Cyclic nonapeptide and its composition and use

Abstract

Disclosed are cyclic nonapeptides, compositions, and uses thereof, relating to the field of polypeptide technology. The disclosed cyclic peptide has a structure of Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met]. Specifically, the present invention relates to the cyclic peptide or its salt, or a composition thereof, and uses thereof in preparing a composition for caring for or treating skin or mucous membranes.

Description

Technical Field

[0001] The present disclosure relates to the field of polypeptide technology, and in particular to cyclic nonapeptides and compositions and uses thereof. Background Art

[0002] Tyrosinase is a key enzyme in the melanin production process. Tyrosine within melanocytes is oxidized by tyrosinase to form dopaquinone, which is then converted into melanin through a series of reactions. Once produced, melanin is transferred to keratinocytes via melanocyte dendrites. Melanin granules in keratinocytes then ascend along epidermal cells to the stratum corneum, affecting skin color or forming spots. Excessive tyrosinase expression leads to excessive melanin production, potentially causing skin problems such as age spots, freckles, chloasma, or melanoma.

[0003] Elastin is a building block of elastic fibers in the extracellular matrix. Slowed elastin production and accelerated degradation are key causes of skin tissue loss of elasticity. Elastase is a proteolytic enzyme found in human cells and tissues, specifically degrading elastin. Overexpression of elastase accelerates elastin degradation, leading to a loss of skin elasticity and signs of aging, such as sagging and loosening.

[0004] Currently, compounds that can inhibit tyrosinase activity include kojic acid and arbutin, while compounds that have an inhibitory effect on elastase include quercetin. In the face of increasingly frequent skin problems, it is necessary to research more effective active ingredients. Summary of the Invention

[0005] The present disclosure relates to a cyclic nonapeptide and a composition and use thereof. The cyclic nonapeptide and the composition containing the cyclic nonapeptide have the effects of caring for or treating skin or mucous membranes.

[0006] In one aspect, the present disclosure provides a cyclic peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, wherein the structure of the cyclic peptide is Cyclo-[Phe-Phe-Pro-Pro-Trp-Val-Arg-Lys-Met].

[0007] The cyclic peptides disclosed herein contain a large number of asymmetric carbon atoms. Those skilled in the art will appreciate that the cyclic peptides disclosed herein have stereoisomers and may exist as stereoisomers or mixtures of stereoisomers, and thus may potentially produce isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbon atoms and the isomers or isomeric mixtures present.

[0008] In some embodiments, the structure of the cyclic peptide disclosed herein is Cyclo-[(D)-Phe-Phe-Pro-Pro-Trp-Val-Arg-Lys-Met], wherein (D)-Phe is a D-amino acid, and the remaining amino acids are L-amino acids.

[0009] In some embodiments, the structure of the cyclic peptide disclosed herein is Cyclo-[Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met], where (D)-Trp is a D-amino acid, and the remaining amino acids are L-amino acids.

[0010] In some embodiments, the structure of the cyclic peptide disclosed herein is Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met], where (D)-Phe and (D)-Trp are D-amino acids, and the remaining amino acids are L-amino acids.

[0011] The present disclosure also includes all suitable isotopic variants of the cyclic peptides. An isotopic variant of a cyclic peptide of the present disclosure is understood herein to mean a cyclic peptide in which at least one atom within the cyclic peptide of the present disclosure is replaced by another atom of the same atomic number, but the atomic mass of the other atom is different from the atomic mass usually or predominantly present in nature. Examples of isotopes that can be incorporated into the cyclic peptides of the present disclosure are those of hydrogen, carbon, nitrogen, oxygen or sulfur, e.g. 2 H (deuterium), 3 H (tritium), 13 C. 14 C. 15 N. 17 O. 18 O. 33 S. 34 S. 35 S or 36 Certain isotopic variants of the cyclic peptides disclosed herein (particularly those into which one or more radioactive isotopes have been incorporated) may be useful, for example, for examining the mechanism of action or distribution of the active compound in vivo; due to their relative ease of preparation and detectability, particularly with 3 H or 14 C isotope-labeled cyclic peptides are suitable for this purpose. In addition, due to the greater metabolic stability of cyclic peptides, the incorporation of isotopes (e.g., deuterium) can produce specific therapeutic benefits, such as an extension of the half-life in vivo or a reduction in the required active dose. Isotopic variants of the cyclic peptides of the present disclosure can be prepared by methods known to those skilled in the art, for example, by the methods further described below and in the examples, by using the respective reagents and / or corresponding isotopic modifications of the starting materials.

[0012] The term "salt" refers to a salt approved for use in animals, and more specifically, humans, including metal salts of the cyclic peptide, the metal including, but not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; including salts formed by the cyclic peptide with an organic base, the organic base including, but not limited to, ethylenediamine, ethanolamine, arginine, lysine, histidine or piperazine; including salts formed by the cyclic peptide with an inorganic acid or an organic acid, the organic acid including, but not limited to, acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate or gluconic acid; the inorganic acid including, but not limited to, hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0013] The nature of the salt is not critical and salts of the cyclic peptide may be obtained by conventional methods known in the art.

[0014] The synthesis of the cyclic peptides disclosed herein, or their stereoisomers, or mixtures of stereoisomers, or salts thereof, can be carried out according to conventional methods known in the art, such as solid phase synthesis, liquid phase synthesis, or a combination of solid phase and liquid phase methods. They can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal, or plant origin.

[0015] For example, a method for obtaining the cyclic peptide disclosed herein comprises the following steps:

[0016] - coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a protected or solid support-bound C-terminus;

[0017] - Elimination of the group protecting the N-terminus;

[0018] - repeating this coupling sequence and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;

[0019] - elimination of the group protecting the C-terminus or cleavage from the solid support;

[0020] - coupling and cyclizing the amino group at the N-terminus of the peptide chain and the carboxyl group at the C-terminus;

[0021] - Elimination of groups protecting side chains.

[0022] In some embodiments, the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising coupling an amino acid having a protected N-terminus and a free C-terminus to an amino acid having a free N-terminus and a C-terminus bound to a polymer support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as required to thereby obtain a peptide of the desired length, followed by cleavage of the synthesized peptide from the initial polymer support and cyclization of the peptide chain by coupling the amino group at the N-terminus to the carboxyl group at the C-terminus.

[0023] The functional groups of the side chains of these amino acids remain adequately protected with temporary or permanent protecting groups throughout the synthesis.

[0024] In some embodiments, solid phase synthesis can be performed by a convergent strategy of coupling a dipeptide or tripeptide to a polymer support or to a dipeptide or amino acid previously bound to a polymer support.

[0025] Due to external use in mammalian bodies, the cyclic peptides of the present disclosure can form part of various types of compositions. Therefore, another aspect of the present disclosure provides a composition comprising an effective amount of the above-mentioned cyclic peptide, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, and at least one excipient and optionally an adjuvant. The composition can be prepared by conventional methods known to those skilled in the art.

[0026] In some embodiments,The adjuvant is selected from the group consisting of: agents that activate Clock expression, analgesics, agents that inhibit PAR-2 ​​activity, agents that regulate PGC-1α synthesis, agents that regulate PPARγ activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents or agents that stimulate fat decomposition, lipolytic agents, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening agents or depigmenting agents, pigmentation promoting agents, self-tanning agents, anti-aging agents, NO-synthase inhibitors, 5α-reductase inhibitors, inhibitors of lysyl hydroxylase and / or prolyl hydroxylase, antioxidants, free radicals Scavengers and / or anti-air pollution agents, active carbonyl scavengers, anti-glycation agents, antihistamines, antivirals, antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, moisture-retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating under-eye bags, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or are capable of inhibiting or preventing their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis agents, agents that stimulate chaperone protein synthesis, agents that stimulate cAMP synthesis, agents that stimulate hyaluronic acid synthesis, agents that stimulate fibronectin synthesis, agents that stimulate deacetylase synthesis, agents that stimulate the synthesis of lipids and stratum corneum components, ceramides, fatty acids, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratosis agents, comedolytic agents, antipsoriatic agents, anti-eczema agents, DNA repair agents, DNA protective agents, stabilizers, antipruritic agents, for the treatment and / or care of allergies agents for sensitive skin, firming agents, tightening agents, restructuring agents, agents against stretch marks, agents regulating sebum production, antiperspirants, agents stimulating healing, agents assisting healing, agents stimulating re-epithelialization, agents assisting re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and / or microcirculation, agents stimulating angiogenesis, agents inhibiting vascular permeability, agents regulating venous tone, agents acting on cellular metabolism, agents for improving the dermal-epidermal junction, agents inducing hair growth, agents inhibiting or retarding hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents obtained from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against ultraviolet A and / or UVB rays, or mixtures thereof.

[0027] The cyclic peptides of the present disclosure have variable solubility in water, depending on the nature of their sequence. Thus, the cyclic peptides of the present disclosure can be incorporated into the composition via aqueous solutions, and those that are insoluble in water can be dissolved in conventional solvents, such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol, or polyethylene glycol, or any combination thereof.

[0028] The effective amount of the cyclic peptides of the present disclosure to be administered, as well as their dosage, will depend on a number of factors, including the age, condition of the user, severity of the condition, route and frequency of administration, and the specific nature of the cyclic peptide to be used.

[0029] "Effective amount" means an amount of the cyclic peptide of the present disclosure that is non-toxic but sufficient to provide the desired effect. The cyclic peptide of the present disclosure is used in the composition of the present disclosure at an effective concentration to obtain the desired effect. In some embodiments, the concentration is between 0.00000001% (by weight) and 20% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.000001% (by weight) and 15% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 10% (by weight) relative to the total weight of the composition; in some embodiments, the concentration is between 0.0001% (by weight) and 5% (by weight) relative to the total weight of the composition.

[0030] Another aspect of the present disclosure provides a delivery system or sustained-release system to achieve better penetration of the active ingredient, which comprises an effective amount of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition.

[0031] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier administered with the cyclic peptides of the present invention, selected from the group consisting of water, oils or surfactants, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol ethers, poloxamers, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin and the like. Those skilled in the art are aware of the diluents that can be used in the different delivery systems in which the cyclic peptides of the present invention can be administered.

[0032] The term "sustained release" is used in its conventional sense to refer to a delivery system that provides for the gradual release of a compound over a period of time. In some embodiments, a sustained release system has a relatively constant level of compound release over a period of time.

[0033] Examples of delivery systems or sustained-release systems include, but are not limited to, liposomes, oleosomes, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion compounds, lipid vesicles, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles, or nanoparticles.

[0034] Another aspect of the present disclosure provides a cosmetic comprising an effective amount of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system.

[0035] In some embodiments, the dosage form of the cosmetic comprises ointment, cream, emulsion, aqueous solution, oil, gel, powder, tablet, mud, patch, film, aerosol, spray, freeze-dried preparation or nano preparation.

[0036] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing a composition for caring for or treating skin or mucous membranes.

[0037] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing a composition for whitening, removing spots, brightening skin color, or eliminating uneven skin color.

[0038] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of a composition for inhibiting tyrosinase activity or inhibiting melanin production.

[0039] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing an anti-aging composition.

[0040] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in preparing a composition for inhibiting elastase activity, or in preparing a composition for increasing skin elasticity and / or improving skin firmness.

[0041] Another aspect of the present disclosure provides a use of the above-mentioned cyclic peptide, or its stereoisomer, or its mixture of stereoisomers, or its salt, or the above-mentioned composition, or the above-mentioned delivery system or sustained-release system in the preparation of cosmetics.

[0042] In the present disclosure, the term "skin" is understood to include the multiple layers comprising it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends inclusive. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes. In the present disclosure, the term "skin" includes the scalp.

[0043] The term "skin care" refers to the maintenance and care of the skin, improving the condition of the skin, making the skin delicate, smooth, tender and healthy.

[0044] The present disclosure has the following advantages and effects:

[0045] 1. The cyclic peptides disclosed herein can effectively inhibit tyrosinase activity. Inhibiting tyrosinase activity can reduce the synthesis of melanin in an organism, thereby achieving whitening and spot-lightening effects. Therefore, the cyclic peptides disclosed herein can reduce melanin synthesis, having the effects of whitening, removing spots, brightening skin color, or eliminating uneven skin tone.

[0046] 2. The cyclic peptide disclosed herein can effectively inhibit the activity of elastase. Inhibiting the activity of elastase can inhibit the hydrolysis of elastin and increase the level of elastin in skin tissue. The loss of elastin is the main cause of sagging, drooping, and fine wrinkles in aging skin. Therefore, the cyclic peptide disclosed herein can inhibit the activity of elastase, thereby increasing skin elasticity, improving skin firmness, and eliminating skin wrinkles, thus having an anti-aging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solution of the present disclosure, the following briefly introduces the drawings required for use in the description of the present disclosure. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0048] Figure 1 The cyclic nonapeptide of Example 1 of the present disclosure (molecular formula C 61 H 84 N 14 O9S) mass spectrum.

[0049] Figure 2 This is a graph showing the results of the tyrosinase activity assay experiment in Example 3 of the present disclosure.

[0050] Figure 3This is a graph showing the results of the elastase activity assay experiment in Example 4 of the present disclosure. DETAILED DESCRIPTION

[0051] To make the objectives, features, and advantages of the present disclosure more readily apparent, the present disclosure is further described below in detail with reference to the accompanying drawings and examples. It should be understood that the described embodiments are only a portion of the embodiments of the present disclosure, and not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present disclosure without inventive effort are intended to fall within the scope of protection of the claims appended to the present disclosure.

[0052] In the present disclosure, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in Eur. J. Biochem. 1984, 138: 9-37.

[0053] Unless otherwise specified, the experimental reagents and materials used in this disclosure can be obtained commercially. The following are the abbreviations of some reagents and materials:

[0054] 2-CTC Resin: a starting resin for peptide synthesis; DCM: dichloromethane; DIPEA: diisopropylethylamine; MeOH: methanol; piperidine: piperidine; DMF: N,N-dimethylformamide; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; TFA: trifluoroacetic acid; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; DMAP: 4-dimethylaminopyridine; TIS: triisopropylsilane; EDT: 1,2-ethanedithiol; Met: methionine; Lys: lysine; Arg: arginine; Val: valine; Trp: tryptophan; Pro: proline; Phe: phenylalanine; Fmoc: 9-fluorenylmethoxycarbonyl; Boc: tert-butoxycarbonyl; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

[0055] Example 1 Preparation of Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met]

[0056] Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met] was prepared by the following steps:

[0057] 1.1 Preparation of Fmoc-Met-2-CTC Resin

[0058] 15.0 g of 2-CTC Resin was weighed and placed in a solid phase synthesis reaction column, swollen with DCM, the resin was washed, and the solvent was removed.

[0059] 13.5 g of Fmoc-Met-OH and 15 mL of DIPEA were added to the swollen resin and reacted for 3 h. The reaction liquid was removed, the resin was washed, and the solvent was removed.

[0060] DCM, MeOH, and DIPEA were added to cap the resin for 0.5 h, and the resin was washed and the solvent was removed.

[0061] 1.2 Removal of Fmoc

[0062] The Fmoc-Met-2-CTC resin was de-Fmocated twice with 20% piperidine / DMF, each time for 10 min. Samples were taken for K-test, and the color was dark blue. The resin was washed with DMF 7 times, and the solvent was removed.

[0063] 1.3 Feeding reaction

[0064] Weigh 16.9 g of Fmoc-Lys(Boc)-OH and 5.6 g of HOBt into a dry Erlenmeyer flask. Dissolve in DMF, seal, and freeze at -18°C for 30 min. Activate with 8.0 mL of DIC for 7 min, avoiding moisture. Add the activated amino acid to the deprotected resin and react for 1 h. Aspirate the reaction solution. A colorless, transparent K-test indicates a complete reaction.

[0065] The N-terminal Fmoc group was deprotected, and 22.7 g of activated Fmoc-Arg(Pbf)-OH was coupled to the peptidyl resin in the presence of 5.6 g HOBt and 8.0 mL DIC using DMF as solvent. The reaction was continued for 1.5 h. The resin was then washed and the Fmoc group deprotection treatment was repeated to couple the next amino acid. In each coupling, 12.0 g of Fmoc-Val-OH, 18.5 g of Fmoc-D-Trp(Boc)-OH, 11.9 g of Fmoc-Pro-OH, 11.9 g of Fmoc-Pro-OH, 13.6 g of Fmoc-Phe-OH, and 13.6 g of Fmoc-D-Phe-OH were sequentially coupled in the presence of 5.6 g HOBt and 8.0 mL DIC using DMF as solvent. After the reaction was complete, the resin was washed and the solvent was removed.

[0066] The N-terminal Fmoc group of the peptidyl resin was deprotected using 20% ​​piperidine / DMF for two 10-min cycles. Samples were taken for K-analysis, revealing a dark blue color. The resin was washed six times with DMF, and the solvent was removed by evacuation. After shrinkage and drying, H-(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met-2-CTC Resin was obtained.

[0067] 1.4 Deresinization

[0068] Measure 4.5 mL of TFA and 445.5 mL of DCM, mix and stir to obtain a cleavage solution. Weigh 30 g of H-(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met-2-CTC Resin and add it to a round-bottom flask. Add the above cleavage solution and stir to react for 0.5 h. Filter with suction, collect the filtrate, spin dry, then add isopropyl ether and wash once, spin dry to obtain 24 g of H-(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met-OH.

[0069] 1.5 loop

[0070] 5g of H-(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met-OH was dissolved in 600mL of DCM. 3g of HBTU, 3g of DIPEA, and 3g of DMAP were then added and the reaction was allowed to proceed for 12 hours. Samples were taken to confirm the complete reaction of the starting material using HPLC. The insoluble material was filtered, and the filtrate was washed twice with 5% potassium bisulfate and twice with saturated sodium chloride, and then dried to obtain Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met].

[0071] 1.6 Cleavage (deprotection group)

[0072] Measure 36 mL of TFA, 1 mL of TIS, 1 mL of pure water, 1 mL of EDT, and 1 mL of thioanisole, mix them evenly to obtain a lysis solution, seal it, and place it in a -18°C refrigerator for later use; place isopropyl ether in a -18°C refrigerator for later use.

[0073] The above Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met] was added to a round-bottom flask, along with the above-mentioned chilled lysate. The reaction was stirred for 2 h. The precipitated solid was then filtered and washed four times with isopropyl ether to obtain 20 g of crude Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met] by wet weight.

[0074] 1.7 Purification

[0075] Weigh 20 g of the crude Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met] and dissolve it in water. After rotary evaporation to remove the isopropyl ether, add an appropriate amount of acetic acid and filter through a 0.45 μm microporous filter membrane to obtain a clear, transparent solution. Purify it by reverse-phase HPLC using the following purification gradient:

[0076] Time (min) Flow rate (mL / min) A% (acetonitrile) B% (pure water) 0 40 8 92 15 40 20 80 30 40 30 70 45 40 40 60 80 40 50 50

[0077] The filtered sample was injected for purification, and the fractions were collected, concentrated, and lyophilized to obtain a 99% pure cyclic peptide Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met], which was designated as cyclic nonapeptide A. The molecular weight was determined to be 1189.1439 by ESI-MS. The mass spectrum is shown in FIG. Figure 1 .

[0078] Linear peptide B: H-(D)-Phe-Phe-Pro-Pro-(D)-Trp(Boc)-Val-Arg(Pbf)-Lys(Boc)-Met-OH in step 1.4 was cleaved, the protecting groups were removed, and the peptide was purified to obtain the linear peptide H-(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met-OH.

[0079] Example 2

[0080] A whitening cream is prepared by the following steps:

[0081]

[0082]

[0083] According to the prescribed dosage, heat the Phase D material in a suitable container to 55-60°C, dissolve completely, and set aside; add Phase A to a stirring pot, stir and heat to 80-85°C; add Phase B to an oil phase pot, stir and heat to 75-80°C, and dissolve completely and become transparent; add Phase B to Phase A, start vacuum, homogenize for 5 minutes, maintain stirring, and keep warm for 20 minutes; start cooling to 60-65°C, add Phase C and pre-dissolved Phase D materials, and homogenize for 2 minutes; cool to 35-40°C, add Phase E materials, and stir for 10-15 minutes.

[0084] Example 3 Tyrosinase activity assay

[0085] 3.1 Reagents and Materials

[0086] Mushroom tyrosinase, L-DOPA.

[0087] 3.2 Instruments

[0088] Microplate reader, clean bench.

[0089] 3.3 Samples to be tested and grouping

[0090] 3.3.1 Samples to be tested

[0091] The test concentration of cyclic nonapeptide A and linear peptide B was 10 ppm.

[0092] 3.3.2 Grouping

[0093] Sample group: test sample, tyrosinase, PBS, L-DOPA;

[0094] Sample zeroing group: test sample, PBS, L-DOPA;

[0095] Blank control group: PBS, tyrosinase, L-DOPA;

[0096] Blank zero adjustment group: PBS, L-DOPA.

[0097] 3.4 Experimental methods

[0098] In a 96-well plate, add 50 μL of the sample to be tested and 50 μL of tyrosinase (25 U / mL) to the sample group; add 50 μL of the sample to be tested and 50 μL of PBS to the sample zero adjustment group; add 50 μL of PBS and 50 μL of tyrosinase (25 U / mL) to the blank control group; and add 100 μL of PBS to the blank zero adjustment group. Incubate at 37°C for 5 minutes, then add 50 μL of PBS and 50 μL of L-DOPA (0.5 mmol / L) to each well and incubate at 37°C for 10 minutes. Measure the OD at 475 nm. 475 value.

[0099]

[0100] Where: A1 is the OD of the sample zeroing group 475 value, A2 is the OD of the sample group 475 A3 is the OD of the blank zero adjustment group. 475 A4 is the OD of the blank control group. 475 value.

[0101] 3.5 Experimental Results

[0102] L-DOPA is a substrate for tyrosinase. Under the catalytic action of tyrosinase, L-DOPA is oxidized to dopaquinones, which absorb visible light at a wavelength of 475 nm. In this experiment, tyrosinase was treated with a test sample and the amount of L-DOPA reacted was measured to determine whether the cyclic nonapeptide A disclosed herein could inhibit tyrosinase activity.

[0103] The results of the test samples' effects on tyrosinase activity inhibition are shown in Figure 2 . The results showed that cyclic nonapeptide A can significantly inhibit the activity of tyrosinase. The inhibition rate of cyclic nonapeptide A on tyrosinase activity at a low concentration of 10 ppm reached 15.6%, while the inhibition rate of linear peptide B on tyrosinase activity at the same concentration was only 8.3%. The experimental results show that cyclic nonapeptide A obtained after the head-to-tail cyclization of linear peptide B has a better effect in inhibiting tyrosinase activity. It can be seen that the cyclic nonapeptide A disclosed in the present invention has an excellent effect of inhibiting tyrosinase activity, can inhibit melanin production, and can be used to improve skin problems such as melanin deposition and chloasma, and achieve the effects of whitening, removing spots, brightening skin color or eliminating uneven skin color.

[0104] Example 4 Elastase activity determination experiment

[0105] 4.1 Reagents and Materials

[0106] PBS buffer, elastase solution, AAAPAN solution.

[0107] 4.2 Instruments

[0108] Microplate reader, electronic balance.

[0109] 4.3 Samples to be tested and grouping

[0110] 4.3.1 Samples to be tested

[0111] The test concentrations of cyclic nonapeptide A and linear peptide B were 100ppm and 200ppm respectively.

[0112] 4.3.2 Grouping

[0113] Sample group: test sample, PBS, elastase, AAAPAN;

[0114] Sample zeroing group: test sample, PBS, AAAPAN;

[0115] Blank control group: PBS, elastase, AAAPAN;

[0116] Blank zero adjustment group: PBS, AAAPAN.

[0117] 4.4 Experimental methods

[0118] In a 96-well plate, add 85 μL PBS, 15 μL of the sample to be tested, and 25 μL of elastase solution (2 mg / mL) to the sample group; add 110 μL PBS and 15 μL of the sample to be tested to the sample zero adjustment group; add 100 μL PBS and 25 μL of elastase solution (2 mg / mL) to the blank control group; and add 125 μL PBS to the blank zero adjustment group. After incubation at 25°C for 15 minutes, add 25 μL of AAAPAN solution (1.015 mmol / L) to each well and incubate at 25°C for 15 minutes. Measure the OD at 410 nm. 410 value.

[0119]

[0120] Where: A1 is the OD of the sample zeroing group 410 value, A2 is the OD of the sample group 410 A3 is the OD of the blank zero adjustment group. 410 A4 is the OD of the blank control group. 410 value.

[0121] 4.5 Experimental Results

[0122] AAAPAN (N-succinyl-alanine-alanine-alanine-p-nitroaniline) is a substrate of elastase. Under the catalytic action of elastase, AAAPAN is decomposed, and the decomposition products absorb visible light at a wavelength of 410 nm. In this experiment, elastase was treated with a test sample and the reaction amount of AAAPAN was measured to determine whether the cyclic nonapeptide A disclosed herein could inhibit elastase activity.

[0123] The results of the test samples' effects on the inhibition of elastase activity are shown in Figure 3 The results showed that the cyclic nonapeptide A disclosed herein can significantly inhibit the activity of elastase. At a concentration of 100 ppm, the inhibition rate of cyclic nonapeptide A on elastase activity reached 62.5%, while the inhibition rate of linear peptide B on elastase activity was only 12.9%. At a concentration of 200 ppm, the inhibition rate of cyclic nonapeptide A on elastase activity reached 75.0%, while the inhibition rate of linear peptide B on elastase activity was 30.1%.

[0124] The experimental results show that the cyclic nonapeptide A obtained by cyclizing the linear peptide B from head to tail has a better inhibitory effect on elastase activity. Therefore, it can be seen that the cyclic nonapeptide A disclosed herein has an excellent effect of inhibiting elastase activity and can be used to improve skin problems such as skin sagging and wrinkles, effectively increase skin elasticity and / or improve skin firmness, and achieve anti-aging, firming, and wrinkle-removing effects.

[0125] In this disclosure, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or terminal device. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or terminal device comprising the element.

[0126] Although the specific embodiments of the present disclosure have been described for illustrative purposes, those skilled in the art may make various modifications or improvements without departing from the spirit and scope of the present disclosure. These modifications or improvements should fall within the scope of the appended claims of the present disclosure.

Claims

1. A cyclic peptide or a salt thereof, characterized in that The structure of the cyclic peptide is Cyclo-[(D)-Phe-Phe-Pro-Pro-(D)-Trp-Val-Arg-Lys-Met].

2. The cyclic peptide or salt thereof according to claim 1, characterized in that The salt includes a metal salt of the cyclic peptide, and the metal includes: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; Alternatively, the salt includes a salt formed by the cyclic peptide and an organic base, and the organic base includes: ethylenediamine, ethanolamine, arginine, lysine, histidine or piperazine; Alternatively, the salt includes a salt formed by the cyclic peptide and an inorganic acid or an organic acid, wherein the organic acid includes: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

3. A composition, characterized in that The invention comprises an effective amount of the cyclic peptide or salt thereof according to claim 1 or 2, at least one excipient and an optional adjuvant.

4. The composition according to claim 3, characterized in that The adjuvant is selected from the group consisting of: agents that activate Clock expression, analgesics, agents that inhibit PAR-2 ​​activity, agents that regulate PGC-1α synthesis, agents that regulate PPARγ activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic agents, lipolytic agents, lipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulators or inhibitors, whitening agents or depigmenting agents, pigmentation promoting agents, self-tanning agents, NO-synthase inhibitors, 5α-reductase inhibitors, inhibitors of lysyl hydroxylase and / or prolyl hydroxylase, antioxidants, agents against air pollution, active carbonyl anti-glycation agents, antihistamines, antivirals, antiparasitics, emulsifiers, emollients, organic solvents, liquid propellants, moisture-retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gelling polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating under-eye bags, keratolytic agents, antimicrobial agents, agents that stimulate the synthesis of dermal or epidermal macromolecules and / or are capable of inhibiting or preventing their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperone synthesis , agents that stimulate cAMP synthesis, agents that stimulate hyaluronic acid synthesis, agents that stimulate fibronectin synthesis, agents that stimulate sirtuin synthesis, agents that stimulate the synthesis of lipids and stratum corneum components, ceramides, fatty acids, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratosis agents, comedolytic agents, anti-psoriatic agents, anti-eczema agents, DNA repair agents, DNA protective agents, stabilizers, antipruritic agents, agents for treating and / or caring for sensitive skin , firming agents, tightening agents, restructuring agents, agents against stretch marks, agents regulating sebum production, antiperspirants, agents stimulating healing, agents assisting healing, agents stimulating re-epithelialization, agents assisting re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and / or microcirculation, agents stimulating angiogenesis, agents inhibiting vascular permeability, venous tone agents, agents acting on cellular metabolism, agents for improving dermal-epidermal junction, agents inducing hair growth, agents inhibiting or retarding hair growth, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents obtained from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against ultraviolet A and / or UVB rays, or mixtures thereof.

5. A delivery system or sustained-release system, characterized in that Comprising an effective amount of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4; The delivery system or sustained-release system includes: liposomes, oleosomes, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, inclusion compounds, lipid vesicles, micelles, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles or nanoparticles.

6. A cosmetic, characterized in that: Comprising an effective amount of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4, or the delivery system or sustained-release system according to claim 5.

7. The cosmetic according to claim 6, characterized in that The dosage forms of the cosmetics include ointments, creams, emulsions, aqueous solutions, oils, gels, powders, tablets, muds, patches, films, aerosols, sprays, freeze-dried preparations or nano preparations.

8. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4, or the delivery system or sustained-release system according to claim 5 in the preparation of a composition for caring for skin or mucous membranes.

9. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4, or the delivery system or sustained-release system according to claim 5 in the preparation of a composition for whitening, removing spots, brightening skin color, or eliminating uneven skin color.

10. The use according to claim 9, characterized in that The whitening includes inhibiting tyrosinase activity or inhibiting melanin production.

11. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4, or the delivery system or sustained-release system according to claim 5 in the preparation of a composition for skin anti-aging, wherein the anti-aging comprises inhibiting elastase activity.

12. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4, or the delivery system or sustained-release system according to claim 5 in the preparation of a composition for increasing skin elasticity and / or improving skin firmness.

13. Use of the cyclic peptide or salt thereof according to claim 1 or 2, or the composition according to claim 3 or 4, or the delivery system or sustained-release system according to claim 5 in the preparation of cosmetics.

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