Drug used for treating hyperpigmentation

Abstract

The present invention relates to the use of annexin A5. Specifically, annexin A5 can be used to prepare a composition, the composition being used for (a) preventing and / or treating skin hyperpigmentation; (b) whitening skin; and / or (c) delaying the progression of melanoma. The annexin A5 can significantly inhibit the synthesis of melanin, and inhibit the synthesis of melanin in melanoma cells, and thus has excellent effects of whitening skin and reducing continuous deposition of pigments in melanoma cells.

Description

A drug for treating pigmentation Technical Field

[0001] The present invention relates to the field of medicines, and in particular to a medicine for treating pigmentation. Background Art

[0002] Skin hyperpigmentation is a common, multi-faceted skin condition that includes post-inflammatory hyperpigmentation, chloasma, age spots, and freckles, primarily manifesting as a darkening of localized patches or spots. These changes in skin color are the result of a combination of internal and external factors, including hormonal changes, inflammation, trauma, skin diseases, medications, and ultraviolet radiation. Its pathological nature is an excess of melanin in the skin layer at the site of the lesion, associated with an increase in total melanin production or a slowed metabolism of melanin granules in the skin layer. Melanin is primarily produced by melanocytes in various layers of the skin. Therefore, changes in the production or distribution of melanocytes can lead to skin hyperpigmentation.

[0003] Although hyperpigmentation is not a life-threatening condition, it significantly impacts patients' quality of life by impairing their psychological well-being. Currently, there are two main clinical approaches to improving skin pigmentation: medication and phototherapy. Medication is further categorized into topical medications, oral medications, and chemical peels. The primary therapeutic mechanisms are inhibition of tyrosinase synthesis, interference with melanin synthesis, removal of damaged skin, and promotion of skin cell regeneration. Hydroquinone (also known as hydroquinone), retinoic acid, and glucocorticoids are three commonly used topical medications; tranexamic acid, glutathione, and polylysine are currently the most commonly used in clinical practice. Despite the wide variety of medications available, their efficacy is uncertain and varies significantly between individuals. Furthermore, medications can also cause various side effects, such as dryness, peeling, or hypopigmentation, which can prolong treatment and lead to poor patient compliance. Chemical peels are also widely used in clinical practice for treating post-inflammatory hyperpigmentation and photochromic spots, but recurrence is common. While photoelectric therapy has achieved good results, it is associated with significant pain during treatment and post-operative side effects (such as redness, heat, swelling, or scab formation at the treated area), which can result in prolonged downtime. Because all traditional therapies have their limitations, finding potential, safer, and more effective treatment options for currently incurable skin pigmentation is a key area of ​​modern medicine's future development.

[0004] Therefore, there is a need in the art to develop a drug with high safety and low side effects that can effectively treat pigmentation. Summary of the Invention

[0005] The purpose of the present invention is to provide a use of annexin A5 in preparing a composition for (a) preventing and / or treating skin pigmentation, (b) whitening skin color, and / or (c) delaying the progression of melanoma.

[0006] In a first aspect of the present invention, there is provided a use of annexin A5 in preparing a composition for one or more of the following purposes: (a) preventing and / or treating skin pigmentation; (b) whitening skin color; and / or (c) delaying the progression of melanoma.

[0007] In another preferred embodiment, the weight fraction of Annexin A5 in the composition is 1-99 wt % of the total weight of the composition, such as 80%, 85%, 90%, 95%, 98% or 99%.

[0008] In another preferred embodiment, the skin pigmentation is selected from the group consisting of nevus of Ota, café au lait spots, post-inflammatory pigmentation, chloasma, age spots, freckles, sun spots, and pigmentation in melanoma (pigmented nevus with malignant changes).

[0009] In another preferred embodiment, the skin pigmentation includes primary skin pigmentation or secondary skin pigmentation.

[0010] In another preferred embodiment, the skin pigmentation is skin pigmentation caused by one or more factors selected from the group consisting of: hormonal changes, inflammation, trauma, skin diseases, drugs, and ultraviolet radiation.

[0011] In another preferred embodiment, the skin pigmentation caused by hormone changes includes skin pigmentation caused by Addison's disease, pregnancy or oral contraceptives.

[0012] In another preferred embodiment, the skin pigmentation caused by inflammation includes skin pigmentation caused by inflammatory skin diseases.

[0013] In another preferred embodiment, the inflammatory skin disease includes lichen planus and / or lichen planus-like drug eruption.

[0014] In another preferred embodiment, the drug-induced skin pigmentation includes skin pigmentation caused by one or more of amiodarone, tetracycline, minocycline, bleomycin, cyclophosphamide, chloroquine, quinine, chlorpromazine and other phenothiazine drugs.

[0015] In another preferred embodiment, the skin pigmentation is the pigmentation produced by the human body itself.

[0016] In another preferred embodiment, the skin pigmentation does not include hemochromatosis.

[0017] In another preferred embodiment, the prevention and / or treatment of skin pigmentation includes one or more features selected from the following group:

[0018] (i) inhibiting the production of melanin in melanocytes;

[0019] (ii) Whitening and / or brightening skin tone.

[0020] In another preferred embodiment, the melanocytes are normal melanocytes or melanoma cells.

[0021] In another preferred embodiment, the delaying of melanoma progression includes delaying melanoma progression by inhibiting melanin synthesis in melanoma cells and reducing the continuous deposition of melanin in melanoma cells.

[0022] In another preferred embodiment, the delaying of melanoma progression does not include inhibiting melanoma cell proliferation.

[0023] In another preferred embodiment, the delaying of melanoma progression includes inhibiting melanin synthesis in melanoma cells and / or reducing the continuous deposition of melanin in melanoma cells.

[0024] In another preferred embodiment, the annexin A5 is annexin A5 from humans or non-human mammals.

[0025] In another preferred embodiment, the non-human mammal includes pigs, monkeys, cows, sheep, rats, mice or rabbits.

[0026] In another preferred embodiment, the composition is a pharmaceutical composition, a cosmetic composition and / or a medical cosmetic composition.

[0027] In another preferred embodiment, the composition further comprises a carrier acceptable in pharmaceuticals, cosmetics or medical cosmetology.

[0028] In another preferred embodiment, the composition further comprises other drugs that can be used to prevent and / or treat skin pigmentation and / or whiten skin color.

[0029] In another preferred embodiment, the other drugs that can be used to prevent and / or treat skin pigmentation and / or whiten skin color are selected from the following group: hydroquinone, retinoic acid, azelaic acid, kojic acid, glycolic acid, arbutin, or a combination thereof.

[0030] In another preferred embodiment, the composition is in the form of a liquid dosage form, a semisolid dosage form or a solid dosage form.

[0031] In another preferred embodiment, the composition is an oral preparation, an external preparation or an injection preparation.

[0032] In another preferred embodiment, the injection preparation is an intravenous injection preparation, an intramuscular injection preparation, a subcutaneous injection preparation or an intraperitoneal injection preparation.

[0033] In another preferred embodiment, the composition is administered orally, externally or by injection, preferably by topical application or local injection.

[0034] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: tablets, lozenges, powders, granules, oral liquids, capsules, microcapsules, powder injections, and injections.

[0035] In another preferred embodiment, the dosage form of the cosmetic composition is selected from the group consisting of solution, gel, cream, lotion, ointment, cream, paste, powder, and patch.

[0036] In another preferred embodiment, the dosage form of the medical cosmetic composition is selected from the following group: microcapsules, powder injections, freeze-dried powders, and injections.

[0037] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0038] (a) Annexin A5; (b) a pharmaceutically acceptable carrier; and optionally (c) other drugs that can be used to prevent and / or treat skin pigmentation and / or whiten skin color.

[0039] In another preferred embodiment, the other drugs that can be used to prevent and / or treat skin pigmentation and / or whiten skin color are selected from the following group: hydroquinone, retinoic acid, azelaic acid, kojic acid, glycolic acid, arbutin, or a combination thereof.

[0040] In another preferred embodiment, the dosage form of the pharmaceutical composition is a liquid dosage form, a semisolid dosage form or a solid dosage form.

[0041] In another preferred embodiment, the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

[0042] In another preferred embodiment, the injection preparation is an intravenous injection preparation, an intramuscular injection preparation, a subcutaneous injection preparation or an intraperitoneal injection preparation.

[0043] In another preferred embodiment, the pharmaceutical composition is administered orally, externally or by injection, preferably by topical application or local injection.

[0044] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: tablets, lozenges, powders, granules, oral liquids, capsules, microcapsules, powder injections, and injections.

[0045] In a third aspect of the present invention, a method for inhibiting melanin synthesis in vitro is provided, comprising the steps of contacting annexin A5 with cells capable of producing melanin, thereby inhibiting melanin synthesis.

[0046] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0047] In another preferred embodiment, the cells capable of producing melanin include normal melanocytes and / or abnormal melanoma cells.

[0048] In another preferred embodiment, the concentration of Annexin A5 is 100 μg / mL or more, preferably 200 μg / mL or more.

[0049] In the fourth aspect of the present invention, a method for (a) preventing and / or treating skin pigmentation; (b) whitening skin color; and / or (c) delaying the progression of melanoma is provided, comprising the steps of administering annexin A5 or a pharmaceutical composition containing annexin A5 as described in the second aspect of the present invention to a subject in need thereof.

[0050] In another preferred embodiment, the subject is a human or non-human mammal or fish, such as a pig, monkey, cow, sheep, rat, mouse, rabbit or zebrafish.

[0051] In another preferred embodiment, the administration concentration of Annexin A5 is 100 μg / mL or higher, preferably 200 μg / mL or higher.

[0052] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows a typical graph of melanin signal intensity in the zebrafish head after sample treatment.

[0054] Figure 2 shows the melanin signal intensity in zebrafish heads after sample treatment. **p<0.01, ***p<0.001 compared with the normal control group.

[0055] Figure 3 shows the proliferation capacity of B16 cells (CCK8) after sample treatment. Compared with the normal control group, there was no significant difference in ns.

[0056] Figure 4 shows the total amount of cytochrome in B16 cells after sample treatment. ***p<0.001 compared with the normal control group. DETAILED DESCRIPTION

[0057] After extensive and intensive research, screening, and testing, the present inventors have developed a drug for treating pigmentation. Through systematic animal and cell experiments, the present inventors have demonstrated that Annexin A5 exhibits significant effects in inhibiting melanin synthesis and regulating tyrosine kinase activity, particularly in inhibiting pigment synthesis and the synthesis of pigments in melanoma cells. Therefore, Annexin A5 is expected to be an effective treatment for whitening skin, treating pigmentation, and treating melanoma. This is the basis for the present invention.

[0058] the term

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0061] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0062] As used herein, "promoting," "enhancing," or "increasing" an indicator means that the indicator is increased by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80% after the use of Annexin A5 compared to the absence of Annexin A5.

[0063] As used herein, "inhibition," "reduction," or "reduction" of an indicator means that the indicator is reduced by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80% after the use of Annexin A5, compared to the absence of Annexin A5.

[0064] As used herein, the term "prevention" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease. "Prevention" as used herein also includes delaying the onset of a disease and / or its attendant symptoms and reducing the risk of a subject acquiring a disease.

[0065] As used herein, the term "treating" includes delaying and stopping the progression of a disease, or eliminating the disease, and does not require 100% inhibition, eradication, and reversal. In some embodiments, the use of Annexin A5 reduces, inhibits, and / or reverses skin pigmentation, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the absence of Annexin A5. In some embodiments, the use of Annexin A5 delays melanoma progression, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the absence of Annexin A5.

[0066] As used herein, the term "treating pigmentation" may include alleviating the progression of the condition, reducing the synthesis of pigments.

[0067] Annexin A5

[0068] Annexins (abbreviated as Anx) are sensors of calcium ions in eukaryotic cells and can reversibly bind to membrane phospholipids under conditions of calcium ion activation. The annexin family is diverse, with a total of 12 different annexin genes (ANXA1-ANXA11 and ANXA13), which are scattered throughout the human genome (chromosomes 1, 2, 4, 5, 8-10 and 15). The annexin family has a unique -COOH core structure composed of 4 highly homologous annexin repeat sequences, each of which contains multiple 5α helical structures. When connected to short loops, they form a slightly curved plane. Annexin and Ca 2+ Annexins bind to the convex surface of the membrane and undergo conformational changes, enabling the hydrophilic sites in the center of the repeats to bind to negatively charged phospholipids on the surrounding cell membrane, participating in the repair and destruction of the cell membrane. Studies have shown that annexins play an important role in various steps of the membrane repair system, mediated by mechanisms such as exocytosis (such as ANXA2), endocytosis (such as ANXA1, 2, 6, and 8), and microparticle shedding (such as ANXA1, 6, and 7).

[0069] AnxA5 is currently mainly used as a reagent for detecting cell apoptosis. In vitro studies have found that although it has multiple functions such as anti-inflammation, promoting fibrinolysis, and anti-thrombosis, its function in the pigment synthesis process is not very clear.

[0070] The annexin that can be used for (a) preventing and / or treating skin pigmentation, (b) whitening skin color, and / or (c) delaying melanoma progression in the present invention is not particularly limited and can be any of ANXA1-ANXA11 and ANXA13, preferably annexin A5.

[0071] Annexin A5 that can be used in the present invention is not particularly limited and can be Annexin A5 derived from any organism, preferably from a mammal (such as a primate), more preferably from a human. In addition, it should be understood that "Annexin A5" includes wild-type or mutant (including truncated) Annexin A5, as long as the mutant Annexin A5 retains or maintains the detoxification activity of wild-type Annexin A5.

[0072] use

[0073] The present invention provides the use of annexin A5 in preparing a composition for (a) preventing and / or treating skin pigmentation, (b) whitening skin color, and / or (c) delaying the progression of melanoma. In particular, the composition has a significant effect in reducing melanin synthesis and whitening skin.

[0074] Skin pigmentation is a common, multi-faceted skin condition that includes post-inflammatory hyperpigmentation, melasma, age spots, and freckles. It manifests primarily as a darkening of localized patches or spots. These skin color changes are the result of a combination of internal and external factors, including hormonal changes, inflammation, trauma, skin diseases, medications, and ultraviolet radiation. Its pathological essence is an excess of melanin in the skin at the site of the lesion, coupled with an increase in total melanin production.

[0075] The present invention discloses that annexin A5 has the effect of inhibiting pigment synthesis. In the present invention, annexin A5 can inhibit the total amount of pigment in the head of zebrafish juveniles and can inhibit the synthesis of melanin in mouse melanoma cells, thereby having a whitening effect.

[0076] Composition and administration

[0077] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, cosmetic compositions and / or medical cosmetic compositions.

[0078] The composition of the present invention may also include a carrier that is acceptable in pharmaceuticals, cosmetics or medical cosmetology. "Acceptable carrier in pharmaceuticals, cosmetics or medical cosmetology" refers to: one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of carriers that are acceptable in pharmaceuticals, cosmetics or medical cosmetology include cellulose and its derivatives, gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, dispersants, moisturizers, UV inhibitors, film-forming agents, oil-soluble gelling agents, organically modified clay minerals, antibacterial agents, flavors, salts, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, rough skin improving agents, blood circulation promoters, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, etc.

[0079] The oil-soluble gelling agent is a gelling agent selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid and α,γ-di-n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, and cyclodextrin 2-ethylhexanoate palmitate; sucrose fatty acid esters such as sucrose palmitate and sucrose stearate; benzyl derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol; organically modified clay minerals such as dimethylbenzyldodecyl ammonium montmorillonite clay and dimethyldioctadecyl ammonium montmorillonite clay, and the like. One or two or more kinds of gelling agents can be used as needed.

[0080] Moisturizers include: glycerin, sorbitol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, etc. Antimicrobial preservatives include: alkyl parahydroxybenzoates, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc. Antimicrobial agents include: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl parahydroxybenzoates, para-chloro-metacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-N-carbanilide, triclosan, photosensitizer, phenoxyethanol, etc.

[0081] Antioxidants include tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and phytic acid. pH adjusters include lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, and ammonium bicarbonate. Chelating agents include alanine, sodium EDTA, sodium polyphosphate, sodium metaphosphate, and phosphoric acid. Cooling agents include L-menthol and camphor. Anti-inflammatory agents include allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, and azulene.

[0082] Skin beautification ingredients include: whitening agents such as placenta extract, arbutin, glutathione, and saxifrage extract; cell activators such as royal jelly, photosensitizer, cholesterol derivatives, and calf blood extract; skin roughness improvers; blood circulation promoters such as valeramide nonanoate, benzyl nicotinate, β-butoxyethyl nicotinate, capsaicin, gingerol, blister beetle tincture, ichthyol, caffeine, tannic acid, α-borneol, tocopheryl nicotinate, inositol hexanicotinate, cyclomandelate, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthin, and γ-oryzanol; skin astringents such as zinc oxide and tannic acid; anti-seborrheic acid agents such as sulfur, etc. Vitamins include: vitamin A oil, rosin oil, rosin oil acetate, rosin oil palmitate and other vitamin A; riboflavin, riboflavin butyrate, flavin adenine nucleotide and other vitamin B2; pyridoxine hydrochloride, pyridoxine dioctanoate, pyridoxine tripalmitate and other vitamin B6; vitamin B12 and its derivatives, vitamin B15 and its derivatives and other vitamin B; L-ascorbic acid, L-ascorbic acid dipalmitate, L-ascorbic acid-2-sulfate sodium, Vitamin C such as dipotassium L-ascorbyl diester phosphate; vitamin D such as ergocalciferol and cholecalciferol; vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, dl-α-tocopheryl acetate, dl-α-tocopheryl nicotinate, and dl-α-tocopheryl succinate; vitamin H; vitamin P; niacin such as niacin, benzyl nicotinate, and niacinamide; pantothenic acids such as calcium pantothenate, D-panthenol, pantothenyl ethyl ether, and acetyl pantothenyl ethyl ether; biotin, etc.

[0083] Amino acids include: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc.

[0084] The pharmaceutical composition of the present invention comprises the above-mentioned annexin as an active ingredient and a pharmaceutically acceptable carrier.

[0085] Typically, Annexin A5 can be prepared into pharmaceutical compositions in the form of tablets, capsules, powders, microgranules, solutions, lozenges, injections, elixirs, suspensions, tinctures, poultices, liniments, lotions, and aerosols. Pharmaceutical compositions can be prepared using commonly known preparation techniques, and suitable pharmaceutical additives can be added to the composition.

[0086] There are no particular limitations on the administration of the composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral (intravenous, intramuscular, intraperitoneal, subcutaneous) injection, and topical administration. The composition of the present invention is in the form of an oral preparation, a topical preparation, or an injection preparation, preferably a topical injection preparation or a topical topical preparation, and more preferably a topical subcutaneous injection preparation.

[0087] Typically, solid dosage forms for oral administration or delivery include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.

[0088] Capsules, tablets, and pills may also contain buffering agents. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents.

[0089] Liquid dosage forms for oral administration or administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, and flavorings. In addition to the active ingredient, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0090] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0091] Dosage forms for topical or topical administration of the composition of the present invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0092] The active ingredient of the present invention can be used alone or in combination with one or more other drugs for pigmentation-related diseases, such as hydroquinone (also known as hydroquinone), tretinoin, glucocorticoids, tranexamic acid, glutathione and polylysine.

[0093] When administering the composition, a safe and effective amount of the acellular fat extract of the present invention is applied to a human or non-human animal (such as rats, mice, dogs, cats, cattle, sheep, chickens, ducks, etc.) in need of treatment, wherein the dosage during administration is an effective dosage that is acceptable in pharmaceuticals, food, or health products. As used herein, the term "safe and effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art will appreciate that the "safe and effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drugs used, the severity of the disease, and other drug combinations. For example, for a person weighing 60 kg, the daily dosage is typically 0.1-1000 mg, preferably 1-600 mg, and more preferably 2-300 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of skilled physicians.

[0094] The main advantages of the present invention include:

[0095] (1) The present invention discloses for the first time the use of annexin A5 in (a) preventing and / or treating skin pigmentation, (b) whitening skin color, and / or (c) delaying the progression of melanoma.

[0096] (2) The present invention discloses that annexin A5 has the effect of inhibiting pigment synthesis.

[0097] (3) The present invention discovered for the first time that annexin A5 can inhibit the synthesis of head pigment in zebrafish larvae and inhibit the synthesis of melanin in mouse melanoma cells.

[0098] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0099] Annexin A5: extracted and purified from human adipose tissue, accession number: P08758:

[0100] Example 1

[0101] 1. Experimental Animals

[0102] Wild-type AB strain zebrafish were bred in natural pair mating. Zebrafish 6 hours after fertilization (6hpf) were used to evaluate the whitening efficacy of the samples. Zebrafish were raised in 28°C fish water (water quality: 200mg of instant sea salt was added to each 1L of reverse osmosis water, the conductivity was 450-550μS / cm; pH was 6.5-8.5; hardness was 50-100mg / L CaCO3). The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2023-7386-01.

[0103] 2. Experimental methods:

[0104] 6hpf wild-type AB strain zebrafish were randomly selected and plated in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (concentrations shown in Tables 1-1, 1-2, and 1-3) were administered, along with a positive control of arbutin (lot number L2208327, Shanghai Aladdin Biochemical Technology Co., Ltd.) at a concentration of 3000 μg / mL. A normal control group was also established, with a volume of 3 mL per well. After two days of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope (SZX7, OLYMPUS, Japan). Data were analyzed and collected using advanced image processing software. The intensity of melanin signal in the zebrafish heads was analyzed, and the whitening efficacy of the samples was evaluated using statistical analysis of this indicator. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated statistical significance.

[0105] 3. Experimental Results

[0106] Table 1. Sample whitening efficacy evaluation test results (n=10)

[0107] The experimental results in Figures 1-2 and Table 1 show that Annexin A5 has a significant inhibitory effect on pigment synthesis in the head of zebrafish larvae (***p<0.001), and has a significant whitening effect. The difference is statistically significant. At similar concentrations, the whitening effect of Annexin A5 is equivalent to that of the positive control group (arbutin).

[0108] Example 2

[0109] 1. Effect of Annexin A5 on the Proliferation of B16 Cells

[0110] After B16 cells were digested, a cell suspension was prepared and plated at 2,000 cells per well in a 96-well plate. The plate was then incubated at 37°C for 24 hours. The next day, the 96-well plate cells were treated with 1,000 ng / ml of annexin A5 or PBS, with three replicates per group, and culture continued for 72 hours. After 72 hours of drug incubation, the 96-well plate was removed, and 10 μl of CCK8 solution was added to each well of cells. The cells were mixed and incubated at 37°C for 1 hour. The cells were then assayed using a microplate reader, and the proliferation capacity of each group of cells was calculated. The results are shown in Figure 3.

[0111] 2. Effect of Annexin A5 on Pigment Synthesis in B16 Cells

[0112] Melanoma B16 cells were washed twice with PBS and then digested with 0.25% trypsin (Gibco). 6.0×10 5 Cells were centrifuged at 4000 x g for 5 minutes, and the pellet was washed twice with PBS. The cell pellet was then dissolved in 120 μl of 1 M NaOH and 10% dimethyl sulfoxide (DMSO). The cell lysate was incubated at 80°C for 2 hours. Subsequently, 100 μl of the lysate was placed in a 96-well plate, with three replicates set up for each concentration group. The absorbance at a wavelength of 400 nm was measured on a microplate reader. Melanin formation rate = A experimental group / A blank group average × 100%. The results are shown in Figure 4.

[0113] The experimental results showed that annexin A5 could not significantly promote or inhibit the proliferation of B16 melanoma cells; however, annexin A5 could significantly reduce the total amount of pigment synthesis in B16 cells, and had the effect of whitening and delaying the progression of melanin.

[0114] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Use of annexin A5 in the preparation of a composition for one or more of the following uses: (a) preventing and / or treating skin pigmentation; (b) whitening skin tone; and / or (c) delaying melanoma progression.

2. The use according to claim 1, characterized in that, The skin pigmentation is selected from the group consisting of: nevus of Ota, café-au-lait spots, post-inflammatory hyperpigmentation, melasma, senile lentigines, freckles, solar lentigines, and pigmentation in melanoma (pigmented nevus with malignant changes).

3. The use according to claim 1, wherein The skin pigmentation is skin pigmentation caused by one or more factors selected from the group consisting of: hormonal changes, inflammation, trauma, skin diseases, drugs, and ultraviolet irradiation.

4. The use according to claim 1, characterized in that, The skin pigmentation is the deposition of pigments produced by the human body itself.

5. The use according to claim 1, characterized in that, The prevention and / or treatment of skin pigmentation includes one or more characteristics selected from the group consisting of: (i) inhibiting the production of melanin in melanocytes; (ii) whitening and / or brightening skin tone.

6. The use according to claim 1, characterized in that, The delaying of melanoma progression includes delaying melanoma progression by inhibiting melanin synthesis in melanoma cells and reducing the continuous deposition of melanin in melanoma cells.

7. The use according to claim 1, characterized in that, The composition is a pharmaceutical composition, a cosmetic composition, and / or a medical aesthetic composition.

8. The use according to claim 1, characterized in that, The dosage form of the composition is a liquid dosage form, a semi-solid dosage form, or a solid dosage form.

9. The use according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is selected from the group consisting of: tablets, lozenges, powders, granules, oral liquids, capsules, microcapsules, powder injections, injections; The dosage form of the cosmetic composition is selected from the group consisting of: solutions, gels, creams, lotions, ointments, creams, pastes, powders, patches; The dosage form of the medical aesthetic composition is selected from the group consisting of: microcapsules, powder injections, lyophilized powders, injections.

10. A method for inhibiting melanin synthesis in vitro, characterized in that, It includes the step of contacting annexin A5 with cells capable of producing melanin to thereby inhibit melanin synthesis.

Images