Use of tolvaptan in a medicament or cosmetic product for inhibiting melanin synthesis

Abstract

The application of a drug or medical aesthetic product containing tolvaptan as an active ingredient for inhibiting melanin synthesis. Cell activity detection and melanin content determination show that tolvaptan can significantly reduce the intracellular melanin content without affecting the cell activity. Tyrosinase activity detection shows that tolvaptan does not affect the catalytic activity of TYR. Western blotting experiment shows that tolvaptan can down-regulate the expression levels of TYR family proteins, microphthalmia-associated transcription factor (MITF), cyclic adenosine monophosphate response element binding protein (CREB) and melanocortin 1 receptor (MC1R).

Description

Technical Field

[0001] This invention relates to a novel use of a compound, particularly to a compound that downregulates the expression of proteins such as cyclic adenosine monophosphate (cAMP) response element-binding protein and melanocortin 1 receptor, thereby inhibiting melanin synthesis. Background Technology

[0002] Melanin is synthesized by melanocytes. Melanin synthesis is a complex mechanism involving multiple enzymatic reactions and chemical processes, resulting in the production of two types of melanin: eumelanin and pheomelanin (Int J Mol Sci. 2023, 24(9): 8305). Eumelanin is an insoluble polymer with a dark brown to black color, while pheomelanin is a soluble polymer with a light red color.

[0003] Skin color depends on the amount of melanin and its distribution in the skin. Under physiological conditions, melanin can protect the skin from the harmful effects of ultraviolet radiation. However, excessive melanin deposition can lead to a range of skin problems, such as melasma, freckles, and post-inflammatory hyperpigmentation (Int J Mol Sci, 2016, 17(7): 1144).

[0004] Most melanin synthesis inhibitors currently used in clinical practice target tyrosinase, achieving therapeutic effects by inhibiting its activity. However, studies have found that long-term use of these inhibitors may cause adverse reactions such as permanent depigmentation and exogenous okra, a limitation that urgently needs to be addressed. Summary of the Invention

[0005] One object of the present invention is to provide a substance having the activity of inhibiting melanin synthesis.

[0006] Another object of the present invention is to provide a compound having the activity of inhibiting melanin synthesis.

[0007] Another object of the present invention is to provide the use of tolvaptan in the preparation of medicaments or medical aesthetic products that are inhibitors of the MC1R / cAMP signaling pathway.

[0008] Another object of the present invention is to provide the use of tolvaptan in the preparation of drugs or medical aesthetic products that inhibit melanin synthesis.

[0009] In the complex signaling pathways of melanin synthesis, the MC1R / cAMP signaling pathway, the Wnt / β-catenin signaling pathway, and the MAPK / EKR signaling pathway are three major signal transduction pathways (J Biol Chem, 2007, 282(38): 27557-27561). All three are involved in the regulation of melanin synthesis by the microphthalmia-associated transcription factor (MITF), which controls the gene expression of three enzymes: tyrosinase (TYR), tyrosinase-associated protein 1 (TRP1), and tyrosinase-associated protein 2 (TRP2). Among these, the MC1R / cAMP signaling pathway primarily promotes melanin synthesis by increasing intracellular cAMP levels.

[0010] Solar ultraviolet radiation is a key external physical factor influencing melanin synthesis in the skin. UV radiation induces keratinocytes to produce alpha-melanocyte-stimulating hormone (α-MSH), which then binds to the melanocortin 1 receptor (MC1R), initiating a cAMP-dependent signaling pathway. MC1R is highly expressed in both normal and malignant melanocytes and is known as the α-MSH receptor due to its high affinity for α-MSH. MC1R is the most upstream G protein-coupled receptor in the cAMP-dependent signaling pathway, and its location determines its important role in regulating melanin production. When MC1R is stimulated by α-MSH, it increases intracellular cAMP levels by activating adenylate cyclase (AC), which in turn activates protein kinase A (PKA) and cyclic adenosine monophosphate response element-binding protein (CREB), thereby regulating gene transcription and increasing MITF expression. MITF then activates the expression of proteases related to melanin formation.

[0011] Tolvaptan (TOL) is an antidiuretic hormone receptor antagonist currently used to treat inappropriate indigestion syndrome (SIAD) and autosomal dominant polycystic kidney disease (ADPKD) (J Clin Endocrinol Metab, 2013, 98(4): 1321-1332).

[0012] The tolvaptan provided by this invention is used as an inhibitor to suppress melanin synthesis. At concentrations not exceeding 30 µM, tolvaptan exhibits no significant cytotoxicity.

[0013] This invention utilizes the dose-dependent ability of tolvaptan to inhibit melanin synthesis. Furthermore, the mechanism by which tolvaptan inhibits melanin synthesis is not by directly inhibiting the catalytic activity of TYR, but rather by affecting complex intracellular signaling pathways, reducing intracellular MITF levels, inhibiting the synthesis of new TYR, and ultimately suppressing intracellular melanin synthesis. MITF plays a central role in regulating melanin synthesis and the transcription of melanin-producing enzymes. Downregulating MITF can inhibit the expression of tyrosinase and other enzyme proteins (such as TRP-1 and TRP-2), thereby suppressing melanin production.

[0014] This invention utilizes tolvaptan to inhibit melanin synthesis by suppressing the MC1R / cAMP signaling pathway. Verification has shown that the tolvaptan provided by this invention effectively reduces the expression levels of CREB and MC1R in B16F10 cells, ultimately inhibiting MITF expression and reducing the transcriptional activity of proteins such as TYR and TRP-2, thereby decreasing melanin synthesis.

[0015] Tolvaptan is used as the active ingredient and mixed with other excipients to make drugs (preparations) or medical aesthetic products (such as skin whitening products or sun protection products) for inhibiting melanin synthesis.

[0016] These pharmaceutical excipients can be those commonly used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants; or they can be selected for use to be compatible with the substances in the formulation, such as emulsifiers, solubilizers, antibacterial agents, analgesics, and antioxidants. These excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the body and enhancing the drug delivery effect of the composition.

[0017] In aqueous injections, excipients generally include isotonic agents and buffer solutions, as well as necessary emulsifiers (such as Tweeen-80, Pluronic, and Poloxamer), solubilizers, and antibacterial agents. In addition, they may include other pharmaceutically acceptable excipients, such as antioxidants, pH adjusters, and analgesics.

[0018] Excipients used in the preparation of oral liquid formulations generally include solvents, as well as necessary flavoring agents, antibacterial agents, emulsifiers, and coloring agents.

[0019] Excipients used in tablet manufacturing generally include fillers (such as starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium), and lubricants (such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate).

[0020] The excipients used in the preparation of emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents.

[0021] The excipients used to produce granules are similar to those used for tablets, but the granulation process is different. Depending on the requirements, the produced granules are mixed with a gliding agent and then encapsulated to obtain capsules.

[0022] Various excipients and compounds are used to formulate dosage forms that facilitate drug delivery, such as, but not limited to, aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release formulations, and controlled-release formulations. Furthermore, excipients may be used to achieve specific drug delivery purposes or methods, such as sustained-release, controlled-release, and pulsatile administration, including, but not limited to, gelatin, albumin, chitosan, polyethers, and polyester polymers, such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and their copolymers. The main manifestations of "facilitating drug delivery" include, but are not limited to, improved therapeutic efficacy, increased bioavailability, reduced toxicity and side effects, and improved patient compliance.

[0023] The tolvaptan of this invention can be combined with other excipients, such as through chemical coupling, to further improve the efficacy of the compound, reduce toxicity, and prolong the dosing cycle. These excipients are typically polymers, such as polyesters, polyethers, and polyamides.

[0024] The tolvaptan of this invention can also be loaded or coated as an active ingredient onto biocompatible materials to form a delivery system, utilizing common materials such as PLA, PLGA, PGA, GelMA, and metals. It can also be mixed with biocompatible biodegradable materials to form microneedles and microneedle arrays, or loaded into metal microneedles to form microneedle chips. When the microneedles are inserted into the skin, tolvaptan is released into the dermis. Attached Figure Description

[0025] Figure 1Figure 1 shows the experimental results of tolvaptan inhibiting melanin synthesis in B16F10 cells. In the figure, A is the structure of tolvaptan, B is the effect of tolvaptan treatment for 48 h on the activity of B16F10 cells, C is the effect of tolvaptan treatment for 48 h on the melanin content of B16F10 cell supernatant, and D is the effect of tolvaptan treatment for 48 h on the melanin content of B16F10 cell lysate.

[0026] Figure 2 The results of tolvaptan regulating TYR and MITF are shown in the figure. Among them, A is the statistical graph of tyrosinase activity detected after 48 h of tolvaptan treatment of B16F10 cells; B is the electrophoresis image of TYR family proteins and MITF after 48 h of treatment of B16F10 cells with different concentrations of tolvaptan; C is the statistical graph of relative protein expression level of TYR; D is the statistical graph of relative protein expression level of TRP1; E is the statistical graph of relative protein expression level of TRP2; and F is the statistical graph of relative protein expression level of MITF.

[0027] Figure 3 The results of tolvaptan regulation of CREB and MC1R are shown in the figure. Among them, A is the electrophoresis image of CREB and MC1R after B16F10 cells were treated with different concentrations of tolvaptan for 48 h, B is the statistical graph of the relative protein expression level of CREB in each group, and C is the statistical graph of the relative protein expression level of MC1R.

[0028] Figure 4 Image showing the melanin signal intensity in the head of zebrafish after treatment with different concentrations of tolvaptan;

[0029] Figure 5 A statistical graph showing the melanin signal intensity in the head of zebrafish after treatment with different concentrations of tolvaptan.

[0030] Figure 6 A statistical chart showing the melanin content of zebrafish after treatment with different concentrations of tolvaptan;

[0031] Figure 7 A statistical graph showing the tyrosinase activity of zebrafish after treatment with different concentrations of tolvaptan.

[0032] Figure 8 This is a statistical graph showing the relative expression levels of the tyr gene in zebrafish after treatment with different concentrations of tolvaptan. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0034] The sources of the main experimental reagents used in the following embodiments of the present invention are specifically described below:

[0035] Tolvaptan was purchased from MedChemExpress (USA); 3,4-dihydroxy-l-phenylalanine (L-DOPA) and Triton X-100 were purchased from Sigma-Aldrich (USA); fetal bovine serum, antibiotic-antifungal agent, phenol red-free DMEM, and trypsin-EDTA were purchased from Gibco (USA); dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd.; thiazolyl blue (MTT), BCA protein quantification kit, WB cell lysis buffer, and antibody diluent were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; ice-free rapid transfer buffer, protein-free rapid blocking buffer, antibody elution buffer, 5×SDS-PAGE protein loading buffer, and protein marker were purchased from Wuhan Sewell Biotechnology Co., Ltd.; rabbit anti-mouse TYR antibody, rabbit anti-mouse TRP1 antibody, rabbit anti-mouse TRP2 antibody, rabbit anti-mouse MITF antibody, rabbit anti-mouse CREB antibody, rabbit anti-mouse MC1R antibody, and rabbit anti-mouse β-actin antibody were purchased from Shanghai Aibmate Pharmaceutical Technology Co., Ltd.

[0036] The specific experimental methods used in the following embodiments of the present invention are described below:

[0037] 1) Cell Culture

[0038] B16F10 mouse melanoma cells from a cell bank (Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. All cells were cultured at 37°C and 5% CO2 saturated humidity. The culture medium was changed every two days on average, and cells were passaged when they reached over 90% confluence. Subsequent experiments were conducted using cells up to passage 12.

[0039] 2) Detection of the effect of tolvaptan on cell viability

[0040] B16F10 cell suspension was seeded into 96-well plates, with 100 µL of cells added to each well (5 x 10⁶ cells). 4 B16F10 cell suspension was administered at a concentration of 1 / ml. After cell attachment, the culture medium was discarded, and culture medium containing different concentrations of tolvaptan (0µM, 10µM, 20µM, 30µM, 40µM, 50µM, 60µM, 70µM, 80µM, and 100µM, with 3 replicates per group) was added. After 48 hours, culture medium containing 0.5 mg / mL MTT was added. After incubation for 3 hours, the culture medium was discarded, and 100 µL LDMSO was added to each well. The cells were shaken on a shaker for 10 minutes, and the absorbance at 540 nm was measured.

[0041] 3) Determination of melanin content and tyrosinase activity in B16F10 cells

[0042] B16F10 cell suspension was seeded into 6-well plates, with 2 mL of cells added to each well (3 × 10⁶ cells). 4 B16F10 cell suspension was administered at a concentration of 1000 mL. After cell attachment, phenol red-free medium containing different concentrations of tolvaptan (0 µM, 10 µM, 15 µM, 20 µM, 25 µM, and 30 µM) was added. After 48 hours of treatment, the supernatant from all wells was collected, and their absorbance at 475 nm was measured. Cells were digested with 0.05% trypsin, collected into centrifuge tubes, and centrifuged at 12,000 rpm for 10 minutes at 4°C. After centrifugation, the cells were resuspended in PBS, centrifuged again, and the precipitate was collected. The precipitate was then dissolved in 1 M sodium hydroxide solution and heated at 80°C for 10 minutes. After the melanin was dissolved, its absorbance at 405 nm was measured.

[0043] B16F10 cell suspension was seeded into 6-well plates, with 2 mL of cells added to each well (3 × 10⁶ cells). 4 B16F10 cell suspension was administered at a concentration of 10 µM / mL. After cell adhesion, phenol red-free medium containing different concentrations of tolvaptan (0 µM, 10 µM, 15 µM, 20 µM, 25 µM, and 30 µM) was added. After 48 hours of culture, the supernatant was discarded, and the cells were washed twice with PBS and lysed using 1% Triton X-100. Cell lysate was scraped off with a pipette tip and transferred to a 1.5 mL EP tube, then centrifuged at 12,000 rpm for 10 minutes at 4°C. 100 µL of sample supernatant was added to each well of a 96-well plate, followed by 100 µL of 2 mM L-DOPA. The plate was incubated at 37°C for 1 hour, and then the absorbance was measured at 475 nm.

[0044] 4) Western Blot

[0045] 2 mL of B16F10 cell suspension (1×10⁻⁶) 5Cells / mL) were seeded in a 6-well cell culture plate. After 24 hours, the medium was replaced with a medium containing tolvaptan (0 µM, 10 µM, 20 µM, and 30 µM), and the cells were cultured for 48 hours. Then, the cells were rinsed twice with pre-cooled PBS and lysed on ice for 10 minutes with RIPA buffer containing protease and phosphatase inhibitors. The protein lysate was collected and centrifuged at 12,000 rpm for 10 minutes at 4 °C, and the supernatant was collected. The total protein content was measured using a BCA protein assay kit. Then, the proteins in the supernatant were mixed with one-quarter volume of supernatant buffer (5-fold) and heated at 95 °C for 10 minutes. Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF). After washing with washing buffer, the membrane was incubated with blocking buffer at room temperature for 15 minutes, and then incubated with primary antibodies such as TYR (1:2000), TRP1 (1:2000), TRP2 (1:2000), MITF (1:2000), CREB (1:2000), MC1R (1:2000), and β-actin (1:1000) overnight at 4 °C, and then incubated with secondary antibody at room temperature for 1 hour. The secondary antibody was fluorescent goat anti-rabbit IgG (1:2000). After washing, the membrane was scanned using an Odyssey Dlx dual-color infrared laser imaging system (Li-Cor, USA), and finally the data was saved and analyzed.

[0046] 6) Experimental animals

[0047] Zebrafish were all raised in fish culture water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, and the conductivity was 450~550 μS / cm; pH was 6.5~8.5; hardness was 50~100 mg / L CaCO3). They were provided by Hangzhou Huante Biotechnology Co., Ltd. The license number for the use of experimental animals was: SYXK (Zhe) 2022-0004. The feeding management complied with the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number was: IACUC-2025-12239-01.

[0048] 7) Evaluation of the intensity of melanin signal in animals (whitening effect)

[0049] Six hpf wild-type AB strain zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (concentrations shown in Table 1) were administered, with arbutin at a positive control concentration of 15.0 mM. A normal control group was also included, with a volume of 3 mL per well. Tolvaptan was used for treatment at 28℃ for 72 h. Ten zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were acquired using NIS-Elements D 3.20 advanced image processing software, and the melanin signal intensity (pixels) in the zebrafish head was analyzed. The statistical analysis results were used to evaluate the effect of the samples on the melanin signal intensity in the head. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, with p < 0.05 indicating statistical significance.

[0050] Table 1 Experimental results of melanin signal intensity (n = 10)

[0051]

[0052] In the table, compared with the normal control group, **p < 0.01, ***p < 0.001.

[0053] 8) Melanin content in animals (whitening effect)

[0054] Six hpf wild-type AB strain zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (concentrations shown in Table 2, n=3) were administered, along with a positive control of arbutin at a concentration of 15.0 mM. A normal control group was also included, with a volume of 3 mL per well. Three biological replicates were performed. Tolvaptan was used for treatment at 28℃ for 72 h before collection. The samples were homogenized using a rapid grinder, centrifuged to remove the supernatant, and melanin standard solutions were prepared at a specific concentration. 350 μL of NaOH solution was added to each tube, and the plates were incubated at 60℃ for 1 h. The standard solutions and test samples were transferred to 96-well plates (100 μL / well), and the absorbance was measured at 405 nm using a microplate reader. The melanin content of each sample group was obtained using a standard curve. The statistical analysis results were used to evaluate the effect of the samples on melanin content. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0055] Table 2. Experimental results of melanin content (n = 3)

[0056]

[0057] In the table, compared with the normal control group, **p < 0.01, ***p < 0.001.

[0058] 9) Evaluation of tyrosinase activity (whitening effect)

[0059] Six hpf wild-type AB strain zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (concentrations shown in Table 3) were administered, along with a positive control of arbutin at 15.0 mM. A normal control group was also included, with a volume of 3 mL per well. Three biological replicates were performed. Tolvaptan was used for treatment at 28℃ for 72 h before collection. The samples were homogenized using a rapid homogenizer, centrifuged, and the supernatant was collected. Protein concentrations in each group were determined using a BCA protein assay kit. 250 μg of total zebrafish protein was added to 1 mM levodopa solution at a 1:1 volume ratio, mixed, and transferred to 200 μL / well in a 96-well plate. The absorbance was measured at 475 nm using a microplate reader. The effect of this indicator on tyrosinase activity was evaluated using statistical analysis. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0060] Table 3. Results of tyrosinase activity experiments (n = 3)

[0061]

[0062] In the table, compared with the normal control group, *p < 0.05, **p < 0.01.

[0063] 10) Evaluation of relative expression levels of the tyr gene (skin whitening effect)

[0064] Six hpf wild-type AB strain zebrafish were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group), and three replicates were set up. Water-soluble samples (concentrations shown in Table 4) were administered, along with a positive control of 15.0 mM arbutin, and a normal control group was also included. The volume of each well was 3 mL. Three biological replicates were set up. Tolvaptan was used for treatment at 28℃ for 72 h before the samples were collected. Total RNA was extracted from each group of zebrafish using an automated nucleic acid extractor, and the concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the instructions of the cDNA first-strand synthesis kit. The expression of β-actin and tyr genes was detected by q-PCR. β-actin was used as an internal control for gene expression, and the relative expression level of the tyr gene was calculated. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0065] Table 4. Total RNA concentration and A260 / A280 ratio (n = 3)

[0066]

[0067] 11) Statistical analysis

[0068] Unless otherwise specified, results are based on three independent experiments. The numbers indicate the significance of the multiple comparisons. Student's t-test was used for comparisons between the two groups. One-way ANOVA was used to measure the significance of multiple comparisons of the composite effect (P < 0.05 was considered significant). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0069] Example 1: Tolvaptan inhibits melanin synthesis in B16F10 cells

[0070] The compound structure of tolvaptan is as follows: Figure 1 A, its results on the viability of B16F10 cells showed that, Figure 1 B. When the concentration reached 40 µM, the cell viability of B16F10 cells treated with tolvaptan dropped below 90%, with the absorbance decreasing from 0.927 ± 0.072 in the 0 µM control group to 0.896 ± 0.093 in the 40 µM treatment group. At concentrations above 60 µM, cell viability significantly decreased, with the absorbance further decreasing to 0.807 ± 0.143 (P < 0.05). However, after 48 hours of treatment with 10 µM, 20 µM, and 30 µM tolvaptan, the cell viability of B16F10 cells remained above 90%. Therefore, in subsequent cell experiments, the concentration of tolvaptan was not higher than 30 µM.

[0071] The results of the cell melanin content determination experiment are as follows: Figure 1 C. It was observed that tolvaptan inhibited melanin synthesis in B16F10 cells in a dose-dependent manner. Specifically, the absorbance of the cell supernatant in the 0 µM control group was 0.249 ± 0.066, while the absorbance of the 10 µM, 15 µM, 20 µM, 25 µM, and 30 µM treatment groups decreased to 0.229 ± 0.074, 0.219 ± 0.075, 0.210 ± 0.083, 0.194 ± 0.085, and 0.173 ± 0.086, respectively. In the 25 µM and 30 µM treatment groups, the relative melanin content of the cell supernatant decreased significantly by 25 ± 16.4% (P < 0.05) and 34 ± 19.3% (P < 0.001), respectively.

[0072] Furthermore, the absorbance of the cell lysate in the 0µM control group was 0.109±0.019, while the absorbance of the 10µM, 15µM, 20µM, 25µM, and 30µM treatment groups decreased to 0.101±0.014, 0.096±0.012, 0.092±0.016, 0.082±0.013, and 0.077±0.02, respectively. In the 20µM, 25µM, and 30µM treatment groups, the relative melanin content of the cell lysate decreased significantly by 15.8±6.9% (P<0.05), 23.7±10.5% (P<0.001), and 28.8±15.4% (P<0.001), respectively (see...). Figure 1 D).

[0073] Example 2: Effects of tolvaptan on TYR family proteins

[0074] like Figure 2 As shown in Figure A, TYR activity was detected after treating B16F10 cells with 10µM, 20µM, and 30µM tolvaptan for 48 hours. The results showed no significant difference among the concentrations, indicating the catalytic activity of tyrosinase in B16F10 cells.

[0075] The expression of TYR family proteins was then detected, and the electrophoresis results are as follows: Figure 2 As shown in B. The results of protein blot quantification are as follows. Figure 2 As shown in C and 2E, the levels of TYR and TRP2 were significantly decreased in B16F10 cells treated with tolvaptan at all concentrations (10 µM, 20 µM, and 30 µM). Specifically, the gray value of TYR in the 0 µM control group was 1.262 ± 0.464, while it decreased to 0.494 ± 0.209, 0.462 ± 0.291, and 0.136 ± 0.12 in the 10 µM, 20 µM, and 30 µM treatment groups, respectively. Compared with the control group, the relative expression levels of TYR in the 10 µM, 20 µM, and 30 µM treatment groups decreased by 61.2 ± 4.9% (P < 0.05), 63.9 ± 21.2%, and 90.4 ± 5% (P < 0.01), respectively. The gray value of TRP2 in the 0µM control group was 0.864±0.496, while it decreased to 0.568±0.23, 0.382±0.276, and 0.366±0.235 in the 10µM, 20µM, and 30µM treatment groups, respectively. The relative expression levels of TRP2 in the 10µM, 20µM, and 30µM treatment groups decreased by 24.9±25.9%, 54.8±14% (P<0.01), and 58.4±13% (P<0.01), respectively.

[0076] There was no significant difference in TRP1 expression levels among the different concentration groups (see...). Figure 2 D). The TYR family are downstream genes of MITF, therefore MITF expression was measured. The results are as follows: Figure 2As shown in Figure F, the protein expression level of MITF in B16F10 cells was significantly reduced after treatment with tolvaptan. Specifically, the gray value of MITF in the 0µM control group was 1.194±0.51, while the gray values ​​in the 10µM, 20µM, and 30µM treatment groups decreased to 1.003±0.55, 0.907±0.613, and 0.603±0.455, respectively. Compared with the control group, the relative expression levels of MITF in the 10µM, 20µM, and 30µM treatment groups decreased by 17.9±15.6%, 28.9±20.1% (P<0.01), and 53.8±15.5% (P<0.001), respectively.

[0077] Downregulates the expression of TYR family proteins in B16F10 cells, but does not inhibit the catalytic activity of tyrosinase in B16F10 cells.

[0078] Example 3: Regulation of CREB and MC1R expression by tolvaptan

[0079] The expression levels of CREB and MC1R in B16F10 cells were detected, validating the intracellular signaling pathway regulating melanin synthesis. Results are as follows: Figure 3 The results showed that the expression levels of CREB and MC1R in B16F10 cells decreased significantly with increasing tolvaptan concentration. Specifically, the gray value of CREB in the 0µM control group was 0.875±0.203, while it decreased to 0.634±0.356, 0.464±0.294, and 0.272±0.189 in the 10µM, 20µM, and 30µM treatment groups, respectively; the gray value of MC1R in the 0µM control group was 0.742±0.007, while it decreased to 0.576±0.148, 0.218±0.101, and 0.13±0.027 in the 10µM, 20µM, and 30µM treatment groups, respectively. Compared with the control group, the relative expression levels of CREB in the 10µM, 20µM and 30µM treatment groups decreased by 29.5±26.4%, 50±25.1% (P<0.05) and 71.2±18.7% (P<0.01), respectively; while the relative expression levels of MC1R decreased by 22.5±19.6%, 70.2±13.8% and 81.9±4.7% (P<0.01), respectively.

[0080] Example 4: Animal Experiment Verification

[0081] In this embodiment, tolvaptan at various concentrations was applied to zebrafish to verify the apparent inhibition of melanin. The results showed that tolvaptan reduced the intensity of melanin signaling in the head (as shown in Table 1). Figure 4 and Figure 5 ), reduce the melanin content of zebrafish (Table 2 and ), Figure 6 ), reducing tyrosinase activity (Table 3 and ), Figure 7), and downregulated the relative expression level of the tyr gene (Table 4 and Figure 8 These results corroborate the in vitro validation experiments described above, confirming that tolvaptan, as a melanin synthesis inhibitor, can be used clinically as a drug for the prevention or treatment of melanin. Furthermore, given its proven safety, it can also be used as an active ingredient in medical aesthetic products, such as cosmetics, specifically skin whitening products to inhibit subcutaneous melanin synthesis, or sunscreens to prevent the increase of melanin caused by ultraviolet radiation.

Claims

1. The use of tolvaptan as an active ingredient in the preparation of a drug for inhibiting diseases related to the MC1R / cAMP signaling pathway, wherein the diseases are melasma, freckles and post-inflammatory hyperpigmentation.

2. The application according to claim 1, characterized in that... The drug also includes pharmaceutical excipients.

3. The application according to claim 1, characterized in that... The drug comprises biocompatible materials and is formulated into microneedles.

4. The application according to claim 3, characterized in that... The biocompatible material is a photosensitive gel.

5. The application according to claim 4, characterized in that... The photosensitive gel includes GelMA.

6. The application according to claim 3, characterized in that... It also includes one or more of the following: pH adjusters, emulsifiers, solubilizers, and antibacterial agents.

7. The application according to claim 3, characterized in that... The formulation also includes one or more of analgesics, antioxidants, and colorants.

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