Micellar bioactive collagen-stimulating lipopeptide

Abstract

A lipopeptide of formula (I): RLP(I) (wherein R is a branched hydrocarbyl group, L is a linker, and P is a peptide containing 2 to 10 amino acid residues).

Description

Detailed description of the invention

[0001] [Technical field] This invention relates to novel lipopeptide molecules, compositions containing such lipopeptides, methods for preparing them, and their uses. In particular, this invention relates to novel bioactive collagen-stimulating lipopeptides that self-assemble to form micelles, offering potential applications in fields including skin care, wound healing, and tissue engineering.

[0002] [Background of the Invention] Collagen is a protein widely distributed in living organisms and is an essential component of connective tissue, skin, and bone. Collagen is considered an effective treatment for improving rough skin, sagging skin, wrinkles, and wounds, as well as for treating conditions such as arthritis and tenosynovitis. Type I collagen is the most abundant protein in the dermis layer of the skin and is responsible for skin elasticity. Type II and Type III collagen are other important types of collagen found in vivo. Therefore, new active ingredients that promote collagen production are constantly being researched.

[0003] Certain peptides (up to 30 amino acids in length) are known to stimulate collagen growth in the extracellular matrix of skin, potentially improving not only skin appearance but also the healing of damaged skin. A major challenge in realizing the potential cosmetic and therapeutic effects of these peptides in humans is transdermal delivery of the peptides across the skin barrier (stratum corneum) to their biological sites of action, such as the extracellular matrix or the underlying muscles (Robinson, et al., 2005, International J. Cosmetic Science 27:155). To achieve transdermal delivery of these peptides in humans, chemical modifications of peptides have been previously performed by adding moieties such as acetyl groups and / or palmitoyl groups (Robinson, et al., see above).

[0004] Several peptide amphiphilic (PA) molecules, such as lipopeptides, have been found to possess beneficial cosmetic and therapeutic effects on the skin and are applied in tissue engineering, regenerative medicine, and cosmetics based on the stimulation of collagen production by fibroblasts. Lipopeptides are a remarkable class of self-assembling molecules that can form peptide-functionalized supramolecular nanostructures.

[0005] One known example of a peptide amphiphilic substance (PA) is KTTKS pentapeptide, which is a subfragment of type I collagen propeptide and is a lipopeptide (peptide amphiphilic substance, PA) from the Matrixyl® family developed by Sederma. 16 -It is incorporated into KTTKS. The KTTKS pentapeptide is based on a sequence obtained from a propeptide derived from human type I collagen. C 16 -KTTKS, or palmitoyl pentapeptide-4 (Pal-Lys-Thr-Thr-Lys-Ser=Pal-KTTKS), contains five amino acids directly linked to a 16-carbon aliphatic chain:

[0006] [ka]

[0007] Peptides KTTKS and C 16 -KTTKS has been reported to be able to promote type I and type II collagen production in vitro (RR Jones, V. Castelletto, CJ Connon and IW Hamley, Molecular Pharmaceutics, 2013, 10, 1063-1069). Multiple studies have shown that C 16-KTTKS has also been suggested to reduce the appearance of facial wrinkles (LR Robinson, NC Fitzgerald, DG Doughty, NC Dawes, CA Berge and DL Bissett, International Journal of Cosmetic Science, 2005, 27, 155-160).

[0008] KTTKS is a low-molecular-weight, highly specific bioactive peptide that has been reported to stimulate elastin and collagen, particularly type I and type II; however, its chemical properties severely limit its ability to be delivered across the skin. From a physicochemical standpoint, KTTKS and palmitoyl-KTTKS clearly possess properties unfavorable for meaningful skin penetration.

[0009] [Overview of the prefecture] According to a first aspect, the present invention relates to formula (I): R-LP (I) (In the formula, R is a hydrocarbon group, L is a linker, and P is a peptide containing 2 to 10 amino acid residues.) Regarding lipopeptides.

[0010] Those skilled in the art will understand that the hydrocarbon group, linker, and peptide are, respectively, separate sections of a lipopeptide.

[0011] Remarkably, cell model studies have shown that the lipopeptides of this invention enhance collagen production by more than twofold compared to conventional irritants. Therefore, lipopeptides have clear applications in cosmetic applications, such as skin treatments. They are suitable for cosmetic use because, for example, they can penetrate deeper into the skin and / or improve skin appearance. Lipopeptides exhibit a beneficial combination of reduced cytotoxicity and enhanced collagen production.

[0012] Conventional lipopeptides, for example, C n which is C 14 ~C 18 C corresponding to the lipid chain n -KTTKS (which also comprises the KTTKS Matrixyl™ sequence) forms tape-like protofibrils with a length of up to several micrometers (R Jones et al., Molecular Pharmaceutics 2013, 10(3), 1063-1069 DOI: 10.1021 / mp300549d).

[0013] The lipopeptide of the present invention is C 16 has improved cytocompatibility compared with -KTTKS. C 16 In the presence of -KTTKS, even at a concentration as low as one-tenth of the concentration used to test the lipopeptide of the present invention (in the range of 0.002 wt% to 0.008 wt%), cell viability decreases by 40% or more. The fact that the lipopeptide of the present invention does not induce either a significant increase or a significant decrease in cell proliferation is a desirable characteristic for the application of this compound as a cosmetic or in clinical practice.

[0014] On the other hand, it has surprisingly been found that the lipopeptide of the present invention self-assembles into micelles, which may be referred to as "nanoparticles". The micellar structure of the lipopeptide of the present invention allows the lipopeptide to acquire a new activation mechanism in skin penetration across the stratum corneum, thereby resulting in enhanced activity in the skin, which is attributed to the fact that small micelles can be easily transported through the pores of this barrier.

[0015] In contrast to "micellar" cosmetics, the micelles of the present invention are not a "formulation" in the usual sense (that is, a mixture of components such as conventional surfactants or liposomes), and micelle formation is an intrinsic self-assembly property inherent to the lipopeptide of the present invention. Conventional cleaning surfactants that form micelles do not contain such a motif.

[0016] It is believed that lipopeptides can acquire a micelle structure through the linker structure, without being constrained by theory. The linker preferably contains a polyether group.

[0017] Lipopeptides have the following structure:

[0018] [ka] It is most preferable that it be represented by this.

[0019] This application describes specific benefits relating to enhanced collagen production, which are thought to be due to the specific structure of the peptide. It is hypothesized that if an alternative peptide with a similar linker structure is incorporated into the structure of a lipopeptide, the resulting lipopeptide will acquire enhanced absorption, thereby enhancing the biological effects of the alternative peptide.

[0020] According to a second aspect, the present invention relates to a composition comprising a) a lipopeptide of formula (I); and b) at least one additional component.

[0021] The composition may be a pharmaceutical composition and / or a cosmetic composition. The composition is preferably a topical composition. Additional components are preferably carriers, such as dermatologically acceptable carriers.

[0022] According to a third aspect, the present invention relates to the cosmetic use of the lipopeptide of the first aspect or the composition of the second aspect. The cosmetic use may be for regenerating skin, treating nails, and / or improving the appearance of hair.

[0023] According to a fourth aspect, the present invention relates to a method of cosmetic prevention and / or treatment, To prepare a lipopeptide according to the first embodiment, Applying lipopeptides to the user's skin to improve the appearance of the skin. This includes methods.

[0024] Compositions suitable for use in cosmetic applications can be formulated as cosmetic formulations. Cosmetic formulations can improve the appearance of the skin by reducing the appearance of scar tissue and / or linear skin atrophy, by reducing the number or severity of wrinkles and / or fine lines, by improving skin elasticity, and / or by making skin color more uniform (for example, by reducing the appearance of age-related blemishes or other conditions related to excessive melanin or hyperpigmentation). Cosmetic formulations can be formulations selected from a list consisting of creams, lotions, gels, semi-solids, dispersants, suspensions, foams, mousses, and sprays.

[0025] The lipopeptides of the present invention have also been found to induce wound healing. Specifically, in fibroblast scratch tests and in mouse wound healing models, the presence of these lipopeptides has been shown to increase the rate of wound healing.

[0026] The lipopeptides of the present invention have also been found to possess potent immunomodulatory activity. Specifically, the presence of these lipopeptides eliminates the increase in nitrite, a class of toxins produced by bacteria, which is typically observed when RAW264.7 cells are exposed to lipopolysaccharides. Thus, these lipopeptides can interact with the immune system to downmodulate certain aspects of the host's immune response. The immune system sometimes initiates an immune response to harmless substances such as dust mites, dust, or pollen, thereby triggering allergies (hypersensitivity T). HA two-mediated response occurs. Furthermore, many physiological disorders, such as hypercholesterolemia and obesity, lead to low-grade inflammatory states. Immune modulation, in the context of abnormal immune responses such as allergies or inflammation, means weakening or counteracting a hypersensitive immune response. The present invention may relate to the primary challenges of stimulating / enhancing immune responses and / or inhibiting “abnormal” immune responses. Therefore, this lipopeptide can be applied to the treatment of inflammation-related conditions.

[0027] Because this lipopeptide can stimulate collagen production, it can be applied to the treatment of collagen-related conditions.

[0028] According to a fifth aspect, the present invention relates to a lipopeptide for pharmaceutical use, wherein the lipopeptide is the lipopeptide of the first aspect.

[0029] According to a sixth aspect, the present invention relates to a lipopeptide for use in methods for preventing, treating or improving wounds in the skin of a subject and / or preventing, treating or improving conditions related to inflammation and / or conditions related to collagen, wherein the lipopeptide is the lipopeptide of the first aspect.

[0030] According to a seventh aspect, the present invention relates to an article comprising a lipopeptide of the first aspect, wherein the lipopeptide is configured to come into contact with a target tissue (e.g., skin) during use. Thus, the article may have the lipopeptide on its outer surface. The article may be a medical device, e.g., a wound dressing, a catheter and / or implant. The article may be a tissue scaffold, which may be for either an in-vivo application, e.g., implantation into a target (e.g., regenerative medicine), or an ex-vivo application, e.g., a tissue model.

[0031] US2009 / 092642A1 discloses a farnesyl-like chain conjugated to a peptide containing two amino acids. This compound is used to enhance sunburn and melanin synthesis in skin or hair. Bhide, Rajeev S. et al., Bioorganic & Medicinal Chemistry Letters, 1994, 4(17), pages 2107-12; DOI: 10.1016 / S0960-894X(01)80111-6 describes a two-substrate inhibitor of ras farnesyl proteintransferase. US6015877A describes an inhibitor of carboxy-terminal proteolysis. GB2323783A describes an inhibitor of farnesyl proteintransferase. However, these documents do not mention linkers containing polyethers (e.g., polyethylene glycol) or the peptide used in this application.

[0032] US2019 / 330141A1 describes compounds having a cooling effect for use in personal care compositions, such as oral care compositions containing flavors / aromas. However, it does not mention the specific use of linkers containing polyethers (e.g., polyethylene glycol), peptides used in this application, or sesquiterpenes as hydrocarbon groups.

[0033] JP H04297495A describes compounds containing saturated hydrocarbon chains linked to octapeptides for use as animal cell adhesion inhibitors and platelet aggregation / adhesion inhibitors. However, it does not mention unsaturated or sesquiterpene hydrocarbon chains, linkers containing polyethers (e.g., polyethylene glycol), or peptides used in this application.

[0034] EP3598982A1 describes safe anti-hyperalgesic or analgesic compounds that are bioconjugates of neuropeptide derivatives. The hydrocarbon group of the compound contains more than 25 carbon atoms. Furthermore, linkers including polyethers (e.g., polyethylene glycol) are not mentioned, nor are the peptides used in this application.

[0035] These documents describe lipopeptides, but none of them describe the specific linker group used in the present invention, nor the specific peptide used in the present invention.

[0036] Furthermore, none of these documents describe any technical effects related to increased collagen production, increased wound healing rates, or reduced inflammatory responses.

[0037] This disclosure includes the subject matter of the following clauses:

[0038] 1. A lipopeptide of formula (I): RLP(I) (wherein R is a branched hydrocarbyl group, L is a linker, and P is a peptide of 2 to 10 amino acid residues).

[0039] 2. A lipopeptide according to clause 1, in which R contains 5 to 25 carbon atoms.

[0040] 3. R ​​is an unsaturated lipopeptide according to clause 1 or 2.

[0041] 4. A lipopeptide according to any of the preceding clauses, wherein R is a terpenoid.

[0042] 5. A lipopeptide according to clause 4, wherein R is a sesquiterpene.

[0043] 6. A lipopeptide according to clause 5, in which R is a farnesyl group.

[0044] 7. A lipopeptide according to any of the preceding clauses, wherein linker L includes a polyether moiety.

[0045] 8. A lipopeptide according to Clause 7, wherein the linker comprises a diethylene glycol moiety.

[0046] 9. Linker L,

[0047] [ka] A lipopeptide according to any of the aforementioned clauses.

[0048] 10. A lipopeptide comprising 2 to 10 amino acids as described in any of the preceding clauses.

[0049] 11. A lipopeptide according to any of the preceding clauses, wherein the peptide is a collagen-promoting peptide.

[0050] 12. A lipopeptide according to clause 11, wherein the peptide is selected from KTTKS, GHK, and AHK.

[0051] 13. A lipopeptide according to Clause 1, wherein the peptide is selected from the amino acid sequence KTTKS, or a fragment or variant thereof.

[0052] 14.Formula:

[0053] [ka] A lipopeptide having any of the aforementioned clauses.

[0054] 15.a) lipopeptides as defined in any one of clauses 1 to 14; and b) a topical composition comprising at least one additional component.

[0055] 16. The topical composition of Clause 15, further comprising several additional components, at least one of which is a dermatologically acceptable carrier.

[0056] 17. Cosmetic use of lipopeptides as defined in any one of clauses 1 to 14 or topical compositions as defined in clause 15 or 16 for the purpose of regenerating skin.

[0057] 18. Cosmetic use of lipopeptides as defined in any one of clauses 1 to 14 or topical compositions as defined in clause 15 or 16 for the treatment of nails.

[0058] 19. Cosmetic use of lipopeptides as defined in any one of clauses 1 to 14 or topical compositions as defined in clause 15 or 16 for the purpose of improving the appearance of hair.

[0059] 20. A lipopeptide as defined in any one of clauses 1 to 14 for use in pharmaceuticals.

[0060] Herein, embodiments of the present invention will be described with reference to the following accompanying drawings as an example. [Brief explanation of the drawing]

[0061] [Figure 1] This is a diagram showing the structure of farnesyl-mercapto(diethylene glycol)-KTTKS (FMdEG-KTTKS). [Figure 2a] This figure shows the self-assembly of lipopeptide FMdEG-KTTKS into micelles at concentrations above the critical aggregation concentration (CAC) and its collagen-stimulating activity at concentrations below the CAC. [Figure 2b] This graph shows the variation in peak intensity (I0) and wavelength (λ0) of the fluorescent dye 8-anilino-1-naphthalenesulfonic acid, along with the lipopeptide concentration. [Figure 3]Figure 3a is a graph showing a circular dichroism spectrum exhibiting disordered peptide conformation. Figure 3b is a graph showing an FTIR spectrum similarly exhibiting disordered peptide conformation. Figure 3c is a graph showing dynamic light scattering (DLS) intensity distribution data showing small aggregates. Figure 3d is a graph showing small-angle X-ray scattering (SAXS) analysis of micelles based on a peptide amphiphilic substance, exhibiting micelle structure at concentrations above CAC. Figure 3e is a typical cryo-TEM image with well-defined, relatively monodisperse micelles having sizes consistent with those obtained by DLS and SAXS. [Figure 4] This graph shows the results of cytotoxicity tests of lipopeptides. [Figure 5a] This graph shows the results of a collagen production assay using lipopeptides. [Figure 5b] This graph shows the collagen production per cell by lipopeptides. [Figure 6] Figure 6a is a polarized light microscope (POM) image of cells stained with Sirius red dye. Figure 6b is a polarized light microscope (POM) image of cells stained with Sirius red dye. Figure 6c is a polarized light microscope (POM) image of cells stained with Sirius red dye. Figure 6d is a polarized light microscope (POM) image of cells stained with Sirius red dye. Figure 6e is a polarized light microscope (POM) image of cells stained with Sirius red dye. [Figure 7] Figure 7a is a bright-field optical microscope image of cells stained with Sirius red dye. Figure 7b is a bright-field optical microscope image of cells stained with Sirius red dye. Figure 7c is a bright-field optical microscope image of cells stained with Sirius red dye. Figure 7d is a bright-field optical microscope image of cells stained with Sirius red dye. Figure 7e is a bright-field optical microscope image of cells stained with Sirius red dye. [Figure 8] This graph shows the results of anti-inflammatory tests using lipopeptides. [Figure 9a]This graph shows the percentage of wound healing over time for a control sample and for lipopeptide(II) in a rat excision wound model. [Figure 9b] Figure 9a shows a graph of the rat's body weight over time during the rat experiment. [Modes for carrying out the invention]

[0062] [Detailed description of the invention] In one embodiment, the present invention relates to novel bioactive lipopeptide molecules. These lipopeptide molecules are conjugates of peptides, linkers, and hydrocarbon groups. These lipopeptides enhance collagen production per cell by more than twofold compared to controls. This activity is observed at low concentrations below the critical aggregation concentration (CAC). Above the CAC, these molecules self-assemble to form clearly defined nanoscale spherical micelles.

[0063] As used herein, the term "bioactivity" encompasses any kind of interaction with other biomolecules, such as proteins, glycoproteins, carbohydrates, etc., but is not limited to these.

[0064] The term "lipopeptide" refers to a conjugation of a peptide with a lipid or aliphatic chain (via an amide linkage, thioether linkage, or thioester linkage, or a linker as defined elsewhere herein). The term lipopeptide is used herein interchangeably with the terms "peptide amphiphilic substance," "amphiphilic molecule," "peptide conjugate," "bioactive peptide conjugate," or "molecule of structure (I)."

[0065] The term "peptide," as used herein, refers to a sequence of amino acids linked by peptide bonds or modified peptide bonds. A dipeptide contains two amino acids, a tripeptide contains three amino acids, and the term oligopeptide is generally used to describe a peptide having 2 to about 50 or more amino acids. In the present invention, the peptide of a lipopeptide must contain 2 to 10 amino acid residues. Preferred peptides contain 3 to 10 amino acids, for example, 4 to 10 amino acids or 5 to 10 amino acids. A peptide may contain 2 to 8 amino acids, for example, 2 to 6 amino acids or 2 to 5 amino acids. It is preferable that the peptide contains 3 to 8 amino acids, for example, 4 to 6 amino acids. It is most preferable that the peptide contains 5 amino acids.

[0066] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function in a manner similar to naturally occurring amino acids. The amino acids are preferably α-amino acids, more preferably naturally occurring α-amino acids.

[0067] Amphiphilic peptides, possessing both hydrophilic and hydrophobic ends, frequently exhibit self-assembly properties and possess a wide range of biological functions. When a specific concentration of amphiphilic peptides is present in an aqueous solution, self-assembly can form nanoparticles. Examples of such nanoparticles include micelles, vesicles, and nanofibers. These secondary structures can directly influence the peptide's functionality.

[0068] The lipopeptides of the present invention have been observed to form micelles under certain conditions. These micelles possess properties such as improved targeted delivery, thereby making them promising carriers for bioactive compounds. The micelles are preferably topologically spherical.

[0069] The concentration at which premicelle aggregates are formed is determined by the CAC. In the context of this invention, the micelle structure is formed at concentrations above the CAC. However, the lipopeptides of this invention have been observed to be active even at concentrations below the CAC.

[0070] As used herein, the terms “hydrocarbon substituent” or “hydrocarbon group” refer to a group having carbon atoms directly bonded to the remainder of the molecule and having primarily hydrocarbon characteristics. Examples of hydrocarbon groups include (i) hydrocarbon substituents, i.e., aliphatic (e.g., alkyl or alkenyl) substituents, alicyclic (e.g., cycloalkyl, cycloalkenyl) substituents, as well as aromatic-substituted substituents, aliphatic-substituted substituents, and alicyclic-substituted substituents, as well as cyclic substituents where the ring is completed through another part of the molecule (e.g., two substituents together forming a ring), and optionally, hydrocarbon polymers such as polyethylene; (ii) substituted hydrocarbon substituents, i.e., substituents containing non-hydrocarbon groups that do not alter the primarily hydrocarbon properties of the substituent in the context of the present invention (e.g., halo (especially chloro and fluoro), hydroxy, alkoxy, mercapto, alkylmercapto, nitro, nitroso, and sulfoxy); and (iii) heterosubstituted substituents, i.e., substituents that, in the context of the present invention, have primarily hydrocarbon characteristics but otherwise contain non-carbon elements in a ring or chain composed of carbon atoms. Examples of heteroatoms include sulfur, oxygen, and nitrogen, and include substituents such as pyridyl, furyl, thienyl, and imidazolyl. Generally, within a hydrocarbyl group, there are two or fewer non-hydrocarbon substituents for every 10 carbon atoms, or one or fewer. Generally, there are no non-hydrocarbon substituents within a hydrocarbyl group.

[0071] As used herein, the term "hydrocarbon" refers to the hydrocarbon group defined above, having an alkyl group (preferably a C1-C6 alkyl group) bonded to a central carbon chain.

[0072] The hydrocarbon group is preferably unsaturated. Where used herein, the term "unsaturated" refers to a hydrocarbon group as defined above having at least one carbon-carbon multiple bond, e.g., a double or triple bond. The hydrocarbon group preferably contains at least one double bond, more preferably at least two double bonds, and most preferably three double bonds.

[0073] In preferred embodiments, the R group is a terpenoid group. As used herein, the term “terpenoid” is derived from terpenes and refers to a class of organic compounds containing terpenes. Terpenes have five carbon isoprene units and additional functional groups, generally oxygen-containing functional groups. Terpenes are a subset of terpenoids. Terpenoids can be characterized in terms of the number of isoprene units, as shown in the table below.

[0074] The hydrocarbon is preferably a branched hydrocarbon. In alternative embodiments, the R group is a branched lipid. Preferred branched lipids include phytol, tocopherol, garcinol, or lipoyl groups. Other preferred branched lipids include geranyl and geranylgeranyl.

[0075] [Table 1]

[0076] The R group is preferably a sesquiterpene group.

[0077] In a particularly preferred embodiment, the R group is a farnesyl group:

[0078] [ka] That is the case.

[0079] Peptide "P" contains 2 to 10 amino acids. In some embodiments, the peptide contains about 5 amino acids, i.e., it is a pentapeptide.

[0080] In preferred embodiments, the peptide is a collagen-promoting peptide. “Collagen-promoting peptide,” as used herein, refers to a class of compounds having the biological activity of enhancing collagen production. According to the present invention, collagen-promoting peptides encompass all natural or synthetic peptides capable of enhancing collagen production in the human body.

[0081] Suitable collagen-promoting peptides can be determined, for example, using collagen production assays described later in this specification.

[0082] Known collagen-promoting peptides include those having the sequences KTTKS, GHK, and AHK. Therefore, in a preferred embodiment of the present invention, peptide P is selected from KTTKS, GHK, and AHK. Other known collagen-promoting peptides include GPKG and LSVD, and therefore these are also preferred embodiments of peptide P.

[0083] Other collagen-promoting peptide sequences may also constitute peptide L in some embodiments. These are listed in the table below.

[0084] [Table 2] TIFF2026530578000009.tif29170

[0085] Details of these peptides are available in Advanced Drug Delivery Reviews, Volume 185, June 2022, 114240, which is incorporated by reference, and in the publications cited therein.

[0086] Therefore, peptides can be selected from the list consisting of KTTKS, GHK, AHK, palmitoyl dipeptide-6, palmitoyl-KT, dipeptide-1, carnosine, dipeptide-2, tripeptide-1 copper, tripeptide-1 manganese, tripeptide-38, RGD, tripeptide-2, tripeptide-10, tripeptide-5, tetrapeptide-7, PKEK, tetrapeptide-2, tetrapeptide-5, tetrapeptide-9, tetrapeptide-11, tetrapeptide-21, tripeptide-56, pentapeptide-3, pentapeptide-18, hexapeptide-3, hexapeptide-10, hexapeptide-11, hexapeptide-12, oligopeptide-20, octapeptide-3, nonapeptide-1, oligopeptide-68, and oligopeptide-10. Therefore, the peptides are KTTKS, GHK, AHK, Lys-Val-diaminohydroxybutyrate, Lys-Thr, Tyr-Arg, β-Ala-His, Val-Trp, Cu-Gly-His-Lys, Gly-His-Lys-Mn 2+, Lys-Met(O2)-Lys, Arg-Gly-Asp, Val-Tyr-Val, Lys-α-Asp-Ile, Lys-Val-Lys, Gly-Gln-Pro-Arg, Pro-Lys-Glu-Lys, Lys-Asp-Val-Tyr, β-Ala-His-Ser-His, Gln-Asp-Va l-His, Pro-Pro-Tyr-Leu, Gly-Glu-Lys-Gly, Lys-His-Gly, Gly-Pro-Arg-Pro-Ala, Tyr-D-Ala-Gly-Phe-Leu, Glu-Glu-Met-Gln-Arg-Arg, Ser-Ile-Lys-Val-Ala-Val, Phe -Val-Ala-Pro-Phe-Pro, Val-Gly-Val-Ala-Pro-Gly, Arg-Arg-Leu-Glu-Met-Tyr-Cys-Ala-Pro-Leu-Lys-Pro, Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp, Met-Pro-D-Phe-Arg- It can be selected from the list consisting of D-Trp-Phe-Lys-Pro-Val, Arg-Asp-Gly-Gln-Ile-Leu-Ser-Thr-Trp-Tyr, and Phe-Ala-Lys-Ala-Leu-Lys-Ala-Leu-Leu-Lys-Ala-Leu-Lys-Ala-Leu. The peptides can be selected from a list consisting of ETTES, GPKG, LSVD, KT, Beta-AH (Beta-A: β-alanine), VW, RGD, RGDS, VYV, KVK, GQPR, PKEK, KQVY, Beta-AHSH, QDVH, PPYL, GEKG, KHG, GPRPA, YaGFL (a: D-alanine), EEMQRR, SIKVAV, FVAPFP, VGVAPG, RRLEMYCAPLKP, EEMERRAD, MPfRwFKPV (w: D-tryptophan; f: D-Phe), RRGQILSTWY, and FAKALKALLKALKAK.

[0087] Peptide P preferably consists of the amino acid sequence KTKKS.

[0088] When used herein, the term "linker" refers to a molecule that functions to link, i.e., conjugate or join, two components of a molecule, namely a peptide P and a (optionally branched) hydrocarbyl group R, preferably by covalent bonds (or multiple bonds), and thus connect the two. In some embodiments, the linker comprises one or more amino acids (e.g., a peptide or a protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical part.

[0089] Linker L preferably contains a polyether moiety, such as polyethylene glycol, polypropylene glycol, or polytetramethylene glycol. The general formula for the polyether group is shown below:

[0090] [ka] In the formula, n and m are integers independently selected from 1, 2, and 3. m is preferably 1, and n is preferably 2.

[0091] In a preferred embodiment, the linker L is of formula:

[0092] [ka] (wherein n and m are as defined above) has the following: In this embodiment, the lipopeptide is of formula:

[0093] [ka] The formula has (wherein R, P, n, and m are as defined above). In this embodiment, it is preferable that m is 1 and n is 2.

[0094] In a very preferred embodiment, the linker L is of formula:

[0095] [ka] It has.

[0096] In this embodiment, the lipopeptide is given by formula:

[0097] [ka] (wherein R and P are as defined above)

[0098] According to a very preferred embodiment, the lipopeptide is of the formula:

[0099] [ka] It has.

[0100] This lipopeptide is referred to herein as FMdEG-KTTKS, and also as lipopeptide(II).

[0101] The present invention also provides micelles formed by the self-assembly of the lipopeptides (or more) of the present invention. Surprisingly, the inventors have found that the lipopeptides of structure (I) spontaneously self-assemble to form micelles. Micelles can also be formed from multiple lipopeptides of structure (I). The lipopeptides of the present invention self-assemble to form micelles at concentrations exceeding a defined CAC. A CAC of approximately 0.04 wt% is preferred.

[0102] Therefore, in one embodiment, the lipopeptide of structure (I) self-assembles to form a certain structure. Such a structure may be, for example, a micelle, a vesicle, or the like. The structure is preferably a micelle. The lipopeptide of the present invention spontaneously self-assembles to form micelle nanoparticles. The diameter of these nanoparticles or micelles can range from about 1.5 nm to 10 nm. The diameter of the nanoparticles is preferably 2 nm to 5 nm.

[0103] In one embodiment, the present invention provides a method for enhancing extracellular matrix protein production in cells. The extracellular matrix protein is collagen. Preferably, the collagen is type I or type II collagen. Therefore, in one embodiment, the present invention provides a method for increasing collagen production in cells.

[0104] In one embodiment, the cells may be cultured cells. This gives rise to another aspect of the present invention, which is a method for enhancing collagen production, comprising the step of culturing cells in the presence of an aqueous medium containing a lipopeptide of structure (I).

[0105] As used herein, the term “enhances collagen production” refers to an increase in the amount of collagen biosynthesized and / or secreted by cells. Compared to a suitable control, the increase in amount may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more. For example, the increase in amount means an increase of at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, or more. Preferably, the increase in amount means an increase of at least 200% or more.

[0106] In the context of this specification, a cell may be a single cell, a group of cells, or a tissue, organ (e.g., skin), or organ system. In the case of cells in vivo, contact may be providing lipopeptides to the target.

[0107] In some embodiments, the composition is impregnated into an article. The article may be a bandage or patch, for example, gauze, lint, absorbent cotton, cloth, a semipermeable film (e.g., polyurethane film), a semipermeable foam (e.g., silicone), a hydrogel (e.g., containing methacrylate and / or polyvinylpyrrolidine), a hydrocolloid (e.g., containing carboxymethylcellulose, gelatin and / or pectin), a hydrofiber (e.g., containing sodium carboxymethylcellulose), or an alginate (e.g., containing sodium alginate and / or calcium).

[0108] In another embodiment, the present invention provides a cosmetic composition comprising a lipopeptide of structure (I) and a physiologically acceptable or pharmaceutically acceptable culture medium.

[0109] Compositions suitable for external administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils, etc. In some embodiments, the composition is applied as an external ointment or cream.

[0110] The composition also typically contains pharmaceutically acceptable concentrations of salts, buffers, preservatives (e.g., antioxidants), adjuvants and cytokines, and optionally other therapeutic agents.

[0111] The present invention provides the use of lipopeptides of structure (I) or compositions comprising lipopeptides of structure (I) in cosmetic or non-therapeutic treatments. Applications may range from skincare for the face, body, and hair to makeup treatments, for example. These may include, but are not limited to, effects on skin texture, fine lines, wrinkles, skin elasticity, suppleness, reconstruction, and radiance, as well as effects on hair growth and scalp condition. Other uses of lipopeptides of structure (I), such as in methods for producing tissue in vitro, are also intended herein.

[0112] In another embodiment, the present invention provides a pharmaceutical composition comprising a lipopeptide of structure (I) and a pharmaceutically acceptable carrier.

[0113] The composition may also conventionally contain, at cosmetically and / or pharmaceutically acceptable concentrations, salts, buffers, preservatives (e.g., antioxidants), auxiliary immunostimulants (e.g., adjuvants and cytokines), and optionally other therapeutic agents.

[0114] The amount of lipopeptide of structure (I) that can be incorporated into the pharmaceutical composition may be 0.0001 wt% to 10 wt% relative to the weight of the pharmaceutical composition, for example, 0.001 wt% to 5 wt%, 0.001 wt% to 1 wt%, 0.002 wt% to 0.5 wt%, or 0.005 wt% to 0.05 wt%.

[0115] In another embodiment, the present invention provides lipopeptides for use in methods for preventing, treating, or improving wounds in target skin. The present invention also provides pharmaceutical compositions for use in methods for treating wounds in target skin.

[0116] Skin wounds may include abrasions (e.g., linear abrasions or scratches, patterned abrasions and grazed or brush abrasions), lacerations (e.g., splits, lacerations, stretch lacerations, perforations, blast wounds, cut lacerations, crush lacerations or degloving injuries), thermal injuries (e.g., burns, radiation burns, chemical burns or electrical burns), surgical incisions, pressure ulcers, skin lacerations, arterial ulcers, venous ulcers, or contusions.

[0117] The present invention also relates to a method for treating wounds in target skin. This method involves administering a lipopeptide (for example, in a therapeutically effective dose) to the wound in the target skin according to the present invention.

[0118] The subject may be a human. The subject may be an animal. The animal may be an animal prone to skin wounds, such as a cat, dog, horse, cow, sheep, goat, pig, or bird.

[0119] Inflammation-related conditions can be T helper cell (TH)-mediated diseases or conditions, for example, T H 1. A disease or condition mediated by T H It may be a 2-mediated disease or condition.

[0120] T H 1. The mediated response is insufficient or low in activity. H1. Transmitted diseases or medical conditions include: rheumatoid arthritis (RA); psoriatic arthritis; psoriasis; inflammatory bowel syndrome (IBD); Crohn's disease; ulcerative colitis; multiple sclerosis (MS); influenza, including generalized influenza; respiratory diseases, such as those caused by viruses, including respiratory syncytial virus (RSV); cystic fibrosis (CF); herpes, including genital herpes; sepsis and septic shock; bacterial pneumonia; bacterial meningitis; dengue hemorrhagic fever; endometriosis; prostatitis; uveitis; uterine ripening; alopecia areata; ankylosing spondylitis; celiac disease; dermatomyositis; type 1 diabetes; Goodpasture syndrome; Graves' disease Diseases; Guillain-Barré syndrome; juvenile idiopathic arthritis; Hashimoto's thyroiditis; idiopathic thrombocytopenic purpura; lupus erythematosus; mixed connective tissue disease; myasthenia gravis; narcolepsy; osteoarthritis; pemphigus vulgaris; pernicious anemia; polymyositis; primary biliary cirrhosis; relapsing polychondritis; Sjögren's syndrome; temporal arteritis; vasculitis; Wegner granulomatosis; age-related macular degeneration; infectious diseases (e.g., infections caused by Mycobacterium tuberculosis (Mt), human immunodeficiency virus (HIV), or coronavirus); autoimmune disorders (e.g., arthritis, multiple sclerosis, or type 1 diabetes); cancer; post-cancer surgery or cancer treatment; and post-immunisation; may be one or more selected from this group. H 1. The vector-borne disease or medical condition is preferably one or more of the following: cystic fibrosis (CF), type 1 diabetes mellitus, age-related macular degeneration, or infectious disease (e.g., infection caused by Mycobacterium tuberculosis (Mt), human immunodeficiency virus (HIV), or coronavirus).

[0121] T H T is a T with hyperactive bimediated response. H2-mediated diseases or medical conditions may be one or more selected from / consisting of the group including allergies (e.g., rhinitis, allergic dermatitis, urticaria), asthma, eczema, hay fever, urticaria, chronic graft-versus-host disease, progressive systemic sclerosis, systemic lupus erythematosus; chronic lung disease; scleroderma; anaphylaxis; atrophy (e.g., muscle); and type 1 hypersensitivity disorders, including graft rejection. H The two-mediated disease or medical condition is preferably one or more of the following: allergies (e.g., rhinitis, allergic dermatitis, urticaria), asthma, eczema, and hay fever.

[0122] Collagen-related conditions can be selected from a list consisting of osteogenesis imperfecta, achondroplasia, Ehlers-Danlos syndrome, Alport syndrome, osteoporosis, and Knobloch syndrome. Collagen-related conditions can be selected from a list consisting of osteogenesis imperfecta, Ehlers-Danlos syndrome, childhood cortical hyperostosis (Caffe's disease), collagen disorders (types II and XI), Dupuytren's contracture, Alport syndrome, Goodpasture syndrome, Ulrich myopathy, Bethlem myopathy, atopic dermatitis, dystrophic epidermolysis bullosa, posterior polymorphic corneal dystrophy 2, EDM2 and EDM3, Schmidt metaphysical dysplasia, bullous pemphigoid, and junctional epidermolysis bullosa.

[0123] The articles of the present invention may include lipopeptides coated and / or immobilized (e.g., bonded) to a material. Techniques that can be used to apply lipopeptides to surfaces include 3D printing, electrospinning, electrophoretic deposition, dip coating, drop casting, sol-gel deposition, biomimetic deposition, plasma spraying, layer-by-layer deposition, physical vapor deposition, anodizing (e.g., K. Kravanja et al., Materials & Design 217(2022)110653), hydrogel coating techniques (e.g., S. Bohara et al., Biomaterials Research (2022)26:26), amphiphilic polymer coatings (e.g., W. Xi et al., Nat. Comm. (2021)12:5473), polyleukocyte and platelet-rich fibrin products, e.g., fibronectin / vitronectin exudates (e.g., M. Lollobrigida et al., BioMed Res. Int., (2018):9031435), and / or nanocoatings (e.g., Ming-qi Chen, Front. Bioeng. Biotechnol. (2022)10:878257) is one example.

[0124] Therefore, lipopeptides can be incorporated into, impregnated into, or coated onto materials such as plastics, metals, wood, and / or textiles. These materials can be used to form all or part of an article. Preferably, these materials can be used to form at least part of the surface of an article. This makes the lipopeptides available for contact with the target tissue.

[0125] The article may be a medical device such as an implant or prosthesis. The article is preferably a bandage (e.g., a wound dressing). The bandage may be adhesive or non-adhesive. The bandage may be or may contain gauze, lint, absorbent cotton, cloth, semipermeable film (e.g., polyurethane film), semipermeable foam (e.g., silicone), hydrogel (e.g., containing methacrylate and / or polyvinylpyrrolidine), hydrocolloid (e.g., containing carboxymethylcellulose, gelatin and / or pectin), hydrofiber (e.g., containing sodium carboxymethylcellulose), or alginate (e.g., containing sodium alginate and / or calcium). The bandage is preferably an impregnated bandage, in which the bandage is impregnated with a composition containing lipopeptides, e.g., lipopeptides and retinoids. Hydroxyethylcellulose is a possible additional or alternative component of the hydrocolloid or hydrogel. The article is preferably a gel, e.g., a hydrogel. Hydrogels can be made from natural materials such as collagen (e.g., derived from jellyfish). Hydrogels may contain hydroxyethylcellulose.

[0126] Articles (e.g., bandages or other medical devices) may be sterile and, optionally, may be provided in sterile packaging to prevent contamination before use. Articles can be selected from permanent implants, e.g., artificial heart valves, voice prostheses, artificial joints, intraocular lenses, stents (e.g., vascular stents), and shunts (e.g., hydrocephalus shunts); as well as non-permanent implants, e.g., pacemakers and pacemaker leads, drainage tubes, endotracheal or gastrointestinal tubes, temporary or trial artificial joints, surgical pins, guidewires, surgical staplers, cannulas, subcutaneous or percutaneous ports, and indwelling catheters and catheter connectors, and contact lenses. Implants may be orthopedic implants. In one embodiment, the article is a catheter. Examples of indwelling catheters include urethral catheters, vascular catheters (e.g., central venous catheters, dialysis catheters, peripheral venous catheters, arterial catheters, and pulmonary Swan-Ganz catheters), peritoneal dialysis catheters, central venous catheters, and needleless connectors.

[0127] It has been found that compositions containing the lipopeptide of the present invention at a concentration of less than 0.05 wt% (e.g., CAC (0.04 wt%) or less) may have lower cytotoxicity than compositions containing higher concentrations of the lipopeptide. Therefore, it is preferable that the lipopeptide composition contains the lipopeptide at a concentration of less than 0.05 wt%, or 0.049 wt%, or 0.039 wt%, or 0.03 wt%, or 0.03 wt%, for example, 0.00001 wt% to 0.049 wt%, or 0.0001 wt% to 0.049 wt%, or 0.001 wt% to 0.049 wt%.

[0128] [Examples] Preparation of farnesyl-mercapto(diethylene glycol)-KTTKS (FMdEG-KTTKS, lipopeptide(II))

[0129] [ka]

[0130] Peptide farnesylation was performed according to the method described in N. McCarthy et al., Biochem. J. (2000) 347, 163-171. A peptide was synthesized, and an Fmoc-protected 9-atom polyethylene glycol spacer (di[ethylene glycol]) was added to its N-terminus. Trityl-protected mercaptopropionic acid was then coupled to this spacer using a standard amino acid coupling method, and the method described in the above paper was used for the addition of the farnesyl lipid chain.

[0131] Self-assembly experiment of lipopeptides (II) As the concentration increases, lipopeptides (II) self-assemble. The minimum concentration at which aggregation occurs is identified as the critical aggregation concentration (CAC). Self-assembly of lipopeptides at concentrations above the CAC was investigated using dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryo-TEM imaging.

[0132] CAC was measured using the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). The fluorescence of this probe is sensitive to the hydrophobicity of its surrounding environment.

[0133] Experiments were conducted using a Varian Model Cary Eclipse spectrofluorometer with a 10.0 mm × 5.00 mm quartz cell. The CAC value was estimated by titration with the fluorescent dye 8-anilino-1-naphthalenesulfonic acid (ANS). (2 × 10⁻⁶) -3 A mother solution of 0.2 wt% FMdEG-KTTKS(II) was prepared using wt% ANS as the solvent. 2 × 10 -3 By weighing and adding wt%ANS, 0.15~2×10 -3 Dilutions in the wt% FMdEG-KTTKS range were prepared from the mother solution. The fluorescence emission spectra of a series of dilutions were recorded between 400 nm and 670 nm, with the sample excited at 356 nm.

[0134] Figure 1b shows the variation in intensity (I0) and wavelength (λ0) of the maximum fluorescence emission peak for solutions containing ANS at different concentrations, plotted as a function of lipopeptide concentration. A clear discontinuity was observed in both quantitative values ​​at the same concentration, leading to the determination that 0.04 wt% corresponds to CAC.

[0135] Conformational analysis of lipopeptides The conformation of lipopeptides at concentrations exceeding CAC was investigated using circular dichroism (CD) spectroscopy and Fourier transform infrared (FTIR) spectroscopy.

[0136] Circular dichroism (CD) spectra were recorded using a Chirascan spectropolarimeter (Applied Photo Physics, Leatherhead, UK). The solution was placed between parallel plates (path length 0.01 mm or 0.1 mm). CD spectra were measured with a step size of 0.5 nm, a bandwidth of 0.5 nm, and an acquisition time of 1 second per step. Background CD signals using water were subtracted from the CD data of the sample solution. The CD signals were smoothed using Chirascan Software for data analysis. To avoid artifacts in the smoothed curve, the calculated residuals were selected to vary around the mean value. The CD spectrum shown in Figure 3a is characteristic of the irregular peptide conformation.

[0137] Fourier transform infrared (FTIR) spectra were recorded using a Thermo-Scientific Nicolet iS5 instrument equipped with a DTGS detector, employing a Specac Pearl liquid cell (with the sample contained between fixed CaF2 plates). The spectra were recorded at 900–4000 cm⁻¹. -1 The range was scanned 116 times. The FTIR spectrum shown in Figure 3b is also 1650 cm⁻¹. -1 The presence of numerous peaks in the vicinity is consistent with similarly irregular conformation. 1672cm -1The peak at this point is due to the bound TFA counterion.

[0138] Aggregates were detected using dynamic light scattering (DLS). Dynamic light scattering measurements were performed using the ALV / CGS-3 Compact Goniometer System with an ALV / LSE-5003 correlator, with perpendicularly polarized incident light at a wavelength of λ=632.8 nm. Measurements were performed at θ=90° relative to the incident beam, and data was collected three times over 30 seconds. The intensity autocorrelation function was analyzed using the regularization method. The size distribution was calculated from the autocorrelation function, weighted by the number of test materials. The hydrodynamic radius RH was measured from the maximum value of the size distribution. Figure 3c shows a typical intensity distribution, where the sharp peak corresponds to a hydrodynamic radius Rh=4 nm, indicating small aggregates.

[0139] Small-angle X-ray scattering (SAXS) provides detailed structural information about aggregates. Synchrotron SAXS experiments on solutions were performed at ESRF (Grenoble, France) using a BioSAXS robot on beamline BM29. Several microliters of sample were injected into a quartz capillary (1.8 nm inner diameter) at a slow, highly reproducible rate under the X-ray beam using an automated sample changer. To avoid parasitic scattering, the quartz capillary was sealed in a vacuum chamber. Once the sample was injected into the capillary and reached the X-ray beam, the flow was stopped while SAXS data was acquired. The q range was 0.004–0.4 Å. -1 The settings were adjusted to λ = 1.03 Å. Images were captured using the Pilatus 1M detector. Data processing (background removal, radial averaging) was performed using the dedicated beamline software ISPYB.

[0140] The data shown in Figure 3d demonstrates a flat low-q intensity behavior (for the lowest concentration) along with clearly defined shape factor oscillations, which are characteristic features of the spherical shape factor. The outer micelle radius is (2.5 ± 0.1) nm, which is consistent with DLS when considering the difference between the hydrodynamic radius and the thermodynamic radius due to SAXS.

[0141] For the two higher concentration solutions tested (1 wt% and 5 wt%, data shown in Figure 3d), a broad structure factor peak was present at low q, which can be described by including rigid sphere structure factor in the fitting to the data (fitting parameters in SI Table S1).

[0142] SAXS model. All fittings were performed using SASfit software. SAXS intensities were fitted using the spherical shell shape factor along with the rigid sphere structure factor. The parameters for the spherical shell model are the total radius of the spherical shell R1, the core radius R2, the scattering contrast μ of the inner core, and the scattering contrast τ of the shell. The parameters for the rigid sphere model are the radius R of the rigid sphere. HS , and the volume fraction Φ of the rigid sphere. The background of all SAXS curves was fitted using a constant term C.

[0143] [Table 3]

[0144] However, for these higher concentrations, high q-shape factor oscillations are retained, which indicates a slight change in the inner micelle structure (as well as indicated by fitting parameters other than τ, which represent the overall scaling factor of the concentration-dependent scattering contrast).

[0145] Cryo-TEM imaging was performed using a field emission cryo-electron microscope (JEOL JEM-3200FSC) operating at 200kV. Images were acquired in bright-field mode using zero-loss energy filtering (omega type) with a slit width of 20eV. Micrographs were recorded using a Gatan Ultrascan 4000 CCD camera. The test material temperature was maintained at -187°C during imaging. The vitrification test material was prepared using an automated FEI Vitrobot device with a Quantifoil 3.5 / 1 holy carbon copper grid with a pore size of 3.5 μm. The grid was plasma-cleaned using a Gatan Solarus 9500 plasma cleaner immediately before use and then transferred to the FEI Vitrobot's environmental chamber at room temperature and 100% humidity. Subsequently, 3 μl of the sample solution was applied to the grid, blotting was performed twice for 5 seconds each, and then vitrification was performed in a 1 / 1 mixture of liquid ethane and propane at a temperature of -180°C. The grid containing the vitrified sample solution was maintained at liquid nitrogen temperature and then transferred to a microscope by cryotransfer.

[0146] Figure 3e shows a typical cryo-TEM image with well-defined, relatively monodisperse micelles having sizes consistent with those obtained by DLS and SAXS. The micelle formation is consistent with the observed irregular conformation of peptide chains on the micelle surface. This behavior is consistent with C in nanotapes. 16 -This is in contrast to the β-sheet structure observed for KTTKS (V. Castelletto, IW Hamley, J. Perez, L. Abezgauz and D. Danino, Chemical Communications, 2010, 46, 9185-9187.)

[0147] Collagen production assay Cell Culture: Human dermal fibroblasts derived from adult donor (HDFa) cells were purchased from Sigma Aldrich (UK, product code 106-05A). For cytotoxicity and collagen production assays, the cells were pre-grown by incubation at 37°C in a 5% CO2 atmosphere in DMEM / F12 (Thermo Fisher Scientific, UK) supplemented with 5% FBS (fetal bovine serum obtained from Thermo Fisher Scientific-UK), Glutamax, and the antibiotics penicillin 100 IU / mL, streptomycin 100 μg / mL, and Gibco® Antibiotic-Antimycotic (Gibco-UK) 0.25 μg / mL.

[0148] Cytotoxicity test MTT Cell Compatibility Assay: MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide-Sigma-UK) is a yellow, water-soluble chemical. When incubated with live cells, MTT is converted into insoluble, chromogenic formazan crystals by mitochondria present in the cytoplasm. Therefore, by using the conversion of MTT to formazan crystals, the presence and number of live cells can be estimated by a colorimetric quantitative assay.

[0149] For the MTT assay, cells were harvested using 0.25% trypsin / EDTA (Gibco-UK) and placed in 96-well plates in DMEM / F12 with 5% FBS, Glutamax, Gibco® Antibiotic-Antimycotic (Gibco-UK) and 5 μg / mL insulin (Sigma-UK), with 1 × 10 cells per well. 4 The cells were seeded at a density of 1. The cells were incubated overnight at 37°C in a 5% CO2 atmosphere. The following day, the medium was replaced with DMEM / F12 with six different concentrations of FMDEG-KTTKS, without supplementation.

[0150] To evaluate the cytotoxicity of conventional stimulants in relation to collagen production, cells were incubated with 1 mM ascorbic acid, 0.025 ng / mL TGFβ1 (Invitrogen, UK), and 0.01 mg / mL insulin, while a control group was incubated in DMEM / F12 alone. After 3 days (72 hours) of incubation, cells were washed three times with PBS and incubated with 0.5 mg / mL MTT (Invitrogen) diluted in DMEM / F12 without phenol red at 37°C for 4 hours. Subsequently, DMSO was added to solubilize the formazan crystals, and the plates were incubated again at 37°C for 15 minutes. After this final incubation, absorbance at 560 nm was read using an Infinite F50 plate reader (Tecan-Switzerland). The data were analyzed using prism 8 software. First, background noise was removed by subtracting the OD of the control well containing only the culture medium from the OD obtained using MTT. The number of cells was then estimated by comparing the absorbance at 560 nm with that of a control incubated with DMEM / F12 alone in a triplicate. Statistical tests for selection were standard one-way ANOVA and Bonferroni correction for multiple comparisons.

[0151] Figure 4 shows data from this assay for peptide conjugates at different concentrations (0.2–0.00625 wt%) above and below CAC, as well as for conventional stimulants, e.g., insulin, ascorbic acid, and TGFβ1, which have been previously used in the literature for stimulating collagen production in fibroblasts by different mechanisms; e.g., insulin, ascorbic acid, and TGFβ1, which result in higher collagen deposition through fibroblast activation and proliferation. (ANOVA, n=3), **p<0.01, ***p<0.001.

[0152] Compared to controls incubated in basic medium alone, a statistically significant growth effect of conventional irritants was observed, and a decrease in viability of approximately 30% (compared to basic medium) was observed in cells incubated with peptide conjugates below CAC (those with a lower CAC concentration) (not statistically significant). Samples incubated with peptide conjugates above CAC showed a very large decrease in cell density compared to controls, indicating high cytotoxicity when exceeding CAC. MTT assays showed much better tolerance to lipopeptides at concentrations below CAC. MTT assays also indicated that lipopeptides (e.g., FMDEG-KTTKS) have good cell viability in monomeric form. 16 -In the case of KTTKS, even at concentrations as low as one-tenth of those used in the present invention (in the range of 0.002 to 0.008 wt%), the cell viability decreased by more than 40%, therefore, C 16 -Cell compatibility is improved compared to sequences based on KTTKS.

[0153] Collagen production assay To estimate collagen production in HDFa cells, similar to MTT cells, 1 × 10 cells per well were fed complete DMEM + 5 μg / mL insulin. 4 Cells were seeded at a density of 1,000 cells and incubated for 3 days in unreplenished DMEM in the presence of different concentrations of peptides and conventional stimulants (1 mM ascorbic acid, 0.025 ng / mL TGFβ1, and 0.01 mg / mL insulin). After incubation, the wells were washed with PBS and fixed with ice-cold 70% ethanol to fix the material deposited on the surface. The plates were transferred to a -80°C cryogenic refrigerator for 10 minutes and washed with water to remove all residual ethanol. After fixation, the collagen was incubated overnight at 4°C with gentle stirring in a solution containing 1 mg / mL Direct Red in picric acid. Finally, the wells were washed with distilled water until all excess dye was removed, and then the collagen and cells were treated with 1 mM NaOH at room temperature for 10 minutes, and the absorbance at 490 nm was measured.

[0154] Polarizing microscope: 5 × 10 for microscopic assays. 4 100 HDFa cells were placed on coverslips and incubated overnight in a 24-well plate in DMEM / F12 with 10% FBS, 2 mM glutamax, antibiotic-antimycotic, and 5 μg / mL insulin. The following day, the cells were washed and incubated for 3 days with lipopeptides and conventional irritants, while controls were incubated in DMEM / F12 alone without any supplements. The coverslips were washed three times with PBS, fixed with cold ethanol, and stained with Sirius red. After incubation with Sirius red, the coverslips were washed with distilled water to remove all unbound dye and placed on glass slides. The slides were imaged using an Olympus BX41 microscope (Olympus Life Sciences, UK) equipped with polarizers.

[0155] Collagen production was evaluated for three lipopeptide concentrations below CAC. Figure 5a shows the average amount of collagen [mg / mL] produced in each well containing HDFa cells in the presence of FMdEG-KTTKS, conventional irritants, and a basic medium without any additives. Figure 5b shows the amount of collagen produced per cell.

[0156] Figure 6 shows polarized light microscopy (POM) images of cells stained with Sirius red. Specifically, Figure 6 shows polarized light microscopy images of Sirius red-stained HDFa cells in the presence of (a) basic medium, (b) conventional irritants, (c) 0.00625 wt% FMdEG-KTTKS, (d) 0.0125 wt% FMdEG-KTTKS, and (e) 0.025 wt% FMdEG-KTTKS. Scale bar = 200 mm.

[0157] Figure 7 shows bright-field microscopy images of Sirius red-stained HDFa cells in the presence of (a) basic medium, (b) conventional irritants, (c) 0.00625 wt% FMdEG-KTTKS, (d) 0.0125 wt% FMdEG-KTTKS, and (e) 0.025 wt% FMdEG-KTTKS. Scale bar = 200 μm.

[0158] The quantitative data in Figure 5a show a slight increase in collagen production in cells incubated with all three concentrations of lipopeptides tested, and a significant increase in cells incubated with conventional stimulants. Figure 5b shows the mean collagen production per cell [% compared to control] (ANOVA, n=5), *p<0.05, **p<0.01. When collagen production per cell is plotted (Figure 5b), a statistically significant increase is clearly observed for all concentrations. For example, incubation in DMEM / F12 with 0.0125% FMdEG-KTTKS(II) showed a 221±41% increase in collagen production per cell compared to the control group incubated with medium alone. In contrast to the results obtained for FMdEG-KTTKS(II), conventional stimulants showed only a minimal increase in collagen production per cell, which was not statistically significant. This suggests that the overall increase in collagen production is mainly attributable to the increased proliferative capacity of dermal fibroblasts under the influence of TGF-β and insulin.

[0159] HDFa immunocytochemical test Immunocytochemical assays were performed on HDFa cells to compare the production of type I collagen and type III collagen using confocal microscopy.

[0160] 9×10 4A quantity of HDFa cells was placed on a 13 mm coverslip in a 24-well plate, using DMEM / F12 medium containing 5% FBS, and incubated for 24 hours in a cell incubator at 37°C in a 5% CO2 atmosphere to allow adhesion to the coverslip. For the assay, cells were incubated for 72 hours in a cell incubator with conventional supplements, DMEM / F12 containing lipopeptide(II), or medium alone. After 72 hours of incubation, cells were washed three times with PBS, fixed with 4% paraformaldehyde (Thermo Fisher Scientific-UK) at room temperature for 10 minutes, washed three more times with PBS, and permeabilized with 0.1% Triton-X 100 (Merck-UK) in PBS for 15 minutes. The coverslip was washed again with PBS and blocked with 1% BSA at room temperature for 1 hour. The cells were washed three times again with PBS, with the final wash lasting 5 minutes, and incubated at 37°C for 1 hour with primary antibodies: COL1A1(E3E1X) mouse mAb for type I collagen and COL3A1(E3K9U) rabbit mAb for type III collagen (both from Cell Signalling-EU). The coverslips were washed three times again with PBS for 5 minutes each, and incubated at 37°C for 1 hour, protected from light, with secondary antibodies: goat anti-mouse IgG-Alexa Fluor® 488 and donkey anti-rabbit IgG-Alexa Fluor® 647 (both from ThermoFisher Scientific-UK). Finally, the cells were washed again, incubated with phalloidin-Texas red (Thermo-Fisher Scientific-UK) for 1 hour, washed three times with PBS, and mounted on glass slides using a mounting medium consisting of 90% glycerol + 10% PBS, pH 8.5 and ascorbic acid to prevent quenching. Immunofluorescence images were acquired using a Nikon AR1 confocal microscope and processed using Fiji-ImageJ.

[0161] Cell nuclei were stained with DAPI to assess cell viability and localization. Cells were also stained with phalloidin-Texas red to reveal their morphology. Phalloidin stains the cytoskeleton, allowing for the differentiation of cytoplasm and extracellular matrix, as well as type I and type III collagen, and thus enabling assessment of collagen production.

[0162] Lipopeptide(II) was investigated at a concentration of 0.0062 wt%. The control sample included DMEM without serum.

[0163] Microscopic images show increased type I collagen production in samples incubated with 0.062 wt% Fn-KTTKS. Specifically, increased collagen production was observed in the extracellular matrix. Microscopic images of the control sample showed minimal collagen production.

[0164] Anti-inflammatory effect Nitric oxide (NO) is a signaling molecule involved in the pathogenesis of inflammation. Many cell types produce NO when stimulated during cytotoxicity or infection, modulating inflammation in various tissues (Coleman JW. Nitric oxide in immunity and inflammation. International immunopharmacology. 2001 Aug 1;1(8):1397-406). NO production can be quantified using the Gries reaction. The Gries reaction is a chemical reaction that detects nitrite and nitrate in biological fluids, where nitrite reacts with primary aromatic amines to form diazonium salts, resulting in a red color (Kampfer A, et al., Chemical Research in Toxicology. 2017 Apr 17;30(4):1030-7.).

[0165] During the inflammatory process, NO is produced by cells and rapidly broken down into nitrite in the culture medium, which can be detected using Grease's reagent. This test has already been used in the literature to evaluate the anti-inflammatory effects of molecules containing KTTKS-related peptides and farnesyl by monitoring the level of nitrite present in the culture medium after stimulation with LPS or other pro-inflammatory agents, and by blocking the ability of these agonists to increase nitrite levels in the sample by enhancing cellular NO production (Fadilah NI, et al., Journal of Saudi chemical society. 2020 Aug 1;24(8):606-19; Wang M et al., Bioorganic & Medicinal Chemistry Letters. 2016;26(14):3342-5).

[0166] To evaluate the anti-inflammatory effect of this lipopeptide, 5 × 10 3RAW264.7 mouse macrophage cells were seeded in 96-well plates and incubated for 24 hours in a cell incubator at 37°C and 5% CO2. The culture medium used was DMEM (ThermoFisher Scientific UK) supplemented with 10% fetal bovine serum (Gibco), Gibco® Antibiotic-Antimycotic, and L-glutamine (Gibco). After the initial 24-hour incubation, the RAW cells were washed three times with PBS and incubated in fresh DMEM without FBS or phenol red, supplemented with 5 μg of E. coli lipopolysaccharide (LPS). Samples were incubated in the presence of 0.025 wt%, 0.0125 wt%, and 0.0062 wt% lipopeptides (II), and control samples were incubated in basal medium containing LPS. To observe normal, expected nitrite production and compare it to enhanced nitrite production resulting from NO release in the medium induced by the addition of LPS to the culture medium, a negative control was cultured in DMEM without LPS. After incubation with LPS, 90 μL of the cell supernatant was collected and transferred to a new 96-well plate. A 10 μL aliquot of Gries reagent solution (Merck, Cas-G4410) was added, and the plate was incubated at room temperature for 10 minutes, protected from light. The absorbance at 550 nm was measured using a Tecan Infinite F50 plate reader, and the data were plotted to track nitrite production. Comparative samples using LPS and conventional stimulants (1 mM ascorbic acid, 0.025 ng / mL TGFβ1, and 0.01 mg / mL insulin) were also investigated.

[0167] It can be confirmed that the nitrite levels for all three compositions containing lipopeptide(II) were equivalent to those of the control.

[0168] Figure 8 shows the percentage of nitrite compared to the control when using 0.0062–0.025% FMdEG-KTTKS.

[0169] Compositions containing lipopeptide(II) completely counteract LPS-induced nitrite production, thereby demonstrating that compositions containing lipopeptide(II) are effective treatments for inflammation.

[0170] Skin healing To simulate skin injury, a scratch assay was performed to evaluate cell migration in response to a "damage" created on a fibroblast monolayer. This assay involved creating a cell monolayer on a multiwell assay plate, creating a cell-free compartment within the monolayer, and quantifying cell migration over 72 hours by monitoring recolonization in the cell-free compartment. For this assay, L929 mouse fibroblasts were grown in T-25 flasks in DMEM containing 10% FBS, Gibco® Antibiotic-Antimycotic, and L-glutamine (Gibco). The cells were detached using trypsin (ThermoFisher Scientific-UK) and grown in 24-well plates until full confluence was reached. Subsequently, to simulate skin injury, a scratch was created on this cell monolayer using a 200 μL pipette tip. After washing the plate three times with PBS to remove serum and dead cells, fresh DMEM without FBS was added. Using a standard optical microscope, images of scratches at the same location in each well were acquired over 72 hours to evaluate the migration of scratches from the boundary into the cell-free compartment created by the artificial "injury."

[0171] Samples containing lipopeptide(II) at concentrations of 0.025 wt%, 0.0125 wt%, and 0.00625 wt% were investigated.

[0172] In control samples incubated in basic culture medium without any additives, only minimal fibroblast migration was observed, if any, and no visible migration toward the center of the wound was observed even after 72 hours.

[0173] However, in samples incubated at 0.0062 wt%, cell migration from the edges of the excised area to the center of the wound was observed after 24 hours, and the fibroblast layer showed visible recovery after 72 hours compared to the control.

[0174] Rat excision wound model Wound healing was investigated using a rat model of diabetic excision wounds. Lipopeptide(II) was compared to a control sample over 25 days. Body weight was measured every 4 days for each group.

[0175] Female albino Wistar rats, 10–12 weeks old and weighing 180–190 g, were maintained in a 12-hour light-dark cycle without restrictions on standard laboratory animal feed and water intake. The rats were fasted overnight, after which diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ; Sigma-Aldrich) at a dose of 45 mg / kg of body weight in 0.1 mol / L sodium citrate buffer, pH 4.5. Blood glucose levels were measured weekly from tail vein samples. Rats with blood glucose levels exceeding 17 mmol / L during the induction period were considered to have stable diabetes and were selected for wound healing experiments. Stable hyperglycemia was achieved in approximately 95% of rats after STZ injection.

[0176] Three weeks after STZ injection, 20 female albino Wistar rats were randomly divided into five treatment groups: PBS control and Pep F (n=10 per group) (Figure 1). General anesthesia was maintained using xylazine and ketamine (Sigma-Aldrich, Germany; 5 mg xylazine and 40 mg ketamine per kg of body weight in sterile PBS). Anesthesia was maintained during surgery by administering a continuous flow of isoflurane (2 mL / min). Before wound creation, the dorsal hair was completely shaved and the skin was sterilized with povidone-iodine. A full-thickness annular wound with a diameter of 8 mm was created on the back using a sterile biopsy punch. A bioactive peptide solution was prepared in PBS immediately before topical application, and 1% (w / v) peptide solution or 300 μL of PBS was applied directly to each wound. To prevent postoperative infection and aid in monitoring, a thin, translucent adhesive film (Tegaderm, 3M, USA) was placed over the wound. The rats were then returned to their cages and carefully monitored for 48 hours for any signs of postoperative stress, secondary infection, or ulceration. The control group in this study consisted of untreated animals.

[0177] The wounds were photographed at regular intervals on days 0, 4, 8, 12, 16, 20, and 24 using a digital camera positioned at a fixed distance and magnification. Wound closure was quantified using ImageJ software with the formula: %wound closure = [(original wound area - current wound area) / original wound area] × 100%. The animals' body weight was monitored and recorded throughout the entire 20-day experimental period.

[0178] For the diabetes model, rats were euthanized 24 days after wound creation. Skin tissue was fixed in 4% paraformaldehyde and immersed in 30% sucrose for 48 hours for cryopreservation. Subsequently, 5 μm thick tissue sections were obtained from OCT-embedded blocks. These sections were analyzed using hematoxylin-eosin (H&E) staining and Masson's trichrome staining. Imaging of the stained sections was then performed using an Olympus BX-53 upright microscope.

[0179] Figure 9a is a graph showing the percentage of wound healing for each sample group over the course of the experiment. Figure 9b is a graph showing the body weight of each treatment group over the course of the experiment.

[0180] As shown in Figure 9a, throughout the experiment, wounds exposed to samples containing lipopeptide(II) healed to a greater extent than wounds exposed to control samples.

[0181] As shown in Figure 9b, the body weight of the rats used in this experiment remained unaffected throughout the experiment. This demonstrates that lipopeptide(II) is safe and well-tolerated by the subjects at effective doses.

[0182] Discussion and Conclusion The designed bioactive collagen-stimulating peptide conjugate FMdEG-KTTKS(II) self-assembles into spherical micelles at concentrations exceeding the strictly defined CAC. The formation of these micelles creates elongated nanostructures, i.e., long nanotapes. 16 -KTTKS and C 18 -This is in contrast to previous reports on KTTKS. This highlights the influence of the chain structure on the resulting nanostructure.

[0183] MTT assays show that FMdEG-KTTKS(II), in monomeric form, induces collagen production by dermal fibroblasts, accompanied by good cell viability. 16 -In the case of KTTKS, even at concentrations as low as one-tenth of those used in this study (ranging from 0.002 to 0.008 wt%), cell viability decreased by more than 40%, therefore C 16 Compared to sequences based on -KTTKS, cytocompatibility is improved. The fact that FMdEG-KTTKS(II) does not induce a significant increase or decrease in cell proliferation is a desirable feature for the cosmetic or clinical application of this compound.

[0184] Even with improved cytocompatibility, FMdEG-KTTKS(II) showed a more than 10-fold increase in collagen per cell compared to the basic medium at a concentration of 0.008 wt% of the peptide amphiphilic substance. 16 -Although to a lower degree than KTTKS, it significantly stimulates collagen production per cell (RR Jones, V. Castelletto, CJ Connon and IW Hamley Molecular Pharmaceutics, 2013, 10, 1063-1069). The substantial increase in cytotoxicity of FMdEG-KTTKS(II) at concentrations above CAC, where molecules self-assemble to form micelles, is noteworthy. This property could underpin applications where cytotoxicity is desirable, such as antimicrobial materials. The type of collagen produced was type I collagen, the main collagen found in many types of tissues, including skin, tendons, bones, and many other structures in the body. Specifically, it has been shown that FMdEG-KTTKS(II) can increase type I collagen production in HDFa cells, particularly in the extracellular matrix.

[0185] FMdEG-KTTKS(II) also shows promising anti-inflammatory effects.

[0186] FMdEG-KTTKS(II) has also been found to enable faster healing of skin wounds in fibroblast and rat models / model rats. In mouse models, FMdEG-KTTKS(II) has been found to be safe and well-tolerated by subjects at effective doses.

Claims

1. Equation (I): R-L-P (I) (In the formula, R is a hydrocarbyl group, L is a linker, P is a peptide containing 2 to 10 amino acid residues. A lipopeptide.

2. The lipopeptide according to claim 1, wherein the linker L includes a polyether portion.

3. The aforementioned polyether portion is given by formula: 【Chemistry 1】 (In the formula, n and m are integers independently selected from 1, 2, and 3.) The lipopeptide according to claim 2, represented by [the specified figure].

4. formula: 【Chemistry 2】 (In the formula, n and m are integers independently selected from 1, 2, and 3. R is a hydrocarbyl group, P stands for peptide. The lipopeptide according to claim 3, having the following characteristics.

5. formula: 【Transformation 3】 (In the formula, R is a hydrocarbyl group, P stands for peptide. The lipopeptide according to claim 4, having the following characteristics.

6. The lipopeptide according to claim 2 or 3, wherein the polyether portion is a polyethylene glycol portion, a polypropylene glycol portion, or a polytetramethylene glycol portion.

7. The lipopeptide according to claim 6, wherein the linker comprises a diethylene glycol portion.

8. The lipopeptide according to any of the above claims, wherein the peptide is a collagen-promoting peptide.

9. The peptide is selected from KTTKS, ETTES, GPKG, LSVD, KT, beta AH (beta A: β-alanine), VW, RGD, RGDS, VYV, KVK, GQPR, PKEK, KQVY, beta AHSH, QDVH, PPYL, GEKG, KHG, GPRPA, YaGFL (a: D-alanine), EEMQRR, SIKVAV, FVAPFP, VGVAPG, RRLEMYCAPLKP, EEMERRAD, MPfRwFKPV (w: D-tryptophan; f: D-Phe), RRGQILSTWY, FAKALKALLKALKAK, GHK, AHK, Lys-Val-diaminohydroxybutyrate, Lys-Thr, Tyr-Arg, β-Ala-His, Val-Trp, Cu-Gly-His-Lys, Gly-His-Lys-Mn ２＋ , Lys-Met (O ２ )-Lys, Arg-Gly-Asp, Val-Tyr-Val, Lys-α-Asp-Ile, Lys-Val-Lys, Gly-Gln-Pro-Arg, Pro-Lys-Glu-Lys, Lys-Asp-Val-Tyr, β-Ala-His-Ser-His, Gln-Asp-Val-His, Pro-Pro-Tyr-Leu, Gly-Glu-Lys-Gly, Lys-His-Gly, Gly-Pro-Arg-Pro-Ala, Tyr-D-Ala-Gly-Phe-Leu, Glu-Glu-Met-Gln-Arg-Arg, Ser-Ile-Lys-Val-Ala-Val, Phe-Val-Ala-Pro-Phe-Pro, Val-Gly-Val-Ala-Pro-Gly, Arg-Arg-Leu-Glu-Met-Tyr-Cys-Ala-Pro-Leu-Lys-Pro, Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp, Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val, Arg-Arg-Gly-Gln-Ile-Leu-Ser-Thr-Trp-Tyr, and Phe-Ala-Lys-Ala-Leu-Lys-Ala-Leu-Leu-Lys-Ala-Leu-Lys-Ala-Leu, according to claim 8, which is the lipopeptide.

10. The lipopeptide according to claim 9, wherein the peptide is selected from ETTES, GPKG, LSVD, KTTKS, GHK, and AHK.

11. The lipopeptide according to claim 10, wherein the peptide is KTTKS.

12. The lipopeptide according to any one of the above claims, wherein the peptide comprises 3 to 10 amino acid residues.

13. The aforementioned hydrocarbyl group a) It is a branch, b) containing 5 to 25 carbon atoms, and / or c) It is unsaturated. The lipopeptide according to any of the above claims.

14. The lipopeptide according to any of the above claims, wherein the hydrocarbyl group is a terpenoid.

15. The lipopeptide according to claim 14, wherein the terpenoid is a sesquiterpene.

16. The lipopeptide according to claim 15, wherein the sesquiterpene is a farnesyl group.

17. formula: 【Chemistry 4】 The lipopeptide according to claim 16, having the following characteristics.

18. a) A lipopeptide according to any one of claims 1 to 17, and b) at least one additional component A composition containing the following:

19. The composition according to claim 18, wherein the additional component is a dermatologically acceptable carrier.

20. Cosmetic use of the lipopeptide according to any one of claims 1 to 17 or the composition according to claim 18 or claim 19.

21. A method of cosmetic prevention and / or treatment, To prepare a lipopeptide according to any one of claims 1 to 17, To apply the lipopeptide to the user's skin in order to improve the appearance of the skin, Methods that include...

22. The cosmetic use according to claim 20 or the method according to claim 21, for the purpose of regenerating skin, treating nails, and / or improving the appearance of hair.

23. A lipopeptide for pharmaceutical use, which is a lipopeptide according to any one of claims 1 to 17.

24. A lipopeptide for use in methods for preventing, treating or improving wounds in target skin and / or for preventing, treating or improving conditions related to inflammation and / or conditions related to collagen, the lipopeptide being as described in any one of claims 1 to 17.

25. An article comprising a lipopeptide according to any one of claims 1 to 17, wherein the lipopeptide is configured to come into contact with a target tissue (e.g., skin) when used.

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