and CD8α in Example 1 of the present invention, wherein Con: normal group, Mon: monobenzone group, Mon+RS: monobenzone + restraint group, Mon+RS+Ver: monobenzone + restraint + Ver group, Mon+RS+tacrolimus: monobenzone + restraint + tacrolimus group;

[0024] Fig. 9 This is the result of the CCL25 content in the serum of the subjects in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

[0027] Example 1

[0028] 1. Animal Experiments

[0029] Female 3-week-old SPFC57BL / 6 mice were purchased. The model group (monobenzone group) was treated with 0.2 ml of 40% monobenzone cream applied on a 2×2 cm 2The back was shaved, and the control group (normal group) was treated with blank cream applied on a 2×2 cm 2 The back was shaved for 8 weeks. The model group was given restraint stress for 18 hours (16:00-10:00) every other day (i.e., the Monobenzone + restraint group). There were 8 mice in each group. The drug-treated group (Monobenzone + restraint + Ver group) was subcutaneously injected with Vercirnon (Ver, 10.0 mg / kg) every other day after restraint stress; the Monobenzone + restraint + tacrolimus group was smeared with 0.1% tacrolimus ointment (10.0 mg / kg) on ​​a 2×2 cm 2 The backs were shaved for 2 weeks. At the end of the experiment, the mice were anesthetized with ether and killed, and the skin tissue and blood of the mice were collected.

[0030] Restraint stress: Use an electric soldering iron to make a hole in the front and back of a 50ml centrifuge tube. The front is the breathing hole for the mouse, and the back is where the tail comes out. Make a row of 3 holes on the side. The 3 rows are the ventilation holes for the mouse. Figure 1 shown.

[0031] 2. Skin lesions: evaluation of depigmentation

[0032] Each exposure site was examined and the degree of depigmentation was estimated as a percentage of the anatomical site. Scoring was performed according to the following scheme:

[0033] No signs of discoloration (0%), 0;

[0034] >5%, 1 point;

[0035] >5-25%, 2 points;

[0036] >25-50%, 3 points;

[0037] >50-75%, 4 points;

[0038] 75~100%, 5 points.

[0039] The depigmentation score was defined as the sum of the scores.

[0040] 3. Immunofluorescence Staining

[0041] The cut paraffin sections were placed in xylene I for 20 min-xylene II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min-50% alcohol for 5 min-25% alcohol for 5 min, and dewaxed by distilled water for 3 to 5 times. After antigen repair and membrane permeabilization, 3% hydrogen peroxide was added to treat the catalase in the tissue, and finally blocked with goat serum. Then, primary antibodies CCR9 (antibody concentration 1:200), CD8α (antibody concentration 1:50), ALOX15 (antibody concentration 1:50), E-cadherin (antibody concentration 1:1000), MelanA (antibody concentration 1:1000) were added for incubation at 4°C overnight. After the target slices were washed with TBST three times, AlexaFluorTM488goatanti-rabbitlgG(H+L) and AlexaFluorTM555goatanti-mouselgG(H+L) fluorescent secondary antibodies (1:1000) were added to the target tissues and incubated at 37°C in the dark for 60 minutes. Subsequently, each target tissue was washed three times with PBS for 5 minutes each time. The researchers stained the target tissues with DAPI (1:1000) for 10 minutes in the dark at 37°C. Similarly, the target tissues were washed three times with PBS for 5 minutes each time. Finally, each target slice was sealed with anti-fluorescence quenching sealing agent, and attention was paid to avoid light and dryness, and the tissue staining was recorded using a laser confocal imaging system. During the operation, continuous slices in the same slice were taken as background controls, and the controls were incubated with goat serum without primary antibody when incubating the primary antibody.

[0042] 4. Elisa kit detection (E-cadherin and CCL25)

[0043] Set up standard wells and sample wells, and add 50μL of standards of different concentrations to each standard well. Set up blank control wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μL of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. Add 100μL of enzyme-labeled reagent to each well, except the blank control well. Seal the plate with a sealing film and incubate at 37℃ for 60 minutes. Then dilute the 20-fold concentrated washing solution with 20-fold distilled water for use. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and discard it, repeat this 5 times, and pat dry. First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color at 37℃ in the dark for 15 minutes. Add 50μL of stop solution to each well to stop the reaction (the blue color turns yellow immediately). Use the blank control well as zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0044] 5. Experimental Results

[0045] The experimental results are as follows Figures 2 to 8 shown.

[0046] Depend on Figure 2 , Figure 3 It can be seen that the Mon group had back hair depigmentation compared to the Con group. After restraint, the back hair depigmentation of Mon+RS was aggravated. Then, on this basis, Ver (10.0 mg / kg) was subcutaneously injected, and the back hair depigmentation of the Mon+RS+Ver group was improved. The back score was taken in the fourth week after administration, and statistics were collected. ( ** P<0.01 compared with Con, ## P<0.01 compared with the Mon group; ^^P<0.01 compared with the Mon+RS group).

[0047] Depend on Figure 4 The results showed that in the epidermis of the skin, compared with the Con group, the expression of E-cadherin and Melan-A in the Mon group was reduced, and the binding expression decreased; after restraint, the negative expression of E-cadherin and Melan-A in the Mon+RS group was aggravated, and the binding expression was even less; after administration, the positive expression of E-cadherin and Melan-A in the Mon+RS+Ver group increased, and the binding expression also increased significantly; the positive expression and binding expression of E-cadherin and Melan-A in the Mon+RS+tacrolimus group also increased, but the effect was average compared with the Mon+RS+Ver group.

[0048] Depend on Figure 5 The results showed that in the serum, the expression of E-cadherin in the Mon group decreased compared with the Con group. After restraint, the negative expression of E-cadherin in the Mon+RS group increased, and the expression of E-cadherin in the Mon+RS+Ver group increased ( * P<0.05 compared with Mon, ## P < 0.01 compared with the Mon + RS group).

[0049] Depend on Figure 6 The results showed that in the epidermis of the skin, the positive expression of ALOX15 in the Mon group was higher than that in the Con group. After restraint, the positive expression of ALOX15 in the Mon+RS group was aggravated, the positive expression of ALOX15 in the Mon+RS+Ver group was reduced, and the positive expression of ALOX15 in the Mon+RS+tacrolimus group was also decreased, but the effect was not as obvious as that in the Mon+RS+Ver group.

[0050] Depend on Figure 7 The results showed that in the serum, the expression of CCL25 in the Mon group was not significantly increased compared with the Con group, but after restraint, the positive expression of CCL25 in the Mon+RS group increased, and the expression of CCL25 in the Mon+RS+Ver group decreased significantly. ( * P<0.05 compared with Mon, ## P < 0.01 compared with the Mon + RS group).

[0051] Depend on Figure 8 The results showed that in the epidermis of the skin, compared with the Con group, the positive expression of CCR9 and CD8α in the Mon group increased, and after restraint, the positive expression of CCR9 and CD8α in the Mon+RS group increased, and the combined expression of the two also increased significantly. After medication, the positive expression of CCR9 and CD8α in the Mon+RS+Ver group decreased, and the combined expression also decreased significantly. The positive expression and combined expression of CCR9 and CD8α in the Mon+RS+tacrolimus group also decreased.

[0052] Example 2

[0053] 91 subjects were randomly selected to measure the CCL25 content in serum. Fig. 9 shown.

[0054] Depend on Fig. 9 The results showed that among the 91 randomly selected subjects, 71 were patients with vitiligo and 20 were normal subjects. The CCL25 concentration in the serum of patients with vitiligo was significantly higher than that of normal subjects. * P<0.05 compared with the normal group)

[0055] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0056] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Viseno in the preparation of drugs for the treatment of vitiligo.

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