Application of ONO-2952 in preparation of medicine / cosmetic for treating and / or preventing hyperpigmentation diseases

Abstract

The invention discloses an application of ONO-2952 or a pharmaceutically acceptable salt thereof in preparation of drugs / cosmetics for treating and / or preventing hyperpigmentation diseases, and belongs to the technical field of medicines. The ONO-2952 disclosed by the invention inhibits melanin synthesis induced by endogenous factors such as alpha-MSH and ACTH and ultraviolet ray UVB, and inhibits melanin synthesis by inhibiting expression of key proteins MITF and Tyrosinase for melanin synthesis in melanocytes. Therefore, the ONO-2952 or the pharmaceutically acceptable salt thereof can be used for preparing medicines / cosmetics for treating and / or preventing hyperpigmentation diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to use of ONO-2952 or a pharmaceutically acceptable salt thereof in preparing medicines / cosmetics for treating and / or preventing hyperpigmentation diseases. Background Art

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

[0003] At present, there are many kinds of whitening products commonly used in the market, and their main active ingredients include vitamin C, niacinamide, arbutin, kojic acid, etc. 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, dryness, etc. Certain ingredients such as kojic acid may cause skin sensitivity or phototoxic reactions, leading to permanent depigmentation. Arbutin is a derivative of hydroquinone and can also cause skin toxic reactions. Therefore, it is particularly important to develop new whitening products to reduce potential risks and meet the needs of different groups for whitening and health.

[0004] ONO-2952 (CAS: 895169-20-7), chemical name 1-((1S)-1-(4-chloro-2-methoxyphenyl)-5-fluoro-1,9-dihydrospiro[β-carboline-4,1'-cyclopropane]-2(3H)-yl)ethanone, is a white to off-white solid. The molecular formula is C 22 H 20 ClFN2O2, molecular weight 398.86. ONO-2952 belongs to the indole alkaloid class, which 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] At present, there are no reports on the effect of ONO-2952 in inhibiting pigment synthesis in domestic and foreign research. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of medicines / cosmetics for treating and / or preventing hyperpigmentation diseases.

[0007] The present invention has found that in vitro and in vivo pharmacodynamic tests have shown that ONO-2952 can inhibit endogenous factors (α-MSH, ACTH) and ultraviolet UVB-induced melanin synthesis, and has the efficacy of treating hyperpigmentation diseases, especially freckles, pregnancy spots, chloasma, coffee spots, nevus, and ultraviolet-induced pigmentation. Therefore, ONO-2952 can be used to prepare drugs / cosmetics for treating and / or preventing hyperpigmentation diseases.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides a use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of a medicine / cosmetic for treating and / or preventing a hyperpigmented disease. The chemical structural formula of ONO-2952 is as follows:

[0010]

[0011] The pigmented disease is freckles, pregnancy spots, chloasma, coffee spots, nevus or ultraviolet-induced pigmentation.

[0012] The ONO-2952 or a pharmaceutically acceptable salt thereof is used as the sole active ingredient to prepare medicines / cosmetics for treating and / or preventing hyperpigmentation diseases.

[0013] The ONO-2952 or its pharmaceutically acceptable salt is used in combination with other active ingredients to prepare medicines / cosmetics for treating and / or preventing hyperpigmentation diseases.

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

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

[0016] The emulsifier is selected from at least one of Tweens, Spans, glycerol fatty acid esters, pectin, agar, sodium alginate or silicon dioxide.

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

[0018] The diluent is selected from at least one of starch, sugar, cellulose or inorganic salts.

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

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

[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, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked polyvinyl pyrrolidone.

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

[0024] The drug of the present invention can be administered in various known ways, such as transdermally, by spraying. The drug of the present invention can be administered alone or in combination with other drugs. The transdermal composition can be in any dosage form acceptable to the skin, including but not limited to creams, ointments, cream preparations, 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 the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, and is non-toxic to the patient. The selected dosage level depends on a variety of factors, including the route of administration, time of administration, rate of excretion, duration of treatment, other drugs, compounds and / or materials used in combination with ONO-2952, age, sex, weight, general health and previous medical history of the patient being treated, and similar factors known in the medical field.

[0026] The present invention studies the effect of ONO-2952 on the synthesis of melanin in B16F10 cells. The experiment shows that within the concentration range of 5-40 μM, ONO-2952 has no significant effect on the proliferation of B16F10 cells, showing low toxicity. ONO-2952 can significantly inhibit the synthesis of melanin in B16F10 cells, and can inhibit the synthesis of melanin by inhibiting the expression of key proteins (MITF, Tyrosinase) in melanin synthesis in B16F10 cells.

[0027] The present invention also studies the effect of ONO-2952 on the pigmentation of the guinea pig back skin induced by ultraviolet UVB. The experiment shows that ONO-2952 can significantly reduce the pigmentation of the guinea pig back skin caused by ultraviolet UVB.

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

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

[0030] Beneficial effects:

[0031] The present invention provides the use of ONO-2952 or a pharmaceutically acceptable salt thereof in preparing a medicine / cosmetic for treating and / or preventing hyperpigmentation diseases, in particular, the use of ONO-2952 or a pharmaceutically acceptable salt thereof in preparing a medicine / cosmetic for treating and / or preventing freckles, pregnancy spots, chloasma, coffee spots, nevus, and ultraviolet-induced pigmentation. Experiments have shown that ONO-2952 can inhibit melanin synthesis by inhibiting the expression of key melanin synthesis proteins (MITF, Tyrosinase) in melanocytes; ONO-2952 can inhibit melanin synthesis in guinea pig skin. The present invention provides a new and effective treatment option for hyperpigmentation diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[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 in B16F10 cells induced by α-MSH.

[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 UVB-induced melanin synthesis in B16F10 cells.

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

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

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

[0039] Figure 8 It is the effect of ONO-2952 on the synthesis of melanin in the back skin of guinea pigs (ammonia silver staining of melanin in the back skin). DETAILED DESCRIPTION

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

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

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

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

[0044] Example 1 Effect of ONO-2952 on Melanin Synthesis in B16F10 Cells

[0045] 1. Effect of ONO-2952 on B16F10 cell proliferation

[0046] B16F10 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) in the exponential growth phase and in good condition were taken and cultured at 5×10 3 The cells were inoculated at a density of 100 μL / well in a 96-well culture plate. Incubated in an incubator at 37°C and 5% CO2 for 24 hours. ONO-2952 was dissolved in DMSO, and different concentrations of ONO-2952 (5, 10, 20, 40 μM) were added to the ONO-2952 treatment group. An equal volume of blank solvent (DMSO) was added to the control group. The culture was continued for 48 hours. 20 μL of MTT solution was added to each well and incubated in a 37°C incubator for 4 hours. After removing the supernatant, 200 μL of DMSO was added to each well and shaken on a flat shaker for 10 minutes. The absorbance of each well was measured at a wavelength of 570 nm using a microplate reader.

[0047] Cell proliferation rate = (absorbance value of ONO-2952 treatment group at different concentrations / absorbance value of control group) × 100%, each experiment was repeated 3 times (n = 3), and the data were 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 significant. The experimental results are shown in Figure 1 shown.

[0048] It can be seen from the figure that compared with the control group, ONO-2952 at a concentration of 5-40 μM had no significant effect on the growth of B16F10 cells (P>0.05).

[0049] It can be seen that within the concentration range of 5-40 μM, ONO-2952 had no significant effect on the proliferation of B16F10 cells and showed low toxicity.

[0050] 2. Effect of ONO-2952 on melanin synthesis in B16F10 cells

[0051] B16F10 cells (Cell Bank, Chinese Academy of Sciences) in exponential growth phase and in good condition were obtained, digested and counted, and the number of cells was calculated according to 1×10 5 The cells were inoculated into 6-well plates at a density of 100 cells / mL. The cells were placed in a 37°C, 5% CO2 incubator for 24 hours. Different concentrations of ONO-2952 (5μM, 10μM, 20μM) prepared in DMSO were added to the cells. After 1 hour, α-MSH (60nM, M118985, Aladdin), ACTH (100nM, A118750, Aladdin) and UVB irradiation (50mJ / cm 2 ) cells. The control group was treated with an equal amount of DMSO. Each group of cells was cultured for 48 hours. The treated cells were collected, and 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, and lysed at 4°C for 20 minutes. Centrifuge at 4°C and 12000 r / min for 10 minutes, add 100 μL NaOH (1 mol / L, containing 10% DMSO) to the lower layer of melanin precipitate, and lyse in an 80°C water bath for 2 hours. The completely dissolved melanin was added to a 96-well plate at 100 μL / well, and the absorbance value was measured at 405 nm. Each experiment was repeated 3 times (n=3), and statistical analysis was performed using GraphPad Prism8.0.2 software. Data were expressed as mean ± SD, and one-way ANOVA was used for inter-group comparisons. P < 0.05 was considered to be significantly different. Figure 2 , Figure 3 and Figure 4 shown.

[0052] Figure 2 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﹤0.05, ## P﹤0.01). Figure 3 and Figure 4 Compared 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 in a dose-dependent manner.

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

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

[0056] B16F10 cells (Cell Bank, Chinese Academy of Sciences) in exponential growth phase and in good condition were obtained, digested and counted, and the number of cells was calculated according to 1×10 5The cells were inoculated into 6-well plates at a density of 100 μL / mL. The cells were placed in a 37°C, 5% CO2 incubator for 24 hours. Different concentrations of ONO-2952 (5μM, 10μM, 20μM) prepared with DMSO were added to the cells. After 1 hour, α-MSH (60nM) was added to the cells, and the control group was added with an equal amount of DMSO. The cells in each group were cultured for 48 hours. 100μL of non-denaturing lysis buffer containing 50mMTris-HCl (pH 7.4), 150mM NaCl, 1% NP-40, 0.1% SDS and 1mM PMSF was added, and the supernatant was taken after centrifugation. The protein concentration was determined using a BCA kit (P0009, Beyotime), and 1 / 4 volume of 5*Loading Buffer (P0015, Beyotime) was added to the supernatant and mixed, and boiled at 95°C for 7 minutes. Then, SDS-PAGE polyacrylamide gel electrophoresis was performed under the conditions of 80V for 30 minutes and 120V for 1 hour. After the electrophoresis, the protein was transferred to the NC membrane with a voltage of 100V for 1 hour and blocked with TBST (0.05% Tween-20) containing 5% skim milk for 1.5 hours. After blocking, it was washed twice with TSBT for 5 minutes each. The membrane was incubated with the primary antibodies MITF (ab303531, Abcam), Tyrosinase (ab315252, Abcam), and β-actin (AF0003, Beyotime) dissolved in TBST solution at 4°C overnight. Wash with TBST for 5 minutes and repeat 4 times. Then, HRP-labeled secondary antibodies (A0208, A0216, Beyotime) were added to bind to the primary antibodies and the membrane was incubated at room temperature for 1 hour. The membrane was removed and washed 4 times with TBST for 5 minutes each. Chemiluminescence detection, the pre-mixed luminescent solution (P0018FS, Biyuntian) was added to the membrane, the gel imaging system was used to take pictures, and the results were analyzed using ImageJ software. Each group of experiments was repeated 3 times (n=3), and statistical analysis was performed using GraphPad Prism 8.0.2 software. Data were expressed as mean ± standard deviation (Mean±SD), and one-way ANOVA was used for inter-group comparison. P<0.05 was considered to be significantly different. Results are shown in Figure 5 .

[0057] It can be seen from the figure that 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 could inhibit the protein expression of MITF and Tyrosinase in B16F10 cells, with significant difference ( ## P﹤0.01, ### P﹤0.001).

[0058] Therefore, ONO-2952 can inhibit melanin synthesis by inhibiting the expression of key proteins (MITF, Tyrosinase) in melanin synthesis 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.) were selected, half male and half female. After adaptive feeding, the long hair on the back of the guinea pigs was cut with scissors, and the short hair was shaved with an electric shaver. The SH4B ultraviolet light therapy device was used to irradiate the skin on the back of the guinea pigs, with a total area of ​​6×6 cm 2 The lamp was 15cm away from the guinea pig's back, and the single irradiation dose was 500mJ / cm 2 , and irradiated continuously for one week. One week after ultraviolet irradiation, stable pigmentation was formed on the back skin of guinea pigs.

[0061] The irradiated area was divided into two application areas: one was smeared with 1% ONO-2952, and the other was smeared with a blank matrix (polyethylene glycol / ethanol = 7 / 3, v / v) as a control group. Each application area was a circle with a diameter of 1 cm, and the two application areas were separated by 0.5 cm. The application was applied twice a day for three weeks. Before the application, the short hair was shaved with an electric shaver. The color change of the guinea pig back skin was measured using a CR-400 colorimeter before ultraviolet irradiation, 1 week after irradiation, and 1, 2, and 3 weeks after the application.

[0062] The color difference value was calculated as each measurement value minus the measurement value before UV exposure.

[0063] Photos were taken for objective comparison. Four weeks later, skin tissue from the guinea pig's back was taken, fixed with 4% paraformaldehyde solution, and routine paraffin sections were made. The tissue sections were stained with Masson-Fontana ammonia silver staining to determine the changes in melanin in the skin. The results are shown in Figure 6 , Figure 7 , Figure 8 .

[0064] Experimental results: Figure 6 This is a photo of the hair loss area on the back of a guinea pig. Compared with the control group, the skin became whiter after three weeks of application of ONO-2952. Figure 7 It is the change of the chromaticity value of the guinea pig's back skin. Ultraviolet radiation reduces the chromaticity value of the guinea pig's skin. ONO-2952 can gradually increase the chromaticity value of the guinea pig's skin. Compared with the control group, * P<0.05. Figure 8This is the result of Masson-Fontana ammonia silver staining of guinea pig skin tissue. Compared with the control group, ONO-2952 can reduce the melanin content in guinea pig skin.

[0065] Thus, ONO-2952 reduces UV-induced melanin pigmentation in the dorsal skin of guinea pigs.

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

Claims

1. Use of ONO-2952 or a pharmaceutically acceptable salt thereof in the preparation of a medicament / cosmetic for treating and / or preventing a hyperpigmented disease, characterized in that: The chemical structure of ONO-2952 is as follows:

2. The use according to claim 1, characterized in that The pigmented disease is freckles, pregnancy spots, chloasma, coffee spots, nevus or ultraviolet-induced pigmentation.

3. The use according to claim 1, characterized in that The ONO-2952 or a pharmaceutically acceptable salt thereof is used as the sole active ingredient to prepare medicines / cosmetics for treating and / or preventing hyperpigmentation diseases.

4. The use according to claim 1, characterized in that The ONO-2952 or a pharmaceutically acceptable salt thereof is used in combination with other active ingredients to prepare medicines / cosmetics for treating and / or preventing hyperpigmentation diseases.

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

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

7. The use according to any one of claims 1 to 4, characterized in that: The ONO-2952 inhibits the melanin synthesis induced by endogenous factors α-MSH, ACTH, and ultraviolet UVB.

8. The use according to any one of claims 1 to 4, characterized in that: The ONO-2952 inhibits the synthesis of melanin by inhibiting the expression of MITF and Tyrosinase, the key proteins for melanin synthesis in melanocytes.

Images