A drug for treating pigmentation

Abstract

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

Description

Technical Field

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

[0002] Skin hyperpigmentation is a common multiple skin disease, including post-inflammatory pigmentation, chloasma, age spots, and freckles, which are mainly manifested by the deepening of the color of local skin patches or spots. This kind of skin color change is the result of the combined effects of many internal and external factors of the subject, and its inducements include hormonal changes, inflammation, trauma, skin diseases, drugs, ultraviolet radiation, etc. Its pathological essence is that there is too much melanin in the skin layer where the lesion is located, which is related to the increase in the total amount of melanin production or the slow metabolism of melanin particles in the skin layer. Melanin is mainly produced by melanocytes in various layers of the skin. Therefore, changes in the production or distribution of melanocytes can lead to skin pigmentation.

[0003] Although pigmentation is not a fatal disease, it greatly affects the patient's quality of life by damaging the patient's mental health. At present, there are two main methods for improving skin melanin in clinical practice, namely drug therapy and phototherapy. Drug therapy is divided into topical drugs, oral drugs and chemical peeling. Among them, inhibiting tyrosinase synthesis, interfering with melanin synthesis, removing damaged skin and promoting skin cell regeneration are the main treatment mechanisms. Hydroquinone (also known as hydroquinone), retinoic acid and glucocorticoids are three commonly used topical drugs; tranexamic acid, glutathione and polylysine are currently used more clinically. Although there are many types of drug therapy, the efficacy is not certain and there are large individual differences. In addition, drug therapy can also produce various side effects, such as dry skin, peeling or hypopigmentation, which leads to prolonged treatment time and poor patient compliance. Chemical peeling has also been widely used in clinical practice to treat post-inflammatory pigmentation, light brown spots, etc., but there are many recurrences after treatment. Although photoelectric therapy has achieved good results, it is very painful during the treatment process, and postoperative reactions (redness, heat, edema, or scabs at the treatment site, etc.) may cause patients to have a long downtime after treatment. Because all traditional therapies have their limitations in treatment, finding potential, safer, and more effective treatment options for skin pigmentation that is currently difficult to cure clinically is the future development direction of modern medicine.

[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 the preparation of 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, wherein the composition is used 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 1-99wt% 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 comprises one or more features selected from the following group:

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

[0019] (ii) Whitening and / or brightening the 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 of human 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, an intramuscular injection, a subcutaneous injection or an intraperitoneal injection.

[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 following group: solution, gel, cream, lotion, ointment, cream, paste, powder, patch.

[0036] In another preferred embodiment, the dosage form of the medical cosmetic composition is selected from the following group: microcapsules, powder injections, lyophilized 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, an intramuscular injection, a subcutaneous injection or an intraperitoneal injection.

[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 more, preferably 200 μg / mL or more.

[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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A typical plot of melanin signal intensity in the zebrafish head after sample processing is shown.

[0054] Figure 2 The melanin signal intensity of zebrafish head after sample treatment is shown. Compared with the normal control group, **p<0.01, ***p<0.001.

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

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

[0057] The inventors have conducted extensive and in-depth research, and through a large number of screenings and tests, provided a drug for pigmentation. The present invention has confirmed through systematic animal experiments and cell experiments that annexin A5 has significant effects in inhibiting the synthesis of melanin, regulating the activity of tyrosine kinase, and especially inhibiting pigment synthesis, and inhibiting the synthesis of melanoma cell pigments. Therefore, annexin A5 is expected to become an effective means for whitening skin color, treating pigmentation, and treating melanoma. The present invention was completed on this basis.

[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 and 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 numerical 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" a certain 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 a certain 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 getting a disease.

[0065] As used herein, the term "treatment" includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, compared with the absence of Annexin A5, 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%. In some embodiments, compared with the absence of Annexin A5, the use of Annexin A5 delays the progression of melanoma, for example, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%.

[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, consisting of 4 highly homologous annexin repeat sequences, each of which contains multiple 5α helical structures, which form a slightly curved plane when connected to a short loop. Annexin and Ca 2+ It binds to its convex surface and undergoes conformational changes, allowing the hydrophilic site in the center of its repeat sequence to bind to the negatively charged phospholipids on the surrounding cell membrane, participating in the damage and repair of the cell membrane. Studies have shown that annexin plays an important role in each step of the membrane repair system, mediated by exocytosis (such as ANXA2), endocytosis (such as ANXA1, 2, 6, 8), and particle shedding (such as ANXA1, 6, 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 the progression of melanoma in the present invention is not particularly limited, and can be any one 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 mammals (such as primates), and more preferably from humans. 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, it has a significant effect in reducing the synthesis of melanin and whitening.

[0074] Skin pigmentation is a common multiple skin disease, including post-inflammatory pigmentation, chloasma, age spots, freckles, and is mainly manifested by the deepening of the color of local skin patches or spots. This kind of skin color change is the result of the combined effects of many internal and external factors of the subject, and its causes include hormone changes, inflammation, trauma, skin diseases, drugs, ultraviolet radiation, etc. Its pathological essence is the excessive melanin in the skin layer where the lesion is located, and the increase in the total amount of 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 fry and can inhibit the synthesis of melanin in mouse melanoma cells, and has the effect of whitening.

[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 pharmaceutically, cosmetically, or medically acceptable carrier. "Pharmacologically, cosmetically, or medically acceptable carrier" 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 pharmaceutically, cosmetically, or medically acceptable carriers 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, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, dispersants, humectants, UV inhibitors, film-forming agents, oil-soluble gelling agents, organic modified clay minerals, antibacterial agents, flavors, salts, pH adjusters, 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; benzylidene sorbitol derivatives such as monobenzylidene sorbitol and dibenzylidene sorbitol; organic modified clay minerals such as dimethylbenzyl dodecyl ammonium montmorillonite clay and dimethyl dioctadecyl 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. Antibacterial preservatives include: alkyl parahydroxybenzoate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc. Antibacterial agents include: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl parahydroxybenzoate, parachlorometacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-N-carbanilide, triclosan, photosensitizer, phenoxyethanol, etc.

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

[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 valerianamide, benzyl nicotinate, β-butoxyethyl nicotinate, capsaicin, gingerol, blister beetle tincture, ichthyol, caffeine, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cycloamycin, 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, acetic acid rosin oil, palmitic acid rosin oil 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-tocopherol acetate, dl-α-tocopherol nicotinic acid, dl-α-tocopherol succinate; Vitamin H; Vitamin P; Nicotinic acid such as niacin, benzyl nicotinate, niacinamide; Pantothenic acid such as calcium pantothenate, D-panthenol, pantothenic acid ethyl ether, acetyl pantothenic acid 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 a pharmaceutical composition, such as a tablet, capsule, powder, microgranule, solution, lozenge, injection, elixir, suspension, tincture, poultice, liniment, lotion and aerosol. The pharmaceutical composition can be prepared by a generally known preparation technique, and a suitable pharmaceutical additive can be added to the drug.

[0086] There is no particular limitation on the administration of the composition of the present invention, and 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, an external preparation, or an injection preparation, preferably a local injection preparation or a local external preparation, and more preferably a local subcutaneous injection preparation.

[0087] Typically, solid dosage forms for oral administration or administration 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 starch 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 conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices. In addition to the active ingredient, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methylate and agar or mixtures of these substances.

[0090] Compositions for parenteral injection may include 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] The dosage forms of the composition of the present invention for topical application or administration 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 if necessary.

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

[0093] When applying the composition, the acellular fat extract of the present invention in a safe and effective amount is applied to the human or non-human animal (such as rat, mouse, dog, cat, cattle, sheep, chicken, duck, etc.) that needs to be treated, wherein the dosage during application is an effective dosage that can be accepted in medicine, food or health products. As used herein, the term "safe and effective amount" refers to an amount that produces function or activity to humans and / or animals and can be accepted by humans and / or animals. It should be understood by those of ordinary skill in the art that the "safe and effective amount" can vary with the form of the pharmaceutical composition, the route of administration, the adjuvant of the drug used, the severity of the disease, and the combination of other drugs. For example, for a person of 60 kg body weight, the daily dosage is usually 0.1-1000 mg, preferably 1-600 mg, and more preferably 2-300 mg. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, and these are within the skill range 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 pigments 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 only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[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 pairs. Zebrafish aged 6 hours after fertilization (6hpf) were used to evaluate the whitening efficacy of the samples. Zebrafish were raised in 28°C fish farming water (water quality: 200 mg of instant sea salt was added to every 1L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3), the experimental animal use license number was: SYXK (Zhejiang) 2022-0004, and the breeding management met the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethics review number was: IACUC-2023-7386-01.

[0103] 2. Experimental methods:

[0104] 6hpf wild-type AB strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Water-soluble samples (concentrations are shown in Tables 1-1, 1-2 and 1-3), positive control arbutin batch number L2208327, Shanghai Aladdin Biochemical Technology Co., Ltd., 3000μg / mL concentration, and a normal control group was set up at the same time, with a capacity of 3mL per well. After 2 days of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and placed under a dissecting microscope (SZX7, OLYMPUS, Japan) for photography, and data were analyzed and collected using advanced image processing software. The melanin signal intensity of the zebrafish head was analyzed, and the whitening effect of the sample was evaluated by the statistical analysis results of this indicator. The statistical analysis results are expressed as mean±SE. SPSS26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0105] 3. Experimental results

[0106]

[0107]

[0108] Table 1 Sample whitening efficacy evaluation test results (n = 10)

[0109] Figure 1-2 The experimental results in Table 1 show that annexin A5 has a significant inhibitory effect on the pigment synthesis of 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).

[0110] Example 2

[0111] 1. Effect of Annexin A5 on the proliferation of B16 cells

[0112] After the B16 cells were digested, a cell suspension was prepared and inoculated into a 96-well plate at a concentration of 2000 cells / well and cultured in a 37°C incubator for 24 hours. The next day, the 96-well plate cells were treated with 1000 ng / ml of annexin A5 or PBS, with 3 replicates per group, and culture continued for 72 hours. After 72 hours of drug incubation, the 96-well plate was removed, 10 ul of CCK8 solution was added to each well of cells, mixed and incubated in a 37°C incubator for 1 hour, then detected using an ELISA instrument and the proliferation capacity of each group of cells was calculated. The results are shown in Figure 3 shown.

[0113] 2. Effect of Annexin A5 on Pigment Synthesis in B16 Cells

[0114] Melanoma B16 cells were washed twice with PBS and then digested with 0.25% trypsin (Gibco). 6.0×10 5 Cells were centrifuged at 4000xg for 5 minutes, and the precipitate was washed twice with PBS. The cell pellet was then dissolved in 120μl of 1M NaOH and 10% dimethyl sulfoxide (DMSO). The cell lysate was incubated at 80°C for 2 hours. Subsequently, 100μl of the lysate was placed in a 96-well plate, and 3 replicate wells were set for each concentration group. The absorbance at a wavelength of 400nm was measured on an ELISA reader. The melanin formation rate = A experimental group / A blank group average value × 100%. The results are shown in Figure 4 shown.

[0115] 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.

[0116] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of annexin A5 in preparing a composition, wherein the composition is used for one or more of the following purposes: (a) preventing and / or treating skin melanin deposition; and / or (b) whitening skin color.

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 melanin deposition, chloasma, age spots, freckles, sun spots, and melanin deposition in melanoma.

3. The use according to claim 1, characterized in that The skin melanin pigmentation includes primary skin melanin pigmentation or secondary skin melanin pigmentation.

4. The use according to claim 1, characterized in that The skin melanin deposition is skin melanin deposition caused by one or more factors selected from the following group: hormonal changes, inflammation, trauma, skin diseases, drugs, and ultraviolet radiation.

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

6. The use according to claim 1, characterized in that The prevention and / or treatment of skin melanin pigmentation comprises one or more features selected from the following group: (i) Inhibit melanin production in melanocytes; (ii) Whitening and / or brightening the skin tone.

7. The use according to claim 6, characterized in that The melanocytes are normal melanocytes or melanoma cells.

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

9. The use according to claim 1, characterized in that The composition further comprises a carrier acceptable in pharmaceuticals, cosmetics or medical cosmetology.

10. The use according to claim 1, characterized in that The composition also includes other drugs that can be used to prevent and / or treat skin pigmentation and / or whiten skin color.

11. The use according to claim 10, characterized in that 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.

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

13. The use according to claim 1, characterized in that The composition is an oral preparation, an external preparation or an injection preparation.

14. The use according to claim 13, characterized in that The injection preparation is an intravenous injection preparation, an intramuscular injection preparation, a subcutaneous injection preparation or an intraperitoneal injection preparation.

15. The use according to claim 8, characterized in that The dosage form of the pharmaceutical composition is selected from the following group: tablets, powders, granules, oral liquids, capsules, microcapsules, and injections.

16. The use according to claim 8, characterized in that The dosage form of the cosmetic composition is selected from the group consisting of solution, gel, emulsion, ointment, cream, paste, powder, and patch.

17. The use according to claim 8, characterized in that The dosage form of the medical cosmetic composition is selected from the following group: microcapsules, freeze-dried powders, and injections.

18. The use according to claim 17, characterized in that The injection is a powder injection.

19. The use according to claim 1, characterized in that The composition is administered orally, topically or by injection.

20. The use according to claim 1, characterized in that The composition is administered by topical application or local injection.

21. A method for inhibiting melanin synthesis in vitro, characterized in that: The method comprises the steps of: contacting annexin A5 with cells capable of producing melanin, thereby inhibiting melanin synthesis; the cells capable of producing melanin include normal melanocytes.

22. The method according to claim 21, characterized in that The method is non-diagnostic and non-therapeutic.

Images