Use of ONO-2952 in the preparation of pharmaceuticals / cosmetics for the treatment and / or prevention of hyperpigmentation disorders

Abstract

This invention discloses the use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of drugs / cosmetics for treating and / or preventing hyperpigmentation disorders, belonging to the field of pharmaceutical technology. ONO-2952 of this invention inhibits melanin synthesis induced by endogenous factors α-MSH, ACTH, and UVB radiation, thereby inhibiting melanin synthesis by suppressing the expression of key melanin synthesis proteins MITF and Tyrosinase in melanocytes. Therefore, ONO-2952 or a pharmaceutically acceptable salt thereof can be used in the preparation of drugs / cosmetics for treating and / or preventing hyperpigmentation disorders.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of medicaments / cosmetics for the treatment and / or prevention of hyperpigmentation disorders. Background Technology

[0002] Melanin is primarily produced by melanocytes in the skin and is found in the epidermis. Its main function is to protect the skin from UV damage by absorbing and scattering ultraviolet (UV) rays, thereby reducing damage to skin cells. However, excessive melanin can lead to pigmentation problems such as age spots and uneven skin tone, and can even cause hyperpigmentation disorders. Excessive melanin not only affects appearance but can also cause psychological stress and decreased self-confidence. In severe cases, it may even be associated with certain skin diseases, such as melanoma.

[0003] Currently, there are numerous whitening products on the market, with their main active ingredients including Vitamin C, niacinamide, arbutin, and kojic acid. These ingredients achieve whitening effects by inhibiting melanin synthesis or accelerating skin metabolism. However, these products are not effective for everyone and may be accompanied by adverse reactions, including skin allergies, irritation, and dryness. Some ingredients, such as kojic acid, may cause skin sensitivity or phototoxic reactions, leading to permanent pigmentation loss. Arbutin, a derivative of hydroquinone, can also cause skin toxicity. Therefore, developing new whitening products to mitigate potential risks and meet the diverse needs of different groups for whitening and health is particularly important.

[0004] ONO-2952 (CAS: 895169-20-7), chemically named 1-((1S)-1-(4-chloro-2-methoxyphenyl)-5-fluoro-1,9-dihydrospiro[β-carboline-4,1'-cyclopropane]-2(3H)-yl)ethyl ketone, is a white to off-white solid. Its molecular formula is C2. 22 H 20 ClFN2O2 has a molecular weight of 398.86. ONO-2952 belongs to the indole alkaloid class and has potential applications in the prevention and / or treatment of multiple sclerosis and fibromyalgia. In addition, ONO-2952 is also used to treat irritable bowel syndrome.

[0005] Currently, there are no reports in domestic or international research on the effect of ONO-2952 on inhibiting pigment synthesis. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide the use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of medicaments / cosmetics for the treatment and / or prevention of hyperpigmentation disorders.

[0007] This invention has found that in vitro and in vivo pharmacodynamic studies have demonstrated that ONO-2952 can inhibit melanin synthesis induced by endogenous factors (α-MSH, ACTH) and ultraviolet B radiation, thus exhibiting therapeutic efficacy for hyperpigmentation disorders, particularly freckles, melasma, age spots, café-au-lait spots, nevi, and ultraviolet-induced pigmentation. Therefore, ONO-2952 can be used to prepare drugs / cosmetics for the treatment and / or prevention of hyperpigmentation disorders.

[0008] Technical solution: The objective of this invention is achieved through the following technical solution:

[0009] This invention provides the use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of medicaments / cosmetics for treating and / or preventing hyperpigmentation disorders, wherein the chemical structural formula of ONO-2952 is as follows:

[0010]

[0011] The hyperpigmentation disorders mentioned are freckles, melasma, chloasma, café-au-lait spots, nevi, or ultraviolet-induced pigmentation.

[0012] The ONO-2952 or its pharmaceutically acceptable salt is used as the sole active ingredient in the preparation of medicines / cosmetics for the treatment and / or prevention of hyperpigmentation disorders.

[0013] The ONO-2952 or its pharmaceutically acceptable salt is used in combination with other active ingredients to prepare medicines / cosmetics for the treatment and / or prevention of hyperpigmentation disorders.

[0014] The drug includes ONO-2952 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0015] The excipients include one or more of emulsifiers, binders, diluents, wetting agents, antioxidants, preservatives, or disintegrants.

[0016] The emulsifier is selected from at least one of Tween-type, Span-type, glycerol fatty acid esters, pectin, agar, sodium alginate, or silica.

[0017] The adhesive is selected from at least one of starch paste, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose, or ethyl cellulose.

[0018] The diluent is selected from at least one of starches, sugars, celluloses, or inorganic salts.

[0019] The wetting agent is selected from at least one of water or ethanol.

[0020] The antioxidant is selected from at least one of ascorbic acid, sulfite, bisulfite, gallic acid and its lipids.

[0021] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts, or parabens.

[0022] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, crospovidone, low-substituted hydroxypropyl cellulose, or crospovidone.

[0023] The dosage form of the drug is a cream, ointment, lotion, gel, film, or spray.

[0024] The medicaments of the present invention can be administered in various known ways, such as transdermal or by spray. The medicaments of the present invention can be administered alone or in combination with other drugs. Transdermal compositions can be any skin-acceptable dosage form, including, but not limited to, creams, ointments, lotions, gels, and films.

[0025] The actual dosage level of the active ingredient in the medicament of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including route of administration, time of administration, excretion rate, duration of treatment, other drugs, compounds, and / or materials used in combination with ONO-2952, the age, sex, weight, general health condition, and medical history of the patient being treated, as well as similar factors known in the medical field.

[0026] This invention investigated the effect of ONO-2952 on melanin synthesis in B16F10 cells. Experiments showed that within the concentration range of 5-40 μM, ONO-2952 had no significant effect on B16F10 cell proliferation, exhibiting low toxicity. ONO-2952 significantly inhibited melanin synthesis in B16F10 cells, and this inhibition was achieved by suppressing the expression of key melanin synthesis proteins (MITF, Tyrosinase) in B16F10 cells.

[0027] This invention also investigated the effect of ONO-2952 on UVB-induced pigmentation of the back skin of guinea pigs. The experiment showed that ONO-2952 can significantly reduce UVB-induced pigmentation of the back skin of guinea pigs.

[0028] The ONO-2952 inhibits melanin synthesis induced by endogenous factors α-MSH, ACTH, and ultraviolet B radiation.

[0029] The ONO-2952 inhibits melanin synthesis by suppressing the expression of key melanin synthesis proteins MITF and Tyrosinase in melanocytes.

[0030] Beneficial effects:

[0031] This invention provides the use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of medicaments / cosmetics for the treatment and / or prevention of hyperpigmentation disorders, particularly for the preparation of medicaments / cosmetics for the treatment and / or prevention of freckles, melasma, age spots, café-au-lait spots, nevi, and UV-induced hyperpigmentation. Experiments have demonstrated that ONO-2952 inhibits melanin synthesis by suppressing the expression of key proteins in melanin synthesis (MITF, Tyrosinase) in melanocytes; ONO-2952 can also inhibit melanin synthesis in guinea pig skin. This invention provides a new and effective treatment option for hyperpigmentation disorders. Attached Figure Description

[0032] Figure 1 The effect of ONO-2952 on the growth of B16F10 cells.

[0033] Figure 2 The effect of ONO-2952 on melanin synthesis induced by α-MSH in B16F10 cells.

[0034] Figure 3 The effect of ONO-2952 on ACTH-induced melanin synthesis in B16F10 cells.

[0035] Figure 4 The effect of ONO-2952 on melanin synthesis in UVB-induced B16F10 cells.

[0036] Figure 5 The effect of ONO-2952 on the expression of MITF and Tyrosinase, key proteins in melanin synthesis, in B16F10 cells.

[0037] Figure 6 This shows the effect of ONO-2952 on melanin synthesis in the back skin of guinea pigs (back skin photo).

[0038] Figure 7 This is a color difference graph showing the effects of ONO-2952 and the control group on the skin on the backs of guinea pigs over time.

[0039] Figure 8 The effect of ONO-2952 on melanin synthesis in the back skin of guinea pigs (ammonia silver staining of melanin in the back skin). Detailed Implementation

[0040] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0043] ONO-2952, purity: 99.51%, CAS: 895169-20-7.

[0044] Example 1: Effect of ONO-2952 on melanin synthesis in B16F10 cells

[0045] I. Effects of ONO-2952 on the proliferation of B16F10 cells

[0046] 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 5 × 10⁻⁶ cells / cells. 3 The ONO-2952 was seeded at a density of 10 cells / well in 96-well plates. The plates were incubated at 37°C and 5% CO2 for 24 hours. ONO-2952 was dissolved in DMSO, and different concentrations of ONO-2952 (5, 10, 20, and 40 μM) were added to the ONO-2952 treatment groups. An equal volume of blank solvent (DMSO) was added to the control group. The plates were incubated for another 48 hours. 20 μL of MTT solution was added to each well, and the plates were incubated at 37°C for 4 hours. After removing the supernatant, 200 μL of DMSO was added to each well, and the plates were shaken on a plate shaker for 10 minutes. The absorbance of each well was measured at 570 nm using a microplate reader.

[0047] Cell proliferation rate = (absorbance value of different concentrations of ONO-2952 treatment group / absorbance value of control group) × 100%. Each experiment was repeated 3 times (n=3). Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was performed using GraphPad Prism 8.0.2 software, and P < 0.05 was considered statistically significant. Experimental results are as follows: Figure 1 As shown.

[0048] As shown in the figure, compared with the control group, the concentration of ONO-2952 at 5-40 μM had no significant effect on the growth of B16F10 cells (P>0.05).

[0049] Therefore, it can be seen that ONO-2952 has no significant effect on the proliferation of B16F10 cells in the concentration range of 5-40 μM, showing low toxicity.

[0050] II. Effects of ONO-2952 on melanin synthesis in B16F10 cells

[0051] B16F10 cells in the exponential growth phase and in good condition (Chinese Academy of Sciences Cell Bank) were digested and counted, and then divided into 1×10⁻⁶ cells. 5 Cells were seeded at a density of [number] cells / mL into 6-well plates. Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Different concentrations of ONO-2952 (5 μM, 10 μM, 20 μM) prepared with DMSO were added to the cells. After 1 hour, the cells were irradiated with α-MSH (60 nM, M118985, Aladdin), ACTH (100 nM, A118750, Aladdin), and UVB (50 mJ / cm²), respectively. 2 Cells were treated. The control group received an equal amount of DMSO. Cells in both groups were cultured for 48 hours. After treatment, cells were collected and lysed in 100 μL of a non-denaturing lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1 mM PMSF for 20 minutes at 4°C. The cells were then centrifuged at 12000 rpm for 10 minutes at 4°C. The lower melanin precipitate was added to 100 μL of NaOH (1 mol / L, containing 10% DMSO) and lysed in an 80°C water bath for 2 hours. The completely dissolved melanin was added at 100 μL per well to a 96-well plate, and the absorbance was measured at 405 nm. Each experiment was repeated three times (n=3), and statistical analysis was performed using GraphPad Prism 8.0.2 software. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for inter-group comparisons, and P < 0.05 was considered statistically significant. Results are as follows... Figure 2 , Figure 3 and Figure 4 As shown.

[0052] Figure 2 In the study, compared with the blank control group, the α-MSH treatment group significantly promoted melanin synthesis. *** P < 0.001); compared with the α-MSH treatment group, ONO-2952 significantly inhibited melanin synthesis (P < 0.001); # P < 0.05 ## P < 0.01). Figure 3 and Figure 4 In comparison with the ACTH-treated group and the UVB-treated group, ONO-2952 significantly inhibited melanin synthesis. *P < 0.05 ** P < 0.01).

[0053] Experimental results: Compared with the α-MSH, ACTH and UVB treatment groups, ONO-2952 significantly inhibited the melanin synthesis induced by these factors, and showed a dose-dependent effect.

[0054] Therefore, ONO-2952 can significantly inhibit melanin synthesis in B16F10 cells.

[0055] III. Effects of ONO-2952 on the expression of MITF and Tyrosinase, key proteins in melanin synthesis, in B16F10 cells.

[0056] B16F10 cells in the exponential growth phase and in good condition (Chinese Academy of Sciences Cell Bank) were digested and counted, and then divided into 1×10⁻⁶ cells. 5Cells were seeded at a density of cells / mL into 6-well plates. Cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Different concentrations of ONO-2952 (5 μM, 10 μM, 20 μM) prepared with DMSO were added to the cells. After 1 hour, α-MSH (60 nM) was added to the cells, while the control group received an equal volume of DMSO. Cells in all groups were cultured for another 48 hours. 100 μL of non-denaturing lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1 mM PMSF was added. After centrifugation, the supernatant was collected. Protein concentration was determined using a BCA kit (P0009, Beyotime). 1 / 4 volume of 5*Loading Buffer (P0015, Beyotime) was added to the supernatant, and the mixture was boiled at 95°C for 7 minutes. SDS-PAGE with polyacrylamide gel electrophoresis was then performed under the following conditions: 80V for 30 minutes; 120V for 1 hour. After electrophoresis, the protein was transferred to a NC membrane at 100V for 1 hour and blocked with TBST (0.05% Tween-20) containing 5% skim milk for 1.5 hours. After blocking, the membrane was washed twice with TSBT for 5 minutes each time. The membrane was then incubated overnight at 4°C with primary antibodies MITF (ab303531, Abcam), Tyrosinase (ab315252, Abcam), and β-actin (AF0003, Beyotime) dissolved in TBST solution. The membrane was washed with TBST for 5 minutes, repeated 4 times. HRP-labeled secondary antibodies (A0208, A0216, Beyotime) were then added to bind the primary antibodies, and the membrane was incubated at room temperature for 1 hour. The membrane was then removed and washed 4 times with TBST for 5 minutes each time. Chemiluminescence detection was performed by adding a pre-mixed luminescent solution (P0018FS, Beyotime) onto the membrane, imaging the images using a gel imaging system, and analyzing the results using ImageJ software. Each experiment was repeated three times (n=3), and statistical analysis was performed using GraphPad Prism 8.0.2 software. Data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for inter-group comparisons, and P < 0.05 was considered statistically significant. Results are shown below. Figure 5 .

[0057] As can be seen from the figure, α-MSH significantly increased the protein expression of MITF and Tyrosinase in B16F10 cells. *** P < 0.001); Compared with the α-MSH treatment group, ONO-2952 can inhibit the protein expression of MITF and Tyrosinase in B16F10 cells, with significant differences. ## P < 0.01, ### P < 0.001).

[0058] Therefore, ONO-2952 can inhibit melanin synthesis by suppressing the expression of key proteins (MITF, Tyrosinase) in B16F10 cells.

[0059] Example 2: Effect of ONO-2952 on UV-induced pigmentation on the back of guinea pigs

[0060] Eight guinea pigs (purchased from Yizheng Anlimao Biotechnology Co., Ltd.), half male and half female, were selected. After acclimatization, the long hair on the backs of the guinea pigs was trimmed with scissors, and the short hair was shaved off completely with an electric shaver. The skin on the backs of the guinea pigs was irradiated with an SH4B ultraviolet light therapy device, covering a total area of ​​6×6cm. 2 The lamp was placed 15cm from the guinea pig's back, and the single irradiation dose was 500mJ / cm². 2 The guinea pigs were exposed to ultraviolet light continuously for one week. After one week of ultraviolet light exposure, stable pigmentation formed on the skin of their backs.

[0061] The irradiation area was divided into two treatment zones: one zone was treated with 1% ONO-2952, and the other zone was treated with a blank matrix (polyethylene glycol / ethanol = 7 / 3, v / v) as a control group. Each treatment zone was a circle with a diameter of 1 cm, and the two treatment zones were spaced 0.5 cm apart. The medication was applied twice daily for three weeks. Before treatment, the guinea pigs' fur was shaved short with an electric razor. The color changes of the skin on the back of the guinea pigs were measured using a CR-400 colorimeter before ultraviolet irradiation, one week after irradiation, and one, two, and three weeks after treatment.

[0062] The color difference value is calculated by subtracting the measurement value before ultraviolet irradiation from each measurement value.

[0063] Photographs were taken for objective comparison. Four weeks later, skin tissue from the backs of guinea pigs was collected, fixed with 4% paraformaldehyde solution, and routinely sectioned in paraffin. The tissue sections were stained using the Masson-Fontana ammoniacal silver staining method to determine changes in melanin in the skin. Results are shown below. Figure 6 , Figure 7 , Figure 8 .

[0064] Experimental results: Figure 6 This is a photo of the hairless area on the back of a guinea pig. Compared with the control group, the skin turned white after three weeks of ONO-2952 application. Figure 7 This refers to changes in the skin color value on the back of guinea pigs. Ultraviolet radiation decreases the skin color value of guinea pigs, while ONO-2952 can gradually increase the skin color value compared to the control group. * P < 0.05. Figure 8The results of Masson-Fontana ammonia silver staining of guinea pig skin tissue show that, compared with the control group, ONO-2952 can reduce the melanin content in guinea pig skin.

[0065] Therefore, ONO-2952 reduces UV-induced melanin deposition in the skin on the back of guinea pigs.

[0066] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of medicaments / cosmetics for the treatment and / or prevention of hyperpigmentation disorders, characterized in that, The chemical structural formula of ONO-2952 is as follows: ； The hyperpigmentation disorders mentioned are freckles, melasma, chloasma, café-au-lait spots, nevi, or ultraviolet-induced pigmentation.

2. The use according to claim 1, characterized in that, The ONO-2952 or its pharmaceutically acceptable salt is used as the sole active ingredient in the preparation of medicines / cosmetics for the treatment and / or prevention of hyperpigmentation disorders.

3. The use according to claim 1, characterized in that, The ONO-2952 or its pharmaceutically acceptable salt is used in combination with other active ingredients to prepare medicines / cosmetics for the treatment and / or prevention of hyperpigmentation disorders.

4. The use according to any one of claims 1-3, characterized in that, The drug includes ONO-2952 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

5. The use according to claim 4, characterized in that, The dosage form of the drug is a cream, ointment, lotion, gel, film, or spray.

6. The use according to any one of claims 1-3, characterized in that, The ONO-2952 inhibits melanin synthesis induced by endogenous factors α-MSH, ACTH, and ultraviolet B radiation.

7. The use according to any one of claims 1-3, characterized in that, The ONO-2952 inhibits melanin synthesis by suppressing the expression of key melanin synthesis proteins MITF and Tyrosinase in melanocytes.

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