Composition for topical administration

Abstract

This provides a new treatment for hyperpigmentation, a skin condition characterized by decreased pigmentation. [Solution] In one aspect, the present invention relates to a topical composition for the treatment of depigmentation skin diseases, comprising staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a composition for topical administration for the treatment of hypopigmentation skin diseases.

Background Art

[0002] There are various ethnic groups in the world, and one of the major indicators for classifying them is skin color. It is known that melanin pigment determines skin color and is synthesized by melanocytes. It is known that the number of melanocytes in the skin does not differ significantly between Caucasians and Blacks, and the difference in the amount of melanin synthesized by melanocytes results in the difference in skin color.

[0003] Normally, within the same ethnic group, skin color is generally the same. However, due to environmental factors such as exposure to sunlight, there is a certain degree of variation even within the same ethnic group, and it is common for the same person to have slightly different skin colors depending on the部位. Such a constant abnormality in skin color that significantly deviates from the general variation in skin color is called dyschromia.

[0004] There are various types of dyschromia. Here, hypopigmentation skin disease is defined as a symptom in which the skin color becomes significantly lighter compared to the original skin color due to abnormalities in melanocytes. A typical example of hypopigmentation skin disease is "vitiligo," in which a part of the skin becomes white. The acquired vitiligo is collectively called "common vitiligo." Common vitiligo is clinically classified into localized type, which occurs only in a part of the body, segmental type, which occurs unilaterally in accordance with a certain nerve innervation area, and generalized type, which is scattered over a relatively wide area. As reasons for the occurrence of common vitiligo, there are various theories such as the disappearance of melanocytes due to autoimmunity and the dysfunction of melanocytes due to neuropathy. However, recent research has shown that it is not a simple disease that can be attributed to a single cause. There are multiple factors and multiple pathways leading to the onset of common vitiligo, and their simultaneous occurrence particularly makes the treatment of refractory common vitiligo patients difficult.

[0005] For pigmentary disorders, particularly vitiligo, ultraviolet therapy and topical steroids have long been used as treatments, and have shown some therapeutic efficacy. In recent years, topical and oral administration of JAK (Janus kinase) inhibitors have also been approved in other countries (e.g., Non-Patent Document 1). Skin grafting is also performed for localized vitiligo. However, there are still a considerable number of refractory cases that are insufficient with the above treatments, and it is difficult to say that a treatment method has been established.

[0006] The cause of depigmented skin diseases is a deficiency of melanin pigment. Melanocytes, which supply melanin pigment, are spindle-shaped when not stimulated, but when activated by stimuli such as ultraviolet light, they change into an amoeba-like form with many dendrites, and supply melanin pigment to surrounding tissues via these dendrites. One of the causes of vitiligo is thought to be when melanocytes become dysfunctional due to external factors such as oxidative stress, and are unable to enter an activated state even when stimulated by ultraviolet light.

[0007] As mentioned earlier, ultraviolet (UV) therapy stimulates melanocytes and promotes melanin synthesis, thus having some effect in treating vitiligo. However, UV light itself is harmful to the human body, limiting the amount of UV light that can be used. Furthermore, it can cause areas other than the vitiligo to darken, creating a contrast that makes the vitiligo more noticeable. Therefore, a method other than UV light, that stimulates melanocytes locally, is necessary for the treatment of vitiligo, but to date, no such method has been established. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] David Rosmarin et al., The Lancet Journal, 2020 Jul 11; 396 (10244): 110-120. doi: 10.1016 / S0140-6736(20)30609-7 [Overview of the project] [Problems that the invention aims to solve]

[0009] In view of the circumstances described above, the present invention aims to obtain a new therapeutic agent for pigment-depleting skin diseases by locally stimulating melanocytes. [Means for solving the problem]

[0010] The inventors conducted studies using normal human skin pigment cells derived from neonatal foreskin and found that staurosporin is effective in activating melanocytes. They also confirmed its effectiveness in enhancing melanin pigment in animal experiments, leading to the completion of the present invention.

[0011] In one aspect, the present invention relates to a topical composition for the treatment of depigmentation skin diseases, comprising staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient. [Effects of the Invention]

[0012] In one aspect, the present invention can provide a novel pharmaceutical composition capable of treating pigment-depleting skin diseases. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows the increase in the number of dendritic structures compared to the control in human normal skin pigment cells (HEMn-MP) derived from neonatal foreskin that were cultured for 24 hours after adding a 300 nM staurosporine solution. [Figure 2] Figure 2 shows the changes in pigment-related proteins compared to a control in human normal skin pigment cells (HEMn-MP) derived from neonatal foreskin, cultured for 24 hours after adding a 300 nM staurosporine solution. [Figure 3]Figure 3 shows the cell viability of human normal cutaneous pigment cells (HEMn-MP) and melanoma cells (G361) derived from neonatal foreskin when the concentration of added staurosporine is varied. [Figure 4] Figure 4 shows the changes in skin color observed after applying the topical administration composition of the present invention to the skin of a guinea pig (KWL:JY-4) for 14 days. [Figure 5] Figure 5 shows the skin color of a guinea pig (KWL:JY-4) after artificially whitening its skin color by applying a 30 wt% rhododenol solution for one month, followed by application of the topical administration composition of the present invention for 14 days, and observation of the skin color thereafter. [Modes for carrying out the invention]

[0014] The present invention will be described in detail below. The topical administration composition of the present invention is characterized by containing staurosporine as an active ingredient. Staurosporine is a compound represented by the following formula ((5S,6R,7R,9S)-6-methoxy-5-methyl-7-(methylamino)-6,7,8,9,15,16-hexahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacene-14-one), and is a non-selective protein kinase C (PKC) inhibitor. Staurosporine is known in research to induce apoptosis and is a type of protein kinase inhibitor, but there have been no cases of clinical application to date.

[0015] [ka]

[0016] When staurosporine is added to normal human epidermal melanocytes (HEMn-MP) derived from neonatal foreskin and cultured for about 24 hours, our study found that dendrites significantly increased compared to the case where only dimethyl sulfoxide (DMSO), the solvent, was added (Figure 1). Also, the concentrations of pigment-related proteins such as PMEL and ENDRB were also increased (Figure 2). Melanocytes are known to extend dendrites to supply melanin pigment to surrounding cells when stimulated by ultraviolet light and the like, and it was shown that staurosporine has an effect similar to ultraviolet light stimulation.

[0017] On the other hand, when comparing the effects of staurosporine on normal human epidermal melanocytes (HEMn-MP) derived from neonatal foreskin and human melanoma cells (G361), the added staurosporine showed cytotoxicity by inducing apoptosis in human melanoma cells (G361), while it did not show cytotoxicity against normal human epidermal melanocytes (HEMn-MP) derived from neonatal foreskin, and rather it was suggested that it might contribute to cell activation (Figure 3). From this experiment, even if melanoma cells are generated in the skin coated with staurosporine, it can be said that the carcinogenic risk during local administration is relatively low because staurosporine acts inhibitory against human melanoma cells.

[0018] Several compounds with a similar skeleton to staurosporine are known, and whether similar compounds have the same effect was examined. Midostaurin is a protein kinase inhibitor synthesized based on staurosporine and is a compound expected to have clinical applications for epidermolysis bullosa and the like (WO2023 / 205504). When comparing the dendrite-inducing ability of staurosporine and midostaurin in normal human epidermal melanocytes (HEMn-MP) derived from neonatal foreskin, no dendrite-inducing effect was observed with midostaurin. From this result, it was considered that the activation ability against normal human epidermal melanocytes derived from neonatal foreskin is a specific effect of staurosporine and is different from the effects reported for midostaurin.

[0019] Staurosporine is known as a compound having an inhibitory effect on a wide range of protein kinases, and due to its lack of enzyme selectivity, it has been considered difficult to clinically apply it for oral drugs or intravenous injections. However, when used as a topical administration agent such as a topical preparation, since the risk of the drug diffusing throughout the body is low, it is possible to prevent exposure to internal organs in the body and solve the problem of enzyme selectivity. Therefore, we decided to study a topical composition containing staurosporine.

[0020] [Composition for topical administration] In one aspect, the present invention relates to a composition for treating hypopigmented skin diseases, containing staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient. According to the present invention, since staurosporine is used as an active ingredient, it is possible to promote the growth of the dendritic structure of melanocytes. By using a topical preparation containing staurosporine, the skin color of guinea pigs can be darkened, and in mice artificially induced with vitiligo, it was possible to cure the disease much faster than natural healing. The topical composition of the present invention can be used for pharmaceuticals for treating "hypopigmentation" such as vitiligo vulgaris in humans. In one or more embodiments, the topical composition of the present invention can be a therapeutic agent that can be an alternative to the treatment method of hypopigmented skin diseases using ultraviolet rays. Alternatively, in one or more embodiments, the topical composition of the present invention can be used for treating the disease together with the treatment method of hypopigmented skin diseases using ultraviolet rays.

[0021] In this invention, "hypochromigmentation" refers to a type of pigment disorder in which a decrease in pigment occurs due to abnormalities in melanocytes, resulting in a condition in which the skin color becomes significantly lighter than what it should be. A typical example of hypochromigmentation is "vitiligo," in which parts of the body's skin turn white in people of African or Asian descent. Acquired vitiligo is collectively called "vitiligo vulgaris." Vitiligo vulgaris is clinically classified into localized type, which occurs only in a part of the body; segmental type, which occurs unilaterally in a specific nerve innervation area; and generalized type, which is scattered over a relatively wide area.

[0022] In the present invention, "treatment of pigment-decreasing skin disease" may, in one or more embodiments, include restoring pigment-decreasing symptoms associated with pigment-decreasing skin disease, slowing the progression of pigment-decreasing skin disease, slowing the worsening of undesirable symptoms associated with pigment-decreasing skin disease, curing pigment-decreasing skin disease, or a combination of two or more of the above.

[0023] In the present invention, "pharmaceutically acceptable salt" refers to a salt that can be used as a pharmaceutical, and in one or more embodiments, examples include acidic salts and basic salts. Examples of acidic salts in one or more embodiments include hydrochloride, hydrobromide, sulfate, nitrate, formate, acetate, benzoate, maleate, fumarate, succinate, tartrate, citrate, lactate, oxalate, glutamate, and aspartate. Examples of basic salts in one or more embodiments include sodium salt, potassium salt, magnesium salt, and calcium salt. Preference pharmaceutically acceptable salts include citrate and lactate.

[0024] Since the active ingredient, staurosporine, is an amine, it can be included in compositions for topical administration in the form of, for example, hydrochloride, and its use in salt form is not particularly restricted. There are also no particular restrictions on its inclusion in the form of a so-called prodrug, which is metabolized in the body to produce staurosporine. However, when used in topical preparations, it does not pass through the liver or gastrointestinal tract, so it is difficult to expect chemical changes due to metabolism, and therefore, inclusion of staurosporine itself is preferred.

[0025] The concentration of staurosporine contained in the topical administration composition of the present invention can be determined by optimizing it after evaluating its efficacy against the disease, cytotoxicity, and / or blood concentration in one or more embodiments. Since the penetration of the active ingredient into the skin differs depending on the base material used in the topical administration composition, the optimal concentration can be appropriately determined depending on the base material. In experiments using neonatal foreskin-derived human normal skin pigment cells (HEMn-MP), cytotoxicity was observed at concentrations of 2000 nM or higher, and no dendritic induction effect was observed at concentrations below 50 nM. Therefore, there are certain limitations on the acceptable concentration from the viewpoint of safety and efficacy, but from the results of animal experiments described below, the concentration of staurosporine contained in the topical administration composition is preferably in the range of 0.005% to 0.1% by weight, and more preferably in the range of 0.025% to 0.075% by weight.

[0026] Since staurosporine is soluble in alcohols, it is preferable to prepare a topical administration composition by dissolving it in alcohol. Examples of alcohols that can be used include ethanol, isopropyl alcohol, 1-butanol, myristyl alcohol, stearyl alcohol, and glycerin in one or more embodiments. There are no particular restrictions on the amount of alcohol used, but for example, a 0.5% by weight ethanol solution of staurosporine can be prepared and then diluted with another base material to obtain a final content of 0.005% by weight to 0.1% by weight. The use of other solvents to improve solubility is not limited. Examples of other solvents that can be used include triacetin, methyl sulfoxide, and lactic acid in one or more embodiments. These may be used alone or in combination with alcohols. In one or more embodiments, staurosporine may be dissolved in dimethyl sulfoxide and then in an alcohol to prepare the composition.

[0027] In the present invention, "local administration" refers to administration via a non-systemic route in which the active ingredient is directly administered to the site where the effect is expected (affected area). Examples of local administration in one or more embodiments include intracutaneous administration, subcutaneous administration, oral administration, rectal administration, ophthalmic administration, and nasal administration. In one or more embodiments, local administration does not include administration via a systemic route such as intravenous administration or oral administration.

[0028] Common dosage forms for topical administration compositions include, in one or more embodiments, subcutaneous injection formulations, oral formulations, suppositories, eye drops, nasal drops, ointments, creams, and lotions. Since the present invention is used for pigment-depleting skin diseases, dosage forms that are applied directly to or patched onto the skin at the disease site are preferred. Examples of skin-administered compositions of the present invention include ointments, lotions, creams, and patches in one or more embodiments. Regarding the dosage form of the topical administration composition of the present invention, since absorption rate and duration of action are generally in a trade-off relationship, the dosage form can be appropriately determined by evaluating the blood concentration of absorbed staurosporine and the therapeutic effect in one or more embodiments. From the viewpoint of absorption rate into the skin, lotions are preferred, and from the viewpoint of duration of effect, patches are superior.

[0029] One embodiment of the topical administration composition of the present invention is a dermal administration composition. The dermatologically administered composition of the present invention further contains a base material in one or more embodiments. The base material used in the dermatologically administered composition of the present invention can be a material commonly used in ointments and creams for topical use. Examples of base materials in one or more embodiments include white petrolatum, stearyl alcohol, glyceryl stearate, squalane, polyethylene glycol, polypropylene glycol, bleached beeswax, and vegetable oils. It is preferable that the base material of the dermatologically administered composition contains vegetable oil to improve the penetration of staurosporine into the skin. As an empirical rule, hydrophilic base materials tend to have weaker skin penetration and the contained components often remain on the epidermis, while lipophilic base materials tend to have strong skin penetration. Certain vegetable oils, in particular, have excellent skin penetration and are used in oil massages. Examples of vegetable oils in one or more embodiments include sesame oil, camellia oil, and olive oil, and it is preferable that these vegetable oils be included in a certain proportion or more of the base material. The content of the base material in the dermatologically administered composition is not particularly limited, but is preferably 20% by weight or more, more preferably 30% by weight or more. There are no specific restrictions on the upper limit of the base material content, but it should be 50% by weight or less.

[0030] In one or more embodiments, the dosage forms of the dermal administration composition of the present invention may include ointments, creams, and lotions, which can be appropriately determined depending on the application site and intended use. The topical administration composition of the present invention may further contain emulsifiers for micelle formation, and other components such as antioxidants, surfactants, gelling agents, preservatives, thickeners, emulsifying stabilizers, and pH adjusters to improve storage stability.

[0031] The topical compositions of the present invention can be manufactured in one or more embodiments according to known methods, etc. In other embodiments, the present invention relates to a method for manufacturing a topical composition for the treatment of depigmentation skin diseases using staurosporine or a pharmaceutically acceptable salt thereof. The manufacturing method of the present invention, in one or more embodiments, includes mixing staurosporine or a pharmaceutically acceptable salt thereof with an alcohol. The manufacturing method of the present invention, in one or more embodiments, includes mixing staurosporine or a pharmaceutically acceptable salt thereof with a solvent, and mixing the resulting mixture with an alcohol, which may optionally include mixing the mixture with the alcohol with a base material. In one or more embodiments, examples of solvents include sulfoxides such as DMSO.

[0032] As described above, the inventors have found that staurosporine can promote the growth of dendritic structures in melanocytes and thereby accelerate the synthesis of melanin pigment. The present invention, in further aspects, relates to a dendrite formation promoter in melanocytes, comprising staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient, depending on the target. The present invention, in further aspects, relates to a melanin pigment synthesis promoter, comprising staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient, depending on the target.

[0033] In other aspects, the present invention relates to a topical composition containing staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient for use in the treatment of depigmentation skin diseases.

[0034] In other aspects, the present invention relates to the use of staurosporine or a pharmaceutically acceptable salt thereof for the preparation of topical compositions for the treatment of depigmentation skin diseases.

[0035] The present invention relates, in other embodiments, to a method for treating depigmentation skin diseases, comprising administering a therapeutically effective amount of staurosporine or a pharmaceutically acceptable salt thereof to a subject requiring treatment. In one or more embodiments, the subject requiring treatment is a subject requiring treatment for depigmentation skin diseases, such as patients with depigmentation skin diseases, patients with vitiligo, and patients with vitiligo vulgaris. The subject may be a human or an animal other than a human.

[0036] The daily dose is approximately 0.01 mg / kg body weight to 100 mg / kg body weight, or approximately 0.1 mg / kg body weight to 20 mg / kg body weight, in one or more embodiments, not limited to this. The number of daily doses is 1 to 4 times, in one or more embodiments.

[0037] The present invention, in yet another aspect, relates to a method for promoting the formation of dendrites in melanocytes, comprising administering staurosporine or a pharmaceutically acceptable salt thereof to a subject of interest. The present invention, in yet another aspect, relates to a method for promoting the synthesis of melanin pigment, comprising administering staurosporine or a pharmaceutically acceptable salt thereof to a subject of interest.

[0038] The present invention further relates to one or more embodiments described below. [1] A topical composition for the treatment of hyperpigmentation, comprising staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient. [2] The topical administration composition described in [1], which is a composition for skin administration. [3] The topical composition according to [1] or [2], wherein the pigment-decreasing skin disease is vitiligo. [4] A topical administration composition according to any one of [1] to [3], comprising 20% ​​by weight or more of vegetable oil and containing an active ingredient in the range of 0.005% by weight to 0.1% by weight.

[0039] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0040] <Reagent procurement> Reagents that did not specifically list the manufacturer were purchased from CELL Signaling Technology. <Preparation of Composition for Local Administration 1> 2.3 mg of reagent-grade staurosporine (CAYMAN Chemical) was dissolved in 2 mL of dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries, Ltd.). After dissolution, reagent-grade ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to make a total volume of 10 mL, preparing an ethanol solution of approximately 500 μMol / L. This solution was mixed with 10 mL of sesame oil (Summit Essential Oils Co., Ltd., for cosmetic use) to prepare a lotion with a concentration of 250 μMol / L (hereafter referred to as μM). Using a similar method, 1000 μM / L and 2000 μM / L ethanol solutions of staurosporine were prepared, and by mixing them with an equal amount of sesame oil, 500 μM and 1000 μM lotions were prepared. 10g of the 1000μM lotion prepared above and 10g of white petrolatum (manufactured by Ken-ei Pharmaceutical Co., Ltd.) were mixed at room temperature to prepare a 500μM ointment. For comparison (Ctrl), a control reagent (hereinafter referred to as Vehicle) was prepared by mixing reagent-grade ethanol and sesame oil in a 1:1 ratio. <Preparation of Composition for Local Administration 2> The solvent for dissolving staurosporine was prepared as follows: 20 g of purified water, 75 g of reagent-grade ethanol, and 5 g of lactic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed to form a solvent. 1 mL of the above solvent was added to 10 mg of reagent-grade staurosporine and stirred at room temperature to completely dissolve it, thereby preparing a staurosporine-lactic acid mixed solution. The concentration of staurosporine was approximately 22 mMol / L (hereinafter referred to as mM). A separate cream base was prepared. 20g of hydrophilic cream "Nikko" (manufactured by Nikko Pharmaceutical Co., Ltd.) and 10g of sesame oil were mixed to create a cream base for a topical administration composition. To 10.5 g of the cream base mentioned above, 0.5 g of the lactic acid solution of staurosporine was added and mixed to prepare a topical administration composition of approximately 1 mM. Also, to 5 g of the cream base, 0.5 g of the lactic acid solution of staurosporine was added and mixed to prepare a topical administration composition of approximately 2 mM.

[0041] <Testing Method> <Tests using cultured cells> <Cell acquisition and culture conditions> The neonatal foreskin-derived human normal skin pigment cells (HEMn-MP) used in the study were purchased from Invitrogen (Thermo Fisher Scientific) along with culture medium (M-254-500) and 1% human melanocyte culture aid (HMGS). Human melanoma cells (G361) were purchased from the Japanese Collection of Research Bioresources and cultured in high-glucose medium. The cultured cells were incubated in a CO2 incubator at 37°C with a CO2 concentration of 5%.

[0042] <Method for counting dendrites> Human normal skin pigment cells (HEMn-MP) derived from neonatal foreskin were placed in a 6-well cell culture plate in a 6x10⁶ format. 5Cells were seeded at a rate of one cell per well. After 24 hours, staurosporine was dissolved in solvent (DMSO) and added to a final concentration of 300 nM. The cells were then cultured in an incubator for 24 hours. For the control, DMSO containing dissolved staurosporine was added. Subsequently, the cultured cells were washed with ice-cold PBS (Phosphate Buffered Saline) and fixed by exposure to 4% paraformaldehyde for 5 minutes. The nuclei of the fixed cultured cells were stained with Hoechst 33342 (Invitrogen, 500-fold dilution) and imaged using a confocal microscope Biozero 8100 (Keyence). The obtained images were analyzed using open-source software (ImageJ, see http: / / rsb.info.nih.gov / ij / index.html) to count the number of dendrites.

[0043] <Evaluation of pigment-related protein expression> Proteins in neonatal foreskin-derived human normal skin pigment cells (HEMn-MP) were extracted from cells based on the method described in the following literature (Yang, F., Yang, L., Wataya-Kaneda, M., Yoshimura, T., Tanemura, A., & Katayama, I. (2018). Uncoupling of ER / Mitochondrial Oxidative Stress in mTORC1 Hyperactivation-Associated Skin Hypopigmentation. J Invest Dermatol, 138(3), 669-678. doi:10.1016 / j.jid.2017.10.007). Protein expression was evaluated by reacting the sample with monoclonal antibodies against each protein, and detecting the remaining amount of monoclonal antibody after the reaction using Western blotting. The manufacturers and dilutions of each monoclonal antibody are as follows. anti-TYRP1 (manufactured by Santa Cruz Biotechnology) was diluted with solvent (PBS / 10% FBS) to a ratio of 1:500. The same procedure was followed for subsequent dilutions. anti-GPNMB (made by Sigma-Aldrich) at a 1:1000 scale anti-PMEL (manufactured by Santa Cruz Biotechnology) at a ratio of 1:500 anti-MelanA (manufactured by Abcam-Aldrich) at a scale of 1:1000 anti-ENDRB (manufactured by Cell Signaling Technology (CST)) at 1:1000 Anti-GAPDH antibody (CST, 1:1000) was used as a control for evaluation. 5 μg of protein extracted from cultured cells was taken and mixed with the monoclonal antibody solution (1000 μL). Western blotting was performed using 15 μL of the supernatant after mixing.

[0044] <Method for evaluating cell viability> Place the target cells (HEMn-MP or G361) in a 96-well cell culture plate in a 2.5x10⁶ format. 4 The cells were placed at a density of [number] cells / well, and each well was treated in an incubator for 24 hours under different test conditions (staurosporin concentration). Afterward, the cells were washed three times with ice-cold PBS. Cell viability was measured using the Cell Count Reagent SF colorimetric assay kit (Nacalai Tesque Co., Ltd.). Specifically, 10 μL of cell counting reagent (Cell Count Reagent SF) was added to each well, and the plate was left in a 37°C incubator for 2 hours. Then, the OD450 value was measured using a microplate reader (Medel 550, Bio-Rad Laboratory), and cell viability was quantified by the change in color. Cell viability was defined as follows: Cell viability (%) = OD450 value of test cells / OD450 value of control cells × 100 <Results and discussion of tests using cultured cells>

[0045] As shown in Figure 1, human normal skin pigment cells (HEMn-MPs) derived from neonatal foreskin stimulated with 300 nM staurosporine showed a significant increase in the number of dendrites after 24 hours compared to the control (solvent). As shown in Figure 2, when the expression of pigment-related proteins was evaluated, staurosporine-stimulated neonatal foreskin-derived human normal skin pigment cells (HEMn-MP) showed enhanced expression of pigment-related proteins such as PMEL (premelanosome protein) and ENDRB (endothelin-B receptor) compared to the control (solvent). As shown in Figure 3, in HEMn-MP stimulated with staurosporine, survival rates did not decrease at any dose, whereas in G361, survival rates decreased in a dose-dependent manner with staurosporine, and a particularly large decrease in survival rates occurred at doses of 1000 nM or higher. These experimental results confirmed that staurosporine increases dendrites and activates HEMn-MP, while exhibiting cytotoxicity against G361. It is known that staurosporine's cytotoxicity against cancer cells is due to the induction of apoptosis, and these experimental results confirm that finding. Furthermore, similar tests were conducted using midostaurin (manufactured by Sigma-Aldrich), which is synthesized from staurosporine. However, in HEMn-MP, only a slight increase in dendrites was observed at concentrations above 1000 nM, and in G361, the ability to induce cell death was considerably lower than that of staurosporine (data not shown). Based on these findings, it is considered that there is no need to further investigate midostaurin or other similar compounds at this time, and that the best course of action is to develop a topical administration composition by optimizing the concentration and base of staurosporine.

[0046] <Animal-based testing> <How to obtain and care for animals> A 6-week-old male black guinea pig (KWL:JY-4) was purchased from Tokyo Laboratory Animals Co., Ltd. and raised in a pathogen-free environment. <Pigmentation-enhancing reaction in normal skin> The hair on the back of experimental guinea pigs (KWL:JY-4) was shaved, and areas #1 to #4 (1cm x 2cm) were marked on the shaved skin. The lotion prepared in <Preparation of Topical Administration Composition 1> was applied to each area. Specifically, 50 μL of lotion containing Vehicle was applied to area #1, 250 μM staurosporine to area #2, 500 μM staurosporine to area #3, and 1000 μM staurosporine to area #4, once a day. Application was continued for 14 days, and the skin color was observed by dermoscopy after 14 days. The lightness (L value), an indicator of skin brightness, was compared between the areas. <Therapeutic effects in animal models of pigment-depleting skin diseases> A 30 wt% rhododenol solution was prepared by dissolving rhododenol (manufactured by Kanebo Cosmetics Inc.) in a solution of reagent-grade ethanol and sesame oil in a 1:1 ratio. The hair on the back of a guinea pig (KWL:JY-4) was shaved, and three areas (2cm x 2cm) were marked on the shaved skin. The 1:1 mixture of reagent-grade ethanol and sesame oil was applied to area #1, and the 30 wt% rhododenol solution was applied to areas #2 and #3 at a rate of 50 μL once daily for one month. This artificially suppressed the melanin pigment synthesis function of melanocytes, creating an animal model of vitiligo (see J Toxicol Sci. 2014;39:615-23). Subsequently, the Vehicle formulation of <Preparation of Topical Administration Composition 1> was applied once daily for 14 days to areas #1 and #2, and the 1000 μM staurosporine-containing lotion formulation of <Preparation of Topical Administration Composition 1> was applied once daily for 14 days to area #3. After 14 days, skin color was observed using dermoscopy.

[0047] <Results and discussion of animal studies> When using guinea pigs with normal skin, it was confirmed that the skin color darkened in a concentration-dependent manner with staurosporine, as shown in Figure 4. Furthermore, tissue samples were taken from the observed area and stained, but no development of malignant melanoma was observed. On the other hand, when using an animal model of vitiligo, as shown in Figure 5, depigmentation was observed in areas #2 and #3, which were treated with rhododenol, compared to area #1. When only Vehicle was applied to the depigmented area #2, not much pigment regeneration was observed, but when staurosporine-containing lotion was applied to area #3, regeneration of black pigment was confirmed. Tissue samples were taken from the observed areas and stained, but no development of malignant melanoma was confirmed. In the animal model described above, when the melanin pigment synthesis function of melanocytes was suppressed with rhododenol, the pigment naturally regenerated about two months after discontinuation of the drug. However, when the topical administration composition of the present invention was used, pigment regeneration was observed to be much faster and more intense than natural regeneration. The results of the above tests showed that staurosporine enhanced black pigmentation in both healthy animals and animals in which pigment-depleting skin disease had been artificially induced. Furthermore, it did not induce abnormal cells such as melanoma. This demonstrated a certain degree of effectiveness in treating pigment-depleting skin disease. [Industrial applicability]

[0048] This could potentially provide novel treatments for pigmentary disorders, particularly vitiligo.

Claims

1. A topical composition for the treatment of hyperpopigmentation, containing staurosporine or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The topical administration composition according to claim 1, which is a composition for skin administration.

3. The topical composition according to claim 1, wherein the pigment-decreasing skin disease is vitiligo.

4. A topical administration composition according to any one of claims 1 to 3, comprising 20% ​​by weight or more of vegetable oil and 0.005% to 0.1% by weight of an active ingredient.

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