Application of XBD-173 in the preparation of drugs for the treatment and / or prevention of pigment loss caused by disorders of pigment synthesis.

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of XBD-173 or its pharmaceutically acceptable salts in the preparation of remedies for treating and / or preventing depigmentation diseases caused by pigment synthesis disorders, particularly in the preparation of remedies for treating and / or preventing vitiligo, leukoderma, pityriasis alba, and albinism. Experiments have shown that XBD-173 can significantly increase melanin synthesis and promote repigmentation in the dorsal lesions of vitiligo mice, while exhibiting no significant toxicity. In summary, XBD-173 has a clear mechanism of action, significant effects, and no significant side effects, demonstrating significant advantages and advancements compared to existing drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to new uses of XBD-173, and more specifically to the use of XBD-173 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of pigment loss diseases caused by pigment synthesis disorders, particularly in the preparation of medicaments for the treatment and / or prevention of vitiligo, leukoderma, pityriasis alba, and albinism. Background Technology

[0002] Melanin is a key pigment synthesized by melanocytes, serving multiple physiological functions, including protecting the skin from damage caused by ultraviolet radiation, participating in the immune system's defense mechanisms, and determining the color of skin, hair, and eyes. Melanin production involves complex biological processes, including synthesis, transport, and distribution. This process is influenced by multiple factors, including genetics, environment, and health conditions, which can lead to disorders in synthesis and its mechanisms. When the number of melanocytes decreases, their synthetic capacity declines, or the synthetic process is blocked, depigmentation disorders can occur, such as vitiligo, leukoderma, pityriasis alba, and albinism. These diseases are widespread globally and significantly impact patients' health and quality of life.

[0003] Currently, commonly used drugs for treating pigmentation disorders include photosensitizing agents (such as psoralen), corticosteroids (such as hydrocortisone), and calcineurin inhibitors (such as tacrolimus). These drugs generally suffer from numerous side effects, unstable efficacy, and poor results in severe cases. For example, psoralen requires combination with other treatments (such as PUVA therapy), but may cause photoaging and skin cancer; hydrocortisone can lead to relapse after discontinuation; and tacrolimus can cause skin itching, burning sensations, or pruritus. Therefore, new therapeutic drugs need to be developed to improve efficacy and reduce side effects.

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

[0005]

[0006] Currently, research on XBD-173 mainly focuses on the treatment of psychological and mental illnesses such as anxiety, panic, and stress-related disorders. However, there are no reports, either domestically or internationally, on its effect of promoting pigment synthesis. Summary of the Invention

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

[0008] Based on this, the present invention also provides a pharmaceutical composition for treating and / or preventing pigment loss diseases caused by pigment synthesis disorders, with XBD-173 or a pharmaceutically acceptable salt thereof as the active ingredient.

[0009] Through extensive modern pharmacological research and animal pharmacodynamic experiments, this invention has demonstrated that XBD-173 can promote melanin synthesis and has the effect of treating pigment loss diseases, especially vitiligo, leukoderma, pityriasis alba, and albinism.

[0010] Currently, the main clinical treatments for vitiligo are hormones and immunosuppressants, which have significant side effects with long-term use and are difficult to tolerate. Phototherapy has a long treatment cycle, requiring patients to visit the clinic or undergo irradiation multiple times, leading to poor adherence and causing photosensitivity, sunburn, and itching. Long-term use also increases the risk of skin cancer. In in vitro experiments, XBD-173 significantly increased melanin synthesis and promoted repigmentation in the dorsal lesions of vitiligo mice, without significant toxicity. In summary, XBD-173 has a clear mechanism of action, significant efficacy, and no significant side effects, representing a significant advantage and advancement compared to existing drugs. Attached Figure Description

[0011] Figure 1 This is a bar chart showing the relative number of B16F10 cells.

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

[0013] Figure 2 This is a bar chart showing the relative melanin content in B16F10 cells.

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

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

[0016] Figure 5 This is a bar chart showing the relative melanin content in the skin tissue 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 significantly promoted the pigment content in the skin lesions of mice in the vitiligo model group. # P<0.05).

[0018] Figure 6 This is an immunoblot image of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the dorsal skin of vitiligo mice. Figure 6 A is the Western blot image of the protein. Figure 6 B is a bar chart showing the relative expression levels of proteins.

[0019] Compared with the control group, the expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in the dorsal skin tissue of mice in the vitiligo model group was significantly reduced. ** 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 Implementation

[0020] 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.

[0021] The following are some of the pharmacodynamic tests and results of this invention:

[0022] Part 1: Effects of XBD-173 on melanin synthesis in B16F10 cells

[0023] I. Effects of XBD-173 on the proliferation of B16F10 cells

[0024] B16F10 cells in the exponential growth phase and in good condition (purchased from the Cell Bank of the Chinese Academy of Sciences) were harvested at a rate of 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of [number] cells / well in 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. Then, different concentrations of XBD-173 (5, 10, 20, and 40 μM) were added to each experimental group, while the control group received an equal volume of DMSO. Incubation continued under the same conditions for another 48 hours. After incubation, 20 μL MTT solution (final concentration 0.5 mg / mL) was added to each well, and the reaction was carried out at 37°C for 4 hours. After removing the supernatant, 200 μL LDMSO was added to each well, and the plate was shaken for 10 minutes to ensure complete dissolution. The absorbance of each well was measured at 570 nm using a microplate reader. Cell proliferation rate was calculated by comparing the absorbance values ​​of the different XBD-173 treatment groups with the control group.

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

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

[0027] II. Effects of XBD-173 on melanin synthesis in B16F10 cells

[0028] Take B16F10 cells that are in the exponential growth phase and in good condition, and administer them at a rate of 2 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 6-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours. After good cell adhesion and growth, different concentrations of XBD-173 (5, 10, and 20 μM) were added to each experimental group, while an equal volume of DMSO was added to the control group. Cells were cultured under the same conditions for another 48 hours. After culture, cells from each group were collected and 100 μL of non-denaturing lysis buffer (containing 1 mM PMSF) was added. Lysis was performed at 4°C for 15 minutes. The lysed samples were centrifuged at 13000 rpm for 15 minutes at 4°C, and the supernatant was used for protein concentration determination (BCA method) to calculate the total protein content. The centrifuged melanin precipitate was added to 100 μL of NaOH solution (1 mol / L, containing 10% DMSO) and lysed in an 80°C water bath for 2 hours to ensure complete dissolution of the melanin. The completely dissolved melanin solution was transferred to a 96-well plate at a volume of 100 μL / well, and the absorbance was measured at 405 nm using a microplate reader.

[0029] Experimental results: such as 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] III. Effects of XBD-173 on tyrosinase activity and expression of melanin synthesis-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells

[0032] Take B16F10 cells that are in the exponential growth phase and in good condition, and administer them at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of cells / well in 6-well cell culture plates and incubated at 37°C and 5% CO2 for 24 hours. After the cells adhered well and grew, different concentrations of XBD-173 (5, 10, and 20 μM) were added to each experimental group, while an equal volume of DMSO was added to the control group. Cells were cultured under the same conditions for another 48 hours. After culture, cells from each group were collected and 100 μL of non-denaturing lysis buffer (containing 1 mM PMSF) was added. The cells were lysed at 4°C for 15 minutes. The lysed samples were centrifuged at 13000 rpm for 15 minutes at 4°C, and the supernatant was used for protein concentration determination (BCA method) to calculate the total protein content.

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

[0034] Western Blot Detection: Add 1 / 4 volume of loading buffer to the protein sample, mix well, and boil at 97°C for 7 minutes for denaturation. Load the treated protein sample onto SDS-PAGE 12% separating gel and 5% stacking gel for electrophoresis at 80V for 30 minutes and 120V for 60 minutes. Transfer the target protein to an NC membrane at 320mA for 1 hour. Block the membrane for 1.5 hours with TBST buffer (containing 0.05% Tween-20) containing 2.5% skim milk. Add Tyrosinase, TRP-1, TRP-2, and β-actin primary antibody (1:1000) and incubate overnight at 4°C. The next day, wash the membrane four times with TBST for 7 minutes each time, add HRP-labeled secondary antibody, and incubate at room temperature for 1 hour. Wash four times with TBST for 7 minutes each time. Add chemiluminescent substrate for development and image the bands using a gel imaging system. The grayscale values ​​of the target protein bands were analyzed using Bio-Rad's Quantity One software, and the relative expression level was calculated using β-actin as an internal reference.

[0035] Experimental results: such as Figure 3 As shown, 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). For example... Figure 4 As shown, compared with the control group, XBD-173 significantly promoted the expression of melanin production-related proteins (Tyrosinase, TRP-1, TRP-2) in B16F10 cells. * P<0.05, ** P<0.01).

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

[0037] Part Two: Effects of XBD-173 on a Hydrogen Peroxide-Induced Vitiligo Mouse Model

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

[0039] C57BL / 6 mice (purchased from Shanghai Bikai Keyi Biotechnology Co., Ltd.) were acclimatized, and one day before the experiment, a 2cm × 2cm area of ​​dorsal hair was removed with an electric shaver, and fine hairs were removed with depilatory cream to induce a synchronized hair follicle growth cycle. The mice were randomly divided into three groups (n=6 per group), numbered sequentially: (Group I) blank control group; (Group II) vitiligo model group (5% hydrogen peroxide); (Group III) vitiligo model group + XBD-173 (20mg / kg) treatment group. Modeling was initiated by applying 5% H2O2 to the depilated area daily for four weeks. After successful modeling, 20mg / kg XBD-173 was applied to the affected skin area for three consecutive weeks. 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 5 As 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 significantly promoted the pigment content 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 a mouse model of vitiligo induced by hydrogen peroxide.

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

[0043] Skin tissue samples from the backs of the experimental mice were collected. The skin tissue (20 mg) was aseptically cut, and pre-cooled tissue lysis buffer (containing protease inhibitors) was added. The samples were homogenized thoroughly using a homogenizer, and the supernatant was extracted by centrifugation to obtain total protein. After quantifying the protein concentration using the BCA method, 1 / 4 volume of loading buffer was added to the protein sample, mixed, and then boiled in a 97°C water bath for 7 minutes to denature the protein. The denatured protein samples were then loaded onto an SDS-PAGE polyacrylamide gel for electrophoresis. After electrophoresis, the protein was transferred to a nitrocellulose membrane (NC membrane) at a constant current of 320 mA. After transfer, the membrane was blocked for 1.5 hours with TBST buffer (containing 0.05% Tween-20) containing 2.5% skim milk to reduce non-specific binding. Subsequently, primary antibodies (Tyrosinase, TRP-1, TRP-2, β-actin) dissolved in the blocking buffer were added, and the membrane was incubated overnight at 4°C. The following day, the membrane was washed four times with TBST buffer for 7 minutes each time. HRP-labeled secondary antibody was then added, and the membrane was incubated at room temperature for 1 hour. The membrane was then washed four more times with TBST buffer for 7 minutes each time. After washing, a chemiluminescent substrate was added to the membrane surface, and band images were captured and saved using a gel imaging system. The grayscale values ​​of the obtained bands were analyzed using Bio-Rad's Quantity One software, with β-actin as an internal control, to calculate the relative expression level of the target protein. Detailed experimental results can be found in [link to results]. Figure 6 .

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

[0046] 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 XBD-173 or a pharmaceutically acceptable salt in the preparation of medicaments for the treatment and / or prevention of pigment loss disorders caused by pigment synthesis disorders, such as vitiligo and pityriasis alba.

2. The use according to claim 1, characterized in that... The pharmaceutically acceptable salt of XBD-173 is the salt formed by its tertiary amine group and an acid.

3. The use according to claim 2, characterized in that... The applicable acid is selected from inorganic or organic acids; the inorganic acid is selected from hydrochloric acid, sulfuric acid or 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.

4. The use according to claim 2, characterized in that... The pharmaceutically acceptable salt is prepared by mixing and reacting the compound with a suitable acid in a polar solvent, and then purified by crystallization.

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