Application of XBD-173 in preparation of medicine for treating and / or preventing depigmentation disease caused by pigment synthesis disorder

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of XBD-173 or pharmaceutically acceptable salts thereof in preparation of medicines for treating and / or preventing pigment decoloring diseases caused by pigment synthesis disorders, in particular to application in preparation of medicines for treating and / or preventing vitiligo, vitiligo, pityriasis albicans and albinism. Experiments prove that XBD-173 can significantly increase melanin synthesis and promote color recovery of pigments in a skin lesion area on the back of a leucoderma mouse, and meanwhile, the XBD-173 has no obvious toxicity. In conclusion, the XBD-173 is clear in action mechanism, remarkable in effect and free of remarkable side effects, and has remarkable advantages and progress compared with an existing medicine.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a new use of XBD-173, and specifically relates to the use of XBD-173 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders, especially in the preparation of a drug for treating and / or preventing vitiligo, leukoplakia, pityriasis alba, and albinism. Background Art

[0002] Melanin is a key pigment synthesized by melanocytes and has multiple physiological functions, including protecting the skin from damage by ultraviolet radiation, participating in the defense mechanism of the immune system, and determining the color of the skin, hair, and eyes. Melanin production involves complex biological processes, including synthesis, transport, and distribution. This process is affected by multiple factors, including genetics, environmental factors, and health conditions, which may lead to disorders in synthesis and synthesis mechanisms. When the number of melanocytes decreases, the synthesis capacity decreases, or the synthesis process is blocked, depigmentation diseases such as vitiligo and leukoplakia, pityriasis alba, and albinism will occur. These diseases are widespread worldwide and have a significant impact on the health and quality of life of patients.

[0003] At present, the drugs commonly used in clinical treatment of depigmentation diseases include photosensitizers (such as psoralen, etc.), corticosteroids (such as hydrocortisone, etc.), calcineurin inhibitors (such as tacrolimus, etc.), etc. These drugs generally have many side effects, unstable efficacy, and poor effects on severe cases. For example, psoralen needs to be used with other treatments (such as PUVA therapy) but may cause photoaging and skin cancer, hydrocortisone relapses after discontinuation, and tacrolimus causes skin itching, burning sensation or itching. Therefore, new therapeutic drugs still need to be developed to improve efficacy and reduce side effects.

[0004] XBD-173 (CAS: 226954-04-7), chemical name is N-ethyl-7,8-dihydro-7-methyl-8-oxo-2-phenyl-N-(phenylmethyl)-9H-purine-9-acetamide; molecular weight is C 23 H 23 N5O2; molecular weight is 401.46; color is white to beige powder. The chemical structure is as follows:

[0005]

[0006] At present, the main research direction of XBD-173 is focused on the treatment of psychological and psychiatric diseases such as anxiety, panic and stress-related disorders. However, there are no reports on its effect of promoting pigment synthesis at home and abroad. Summary of the invention

[0007] The technical problem to be solved by the present invention is that the existing drugs for treating depigmentation diseases caused by pigment synthesis disorders generally have many side effects, unstable efficacy and poor effects in severe cases. Provided is a use of XBD-173 and a pharmaceutically acceptable salt of XBD-173 in the preparation of drugs for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders, especially in the preparation of drugs for treating vitiligo, leukoplakia, pityriasis alba and albinism.

[0008] On this basis, the present invention also provides a pharmaceutical composition containing XBD-173 or a pharmaceutically acceptable salt thereof as an effective ingredient for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders.

[0009] The invention has been proved through a large number of modern pharmacological scientific studies and animal pharmacodynamic experiments that XBD-173 can promote melanin synthesis and has the efficacy of treating depigmentation diseases, especially vitiligo, leukoplakia, pityriasis alba and albinism.

[0010] At present, the main drugs for the treatment of vitiligo in clinical practice are hormones and immunosuppressants. Long-term use has significant side effects and is difficult to tolerate in the long term. The phototherapy treatment cycle is long, and patients need to visit the doctor or receive irradiation many times, with poor compliance, which can cause skin photosensitivity, sunburn, and itching. Long-term use increases the risk of skin cancer. In in vitro experiments, XBD-173 can significantly increase melanin synthesis and promote the restoration of pigmentation in the lesion area on the back of vitiligo mice without obvious toxicity. In summary, XBD-173 has a clear mechanism of action, significant effects and no significant side effects, and has significant advantages and improvements over existing drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a bar graph of the relative number of B16F10 cells.

[0012] Compared with the blank control group, 5-20 μM concentration of XBD-173 had no significant effect on the growth of B16F10 cells (P>0.05), while 40 μM concentration of XBD-173 inhibited the proliferation of B16F10 cells ( * P<0.05).

[0013] Figure 2 It is a bar graph of the relative content of melanin in B16F10 cells.

[0014] Compared with the control group, different concentrations of XBD-173 (10, 20 μM) significantly increased the melanin content in B16F10 cells ( ** P < 0.01; *** P<0.001). Figure 3It is a bar graph of the relative activity of tyrosinase in B16F10 cells. Compared with the control group, different concentrations of XBD-173 (10, 20 μM) significantly increased the tyrosinase activity of B16F10 cells ( ** P < 0.01; *** P<0.001). Figure 4 This is an immunoblot of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells. Figure 4 A is a protein immunoblot. Figure 4 B is a bar graph of relative protein expression.

[0015] Compared with the control group, XBD-173 significantly promoted the expression of melanin-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells. * P<0.05, ** P<0.01).

[0016] Figure 5 It is a bar graph of the relative content of melanin in the skin tissue of the lesion area of ​​the vitiligo mouse model.

[0017] Compared with the blank control group, the epidermal melanin in the skin of mice in the vitiligo model group was significantly reduced (*P<0.05). Compared with the vitiligo model group, the XBD-173 group could significantly promote the content of pigment in the skin lesions of mice in the vitiligo model group ( # P<0.05).

[0018] Figure 6 This is an immunoblot of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the back skin of vitiligo mice. Figure 6 A is a protein immunoblot. Figure 6 B is a bar graph of relative protein expression.

[0019] Compared with the control group, the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the back skin tissue of mice in the vitiligo model group was significantly decreased ( ** P<0.01). Compared with the vitiligo model group, the XBD-173 treatment group significantly increased the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) ( ## P<0.01). DETAILED DESCRIPTION

[0020] 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.

[0021] Below are some pharmacodynamic tests and results of the present invention:

[0022] Part I: Effects of XBD-173 on melanin synthesis in B16F10 cells

[0023] 1. Effect of XBD-173 on B16F10 cell proliferation

[0024] B16F10 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) in the exponential growth phase and in good condition were taken and cultured at a rate of 1×10 4 The cells were inoculated at a density of 100 μM / well in a 96-well cell culture plate and incubated in an incubator at 37°C and 5% CO2 for 24 hours. Then, different concentrations of XBD-173 (5, 10, 20, 40 μM) were added to each experimental group, and the same amount of DMSO was added to the control group. Continue to incubate for 48 hours under the same conditions. After the culture, 20 μL MTT solution (final concentration 0.5 mg / mL) was added to each well and reacted at 37°C for 4 hours. After removing the supernatant, 200 μL DMSO was added to each well and placed on a flat plate oscillator for 10 minutes to ensure full dissolution. The absorbance of each well was measured at a wavelength of 570 nm using a microplate reader. The cell proliferation rate was calculated by comparing the absorbance of the XBD-173 treatment group with the control group at different concentrations.

[0025] Experimental results: Figure 1 As shown in the figure, compared with the blank control group, 5-20 μM concentration of XBD-173 had no significant effect on the growth of B16F10 cells (P>0.05), while 40 μM concentration of XBD-173 inhibited the proliferation of B16F10 cells ( * P<0.05).

[0026] Experimental conclusion: In the concentration range of 5-20μM, XBD-173 had no significant effect on the proliferation of B16F10 cells, but showed a certain inhibitory effect at 40μM.

[0027] 2. Effect of XBD-173 on melanin synthesis in B16F10 cells

[0028] B16F10 cells in exponential growth phase and in good condition were taken and 2×10 5The cells were inoculated at a density of 100 μL / well in a 6-well cell culture plate and incubated in an incubator at 37°C and 5% CO2 for 24 hours. After the cells adhered to the wall and grew well, different concentrations of XBD-173 (5, 10, 20 μM) were added to each experimental group, and the control group was added with an equal amount of DMSO as a control, and continued to be cultured under the same conditions for 48 hours. After the culture was completed, the cells of each group were collected and 100 μL of non-denaturing lysis solution (containing 1 mMPMSF) was added, and lysed at 4°C for 15 minutes. The lysed samples were centrifuged at 4°C and 13000 r / min for 15 minutes, and the supernatant was taken for protein concentration determination (BCA method) to calculate the total protein content. The melanin precipitate after centrifugation was added with 100 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 100 μL / well, and the absorbance value was measured at a wavelength of 405 nm using an ELISA reader.

[0029] Experimental results: Figure 2 As shown, compared with the control group, different concentrations of XBD-173 (10, 20 μM) significantly increased the melanin content in B16F10 cells ( ** P < 0.01; *** P<0.001).

[0030] Experimental conclusion: XBD-173 can increase melanin synthesis in B16F10 cells in a dose-dependent manner.

[0031] Effects of XBD-173 on tyrosinase activity and expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells

[0032] B16F10 cells in exponential growth phase and in good condition were taken and 2×10 5 The cells were inoculated at a density of 100 μL / well in a 6-well cell culture plate and incubated in an incubator at 37°C and 5% CO2 for 24 hours. After the cells adhered to the wall and grew well, different concentrations of XBD-173 (5, 10, 20 μM) were added to each experimental group, and the control group was added with an equal amount of DMSO as a control, and continued to be cultured under the same conditions for 48 hours. After the culture was completed, the cells in each group were collected and 100 μL of non-denaturing lysis solution (containing 1 mM PMSF) was added, and lysed at 4°C for 15 minutes. The lysed samples were centrifuged at 4°C and 13000 r / min for 15 minutes, and the supernatant was taken for protein concentration determination (BCA method) to calculate the total protein content.

[0033] Tyrosinase activity detection: 40 μg of protein was mixed with 0.1 mol / L PBS to a final volume of 100 μL, 100 μL of 0.1% L-DOPA solution was added, and incubated at 37°C for 30 minutes. The absorbance value was measured at a wavelength of 475 nm using an ELISA reader, and the results were expressed as the ratio of the XBD-173 treatment group with different concentrations to the control group.

[0034] Western Blot detection: Add 1 / 4 volume of Loading Buffer to the protein sample, mix well and boil in a 97℃ water bath for 7 minutes for denaturation treatment. Load the treated protein sample on SDS-PAGE 12% separation gel and 5% concentration gel for protein electrophoresis, the electrophoresis conditions are 80V, 30 minutes and 120V, 60 minutes. The transfer condition is 320mA, 1 hour, and the target protein is transferred to the NC membrane. After blocking the membrane with TBST buffer containing 2.5% skim milk (containing 0.05% Tween-20) for 1.5 hours, add Tyrosinase, TRP-1, TRP-2 and β-actin primary antibodies (1:1000) and incubate at 4℃ overnight. The next day, wash the membrane 4 times with TBST, 7 minutes each time, add HRP-labeled secondary antibody, and incubate at room temperature for 1 hour. Wash 4 times with TBST, 7 minutes each time. Add chemiluminescent substrate for development, and take band images with a gel imaging system. The gray value of the target protein band was analyzed by Bio-Rad's Quantity One software, and the relative expression level was calculated using β-actin as an internal reference.

[0035] Experimental results: Figure 3 As shown in Figure 2, compared with the control group, different concentrations of XBD-173 (10, 20 μM) significantly increased the tyrosinase activity of B16F10 cells ( ** P < 0.01; *** P<0.001). Figure 4 As shown in the figure, compared with the control group, XBD-173 can significantly promote the expression of melanin-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells ( * P<0.05, ** P<0.01).

[0036] Experimental conclusion: XBD-173 can increase the tyrosinase activity and the expression of melanin production-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells in a dose-dependent manner, thereby promoting melanin synthesis.

[0037] Part II: Effects of XBD-173 on hydrogen peroxide-induced vitiligo mouse model

[0038] 1. Effect of XBD-173 on the melanin content in the back skin of a mouse model of vitiligo induced by hydrogen peroxide

[0039] After C57BL / 6 mice (purchased from Shanghai Bikeko Wing Biotechnology Co., Ltd.) were adapted to feeding, the back hair of the 2cm×2cm area was removed with an electric shaver one day before the experiment, and the small hairs were removed with a depilatory cream to induce a synchronized hair follicle growth cycle. 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 + XBD-173 (20 mg / kg) administration group. The model was established by applying 5% H2O2 to the depilatory area every day, and the modeling period was four weeks. After the model was successfully established, 20 mg / kg XBD-173 was applied to the mouse skin lesions for three consecutive weeks. 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 5 shown.

[0040] 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<0.05). Compared with the vitiligo model group, the XBD-173 group could significantly promote the content of pigment in the skin lesions of mice in the vitiligo model group ( # P<0.05).

[0041] Experimental conclusion: XBD-173 can effectively increase the melanin content in the back skin of the hydrogen peroxide-induced vitiligo mouse model.

[0042] Effects of XBD-173 on the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the back skin of a mouse model of vitiligo induced by hydrogen peroxide

[0043] The skin tissue samples from the back of the above-mentioned experimental mice were taken. After the skin tissue (20 mg) was aseptically sheared, pre-cooled tissue lysis solution (containing protease inhibitors) was added, and the homogenizer was used to fully homogenize, and the supernatant was extracted by centrifugation to obtain total protein. After quantitative determination of protein concentration using the BCA method, 1 / 4 volume of Loading Buffer was added to the protein sample, mixed and placed in a 97°C water bath and boiled for 7 minutes for protein denaturation. The denatured protein sample was loaded on an SDS-PAGE polyacrylamide gel for electrophoresis separation. After the electrophoresis was completed, the protein was transferred to a nitrocellulose membrane (NC membrane) under a constant current condition of 320 mA. After transfer, TBST buffer containing 2.5% skim milk (containing 0.05% Tween-20) was used for blocking for 1.5 hours to reduce nonspecific binding. Subsequently, primary antibodies (Tyrosinase, TRP-1, TRP-2, β-actin) dissolved in the blocking solution were added respectively and incubated overnight at 4°C. The next day, the membrane was washed 4 times with TBST buffer for 7 minutes each time, then HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour, and the membrane was washed again with TBST for 4 times for 7 minutes each time. After washing, chemiluminescent substrate was added to the membrane surface, and the band image was captured and saved by the gel imaging system. The gray value of the obtained band was analyzed by Bio-Rad's Quantity One software, and β-actin was used as an internal reference to calculate the relative expression of the target protein. For detailed experimental results, see Figure 6 .

[0044] Experimental results: Compared with the control group, the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the back skin tissue of mice in the H2O2 treatment group was significantly reduced ( ** P<0.01). Compared with the H2O2 treatment group, the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the H2O2+XBD-173 treatment group was significantly increased ( ## P<0.01).

[0045] Experimental conclusion: XBD-173 can effectively increase the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the back skin of the hydrogen peroxide-induced vitiligo mouse model.

[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 XBD-173 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders.

2. The use according to claim 1, characterized in that: The depigmentation diseases caused by the pigment synthesis disorder are vitiligo, leukoplakia, pityriasis alba, and albinism.

3. Use of a pharmaceutically acceptable salt of XBD-173 in the preparation of a drug for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders.

4. The pharmaceutically acceptable salt according to claim 3, characterized in that: XBD-173 contains a tertiary amine group that is capable of forming salts with acids.

5. The pharmaceutically acceptable salt according to claim 3, characterized in that The acid includes but is not limited to inorganic acid and organic acid; the inorganic acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, and the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, oxalic acid, tartaric acid, lactic acid, acetic acid or citric acid.

6. The pharmaceutically acceptable salt according to claim 3, characterized in that The pharmaceutically acceptable salt can be prepared by mixing and reacting a compound with an appropriate acid in a polar solvent, and then purified by crystallization.

7. The use according to claim 3, characterized in that: The depigmentation diseases caused by the pigment synthesis disorder are vitiligo, leukoplakia, pityriasis alba, and albinism.

8. A pharmaceutical composition for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders, characterized in that: The pharmaceutical composition contains XBD-173 or a pharmaceutically acceptable salt thereof.

9. The pharmaceutical composition according to claim 8, characterized in that: The depigmentation diseases caused by the pigment synthesis disorder are vitiligo, leukoplakia, pityriasis alba, and albinism.

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