Application of Ro5-4864 or pharmaceutically acceptable salt in preparation of medicine for treating and / or preventing melanocyte degeneration disease caused by inflammatory immune response induced by oxidative stress

Abstract

The invention relates to application of Ro5-4864 or pharmaceutically acceptable salt in preparation of a medicine for treating and / or preventing melanocyte degeneration diseases caused by inflammatory immune response induced by oxidative stress. The melanocyte degeneration diseases caused by inflammatory immune response induced by oxidative stress comprise vitiligo, leukoplakia and pityriasis albicans. The Ro5-4864 and the pharmaceutically acceptable salt of the Ro5-4864, disclosed by the invention, inhibit continuous loss of melanocytes from oxidative stress, are strong in targeting effect and relatively small in side effect, and have remarkable advantages and progress compared with existing medicines.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to new uses of Ro5-4864, specifically the use of Ro5-4864 or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating and / or preventing melanocyte loss diseases caused by oxidative stress-induced inflammatory immune responses, particularly in the preparation of medicaments for treating and / or preventing vitiligo, leukoderma, and pityriasis alba. Background Technology

[0002] Vitiligo is a common acquired depigmentation disorder characterized by the gradual loss of function or dysfunction of melanocytes, resulting in well-defined white patches on the skin, severely impacting patients' appearance, mental health, and quality of life. Although the etiology is complex, oxidative stress is increasingly being recognized as a core driver of the disease's pathogenesis. In melanocytes, excessive accumulation of reactive oxygen species (ROS) disrupts the redox balance, activates the inflammatory cascade, and promotes autoimmune-mediated melanocyte destruction.

[0003] A research team led by Chen Ting published a paper in Nature titled "Anatomically distinct fibroblast subsets determine skin autoimmune patterns." Nature 601, 118–124 (2022). https: / / doi.org / 10.1038 / s41586-021-04221-8. This study expands upon this theoretical framework, highlighting the crucial role of dermal fibroblasts in the pathophysiology of vitiligo. In lesioned skin, fibroblasts exhibit elevated levels of reactive oxygen species (ROS) and secrete various pro-inflammatory and pro-aging mediators, including chemokines, adhesion molecules, and signaling regulators. These alterations maintain chronic inflammation, disrupt the adhesion function of melanocytes, and ultimately lead to pigmentary disorders. Increasing evidence suggests that this inflammatory phenotype is closely related to oxidative stress. Persistent ROS accumulation weakens the intrinsic antioxidant capacity of fibroblasts, creating a vicious cycle of oxidative stress and inflammation.

[0004] Currently, commonly used drugs for treating pigment loss disorders include photosensitizing agents, glucocorticoids, calcineurin inhibitors, and immunosuppressants. However, these generally suffer from numerous side effects, unstable efficacy, and poor results in severe cases. It is noteworthy that ultraviolet phototherapy remains valuable, with narrow-band UVB (NB-UVB) appearing superior to topical psoralen combined with UVA (PUVA) or excimer laser therapy. Furthermore, combining phototherapy with systemic antioxidants (such as extracts from *Polygonum aviculare* or gluten-protective superoxide dismutase) shows great promise. Meanwhile, recently approved topical JAK inhibitors (such as ruxotetinib) have also demonstrated significant efficacy in clinical trials. Therefore, combating oxidative stress and subsequent inflammatory and immune responses provides insights for developing new therapeutic agents.

[0005] Ro5-4864 (4'-chlorodiazepam, CAS: 14439-61-3), chemically named 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepine-2-one; molecular formula is C 16 H 12 Cl₂N₂O; molecular weight 319.19; white to pale yellow solid. Chemical structural formula as follows: Currently, the main research focus of Ro5-4864 is on psychological and mental illnesses such as anxiety, seizures, regulation of GABA-A receptors, and stress-related disorders. However, there are currently no reports, either domestically or internationally, on its role in inhibiting oxidative stress and preventing melanocyte loss. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that existing drugs for treating pigmentation disorders generally have many side effects, unstable efficacy, and poor results in severe cases. The present invention provides the use of Ro5-4864 and pharmaceutically acceptable salts of Ro5-4864 in the treatment and / or prevention of melanocyte loss caused by oxidative stress-induced inflammatory immune response, particularly for the treatment of vitiligo, leukoderma, and pityriasis alba.

[0007] Based on this, the present invention also provides a pharmaceutical composition for treating and / or preventing melanocyte loss diseases caused by oxidative stress-induced inflammatory immune responses, with Ro5-4864 or a pharmaceutically acceptable salt thereof as the active ingredient.

[0008] This invention demonstrates through extensive modern pharmacological research and animal pharmacodynamic experiments that Ro5-4864 can promote the activation of the antioxidant system, inhibit oxidative stress-induced inflammatory immune responses, and has therapeutic effects on pigment loss diseases, especially vitiligo, leukoderma, and pityriasis alba.

[0009] Currently used drugs for treating pigment loss disorders generally suffer from numerous side effects, unstable efficacy, and poor results in severe cases. The Ro5-4864 and its pharmaceutically acceptable salts described in this invention inhibit the continuous loss of melanin from oxidative stress, exhibiting strong targeting and fewer side effects, representing significant advantages and advancements compared to existing drugs. Attached Figure Description

[0010] Figure 1 This is a bar chart showing the relative proliferation activity of NFDFs cells after treatment.

[0011] Figure 1 The results showed that, compared with the control group, 500 μM H2O2 significantly inhibited the activity of NHDFs cells (**P<0.01), while 20 μM Ro5-4864 pretreatment significantly improved the activity of NHDFs cells (**P<0.01).

[0012] Figure 2 This is a flow cytometry image showing the effect of apoptosis detection after NFDFs cell treatment. Among them, Figure 2 A is a scatter plot of cell clusters. Figure 2 B is a bar chart showing the proportion of apoptotic cells.

[0013] Figure 2 Scatter plots A (top left and top right) show that, compared with the control group, treatment with 20 μM Ro5-4864 alone had no significant effect on the apoptosis rate in the Q3 quadrant of NHDFs cells. Figure 2 Scatter plots at the bottom left and bottom right show that 500 μM H2O2 significantly increased the apoptosis rate in the Q3 quadrant of NHDFs cells (***P<0.001), while 20 μM Ro5-4864 pretreatment significantly reduced the apoptosis rate in the Q3 quadrant of NHDFs cells (**P<0.01).

[0014] Figure 3 This is an immunoblot image of antioxidant-related proteins Nrf2 and HO-1, and inflammation-related factors IL-1β, CXCL9, and CXCL10 in NHDF cells. Figure 3 A is the Western blot image of the protein. Figure 3 B is a bar chart showing the relative expression levels of proteins.

[0015] Figure 3The results showed that pretreatment with different concentrations of Ro5-4864 (5, 10, 20 μM) upregulated the expression of Nrf2 and HO-1 compared with the control group (**P<0.01, ***P<0.001). Importantly, activation of the Nrf2 / HO-1 axis was accompanied by a significant reduction in oxidative stress-induced overexpression of IL-1β, CXCL9, and CXCL10 (*P<0.05, **P<0.01, ***P<0.001).

[0016] Figure 4 These are images of melanin stained with ammoniacal silver in the skin tissue of a mouse model of vitiligo. Figure 4 A shows the ammoniacal silver staining of melanin in skin tissue from different treatment groups. Figure 4 B is a bar chart showing the relative melanin content.

[0017] Figure 4 The left and middle images show that, compared with the control group, the melanin content in the dorsal skin of mice in the vitiligo model group was significantly reduced (***P<0.001). Figure 4 The middle and right figures in Figure A show that, compared with the H2O2 treatment group, the melanin content in the skin of the H2O2+ Ro5-4864 treatment group was significantly increased (***P<0.001).

[0018] Figure 5 This is an immunoblot image of the antioxidant-related proteins Nrf2 and HO-1, and the inflammatory factors IL-1β, CXCL9, and CXCL10 in the skin tissue of the lesion area of ​​a vitiligo mouse model. 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.

[0019] Figure 5 The results showed that, compared with the vitiligo model group, Ro5-4864 significantly upregulated the expression of antioxidant-related proteins Nrf2 and HO-1 in the skin lesion area of ​​the vitiligo mouse model (***P<0.001) and inhibited the expression of inflammatory-related factors IL-1β, CXCL9 and CXCL10 (*P<0.05).

[0020] Figure 6 This is an immunofluorescence image of the antioxidant protein HO-1 in fibroblasts in the skin lesions of a vitiligo mouse model. Among them, Figure 6 A is an immunofluorescence image of the antioxidant protein HO-1 in fibroblasts. Figure 6 B is a bar chart of the relative fluorescence intensity of HO-1.

[0021] Figure 6A represents, in order: DAPI labeling of cell nuclei, Vimentin labeling of fibroblasts, HO-1 antibody labeling of HO-1 protein, and the combined three-channel diagram. The middle and lower images of the combined channels show that, compared with the vitiligo model group, Ro5-4864 treatment significantly upregulated the fluorescence intensity of the antioxidant-related protein HO-1 in fibroblasts in the lesion area of ​​the vitiligo mouse model (***P<0.001).

[0022] Figure 7 CD8 in the skin tissue of the vitiligo mouse model + Immunofluorescence images of T cells. Among them, Figure 7 A is CD8 + Immunofluorescence image of T cells Figure 7 B is CD8 + Bar chart showing the relative number of T cells.

[0023] Figure 7 A represents, in order: DAPI labeling of the cell nucleus, CD8 antibody labeling of CD8. + T cells, and a combined image of the two channels. The top and middle images of the combined channels show: CD8+ in the skin lesions of a vitiligo mouse model. + T cells were recruited to infiltrate melanocytes (***P<0.001). The middle and lower figures show that Ro5-4864 significantly inhibited CD8 compared to the vitiligo model group. + T cell recruitment (***P<0.001) protects normal melanocytes from damage. Detailed Implementation

[0024] 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.

[0025] The following are the pharmacodynamic tests and results of this invention: Part 1: Effects of Ro5-4864 on a H2O2-induced fibroblast oxidative stress model I. Ro5-4864 improves H2O2-induced decrease in fibroblast activity Cell viability was assessed using the MTT assay. Healthy, normal human dermal fibroblasts (NHDFs) in the exponential growth phase were cultured at 5 × 10⁶ cells per well. 3Cells were seeded at a density of 1000 μL in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. Then, different concentrations of Ro5-4864 (0, 5, 10, and 20 μM) were added to each experimental group for pretreatment for 6 hours. Following this, 500 μM H2O2 was added, and the cells were cultured for another 24 hours. After pretreatment, 10 μL of MTT solution (final concentration 5 mg / mL) was added to each well, and the cells were cultured at 37°C for another 4 hours. Then, 100 μL of formazan dissolving solution was added to completely dissolve the formazan crystals. The absorbance of each well was measured at 570 nm using a microplate reader, and cell viability was expressed as a percentage relative to the control group.

[0026] Experimental results: such as Figure 1 As shown, compared with the control group, 500 μM H2O2 significantly inhibited the activity of NHDFs cells (**P<0.01), while 20 μM Ro5-4864 pretreatment significantly improved the activity of NHDFs cells (**P<0.01).

[0027] Experimental conclusions: At a concentration of 20 μM, Ro5-4864 alone had no significant effect on the proliferation of NHDFs cells, while 20 μM Ro5-4864 pretreatment significantly enhanced fibroblast activity under oxidative stress.

[0028] II. Ro5-4864 ameliorates H2O2-induced fibroblast apoptosis Normal human dermal fibroblasts (NHDFs) in the exponential growth phase and in good condition were subjected to a 2×10⁻⁶ dose. 5 Cells were seeded at a density of [number] cells / mL in 6-well cell culture plates and cultured at 37°C and 5% CO2 for 24 hours. After good cell adhesion and growth, different concentrations of Ro5-4864 (0, 5, 10, and 20 μM) were added to each experimental group for pretreatment for 6 hours. Then, 500 μM H2O2 was added for further culture for 24 hours. After culture, adherent cells were collected by digestion and centrifugation, and gently resuspended in PBS. After centrifugation and discarding the supernatant, 195 μl Annexin V-FITC binding solution, 5 μl Annexin V-FITC, and 10 μl propidium iodide staining solution were added sequentially, and the mixture was gently mixed. The cells were incubated at room temperature (20-25°C) in the dark for 10-20 minutes, then placed on ice and immediately analyzed using a BDAccuri C6 plus flow cytometer.

[0029] Experimental results: such as Figure 2As shown, compared with the control group, 500 μM H2O2 significantly increased the apoptosis rate of NHDFs cells (***P<0.001), while 20 μM Ro5-4864 pretreatment significantly protected NHDFs cells from apoptosis caused by oxidative stress (**P<0.01).

[0030] Experimental conclusions: At a concentration of 20 μM, Ro5-4864 alone had no significant effect on the apoptosis rate of NHDFs cells. At the same time, Ro5-4864 pretreatment could significantly protect fibroblasts from damage caused by oxidative stress and reduce the apoptosis rate of fibroblasts.

[0031] III. Ro5-4864 activates the Nrf2 / HO-1 antioxidant pathway in fibroblasts and inhibits the expression of H2O2-induced inflammation-related factors. Normal human dermal fibroblasts (NHDFs) in the exponential growth phase and in good condition were subjected to a 2×10⁻⁶ dose. 5 Cells were seeded at a density of [number] cells / mL in 6-well cell culture plates and cultured at 37°C and 5% CO2 for 24 hours. After good cell adhesion and growth, different concentrations of Ro5-4864 (0, 5, 10, and 20 μM) were added to each experimental group for pretreatment for 6 hours. Then, 500 μM H2O2 was added for further culture for 24 hours. After culture, cells from each group were collected and lysed with 100 μL of non-denaturing lysis buffer (containing 1 mM PMSF) at 4°C for 15 minutes. The lysed samples were centrifuged at 13000 r / min for 15 minutes at 4°C, and the supernatant was used for protein concentration determination (BCA method) to calculate the total protein content.

[0032] Western Blot Analysis: Add 1 / 4 volume of Loading Buffer to the protein sample, mix well, and denature by boiling in a 97°C metal bath for 7 minutes. Load the denatured protein sample onto SDS-PAGE 12% separating gel and 5% stacking gel for electrophoresis at 80 V for 30 minutes and 120 V for 60 minutes. Transfer the target protein to an NC / PVDF membrane at 320 mA for 1 hour. Block the membrane with TBST buffer (containing 0.05% Tween-20) containing 5% skim milk at room temperature for 1.5 hours, then incubate overnight at 4°C with primary antibodies Nrf2, HO-1, IL-1β, CXCL9, CXCL10, and β-actin. The next day, wash the membrane four times with TBST for 7 minutes each time, then incubate with HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG at room temperature for 1 hour. Wash again with TBST four times for 7 minutes each time. Chemiluminescent substrate and developing solution were added, and protein bands were developed using an ECL chemiluminescence detection system. The grayscale values ​​of the target protein bands were analyzed using ImageJ software, and the relative expression level was calculated using β-actin as an internal reference.

[0033] Experimental results: such as Figure 3 As shown, Western blot analysis revealed that pretreatment with different concentrations of Ro5-4864 (5, 10, 20 μM) upregulated the expression of Nrf2 and HO-1 compared to the control group (**P<0.01, ***P<0.001). Importantly, activation of the Nrf2 / HO-1 axis was accompanied by a significant reduction in oxidative stress-induced overexpression of IL-1β, CXCL9, and CXCL10 (*P<0.05, **P<0.01, ***P<0.001).

[0034] Experimental conclusion: Ro5-4864 can activate the Nrf2 / HO-1 antioxidant system in NHDFs cells in a dose-dependent manner, thereby reducing the expression of IL-1β, CXCL9 and CXCL10 induced by oxidative stress, indicating that Ro5-4864 can not only enhance antioxidant defense capabilities, but also inhibit inflammatory signal transduction.

[0035] Part 2: Effects of Ro5-4864 on a H2O2-induced vitiligo mouse model I. Ro5-4864 treatment promotes skin pigmentation regeneration in a H2O2-induced vitiligo mouse model. All animal experiments were approved by the University of California, Berkeley Animal Care and Use Committee (Approval No.: 202402280052-2) and conducted in accordance with institutional guidelines. Female C57BL / 6 mice (5 weeks old, weighing 20±2 g) were purchased from Shanghai Bikai Biotechnology Co., Ltd. and housed in a specific pathogen-free (SPF) environment with a 12-hour light / 12-hour dark cycle and free access to food and water. The mice were randomly divided into three groups (n=6 per group): (1) PBS-treated control group; (2) H2O2-induced vitiligo model group; (3) H2O2+Ro5-4864 treatment group. After a 1-week acclimatization period, the hair in a 2×2 cm area on the back of the mice was removed with electric clippers. Two days later, 5% (w / v) H2O2 solution was applied topically (twice daily for 4 weeks) to induce vitiligo-like lesions, while the control group was treated with sterile PBS. After the skin lesion induction was completed, mice in the H2O2+Ro5-4864 treatment group received intradermal injections of Ro5-4864 (2 mg / kg, once daily for 3 weeks). Ro5-4864 was prepared using a solvent containing 10% DMSO, 90% saline and 20% (w / v) sulfobutyl ether-β-cyclodextrin (SBE-β-CD) to a final concentration of 4 mg / mL.

[0036] Melanin in formalin-fixed mouse skin tissue sections was detected using the Masson-Fontana ammoniacal silver staining method. The tissue sections were washed three times with deionized water and then immersed in ammoniacal silver solution at room temperature for 12 hours. After thorough rinsing with deionized water, they were fixed with thiophanate-methyl for 5 minutes, rinsed thoroughly again, and counterstained with neutral red for 5 minutes. Finally, the tissue sections were rinsed and observed under a microscope.

[0037] Experimental results: such as Figure 4 As shown, compared with the control group, the melanin content in the dorsal skin of mice treated with H2O2 was significantly reduced (***P<0.001). Compared with the H2O2 treatment group, the melanin content in the skin of mice treated with H2O2+ Ro5-4864 was significantly increased (***P<0.001).

[0038] Experimental conclusion: Ro5-4864 treatment can effectively restore the melanin content in the skin of a mouse model of H2O2-induced vitiligo.

[0039] II. Ro5-4864 treatment activates the Nrf2 / HO-1 antioxidant pathway in the skin of a vitiligo mouse model and inhibits the expression of inflammatory factors. Skin tissue samples from the backs of the experimental mice were collected. Each group of skin tissue (60 mg) was aseptically minced, and 1 ml of pre-cooled tissue lysis buffer (containing protease inhibitors) was added. The tissue was thoroughly homogenized using a high-speed mechanical homogenizer at 12000 rpm. 0.5 ml of the tissue homogenate was taken, and 2 volumes (1 ml) of extraction reagent were added. After thorough mixing, the mixture was incubated at 4 °C for 10 minutes. The mixture was then centrifuged at 10000 g for 10 minutes at 4 °C, and the upper and lower layers were removed separately, allowing the intermediate protein membrane to adhere to the centrifuge tube wall. After drying in the open for 10 minutes, 200 μL of 2% SDS was added, and the mixture was boiled at 95 °C for 10 minutes, followed by incubation at room temperature for 30 minutes to fully dissolve the precipitate. The mixture was centrifuged at 12000 rpm for 5 minutes, and the protein supernatant was collected for protein quantification using the BCA method.

[0040] Add 1 / 4 volume of loading buffer to the protein sample, mix well, and denature by boiling in a metal bath at 97 °C for 7 minutes. Load the denatured protein sample onto SDS-PAGE 12% separating gel and 5% stacking gel for electrophoresis at 80 V for 30 minutes and 120 V for 60 minutes. Transfer the target protein to an NC / PVDF membrane at 320 mA for 1 hour. Block the membrane with TBST buffer (containing 0.05% Tween-20) containing 5% skim milk at room temperature for 1.5 hours, then incubate overnight at 4 °C with primary antibodies Nrf2, HO-1, IL-1β, CXCL9, CXCL10, and β-actin. The next day, wash the membrane four times with TBST for 7 minutes each time, then incubate with HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG at room temperature for 1 hour. Wash again with TBST four times for 7 minutes each time. Chemiluminescent substrate and developing solution were added, and protein bands were developed using an ECL chemiluminescence detection system. The grayscale values ​​of the target protein bands were analyzed using ImageJ software, and the relative expression level was calculated using β-actin as an internal reference.

[0041] Experimental results: such as Figure 5 As shown, compared with the vitiligo model group, Ro5-4864 significantly upregulated the expression of antioxidant-related proteins Nrf2 and HO-1 in the skin lesion area of ​​the vitiligo mouse model (***P<0.001) and inhibited the expression of inflammatory-related factors IL-1β, CXCL9 and CXCL10 (*P<0.05).

[0042] Experimental conclusion: Ro5-4864 can effectively activate the antioxidant system and inhibit the expression of inflammatory factors and chemokines in the dorsal skin lesion area of ​​a hydrogen peroxide-induced vitiligo mouse model.

[0043] III. Ro5-4864 treatment promotes HO-1 expression in skin fibroblasts of a vitiligo mouse model. For tissue sections, paraffin-embedded mouse skin samples underwent antigen retrieval for 15 minutes in citrate buffer at 95°C. Mouse skin sections were then labeled with vimentin antibody to identify fibroblasts and co-stained with HO-1 antibody overnight at 4°C. The samples were subsequently co-incubated with species-matched fluorescently labeled secondary antibody at room temperature for 1 hour. After counterstaining the cell nuclei with DAPI (1 μg / mL), images were acquired using a confocal laser scanning microscope.

[0044] Experimental results: such as Figure 6 Immunofluorescence showed that, compared with the vitiligo model group, Ro5-4864 treatment significantly upregulated the expression of the antioxidant-related protein HO-1 in fibroblasts in the skin lesion area of ​​the vitiligo mouse model (***P<0.001).

[0045] Experimental conclusion: Ro5-4864 can effectively activate the expression of antioxidant-related proteins in the skin fibroblasts of a vitiligo mouse model.

[0046] IV. Ro5-4864 treatment inhibited CD8 in the skin of a mouse model of vitiligo. + T cells persist infiltrated For tissue sections, paraffin-embedded mouse skin samples underwent antigen retrieval for 15 minutes in citrate buffer at 95°C. To detect cytotoxic T cells, mouse skin tissue was incubated overnight with anti-CD8 antibody at 4°C. The samples were then co-incubated with species-matched fluorescently labeled secondary antibody at room temperature for 1 hour. After counterstaining the cell nuclei with DAPI (1 μg / mL), images were acquired using a confocal laser scanning microscope.

[0047] Experimental results: such as Figure 7 Immunofluorescence showed that CD8+ in the skin lesions of a vitiligo mouse model + T cells continuously infiltrated and killed melanocytes; compared with the vitiligo model group, Ro5-4864 significantly inhibited CD8. + T cell recruitment and maintenance of normal melanocyte function showed significant differences (***P<0.001).

[0048] Experimental conclusion: Ro5-4864 can effectively reduce CD8 levels in the skin of a mouse model of vitiligo. + T-cell infiltration prevents the continued loss of melanocytes.

[0049] 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 Ro5-4864 or a pharmaceutically acceptable salt in the preparation of medicaments for the treatment and / or prevention of melanocyte loss diseases caused by oxidative stress-induced inflammatory immune responses, wherein the melanocyte loss diseases caused by oxidative stress-induced inflammatory immune responses are vitiligo, leukoderma, pityriasis alba.

2. The use according to claim 1, characterized in that... The pharmaceutically acceptable salts of Ro5-4864 are formed by the formation of a salt between a tertiary amine group and a pharmaceutically acceptable acid.

3. The use according to claim 2, characterized in that... The applicable acids are selected from inorganic or organic acids. Inorganic acids are selected from hydrochloric acid, sulfuric acid, or phosphoric acid, and organic acids are 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, followed by crystallization purification.

5. A pharmaceutical composition for treating and / or preventing melanocyte loss caused by oxidative stress-induced inflammatory immune responses, characterized in that: The pharmaceutical composition contains Ro5-4864 or a pharmaceutically acceptable salt thereof, and the melanocyte loss disease caused by the oxidative stress-induced inflammatory immune response is vitiligo, leukoderma, or pityriasis alba.

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