A drug for treating pigmentation

Abstract

The present invention relates to a use of pyruvate kinase. Specifically, the pyruvate kinase of the present invention can be used to prepare a composition for (a) preventing and / or treating skin pigmentation; (b) whitening skin tone; and / or (c) delaying melanoma progression. The pyruvate kinase of the present invention can significantly inhibit melanin synthesis, including melanoma cell synthesis, and has excellent skin whitening effects and reduces persistent pigment deposition in melanoma cells.

Description

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 object of the present invention is to provide a use of pyruvate kinase 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 pyruvate kinase in preparing a composition for one or more of the following uses: (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 pyruvate kinase includes M-type pyruvate kinase, L-type pyruvate kinase, and R-type pyruvate kinase, preferably M-type pyruvate kinase.

[0008] In another preferred embodiment, the M-type pyruvate kinase includes M1 subtype pyruvate kinase and / or M2 subtype pyruvate kinase.

[0009] In another preferred embodiment, the weight fraction of pyruvate kinase in the composition is 1-99 wt % of the total weight of the composition, such as 80%, 85%, 90%, 95%, 98% or 99%.

[0010] 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).

[0011] In another preferred embodiment, the skin pigmentation includes primary skin pigmentation or secondary skin pigmentation.

[0012] 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.

[0013] In another preferred embodiment, the skin pigmentation caused by hormone changes includes skin pigmentation caused by Addison's disease, pregnancy or oral contraceptives.

[0014] In another preferred embodiment, the skin pigmentation caused by inflammation includes skin pigmentation caused by inflammatory skin diseases.

[0015] In another preferred embodiment, the inflammatory skin disease includes lichen planus and / or lichen planus-like drug eruption.

[0016] 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.

[0017] In another preferred embodiment, the skin pigmentation is the pigmentation produced by the human body itself.

[0018] In another preferred embodiment, the skin pigmentation does not include hemochromatosis.

[0019] In another preferred embodiment, the prevention and / or treatment of skin pigmentation includes one or more features selected from the following group:

[0020] (i) inhibiting the production of melanin in melanocytes;

[0021] (ii) Whitening and / or brightening skin tone.

[0022] In another preferred embodiment, the melanocytes are normal melanocytes or melanoma cells.

[0023] 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.

[0024] In another preferred embodiment, the delaying of melanoma progression does not include inhibiting melanoma cell proliferation.

[0025] 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.

[0026] In another preferred embodiment, the pyruvate kinase is pyruvate kinase of human or non-human mammals.

[0027] In another preferred embodiment, the non-human mammal includes pigs, monkeys, cows, sheep, rats, mice or rabbits.

[0028] In another preferred embodiment, the composition is a pharmaceutical composition, a cosmetic composition and / or a medical cosmetic composition.

[0029] In another preferred embodiment, the composition further comprises a carrier acceptable in pharmaceuticals, cosmetics or medical cosmetology.

[0030] 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.

[0031] 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.

[0032] In another preferred embodiment, the composition is in the form of a liquid dosage form, a semisolid dosage form or a solid dosage form.

[0033] In another preferred embodiment, the composition is an oral preparation, an external preparation or an injection preparation.

[0034] 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.

[0035] In another preferred embodiment, the composition is administered orally, externally, by microneedle or injection, preferably by topical application or local injection.

[0036] 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, injections, ointments, creams, tinctures, oral liquids, powder sprays, aerosols or sprays.

[0037] 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.

[0038] 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.

[0039] In a second aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0040] (a) pyruvate kinase; (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.

[0041] 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.

[0042] 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.

[0043] In another preferred embodiment, the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

[0044] 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.

[0045] In another preferred embodiment, the pharmaceutical composition is administered orally, externally, by microneedle or injection, preferably by topical application or local injection.

[0046] 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, injections, ointments, creams, tinctures, oral liquids, powder sprays, aerosols or sprays.

[0047] In a third aspect of the present invention, a method for inhibiting melanin synthesis in vitro is provided, comprising the steps of contacting pyruvate kinase with cells capable of producing melanin, thereby inhibiting melanin synthesis.

[0048] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0049] In another preferred embodiment, the cells capable of producing melanin include normal melanocytes and / or abnormal melanoma cells.

[0050] In another preferred embodiment, the concentration of the pyruvate kinase is 10 μg / mL or more, preferably 100 μg / mL or more.

[0051] In a 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 pyruvate kinase or a pharmaceutical composition containing pyruvate kinase as described in the second aspect of the present invention to a subject in need thereof.

[0052] 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.

[0053] In another preferred embodiment, the administration concentration of the pyruvate kinase is 10 μg / mL or higher, preferably 100 μg / mL or higher.

[0054] 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

[0055] Figure 1 Shown are typical plots of melanin signal intensity in zebrafish heads after sample processing.

[0056] Figure 2 The melanin signal intensity in zebrafish heads after sample treatment is shown. **p<0.01, ***p<0.001 compared with the normal control group.

[0057] 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.

[0058] Figure 4 The total amount of cytochrome in B16 cells after sample treatment is shown. **p<0.01 compared with the normal control group. DETAILED DESCRIPTION

[0059] 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 pyruvate kinase has 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, pyruvate kinase is expected to be an effective means for whitening skin, treating pigmentation, and treating melanoma. This is the basis for the present invention.

[0060] the term

[0061] 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.

[0062] 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."

[0063] 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.).

[0064] 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 pyruvate kinase compared to the absence of pyruvate kinase.

[0065] 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 administration of pyruvate kinase compared to the absence of pyruvate kinase.

[0066] 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.

[0067] 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, elimination, and reversal. In some embodiments, the use of pyruvate kinase 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 pyruvate kinase. In some embodiments, the use of pyruvate kinase slows 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 pyruvate kinase.

[0068] As used herein, the term "treating pigmentation" may include alleviating the progression of the condition, reducing the synthesis of pigments.

[0069] Pyruvate kinase

[0070] Pyruvate kinase (PKM) is an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP during glycolysis, regulating cellular metabolism. In the final step of glycolysis, PKM catalyzes the irreversible conversion of ADP and phosphoenolpyruvate to ATP and pyruvate, both of which are essential for cellular metabolism. Therefore, PKM plays a key role in controlling metabolic flux and ATP production. Four PKM isoforms have been identified in mammals: muscle (M1), liver (L), erythrocyte (R), and the ubiquitous M2. Each of these four isoenzymes has distinct tissue expression patterns and regulatory properties. Currently, the function of PKM in pigment synthesis is not well understood.

[0071] The pyruvate kinase that can be used for the purposes of (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 of the M-type (including the M1 subtype and the M2 subtype), the L-type, and the R-type, preferably the M-type pyruvate kinase, and more preferably the M2 type used in the examples.

[0072] The pyruvate kinase that can be used in the present invention is not particularly limited and can be pyruvate kinase derived from any organism, preferably from a mammal (such as a primate), and more preferably from a human. In addition, it should be understood that "pyruvate kinase" includes wild-type or mutant (including truncated) pyruvate kinase, as long as the mutant pyruvate kinase retains or maintains the detoxification activity of the wild-type pyruvate kinase.

[0073] use

[0074] The present invention provides the use of pyruvate kinase in preparing a composition for (a) preventing and / or treating skin pigmentation, (b) whitening skin color, and / or (c) delaying the progression of melanoma. The composition has a particularly significant effect in reducing melanin synthesis and whitening skin.

[0075] 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.

[0076] The present invention discloses that pyruvate kinase has the effect of inhibiting pigment synthesis. In the present invention, pyruvate kinase can inhibit the total amount of pigment in the head of zebrafish larvae and can inhibit the synthesis of melanin in mouse melanoma cells, having the effects of whitening and delaying the progression of melanoma.

[0077] Composition and administration

[0078] The compositions of the present invention include (but are not limited to): pharmaceutical compositions, cosmetic compositions and / or medical cosmetic compositions.

[0079] 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 pharmaceutically 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, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, dispersants, etc. Some examples of acceptable carriers in cosmetics or medical cosmetology include moisturizers, UV inhibitors, film-forming agents, oil-soluble gelling agents, organically 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] Amino acids include: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc.

[0085] The pharmaceutical composition of the present invention comprises the above-mentioned pyruvate kinase as an active ingredient and a pharmaceutically acceptable carrier.

[0086] Typically, pyruvate kinase can be prepared into pharmaceutical compositions, such as tablets, capsules, powders, microgranules, solutions, lozenges, injections, elixirs, suspensions, tinctures, poultices, liniments, lotions, and aerosols. Pharmaceutical compositions can be prepared by commonly known manufacturing techniques, and suitable pharmaceutical additives can be added to the medicine.

[0087] 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 can be in the form of an oral preparation, a topical preparation, or an injectable preparation.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 a pharmaceutically acceptable effective dosage. 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 the combination with other drugs. 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.

[0095] The main advantages of the present invention include:

[0096] (1) The present invention discloses for the first time the use of pyruvate kinase in (a) preventing and / or treating skin pigmentation, (b) whitening skin color, and / or (c) delaying the progression of melanoma.

[0097] (2) The present invention discloses that pyruvate kinase has the effect of inhibiting pigment synthesis.

[0098] (3) The present invention discovered for the first time that pyruvate kinase can inhibit the synthesis of head pigment in zebrafish larvae and the synthesis of melanin in mouse melanoma cells.

[0099] 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.

[0100] Example 1

[0101] 1. Experimental Animals

[0102] Wild-type AB strain zebrafish were bred in natural pairs. 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 / LCaCO3). 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 of 10 μg / mL and 100 μg / mL pyruvate kinase, respectively) 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 (3 mL per well) was also established. 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 on the zebrafish head 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. P < 0.05 indicated statistical significance.

[0105] 3. Experimental Results

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

[0107]

[0108]

[0109] Compared with the normal control group, *p<0.05, ***p<0.001

[0110] Depend on Figure 1-2 The experimental results shown in Table 1 show that pyruvate kinase significantly inhibits pigment synthesis in the heads of zebrafish larvae (***p<0.001), demonstrating a significant whitening effect. Furthermore, the concentration of the positive control significantly exceeded the dose of pyruvate kinase PKM. Therefore, at similar concentrations, pyruvate kinase's inhibitory effect on pigment synthesis was even more potent than that of the positive control (arbutin).

[0111] Example 2

[0112] Cell experiments

[0113] 1. Effect of pyruvate kinase on the proliferation of B16 cells:

[0114] After the B16 cells were digested, a cell suspension was prepared and inoculated into a 96-well plate at a density of 2000 cells / well, and cultured in a 37°C incubator for 24 hours. The next day, 1000 ng / ml of pyruvate kinase or PBS was used to treat the 96-well plate cells, with 3 replicates per group, and the culture was 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. The cells were then detected using an enzyme-linked microplate reader, and the proliferation capacity of each group of cells was calculated. The results are shown in Figure 2. Figure 3 shown.

[0115] 2. Effect of pyruvate kinase on pigment synthesis in B16 cells

[0116] 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 pellet 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 lysate was placed in a 96-well plate, 3 replicates were set for each concentration group, and the absorbance at a wavelength of 400nm was measured on a microplate reader. The melanin formation rate = A experimental group / A blank group average value × 100%. The results are shown in Figure 2. Figure 4 shown.

[0117] The experimental results showed that pyruvate kinase could not significantly promote or inhibit the proliferation of B16 melanoma cells; however, pyruvate kinase could significantly reduce the total amount of pigment synthesis in B16 cells, and had the effect of whitening and delaying the progression of melanoma.

[0118] 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 M2 subtype pyruvate kinase in the preparation of a composition for treating skin pigmentation, In the composition, the concentration of M2 subtype pyruvate kinase is 10 μg / mL-100 μg / mL; And the pigment is melanin.

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 pigmentation, melasma, age spots, freckles, sun spots, and pigmentation in melanoma.

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

4. 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 radiation.

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

6. The use according to claim 1, characterized in that The composition may also include other drugs useful for treating skin pigmentation.

7. The use according to claim 6, characterized in that The other drugs that can be used to treat skin pigmentation are selected from the following group: hydroquinone, tretinoin, azelaic acid, kojic acid, glycolic acid, arbutin or a combination thereof.

8. The use according to claim 1, characterized in that The composition is an external preparation.

9. The use according to claim 1, characterized in that The composition is a pharmaceutical composition or a cosmetic composition.

10. The use according to claim 1, characterized in that The composition is a medical cosmetic composition.

11. The use according to claim 9, characterized in that The dosage form of the pharmaceutical composition is powder or aerosol.

12. The use according to claim 9, characterized in that The dosage form of the cosmetic composition is selected from the group consisting of solution, gel, emulsion, and ointment.

13. The use according to claim 9, characterized in that The dosage form of the cosmetic composition is selected from the group consisting of cream, paste, powder, and patch.

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