Micellar bioactive collagen-stimulating lipopeptide

Abstract

A lipopeptide of formula (I): R – L – P (I); wherein: R is a branched hydrocarbyl group; L is a linker; and P is a peptide comprises from 2 to 10 amino acid residues.

Description

[0001] MICELLAR BIOACTIVE COLLAGEN-STIMULATING LIPOPEPTIDE

[0002] Field of the Invention

[0003] The invention relates to a novel lipopeptide molecules, to compositions comprising such lipopeptides, to methods for their preparation and to uses thereof. In particular, the invention pertains to novel bioactive collagen stimulating lipopeptides that self-assemble into micelles, offering potential applications in fields including skincare, wound healing and tissue engineering.

[0004] Background of the Invention

[0005] Collagen is a widely distributed protein in living organisms, and is an essential component of connective tissue, skin, bone, and the like. Collagen is considered to be an effective treatment in improving rough skin, sagging skin, wrinkles, wounds, etc., and in treating arthritis, tenosynovitis, and the like. Collagen I is the most abundant protein in the dermis layer of the skin and is responsible for skin firmness. Collagen II and collagen III are other important types of collagen found in vivo. So, new active ingredients that promote the production of collagen are always researched for.

[0006] Certain peptides (of length up to 30 amino acids) are known to stimulate collagen growth in the extra-cellular matrix of the skin, which may improve the appearance of skin as well as improve healing of damaged skin. A major problem in realising the potential cosmetic and therapeutic effects of these peptides in humans is the transdermal delivery of the peptides across the outer skin barrier (stratum comeum) to the site of biological action, e.g., the extra-cellular matrix or underlying muscle (Robinson, et al., 2005, International J. Cosmetic Science 27: 155). To achieve transdermal delivery of these peptides in humans, the peptides have previously been chemically modified by the addition of moieties such as acetyl and / or palmitoyl groups (Robinson, et al., supra).

[0007] Several peptide amphiphile (PA) molecules, such as lipopeptides, are known to have beneficial cosmetic and therapeutic effects on the skin and find applications in tissue engineering, regenerative medicine and cosmetics based on the stimulation of collagen production by fibroblasts. Lipopeptides are a remarkable class of self-assembling molecule that are able to form peptide-fiinctionalized supramolecular nanostructures. One of the known peptide amphiphiles (PA) is KTTKS pentapeptide, a subfragment of type I collagen propeptide, which has been incorporated into the lipopeptide (peptide amphiphile, PA) Cie-KTTKS within the Matrixyl™ family, developed by Sederma. The KTTKS pentapeptide is based on a sequence taken from a propeptide from Human type I collagen. Cie-KTTKS or Palmitoyl pentapeptide-4 (Pal-Lys-Thr-Thr-Lys-Ser = Pal-KTTKS) contains 5 amino acids directly linked to a 16-carbon aliphatic chain:

[0008] Ci6 - KTTKS

[0009] It has been reported that both the peptide KTTKS and Cie-KTTKS are able to promote type I and type II collagen production in vitro (R. R. Jones, V. Castelletto, C. J. Connon and I. W. Hamley, Molecular Pharmaceutics, 2013, 10, 1063-1069). Studies also suggest that Cie-KTTKS can reduce the appearance of facial wrinkles (L. R. Robinson, N. C. Fitzgerald, D. G. Doughty, N. C. Dawes, C. A. Berge and D. L. Bissett, International Journal of Cosmetic Science, 2005, 27, 155-160).

[0010] While KTTKS is a small, highly specific biologically active peptide which has been reported to produce stimulation of elastin and collagens, specifically types I and II, its chemical nature places severe restrictions on its deliverability across skin. From a physicochemical standpoint it is clear that KTTKS and palmitoyl-KTTKS possess unfavourable properties for significant skin permeation.

[0011] Summary of the Invention

[0012] According to a first aspect, the invention relates to a lipopeptide of formula (I):

[0013] R - L - P

[0014] (I)

[0015] Wherein: R is a hydrocarbon group; L is a linker; and P is a peptide comprising from 2 to 10 amino acid residues.

[0016] The skilled person will appreciate that the hydrocarbon group, the linker and the peptide are each discrete sections of the lipopeptide. Surprisingly, the lipopeptide of the invention has been found to enhance collagen production in cell model studies by more than double compared to conventional stimulants. Therefore, the lipopeptide has clear uses in cosmetic applications, such as skin treatments. The lipopeptide will find application in cosmetic uses for example due to its ability to penetrate deeper into the skin and / or improve the appearance of the skin. The lipopeptide displays a beneficial combination of reduced cytotoxicity and collagen production enhancing effects.

[0017] Conventional lipopeptides such as Cn-KTTKS (also including KTTKS Matrixyl™ sequence) with Cn corresponding to Cu - Cis lipid chains, forms tape-like fibrils up to micrometers in length (R Jones et al., Molecular Pharmaceutics 2013, 10 (3), 1063-1069 DOI: 10.1021 / mp300549d).

[0018] Compared to Cie-KTTKS, the lipopeptide of the invention has improved cytocompatibility. Cell viability in the presence of Cie-KTTKS is reduced by 40% or more, even at concentrations up to ten times lower (in the range 0.002 - 0.008 wt%) than have been used to study the lipopeptide of the invention. The fact that the lipopeptide of the invention does not induce significant increase or decrease in cell proliferation is a desirable trait for the application of this compound as a cosmetic or in the clinic.

[0019] By contrast, lipopeptides of present invention have surprisingly been found to self-assemble into micelles, which may be termed ‘nanoparticles’. The micellar structure of the lipopeptides of the invention allows the lipopeptides to achieve a new mode of activity in skin penetration across the stratum comeum, leading to enhanced activity in skin, since the small micelles can readily be transported through pores across this barrier.

[0020] In contrast to ‘micellar’ cosmetics, the micelles of the present invention are not ‘formulations’ in the usual sense (i.e., a mixture of ingredients such as conventional surfactants or liposomes); the formation of micelles is an intrinsic self-assembly property inherent to the lipopeptides of the invention. Conventional cleansing surfactants that form micelles do not contain such motifs.

[0021] Without being bound by theory it is believed that the structure of the linker enables the lipopeptide to achieve the micellar structure. Preferably the linker comprises a polyether group.

[0022] Most preferably the lipopeptide is represented by the structure:

[0023]

[0024] The present application describes specific benefits relating to the enhancement of collagen production, which are believed to result from the specific structure of the peptide. Where an alternative peptide, which provides an alternative biological effect, is incorporated into the structure of the lipopeptide with a similar linker structure, it is envisaged that the resulting lipopeptide will achieve enhanced absorption and thereby enhance the biological effect of the alternative peptide.

[0025] According to a second aspect, the invention relates to a composition comprising: a) a lipopeptide of formula (I); and b) at least one additional ingredient.

[0026] The composition may be a pharmaceutical composition and / or a cosmetic composition. The composition is preferably atopical composition. Preferably the additional ingredient is a carrier, such as a dermatologically acceptable carrier.

[0027] According to a third aspect, the invention relates to a cosmetic use of a lipopeptide of the first aspect or a composition of the second aspect. The cosmetic use may be for regenerating skin, for the treatment of nails, and / or for improving the appearance of hair.

[0028] According to a fourth aspect the invention relates to a method of cosmetic prevention and / or treatment, wherein the method comprises: providing the lipopeptide of the first aspect, and applying the lipopeptide to the skin of a user in order to enhance the appearance of the skin.

[0029] A composition suitable for use in cosmetic applications may be formulated as a cosmetic formulation. The cosmetic formulation may enhance the appearance of the skin by reducing the appearance of scar tissue and / or stretch marks, reducing the number or severity of wrinkles and / or fine lines, improving skin elasticity, and / or by making skin tone more even (e.g. by reducing the appearance of age spots or other conditions relating to excess melanin or hyperpigmentation). The cosmetic formulation may be in a formulation selected from the list consisting of: cream, lotion, gel, semi-solid, dispersion, suspension, foam, mousse and spray.

[0030] The lipopeptide of the invention has also been found to provide wound healing effects. Specifically, the presence of the lipopeptide has been shown to increase the rate of wound healing in a fibroblast scratch test and in a murine wound healing model.

[0031] The lipopeptide of the invention has also been found to have potent immunomodulatory activity. Specifically, the presence of the lipopeptide has been found to negate the usual increase in nitrite that would be seen when RAW 264.7 cells are exposed to lipopolysaccharide, a class of toxin produced by bacteria. Thus, the lipopeptide may interact with the immune system to downregulate specific aspects of a host’s immune response. The immune system sometimes mounts an immune response against innocuous substances, like house mite, dust or pollen, resulting in allergy (an overactive TH2-mediated response). In addition, many physiological disorders, like hypercholesterolemia and obesity, result in a low-grade inflammatory status. Immune modulation in the context of abnormal immune responses, like allergy or inflammation, means dampening or counteracting the hypersensitivity immune response. The present invention may relate to the primary task of the stimulating / enhancing immune responses, and / or the task of inhibiting the ‘abnormal’ immune response. Therefore, the lipopeptide may find applications in treating inflammation-related conditions.

[0032] Due to its ability to stimulate collagen production, the lipopeptide may find applications in treating collagen-related conditions.

[0033] According to a fifth aspect, the invention relates to a lipopeptide for use in medicine, wherein the lipopeptide is of the first aspect.

[0034] According to a sixth aspect, the invention relates to a lipopeptide for use in a method of prevention, treatment or amelioration of a wound in the skin of a subject and / or in a method of prevention, treatment or amelioration of an inflammation-related condition and / or a collagen- related condition, wherein the lipopeptide is of the first aspect.

[0035] According to a seventh aspect, the invention relates to an article comprising a lipopeptide of the first aspect, wherein the article is configured such that, in use, the lipopeptide can contact a tissue (e.g. the skin) of a subject. Therefore, the article may have the lipopeptide on an external surface of the article. The article may be a medical device, such as a wound dressing, catheter and / or implant. The article may be a tissue scaffold, either for in-vivo applications, such as implantation into a subject (e.g. regenerative medicine) or for ex-vivo applications such as tissue models.

[0036] US2009 / 092642 Al discloses famesyl-like chains bonded to peptides that include two amino acids. The compounds are used to intensify tanning of the skin and melanin synthesis in the skin orthe 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 bisubstrate inhibitors of ras famesyl protein transferase. US6015877A describes inhibitors of carboxy terminal proteolysis. GB2323783 A describes inhibitors of famesyl protein transferase. However, there is no mention in these documents of the linker including a polyether (e.g. a polyethylene glycol) or of the peptides used in the present application.

[0037] US 2019 / 330141 Al describes compounds that have a cooling effect, for use in personal care compositions, such as oral care compositions, containing a flavour / perfume system. However, there is no mention of the linker including a polyether (e.g. a polyethylene glycol) or of the peptides used in the present application, or specifically using a sesquiterpene as the hydrocarbon group.

[0038] JP H04297495 A describes compounds containing a saturated hydrocarbon chain linked to an octapeptide for use as an animal cell adhesion inhibitor and platelet aggregation / adhesion inhibitor. However, there is no mention of the hydrocarbon chain being unsaturated or a sesquiterpene, or of the linker including a polyether (e.g. a polyethylene glycol), or of the peptides used in the present application.

[0039] EP3598982 Al describes safe anti-hyperalgesic or analgesic compounds that are bioconjugates of neuropeptide derivatives. The hydrocarbon groups of the compounds contain more than 25 carbon atoms. In addition, there is no mention of the linker including a polyether (e.g. a polyethylene glycol), or of the peptides used in the present application.

[0040] While these documents describe lipopeptides, none of these documents describe the specific linker groups that are used in the present invention or the specific peptides that are used in the present invention.

[0041] Furthermore, none of these documents describe technical effects relating to increasing the production of collagen, increasing the rate of wound healing, or decreasing inflammatory responses. The present disclosure includes the subject-matter of the following clauses:

[0042] 1. A lipopeptide of formula (I): R - L - P (I) wherein R is a branched hydrocarbyl group; L is a linker; and P is a peptide from 2 to 10 amino acid residues.

[0043] 2. A lipopeptide according to clause 1 wherein the R comprises from 5 to 25 carbon atoms.

[0044] 3. A lipopeptide according to clause 1 or 2 wherein R is unsaturated.

[0045] 4. A lipopeptide according to any preceding clause wherein R is a terpenoid.

[0046] 5. A lipopeptide according to clause 4, wherein R is a sesquiterpene.

[0047] 6. A lipopeptide according to clause 5 wherein R is a famesyl group.

[0048] 7. A lipopeptide according to any preceding clause wherein the linker L comprises a polyether moiety.

[0049] 8. A lipopeptide according to clause 7, wherein the linker comprises a diethylene glycol moiety.

[0050] 9. A lipopeptide according to any preceding clause wherein the linker L is

[0051] 10. A lipopeptide according to any preceding clause, wherein the peptide comprises from 2 to 10 amino acids.

[0052] 11. A lipopeptide according to any preceding clause wherein the peptide is a collagen promoting peptide.

[0053] 12. A lipopeptide according to clause 11, wherein the peptide is selected from KTTKS, GHK and AHK.

[0054] 13. A lipopeptide according to clause 1, wherein the peptide is selected from the amino acid sequence KTTKS, or a fragment or variant thereof.

[0055] 14. A lipopeptide according to any preceding clause having the formula:

[0056] 15. A topical composition comprising: a) a lipopeptide as defined in any one of clauses 1 to 14; and b) at least one additional ingredient.

[0057] 16. The topical composition of clause 15 further comprising plurality of additional ingredients and wherein at least one of said additional ingredients is a dermatologically acceptable carrier.

[0058] 17. A cosmetic use of a lipopeptide as defined in any one of clauses 1 to 14 or a topical composition as defined in clause 15 or 16 for regenerating skin. 18. A cosmetic use of a lipopeptide as defined in any one of clauses 1 to 14 or a topical composition as defined in clause 15 or 16 for the treatment of nails.

[0059] 19. A cosmetic use of a lipopeptide as defined in any one of clauses 1 to 14 or a topical composition as defined in clause 15 or 16 for improving the appearance of hair.

[0060] 20. A lipopeptide as defined in any one of clauses 1 to 14 for use in medicine.

[0061] Brief description of the Figures

[0062] An embodiment of the present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0063] Figure 1 shows the structure of famesyl-mercapto(diethylene glycol)-KTTKS (FMdEG-KTTKS);

[0064] Figure 2a represents self-assembly of lipopeptide FMdEG-KTTKS into a micelle above a critical aggregation concentration (CAC) and collagen-stimulating activity below CAC;

[0065] Figure 2b represents the variation of the intensity of the fluorescent dye 8-anilino-l- naphthalene sulfonic acid peak maximum (Io) and wavelength (Xo) with lipopeptide concentration;

[0066] Figure 3a represents circular dichroism spectra which show an unordered peptide conformation.

[0067] Figure 3b represents FTIR spectra, also showing unordered peptide conformation;

[0068] Figure 3c represents dynamic light scattering (DLS) intensity distribution data, which indicates small aggregates;

[0069] Figure 3d represents small-angle x-ray scattering (SAXS) analysis of the peptide amphiphile based- micelles, showing micellar structure above the CAC;

[0070] Figure 3e represents a typical cryo-TEM image with well-defined relatively monodisperse micelles with a size consistent with that obtained from DLS and SAXS; Figure 4 represents cytotoxicity studies of lipopeptide;

[0071] Figure 5a represents collagen production assay by lipopeptide;

[0072] Figure 5b represents collagen production per cell by lipopeptide;

[0073] Figure 6a-e represents polarized optical microscopy (POM) images of the cells stained with dye Sirius red;

[0074] Figure 7a-e represents Bright field optical microscopy images of the cells stained with dye Sirius red;

[0075] Figure 8 represents anti-inflammatory studies of lipopeptide; Figure 9a shows a graph of percentage wound healing over time for a control sample and for lipopeptide (II) in a rat excisional wound model; and

[0076] Figure 9b shows a graph of body weight over time during the rat experiments shown in Figure 9a.

[0077] Detailed description of the Invention

[0078] The invention, in one aspect, is directed to novel bioactive lipopeptide molecules. The lipopeptide molecules are conjugates of a peptide, a linker and a hydrocarbon group. These lipopeptides enhance collagen production per cell by more than double as compared to a control. This activity is observed at low concentrations, below the critical aggregation concentration (CAC). Above the CAC, these molecules self-assemble into well-defined spherical micelles of a nano scale.

[0079] The term “bioactive” as used herein includes, but is not limited to, any type of interaction with another biomolecule, such as a protein, glycoprotein, carbohydrate etc.

[0080] The term “lipopeptides” refers to peptides that are conjugated (via amide, thioether, or thioester linkages, or a linker, as defined elsewhere herein) to lipids or aliphatic chains. The term lipopeptide is used herein interchangeably with the term “peptide amphiphile” or "amphiphilic molecule" or “peptide conjugate” or “bioactive peptide conjugate” or “molecule of structure (I)”.

[0081] The term “peptide” as used herein refers to a sequence of amino acids linked together by peptide bonds or by modified peptide bonds. A dipeptide contains two amino acids; a tripeptide contains three amino acids; and the term oligopeptide is typically used to describe peptides having between 2 and about 50 or more amino acids. The present invention requires that the peptide of the lipopeptide comprises from 2 to 10 amino acid residues. Preferred peptides contain 3 to 10 amino acids, for example from 4 to 10 or from 5 to 10 amino acids. The peptide may comprise from 2 to 8 amino acids, such as from 2 to 6 amino acids, or from 2 to 5 amino acids. Preferably the peptide comprises from 3 to 8 amino acids, such as from 4 to 6 amino acids. Most preferably the peptide comprises 5 amino acids.

[0082] The term “amino acids,” as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Preferably, the amino acids are a-amino acids, more preferably naturally-occurring a-amino acids. Amphiphilic peptides, which possess a hydrophilic end and hydrophobic end, frequently have self-assembling properties, and a wide range of biological functions. When there is a certain concentration of amphiphilic peptides in aqueous solution, nanoparticles are formed by selfassembly. Such nanoparticles include micelles, vesicles and nanofibers. These secondary structures can directly affect peptide functionality.

[0083] The lipopeptides of the present invention are observed to form micelles under certain conditions. These micelles possess properties such as improved targeted delivery, making them promising carriers for bioactive compounds. The micelles are preferably spherical in topology.

[0084] The CAC determines the concentration at which premicellar aggregates are formed. In the context of the present invention, the micelle structure is formed above the CAC. However, it has been observed that the lipopeptides of the invention are active even at concentrations below the CAC.

[0085] As used herein, the term “hydrocarbon substituent” or “hydrocarbon group” refers to a group having a carbon atom directly attached to the remainder of the molecule and having predominantly hydrocarbon character. Examples of hydrocarbon groups include: (i) hydrocarbon substituents, that is, aliphatic (e.g., alkyl or alkenyl), alicyclic (e.g., cycloalkyl, cycloalkenyl) substituents, and aromatic-, aliphatic-, and alicyclic-substituted aromatic substituents, as well as cyclic substituents wherein the ring is completed through another portion of the molecule (e.g., two substituents together form a ring), optionally also hydrocarbon polymers such as polyethylene; (ii) substituted hydrocarbon substituents, that is, substituents containing non-hydrocarbon groups which, in the context of this invention, do not alter the predominantly hydrocarbon nature of the substituent (e.g., halo (especially chloro and fluoro), hydroxy, alkoxy, mercapto, alkylmercapto, nitro, nitroso, and sulfoxy); (iii) hetero substituents, that is, substituents which, while having a predominantly hydrocarbon character, in the context of this invention, contain other than carbon in a ring or chain otherwise composed of carbon atoms. Heteroatoms include sulfur, oxygen, nitrogen, and encompass substituents as pyridyl, furyl, thienyl and imidazolyl. In general, no more than two, or no more than one, nonhydrocarbon substituent will be present for every ten carbon atoms in the hydrocarbyl group; typically, there will be no non- hydrocarbon substituents in the hydrocarbyl group.

[0086] As used herein, the term “hydrocarbon” refers to a hydrocarbon group as defined above which has alkyl groups (preferably C1-C6 alkyl groups) bonded to the central carbon chain. Preferably, the hydrocarbon group is unsaturated. The term “unsaturated” as used herein refers to a hydrocarbon group as defined above having at least one carbon-carbon multiple bond, such as a double bond or triple bond. Preferably, the hydrocarbon group comprises at least one double bond, more preferably at least two double bonds, most preferably three double bonds.

[0087] In preferred embodiments, group R is a terpenoid group. As used herein, the term “terpenoid” refers to a class of organic compounds derived from terpenes and includes terpenes. Terpenes have a five-carbon isoprene unit and contain additional functional groups, typically oxygencontaining functional groups. Terpenes are a subset of terpenoids. Terpenoids may be characterized in terms of the number of isoprene units, as shown in the table below.

[0088] The hydrocarbon is preferably a branched hydrocarbon. In alternative embodiments, the group R is a branched lipid. Preferred branched lipids include the groups phytol, tocopherol, garcinol or lipoyl. Other preferred branched lipids include geranyl and geranylgeranyl.

[0089] Preferably, group R is a sesquiterpene group.

[0090] In a particularly preferred embodiment, group R is a famesyl group:

[0091] The peptide ‘P’ comprises from 2 to 10 amino acids. In some embodiments, the peptide contains about 5 amino acids i.e. is a pentapeptide. In a preferred embodiment, the peptide is a collagen promoting peptide. “Collagen promoting peptide” as used herein, refers to a class of compounds that possess the biological activity of enhancing the production of collagen. Collagen promoting peptides, according to the present invention, include all natural or synthetic peptides that are capable of enhancing the production of collagen in the human body.

[0092] Suitable collagen promoting peptides may be determined, for example, using the collagen production assays described hereinafter.

[0093] Known collagen promoting peptides include those having the sequence KTTKS, GHK and AHK. Accordingly, in preferred aspects of the invention, peptide P is selected from KTTKS, GHK and AHK. Other known collagen promoting peptides include GPKG and LSVD; as such these are also preferred embodiments of peptide P.

[0094] Other collagen promoting peptide sequences can also, in some embodiments, constitute peptide L. These are listed in the table below,

[0095] Details of these peptides are divulged in Advanced Drug Delivery Reviews, Volume 185, June 2022, 114240, the contents of which are incorporated by reference, and the publications cited therein.

[0096] The peptide may, therefore, be selected from the list consisting of: KTTKS, GHK, AHK, Palmitoyl dipeptide-6, Palmitoyl -KT, Dipeptide- 1, Camosine, Dipeptide-2, Copper Tripeptide- 1, Manganese tripeptide-1, 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 peptide may be selected from the list consisting of: KTTKS, GHK, AHK, Lys-Val-Diaminohydroxybutyrate, Lys-Thr, Tyr-Arg, P-Ala-His, Val-Trp, Cu-Gly-His-Lys, Gly-His-Lys-Mn2+, Lys-Met(O2)-Lys, Arg- Gly-Asp, Val-Tyr-Val, Lys-a-Asp-Ile, Lys-Val-Lys, Gly-Gln-Pro-Arg, Pro-Lys-Glu-Lys, Lys- Asp-Val-Tyr, P-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-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 peptide may be selected from the list consisting of: ETTES, GPKG, LSVD, KT, betaAH (betaA: (3-alanine), VW, RGD, RGDS, VYV, KVK, GQPR, PKEK, KQVY, betaAHSH, QDVH, PPYL, GEKG, KHG, GPRPA, YaGFL (a: D-alanine), EEMQRR, SIKVAV, FVAPFP, VGVAPG, RRLEMYCAPLKP, EEMERRAD, MPfRwFKPV (w: D-tryptophan; f: D-Phe), RRGQILSTWY, FA I<ALI<ALLI<A LEAK.

[0097] Preferably, peptide P consists of amino acid sequence KTKKS.

[0098] The term “linker” as used herein refers to molecules that function to link, i.e. conjugate or join, two or components of a molecule together, preferably by a covalent bond(s), i.e., peptide P and (optionally branched) hydrocarbyl group Rthus connecting the two. In some embodiments, the linker comprises an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety.

[0099] Preferably, the linker L comprises a polyether moiety, such as a polyethylene glycol, polypropylene glycol or polytetramethylene glycol. A generalised formula for a poly ether group is given below: wherein n and m are integers independently selected from 1, 2 and 3. Preferably m is 1. Preferably, n is 2.

[0100] In a preferred embodiment, the linker L has the formula: wherein n and m are as defined above. In this embodiment, the lipopeptide has the formula: wherein R, P, n, and m are as defined above. In this embodiment, preferably m is 1. Preferably, n is 2. In a very preferred embodiment, the linker L has the formula:

[0101] In this embodiment, the lipopeptide has the formula: wherein R and P are as defined above.

[0102] According to a highly preferred embodiment, the lipopeptide has the formula:

[0103] This lipopeptide is referred to herein as FMdEG-KTTKS and as lipopeptide (II).

[0104] The present invention also provides micelles, formed by the self-assembly of the inventive lipopeptide(s). The inventors have surprisingly found that lipopeptides of structure (I) spontaneously assemble into micelles. The micelle can also be formed from a plurality of lipopeptides of structure (I). The lipopeptide of the invention self-assembles into micelles above a defined CAC. Preferably, the CAC is about 0.04 wt%.

[0105] Accordingly, in one embodiment, lipopeptide of structure (I) self-assembles into a structure. Such a structure may, for example, be a micelle, a vesicle, or like. Preferably, the structure is a micelle. The lipopeptides of the invention spontaneously self-assemble into micelle nanoparticles. The diameter of these nanoparticles or micelles can range from about 1.5 to 10 nm. Preferably, the diameter of the nanoparticle is from 2 to 5 nm.

[0106] In one embodiment, the present invention provides a method of enhancing extracellular matrix protein production in a cell. The extracellular matrix protein is collagen. Preferably the collagen is type I or type II collagen. Accordingly, in one embodiment, the present invention provides a method of increasing collagen production in cells.

[0107] In one example, the cells can be cultured cells. This gives rise to a further aspect of the invention, which relates to a method of enhancing collagen production, the method comprising the step of culturing cells in the presence of an aqueous medium comprising a lipopeptide of structure (I).

[0108] As used herein, the term "enhancing collagen production" refers to an increase in the amount of collagen biosynthesized and / or secreted by a cell. Compared to a suitable control, the increased amount can 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, it may mean 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, it means an increase of at least 200% or more.

[0109] In the context of this specification, a cell can be a single cell or a population of cells, or a tissue, organ (e.g. skin) or organ system. In the case of in vivo cells, the contacting may be to provide a lipopeptide to the subject.

[0110] In some embodiments, the composition is impregnated within an article. The article may be a dressing or patch, such as gauze, lint, cotton wool, cloth, semi-permeable film (e.g. polyurethane film), semi-permeable foam (e.g. silicone), hydrogel (e.g. comprising methacrylate and / or polyvinyl pyrrolidine), hydrocolloid (e.g. containing carboxymethylcellulose, gelatin and / or pectin), hydrofiber (e.g. containing sodium carboxymethyl cellulose), or alginate (e.g. containing sodium and / or calcium alginate).

[0111] In another embodiment, the invention provides a cosmetic composition comprising the lipopeptide of the structure (I) and a physiologically acceptable medium or pharmaceutically acceptable medium.

[0112] Compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils or the like. The compositions adapted for topical administration may be formulated as liquids, balms, ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, oils or the like. In some embodiments, the composition is applied as a topical ointment or cream. The compositions may also routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives (eg, antioxidants), supplemental immune enhancing agents such as adjuvants and cytokines, and optionally other therapeutic agents.

[0113] The present invention provides the use of the lipopeptide of structure (I) or composition comprising the lipopeptide of structure (I) in cosmetics or non-therapeutic treatment. The applications can range from for example skin care for face, body, hair and ranges of make-up treatments. These include but are not limited to improvement in skin texture, fine lines, wrinkles, skin elasticity, flexibility, restructuring, radiance, effect on hair growth and scalp condition etc. Also contemplated herein are other uses of lipopeptides of structure (I) such in methods of producing tissue in vitro.

[0114] In another embodiment, the invention provides a pharmaceutical composition comprising the lipopeptide of structure (I) and a pharmaceutically acceptable carrier.

[0115] The compositions may also routinely comprise cosmetically and / or pharmaceutically acceptable concentrations of salts, buffers, preservatives (e.g., antioxidants), supplemental immune enhancing agents such as adjuvants and cytokines, and optionally other therapeutic agents.

[0116] The lipopeptide of structure (I) may be incorporated into the pharmaceutical composition in an amount of from 0.0001wt% to 10wt%, such as from 0.001wt% to 5wt%, from 0.001wt% to lwt%, from 0.002wt% to 0.5wt%, or from 0.005wt% to 0.05wt% relative to the weight of the pharmaceutical composition.

[0117] In another embodiment, the invention provides a lipopeptide for use in a method of prevention, treatment or amelioration of a wound in the skin of a subject. The invention also provides a pharmaceutical composition for use in a method of treatment of a wound in the skin of a subject.

[0118] The skin wound may be an abrasion (such as a linear or scratch abrasion, patterned abrasions and grazed or brush abrasions), laceration (such as a split laceration, tom laceration, stretch laceration, perforated laceration, blast laceration, cut laceration, crush laceration or de-gloved laceration), bum wound (such as a thermal bum, radiation bum, chemical bum or electrical bum), surgical incision, pressure injury, skin tear, arterial ulcer, venous ulcer, or bruise. The present invention also relates to a method of treating a wound in the skin of a subject. The method comprises administering to a wound in the skin of said subject the lipopeptide (e.g. in a therapeutically effective amount) according to the present invention.

[0119] The subject may be a human. The subject may be an animal. The animal may be an animal susceptible to skin wounds, such as such as cats, dogs, horses, cattle, sheep, goats, pigs, or birds.

[0120] Inflammation-related conditions may be T helper cell (TH)-mediated diseases or conditions, for example may be a TH 1 -mediated disease or condition, or a TH2 -mediated disease or condition.

[0121] A TH 1 -mediated disease or medical condition in which the TH 1 -mediated response is ineffective or underactive may be one or more selected from the group comprising / consisting of rheumatoid arthritis (RA); psoriatic arthritis; psoriasis; inflammatory bowel syndrome (IBD); Crohn’s disease; ulcerative colitis; multiple sclerosis (MS); flu, including pandemic flu; respiratory disorders, for example those caused by viruses, such as 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; coeliac disease; dermatomyositis; diabetes mellitus Type 1; Goodpasture’s syndrome; Graves’ disease; Guillain-Barre syndrome; juvenile idiopathic arthritis; Hashimoto’s thyroiditis; idiopathic thrombocytopenic purpura; Lupus erythematosus; mixed connective tissue disease; myasthenia gravis; narcolepsy; osteoarthritis; pemphigus vulgaris; pernicious anaemia; polymyositis; primary biliary cirrhosis; relapsing polychondritis; Sjogren’s syndrome; temporal arteritis; vasculitis; Wegener’s granulomatosis; age-related macular degeneration, an infectious disease (e.g. infection with Mycobacterium tuberculosis (Mt), human immunodeficiency virus (HIV) or a coronavirus)); an autoimmune disorder (e.g. arthritis, multiple sclerosis or type 1 diabetes); a cancer; postcancer surgery or cancer treatment; and post-immunisation. Preferably the TH 1 -mediated disease or medical conditions are one or more of cystic fibrosis (CF), diabetes mellitus Type I, age-related macular degeneration, an infectious disease (e.g. infection with Mycobacterium tuberculosis (Mt), human immunodeficiency virus (HIV) or a coronavirus).

[0122] A TH2 -mediated disease or medical condition in which the TH2 -mediated response is overactive may be one or more selected from the group comprising / consisting of type 1 hypersensitivity disorders, including an allergy (e.g. rhinitis, allergic dermatitis, uticaria), asthma, eczema, hay fever, urticarial, chronic graft-versus-host disease, progressive systemic sclerosis, systemic lupus erythematosus; a chronic lung disease; scleroderma; anaphylaxis; atrophy (e.g. muscle); and transplant rejection. Preferably the TH2 -mediated disease or medical conditions are one or more of allergy (e.g. rhinitis, allergic dermatitis, uticaria), asthma, eczema and hay fever.

[0123] Collagen-related conditions may be selected from the list consisting of osteogenesis imperfecta, chondrodysplasias, Ehlers-Danlos Syndrome, Alport syndrome, osteoporosis, and Knobloch syndrome. Collagen-related conditions may be selected from the list consisting of osteogenesis imperfecta, Ehlers-Danlos syndrome, infantile cortical hyperostosis (Caffey's disease), collagenopathy (types II and XI), Dupuytren's contracture, Alport syndrome, Goodpasture's syndrome, Ulrich myopathy, Bethlem myopathy, atopic dermatitis, epidermolysis bullosa dystrophica, posterior polymorphous comeal dystrophy 2, EDM2 and EDM3, Schmid metaphyseal dysplasia, Bullous pemphigoid, and junctional epidermolysis bullosa.

[0124] The article of the invention may comprise the lipopeptide coated and / or fixed (e.g. adhered) to a material. Techniques that may be used to apply the lipopeptide to a surface include 3D printing, electrospinning, electrophoretic deposition, dip-coating, drop casting, sol-gel deposition, biomimetic deposition, plasma spraying, layer-by-layer deposition, physical vapour deposition, anodization (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 polymeric coating (e.g. W. Xi et al., Nat. Comm. (2021) 12:5473), leucocyte- and platelet-rich fibrin products such as fibronectin / vitronectin exudate (e.g. M. Lollobrigida et al., BioMed Res. Int., (2018):9031435) and / or nanocoating (e.g. Ming-qi Chen, Front. Bioeng. Biotechnol. (2022) 10:878257).

[0125] Thus the lipopeptide may be included in, impregnated within, or coated upon a material, such as a plastic, a metal, wood, and / or fabric. The material may be used to form all or part of the article. Preferably the material, is used to form at least part of a surface of the article. This allows the lipopeptide to be available to contact a tissue of a subject.

[0126] The article may be a medical device such as an implant or prosthesis. Preferably the article is a dressing (e.g. a wound dressing). The dressing may be adhesive or non-adhesive. The dressing may be, or comprise, gauze, lint, cotton wool, cloth, semi-permeable film (e.g. polyurethane film), semi-permeable foam (e.g. silicone), hydrogel (e.g. containing methacrylate and / or polyvinyl pyrrolidine), hydrocolloid (e.g. containing carboxymethylcellulose, gelatin and / or pectin), hydrofiber (e.g. containing sodium carboxymethyl cellulose) or alginate (e.g. containing sodium and / or calcium alginate). Preferably the dressing is an impregnated dressing, wherein the dressing is impregnated with the lipopeptide, for example a composition comprising the lipopeptide and a retinoid. Hydroxyethylcellulose is a possible additional or alternative component of a hydrocolloid or hydrogel. Preferably the article is a gel, for example a hydrogel. The hydrogel may be made of natural materials such as collagen (e.g. derived from jellyfish). The hydrogel may contain hydroxyethyl cellulose.

[0127] The article (e.g. dressing or other medical device) may be sterile, and may optionally be provided in a sterile package to prevent contamination prior to use of the article. The article may be selected from permanent implants, such as artificial heart valves, voice prostheses, prosthetic joints, implanted artificial lenses, stents (e.g. vascular stents), and shunts (e.g. hydrocephalus shunts); and non-permanent implants, such as pacemakers and pacemaker leads, drain tubes, endotracheal or gastrointestinal tubes, temporary or trial prosthetic joints, surgical pins, guidewires, surgical staples, cannulas, subcutaneous or transcutaneous ports, and indwelling catheters and catheter connectors, and contact lenses. The implant may be an orthopaedic implant. In one embodiment the article is a catheter. Examples of indwelling catheters include urinary catheters, vascular catheters (e.g. central venous catheters, dialysis catheters, peripheral venous catheters, arterial catheters and pulmonary artery Swan-Ganz catheters), peritoneal dialysis catheters, central venous catheters and needleless connectors.

[0128] It has been found that compositions containing the lipopeptide of the invention at concentrations below 0.05 wt% (e.g. at or below the CAC (0.04 wt%)) can be less cytotoxic than compositions containing higher concentrations of the lipopeptide. Therefore, preferably compositions of the lipopeptide comprise the lipopeptide in a concentration of lower than 0.05 wt%, or 0.049 wt% or lower, such as 0.040 wt% or lower, or 0.039 wt% or lower, or 0.03 wt% or lower, for example from 0.00001 wt% to 0.049 wt%, or from 0.0001 wt% to 0.049 wt%, or from 0.001 wt% to 0.049 wt%.

[0129] Examples

[0130] Preparation of famesyl-mercapto(diethylene glycol)-KTTKS (FMdEG-KTTKS. lipopeptide am Peptide famesylation was carried out as per the method in N. McCarthy et al., Biochem. J. (2000) 347, 163-171. The peptide was synthesized with the addition of an Fmoc protected 9 atom polyethylene glycol spacer (di [ethylene glycol]) at the N terminus to which trityl protected mercaptopropionic acid was coupled via standard amino acid coupling methodology and the method in the above paper was utilised for the addition of the famesyl lipid chain.

[0131] Studies into the self-assembly of lipopeptide (II)

[0132] On increasing concentration, lipopeptide (II) undergoes self-assembly. The minimum concentration at which aggregation occurs is identified as the critical aggregation concentration (CAC). Self-assembly of lipopeptides above the CAC was examined using dynamic light scattering (DLS), Small-angle x-ray scattering (SAXS) and Cryogenic-TEM (Cryo-TEM) Imaging.

[0133] CAC was measured by using the fluorescent probe 8-anilino-l -naphthalene sulfonic acid (ANS), the fluorescence of which is sensitive to the hydrophobicity of its surrounding environment.

[0134] Experiments were carried out using a 10.0 mm x 5.00 mm quartz cell in a Varian Model Cary Eclipse spectrofluorometer. The determination of the CAC value was estimated by titration of the fluorescent dye 8-anilino-l -naphthalene sulfonic acid (ANS). A mother solution of 0.2 wt% FMdEG-KTTKS (II) was prepared using 2x1 O’3wt% ANS as a solvent. Dilutions in the rage 0.15-2X10-3wt% FMdEG-KTTKS where prepared from the mother solution, by adding weighted amounts of 2xl0-3wt% ANS. The fluorescence emission spectra of the sequence of dilutions were recorded between 400 nm and 670 nm exciting the sample at 356 nm.

[0135] Figure lb shows the variation of the intensity of the fluorescence emission peak maximum (Io) and wavelength (Xo) of solutions containing ANS with concentration, plotted as a function of the lipopeptide concentration. There was seen a clear discontinuity in both quantities at the same concentration, 0.04 wt% was determined to be the CAC.

[0136] Conformational analysis of lipopeptide

[0137] The conformation of lipopeptide above the CAC was investigated by using Circular Dichroism (CD) Spectroscopy and Fourier Transform Infrared (FTIR) Spectroscopy. Circular Dichroism (CD) spectra were recorded using a Chirascan spectropolarimeter (Applied Photo Physics, Leatherhead, UK). Solutions were placed between parallel plates (0.01 mm or 0.1 mm path length). CD Spectra were measured with a 0.5 nm step, 0.5 nm bandwidth, and Is collection time per step. The CD signal from the water background was subtracted from the CD data of the sample solutions. CD signal was smoothed using the Chirascan Software for data analysis. The residue of the calculation was chosen to oscillate around the average, to avoid artifacts in the smoothed curve. The CD spectrum shown in Figure 3a is characteristic of an unordered peptide conformation.

[0138] The Fourier Transform Infrared (FTIR) spectra were recorded using a Thermo-Scientific Nicolet iS5 instrument equipped with a DTGS detector, with a Specac Pearl liquid cell (sample contained between fixed CaF2 plates). Spectra were scanned 116 times over the range 900-4000 FTIR spectra shown in Figure 3b are also consistent with a disordered conformation due to the presence of a broad peak near 1650 cm1. The peak at 1672 cm'1is due to bound TFA counterions.

[0139] Dynamic light scattering (DLS) was used to detect aggregates. Dynamic light scattering measurements were carried out using an ALV / CGS-3 Compact Goniometer System with ALV / LSE-5003 correlator using vertical polarized incident light of wavelength X= 632.8 nm. Measurements were performed at 0= 90° to the incident beam, and data were collected three times for 30 sec. The intensity autocorrelation functions were analysed by a regularization method. The size distribution was calculated from the autocorrelation function, as weighted by the number of specimens. The hydrodynamic radius, RH, was measured from the maxima in the size distribution. Figure 3c shows a representative intensity distribution, with a sharp peak corresponding to a hydrodynamic radius Rh = 4 nm, which indicates small aggregates.

[0140] Small-angle x-ray scattering (SAXS) provides details on the structure of the aggregates. Synchrotron SAXS experiments on solutions were performed using BioSAXS robots on beamline BM29 at the ESRF (Grenoble, France). A few microlitres of samples were injected via an automated sample exchanger at a slow and very reproducible rate into a quartz capillary (1.8 nm internal diameter), in the X-ray beam. The quartz capillary was enclosed in a vacuum chamber, in order to avoid parasitic scattering. After the sample was injected in the capillary and reached the X-ray beam, the flow was stopped during the SAXS data acquisition. The q range was set to 0.004-0.4 A1, with = 1.03 A. The images were captured using a Pilatus IM detector. Data processing (background subtraction, radial averaging) was performed using dedicated beamline software ISPYB. The data shown in Figure 3d shows well-defined form factor oscillations along with a flat low q intensity behaviour (for the lowest concentration), these being characteristic features of a sphere form factor. The outer micelle radius is (2.5 ± 0.1) nm, which is consistent with DLS considering the difference between hydrodynamic radii and thermodynamic radii from SAXS.

[0141] For the two higher concentration solutions studied (1 and 5 wt%, data shown in Figure 3d) there is a broad structure factor peak at low q, which could be described by including a hard sphere structure factor in the fits to the data (fit parameters in SI Table SI).

[0142] SAXS models. All fitting was done using the software SASfit. The SAXS intensity was fitted using the form factor for a spherical shell, together with the structure factor for a hard sphere. The parameters for the spherical shell model are the overall radius of the spherical shell, Ri, the radius of the core, Ab. the scattering contrast of the inner core, / , and the scattering contrast of the shell, r. The parameters for the hard sphere model were the hard sphere radius, Rus, and the volume fraction of hard spheres, (|). The background for all the SAXS curves was fitted with a constant term, C.

[0143] Table SI. Parameters extracted from the fitting of the SAXS data in Figure 2d, using the form factor for a spherical shell, together with the structure factor for a hard sphere.

[0144] Key: Spherical Shell Form Factor: overall radius of the spherical shell, Ri, radius of the core, R2, scattering contrast of the inner core, p, scattering contrast of the shell, T. Hard Sphere Structure Factor: hard sphere radius, Rus, volume fraction of hard spheres, (|).

[0145] For these higher concentrations, however, the high q form factor oscillations are retained, indicating that the internal micelle structure is little changed (as also shown by the fit parameters, except r which represents an overall scale factor of scattering contrast, dependent on concentration).

[0146] Cryogenic-TEM (Cryo-TEM) Imaging was carried out using a field emission cryo-electron microscope (JEOL JEM-3200FSC), operating at 200 kV. Images were taken in bright field mode and using zero loss energy filtering (omega type) with a slit width of 20 eV. Micrographs were recorded using a Gatan Ultrascan 4000 CCD camera. The specimen temperature was maintained at -187 °C during the imaging. Vitrified specimens were prepared using an automated FEI Vitrobot device using Quantifoil 3.5 / 1 holey carbon copper grids with a hole size of 3.5 pm. Just prior to use, grids were plasma cleaned using a Gatan Solarus 9500 plasma cleaner and then transferred into the environmental chamber of a FEI Vitrobot at room temperature and 100 % humidity. Thereafter 3 pl of sample solution was applied on the grid and it was blotted twice for 5 seconds and then vitrified in a 1 / 1 mixture of liquid ethane and propane at temperature of -180 °C. The grids with vitrified sample solution were maintained at liquid nitrogen temperature and then cryo-transferred to the microscope.

[0147] Figure 3e shows atypical cryo-TEM image with well-defined relatively monodisperse micelles with a size consistent with that obtained from DLS and SAXS. The formation of micelles is consistent with the observed disordered conformation of the peptide chains at the surface of the micelles. This behaviour is in contrast to the -sheet structure observed for Cie-KTTKS within nanotapes (V. Castelletto, I. W. Hamley, J. Perez, L. Abezgauz and D. Danino, Chemical Communications, 2010, 46, 9185-9187.)

[0148] Collagen production assay

[0149] Cell culture: Human Dermal Fibroblasts from adult donors (HDFa) cells were commercially obtained from Sigma Aldrich (U.K, product code 106-05 A). For the cytotoxicity and collagen production assays, cells were previously cultivated in DMEM / F12 (Thermo Fisher Scientific, U.K) complemented with 5% of FBS (fetal bovine serum obtained from Thermo Fisher Scientific - U.K), Glutamax, and the antibiotics penicillin 100 lU / mL, streptomycin 100 pg / mL and 0.25 pg / mL of Gibco™ Antibiotic-Antimycotic (Gibco-UK)and incubated at 37 °C with an atmosphere of 5% CO2. Cytotoxicity test

[0150] MTT cytocompatibility assays: The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide - obtained from Sigma - UK) is a yellow, water-soluble chemical. When incubated with living cells, the MTT is converted into insoluble chromogenic formazan crystals by the mitochondria present in the cytoplasm. Thus, the presence and number of living cells can be estimated by colorimetric assays using the conversion of MTT into formazan crystals.

[0151] For the MTT assay, cells were harvested with 0.25% trypsin / EDTA (Gibco - U.K) and seed in a density of 1 x 104cells / well into 96 well plates in DMEM / F12 with 5% FBS, Glutamax, Gibco™ Antibiotic-Antimycotic (Gibco-UK)and 5 pg / mL of insulin (Sigma - U.K.). Cells were incubated overnight at 37 °C with an atmosphere of 5% CO2. The following day, the media was replaced by DMEM / F12 with no supplements and six different concentrations of FMDEG- KTTKS.

[0152] For evaluation of cytotoxicity of conventional stimulants for collagen production (cells were incubated with ascorbic acid 1 mM, TGFpi 0.025 ng / mL (Invitrogen, U.K.) and 0.01 mg / mL of Insulin and the control group was incubated only in DMEM / F12). After 3 days (72 hours) of incubation, cells were washed 3X with PBS, and incubated for 4 hours with 0.5 mg / mL of MTT diluted in DMEM / F12 without phenol red (Invitrogen) at 37 °C. After this, DMSO was added for the solubilization of the formazan crystals and the plate was incubated again for 15 minutes at 37 °C. After this final incubation, the absorbance at 560 nm was read using an Infinite F50 plate reader (Tecan - Switzerland). The data was analysed using the software prism 8, first removing the background by subtracting the OD of control wells containing only the media with MTT and estimating the number of cells by comparing the absorbance at 560 nm with controls incubated only in DMEM / F12 in triplicate. The statistical test of choice was the ordinary one-way ANOVA with Bonferroni’s correction for multiple comparisons.

[0153] Figure 4 shows data from this assay for different concentrations of peptide conjugate (from 0.2 to 0.00625wt%), above and below the CAC, as well as the conventional stimulants such as insulin, ascorbic acid and TGFpi which have been previously used in the literature for stimulating the collagen production in fibroblasts by different mechanisms; such as fibroblast activation and proliferation resulting in higher collagen deposition. (ANOVA, n = 3), ** p < 0.01, *** p < 0.001. A statistically significant proliferative effect of the conventional stimulants is observed compared to control incubated only with basal media, and a decrease in viability of around 30% for cells (compared to basal media) incubated with peptide conjugate below the CAC (without statistical significance). Samples incubated with the peptide conjugate above the CAC presented a very large decrease in cell density, when compared with the control, indicating high cytotoxicity above the CAC. MTT assay indicated a much better tolerance for the lipopeptide for concentrations below the CAC. The MTT assay shows that, in monomeric form, lipopeptide (e.g., FMDEG-KTTKS) has good cell viability. Compared with Cie-KTTKS based sequences, the cytocompatibility is improved since cell viability for Cie-KTTKS is reduced by 40% or more, even at concentrations up to ten times lower (in the range 0.002 - 0.008 wt%) than used in present invention.

[0154] Collagen production assays

[0155] For estimating the production of collagen for the HDFa cells, like the MTT cells were seeded at a density of 1 x 104 cells / well in complete DMEM plus 5 pg / mL of insulin and incubated for three days in DMEM without supplements in the presence of different concentrations of peptide and the conventional stimulants (ascorbic acid 1 mM, TGFpi 0.025 ng / mL and 0.01 mg / mL of Insulin). After this incubation, the wells were washed with PBS and fixed with ice cold ethanol 70% to fix deposited material at the surface. This plate was transferred to a -80°C ultra-freezer for 10 minutes and washed with water for removing any residual ethanol. After fixation, the collagen was incubated overnight in a solution containing Direct red 1 mg / mL in picric acid at 4°C under gently agitation. Finally, the wells were washed with distilled water until all exceeding dye was removed, the collagen and cells were then treated with NaOH ImM for 10 minutes at room temperature and absorbance was measured at 490 nm.

[0156] Polarized microscopy: For the microscopy assays, 5 x 104HDFa cells were deposited on coverslips and incubated in DMEM / F12 with 10% FBS, 2 mM of glutamax. Antibiotic- antimycotic and 5 pg / mL of insulin overnight in 24 well plates. The next day, the cells were washed and incubated for 3 days with the lipopeptide, conventional stimulants and controls were incubated with only DMEM / F12 without any supplements. The coverslips were washed 3 times with PBS, fixed with cold ethanol and stained with Sirius red. After the incubation with Sirius red, the coverslips were washed with distilled water to remove any unbound dye and mounted in glass slides. The slides were imaged with an Olympus BX41 microscope (Olympus Life Sciences, UK) equipped with polarizers. Collagen production was evaluated for three concentrations of lipopeptide below the CAC. Figure 5a shows the average amount of collagen [mg / mL] produced by each well containing HDFa cells in the presence of FMdEG-KTTKS, conventional stimulants and basal media without any additives. Figure 5b shows the amount of collagen produced per cell.

[0157] Figure 6 shows polarized optical microscopy (POM) images of the cells stained with Sirius red. Specifically, Figure 6 shows polarized optical microscopy images of HDFa cells stained with Sirius red in the presence of (a) Basal media, (b) Conventional stimulants, (c) 0.00625 wt% FMdEG-KTTKS, (d) 0.0125 wt% FMdEG-KTTKS, (e) 0.025 wt% FMdEG-KTTKS. Scale bar = 200 mm.

[0158] Figure 7 shows brightfield microscopy images of HDFa cells stained with Sirius red in the presence of (a) Basal media, (b) Conventional stimulants, (c) 0.00625 wt% FMdEG-KTTKS, (d) 0.0125 wt% FMdEG-KTTKS, (e) 0.025 wt% FMdEG-KTTKS. Scale bar = 200 pm.

[0159] The quantitative data in Figure 5a indicate a slight increase in collagen production by cells incubated with lipopeptide for all three concentrations studied, and a major increase for cells incubated with the conventional stimulants. Figure 5b shows average collagen production per cell [% relative to control]. (ANOVA, n = 5), * p < 0.05, ** p < 0.01. Plotting the collagen production per cell (Figure 5b), it becomes clear that there is a statistically significant increase for all concentrations. For example, incubating DMEM / F12 with 0.0125% of FMdEG-KTTKS (II) presented an increase in collagen production per cell of 221 ± 41% when compared with the control group incubated only in media. Contrary to the result obtained for the FMdEG- KTTKS (II), the conventional stimulants presented only a minimal increase in the production of collagen / cell, without statistical significance. This indicates that the overall increase in collagen production is mostly due the proliferative increase of dermal fibroblasts under the influence of TGF-P and insulin.

[0160] HDFa Immunocytochemistry

[0161] An immunocytochemistry assay was performed on HDFa cells to compare the production of type I collagen and type III by confocal microscopy.

[0162] A quantity 9xl04HDFa cells were incubated in 13 mm coverslips using 24 well plates using DMEM / F12 media containing 5% FBS and left inside a cell incubator at 37 °C in an atmosphere of 5% CO2 over 24, to attach to the coverslips. For the assay, cells were incubated with DMEM / F12 containing conventional supplements, lipopeptide (II) or only media for 72 hours inside the cell incubator. After the 72 hour incubation, cells were washed 3 times with PBS and fixed using Paraformaldehyde 4% (Thermo Fisher Scientific - UK) for 10 minutes at room temperature, washed 3 times with PBS again and permeabilized with Triton-X 100 at 0.1 % in PBS (Merck-UK) for 15 minutes. The coverslips were again washed with PBS and blocked with BSA 1% for 1 hour at room temperature. Cells were washed again 3 times with PBS, with the last wash for 5 minutes, and incubated with primary antibodies COL1A1 (E3E1X) Mouse mAb for type I collagen and COL3A1 (E3K9U) Rabbit mAb for collagen type III (both from Cell signalling - EU) at 370C for 1 hour. The coverslips were again washed 3 times with PBS for 5 minutes and incubated with secondary antibodies Goat anti-Mouse IgG -Alexa Fluor™ 488 and Donkey anti-Rabbit IgG - Alexa Fluor™ 647 (both bought from ThermoFisher Scientific - UK) for one hour at 37 °C protected from the light. Finally, cells were washed again and incubated for 1 hour with Phalloidin - Texas red (Thermo-Fisher Scientific - UK), washed 3 times with PBS one last time and mounted on glass slides using mounting media consisting of 90% glycerol + 10% PBS at pH 8.5 and ascorbic acid to prevent quenching. The images for immunofluorescence were acquired with a Nikon ARI Confocal microscope, and processed using Fiji-ImageJ.

[0163] The nuclei of cells were stained with DAPI to assess whether the cells were alive and for localization. The cells were also stained with phalloidin-Texas red to reveal cell morphology: phalloidin stains the cytoskeleton of cells and allows the cytoplasm to be differentiated from the from the extracellular matrix and type I and type III collagen, to evaluate the collagen production.

[0164] Lipopeptide (II) was examined at a concentration of 0.0062 wt%. A control sample included DMEM without serum.

[0165] The micrographs show an increase in the production of collagen type I in samples incubated with 0.062 wt% Fn-KTTKS. Specifically, increased production of collagen was observed in the extracellular matrix. Micrographs for the control sample showed minimal collagen production.

[0166] Anti-inflammatory Effects

[0167] Nitric oxide (NO) is a signalling molecule involved in the pathogenesis of inflammation. Many cell types will produce NO after stimulus during cellular injury or infections and will modulate inflammation in different tissues (Coleman JW. Nitric oxide in immunity and inflammation. International immunopharmacology. 2001 Aug 1 ; 1(8): 1397-406). NO production can be quantified using Griess reagent. The Griess reaction is a chemical reaction that detects nitrite and nitrate in biological fluids resulting in a red coloration after forming diazonium salts when nitrite reacts with a primary aromatic amine (Kampfer A, et al., Chemical Research in Toxicology. 2017 Apr 17;30(4): 1030-7.).

[0168] During an inflammation process, NO is produced by cells and quickly degrades into nitrite in the medium, which can be detected using the Griess reagent. This test has already been used in the literature to evaluate the ant inflammatory effect of KTTKS -related peptides and molecules containing famesyl by monitoring the nitrite levels present at the medium after stimulus using LPS or other pro-inflammatory agents and blocking the capacity of these agents to raise nitrite levels in the sample due to enhanced NO production by cells. (Fadilah NI, et al., Journal of Saudi chemical society. 2020 Aug l;24(8):606-19; Wang M et al., Bioorganic & Medicinal Chemistry Letters. 2016; 26(14):3342-5).

[0169] To evaluate the anti-inflammatory effect of this lipopeptide, 5xl03RAW 264.7 mouse macrophage cells were seed in 96 well plates and incubated inside a cell incubator at 37 °C and 5% of CO2 for 24 hours. The medium used was DMEM (ThermoFisher Scientific UK) supplemented with 10% of fetal bovine serum (Gibco), Gibco™ Antibiotic-Antimycotic and L-glutamine (Gibco). After the initial 24 hours incubation, RAW cells were washed 3 times with PBS and incubated in fresh DMEM without FBS or phenol red + 5pg of E. Coll lipopolysaccharide (LPS). Samples were incubated in the presence of lipopeptide (II) at 0.025, 0.0125 and 0.0062 wt% and control samples only in basal media containing LPS. Negative controls were cultured in DMEM without LPS to observe the normal expected nitrite production and compared with the enhanced nitrite production due to NO liberation in the media induced by adding LPS to the media. After LPS incubation, 90 pL of the supernatant of the cells was collected and transferred to a new 96 well plate. An aliquot 10 pL of Griess reagent solution (Merck, Cas-G4410) was added and the plate was incubated at room temperature for 10 minutes protected from the light. The resulting absorbance was measured at 550 nm using an Infinite F50 plate reader from Tecan and data were plotted to track the nitrite production. A comparative sample with LPS and conventional stimulants (ascorbic acid 1 mM, TGFpi 0.025 ng / mL and 0.01 mg / mL of Insulin) was also examined.

[0170] It can be seen that the nitrite levels of all three compositions comprising lipopeptide (II) were comparable to the control.

[0171] Figure 8 shows the percentage of nitrites compared to control with FMdEG-KTTKS from 0.0062% to 0.025. The compositions comprising lipopeptide (II) completely counteract LPS-induced nitrite production, demonstrating that this is an effective treatment of inflammation.

[0172] Skin healing

[0173] A scratch assay was performed to evaluate cellular migration over an "injury" made in a mono cellular layer of fibroblasts, to simulate skin injury. This involved creating a cell monolayer on a multi-well assay plate; creating a cell-free zone in the monolayer; and monitoring the recolonization of the cell free zone to quantity cell migration over a period of 72 hours.

[0174] For this assay, L929 murine fibroblasts were cultivated in DMEM containing 10% FBS, Gibco™ Antibiotic-Antimycotic and L-glutamine (Gibco) inside a T-25 flask. Cells were detached with Trypsin (ThermoFisher Scientific - UK) and cultivated in 24 well plates until reaching complete confluence. After this, a scratch was made into this cell monolayer using a 200 uL pipette tip to simulate a skin injury. The plate was washed 3 times with PBS to remove serum and dead cells before adding fresh DMEM without FBS. A standard optical microscope was used to acquire pictures of the scratches on each well at the same position over 72 h to evaluate migration from the borders of the scratch to the free cell zone created due to the artificial "injury".

[0175] Samples were examined containing lipopeptide (II) at concentrations of 0.025 wt%, 0.0125 wt% and 0.00625 wt%.

[0176] The control sample incubated only in basal media without any additives showed minimal, if any, migration of the fibroblast layer, where even after 72 hours we do not observe any visible migration to the centre of the wound.

[0177] However, for samples incubated at 0.0062 wt% the migration of cells from the edges of the sectioned area to the middle of the wound starting after 24 h was observed, visibly restoring the fibroblast layer after 72 h when compared to the control.

[0178] Rat excisional wound model

[0179] Wound healing was examined using a rat diabetic excisional wound model. Lipopeptide (II) was compared to a control sample over a period of 25 days. Body weight was measured for each group every four days. Female Albino Wistar rats, aged 10 to 12 weeks and weighing between 180 and 190 g, were kept under a 12-hour light-dark cycle with unrestricted access to a standard laboratory diet and water. Diabetes was induced by fasting the rats overnight followed by an intraperitoneal injection of streptozotocin (STZ; Sigma-Aldrich) at a dose of 45 mg / kg body weight in 0.1 mol / L sodium citrate buffer, pH 4.5. Weekly blood glucose levels were measured from tail vein samples. Rats with blood glucose levels above 17 mmol / L during the induction period were deemed stably diabetic and selected for wound healing experiments, with approximately 95% achieving stable hyperglycemia post-STZ injection.

[0180] Three weeks post-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 anaesthesia was maintained using xylazine and ketamine (Sigma-Aldrich, Germany; 5 mg / kg body weight xylazine and 40 mg / kg body weight ketamine in sterile PBS). During surgery, a continuous flow of isoflurane (2 mL / min) was administered to sustain anaesthesia. Prior to wound creation, the dorsal hair was completely shaved, and the skin was sterilized with povidone-iodine. Using sterile biopsy punches, full-thickness circular wounds, 8 mm in diameter, were made on the dorsum. Bioactive peptide solutions were prepared in PBS just before topical application, and 300 pL of 1% (w / v) peptide solution or PBS was applied directly to each wound. To prevent postsurgical infections and aid in monitoring, a thin, translucent adhesive film (Tegaderm, 3M, USA) was placed over the wounds. Rats were then returned to their cages and monitored closely for 48 hours for any signs of postsurgical stress, secondary infections, or ulcers. The controls for the studies were untreated animals.

[0181] Wounds were photographed at fixed intervals on days 0, 4, 8, 12, 16, 20 and 24 using a digital camera set at a constant distance and magnification. Wound closure was quantified using ImageJ software with the formula: % Wound Closure = [(original wound area - current wound area) / original wound area] x 100%. The weight of the animals was monitored and recorded throughout the 20-day experimental period.

[0182] In the diabetic model, rats were euthanized on day 24 after wound creation. Skin tissues were fixed in 4% paraformaldehyde and immersed in 30% sucrose for 48 hours for cryopreservation. Subsequently, 5 pm thick tissue sections were obtained from OCT-embedded blocks. These sections were analysed using Hematoxylin and Eosin (H&E) staining as well as Masson’s trichrome staining. The stained sections were then imaged using an Olympus BX-53 upright microscope. Figure 9a is a graph showing the percentage wound healing for each sample group over the course of the experiment. Figure 9b is a graph showing the body weight for each treatment group over the course of the experiment.

[0183] As shown by Figure 9a, the wounds exposed to the sample containing lipopeptide (II) were more healed than the wounds exposed to the control sample throughout the experiment.

[0184] As shown by Figure 9b, the body weight of rats used in the experiment were not affected over the course of the experiment. This illustrates that lipopeptide (II) is safe and well tolerated by the subjects at effective doses.

[0185] Discussion and Conclusions

[0186] The designed bioactive collagen-stimulating peptide conjugate FMdEG-KTTKS (II) undergoes self-assembly into spherical micelles above a sharply defined CAC. The formation of micelles is in contrast to previous reports on Cie-KTTKS and Cix-KTTKS. which form extended nanostructures, i.e., long nanotapes. This highlights the influence of the chain structure on the nanostructure formed.

[0187] The MTT assay shows that, in monomeric form, FMdEG-KTTKS (II) induces the production of collagen by dermal fibroblasts with good cell viability. Compared with Cie-KTTKS based sequences, the cytocompatibility is improved since cell viability for Cie-KTTKS is reduced by 40% or more, even at concentrations up to ten times lower (in the range 0.002 - 0.008 wt%) than used in the present work. The fact that FMdEG-KTTKS (II) does not induce significant increase or decrease in cell proliferation is a desirable trait for the application of this compound as a cosmetic or in the clinic.

[0188] Even with improved cytocompatibility, FMdEG-KTTKS (II) significantly stimulates collagen production per cell, although to a lower extent than Cie-KTTKS for which a more than ten-fold increase in collagen / cell compared to basal media is observed at concentration 0.008 wt% peptide amphiphile (R. R. Jones, V. Castelletto, C. J. Connon and I. W. Hamley Molecular Pharmaceutics, 2013, 10, 1063-1069). The substantial increase in cytotoxicity of FMdEG- KTTKS (II) above the CAC, where the molecules are self-assembled into micelles, is notable. This property could be the basis for applications where cytotoxicity is desirable, for example in antimicrobial materials. The type of collagen produced was type I collagen which is the predominant collagen found in many types of tissue, including skin, tendons, bones and many other structures in the body. Specifically, it has been shown that FMdEG-KTTKS (II) enables increased type I collagen production in HDFa cells, in particular increasing production of type I collagen in the extracellular matrix.

[0189] FMdEG-KTTKS (II) additionally demonstrates promising anti-inflammatory effects.

[0190] FMdEG-KTTKS (II) has also been found to enable the faster healing of skin wounds through fibroblast and rat models. In the murine model, FMdEG-KTTKS (II) was found to be safe and well tolerated by the subjects at effective doses.

Claims

CLAIMS1. A lipopeptide of formula (I):R - L - P(I) wherein:R is a hydrocarbyl group;L is a linker; andP is a peptide comprising from 2 to 10 amino acid residues.

2. The lipopeptide of claim 1, wherein the linker L comprises a polyether moiety.

3. The lipopeptide of claim 2, wherein the polyether moiety is represented by the formula:wherein n and m are integers independently selected from 1, 2 and 3.

4. The lipopeptide of claim 3, wherein the lipopeptide has the formula:wherein: n and m are integers independently selected from 1, 2 and 3;R is the hydrocarbyl group; andP is the peptide.

5. The lipopeptide of claim 4, wherein the lipopeptide has the formula:wherein:R is the hydrocarbyl group; and P is the peptide.

6. The lipopeptide of claim 2 or claim 3, wherein the polyether moiety is a polyethylene glycol moiety, polypropylene glycol moiety or polytetramethylene glycol moiety.

7. The lipopeptide of claim 6, wherein the linker comprises a diethylene glycol moiety.

8. The lipopeptide of any preceding claim, wherein the peptide is a collagen promoting peptide.

9. The lipopeptide of claim 8, wherein the peptide is selected from KTTKS, ETTES, GPKG, LSVD, KT, betaAH (betaA: (3-alanine), VW, RGD, RGDS, VYV, KVK, GQPR, PKEK, KQVY, betaAHSH, 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, P-Ala-His, Val-Trp, Cu-Gly-His-Lys, Gly-His-Lys-Mn2+, Lys-Met(C>2)-Lys, Arg-Gly-Asp, Val-Tyr-Val, Lys-a-Asp-Ile, Lys-Val-Lys, Gly-Gln-Pro-Arg, Pro-Lys- Glu-Lys, Lys-Asp-Val-Tyr, P-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-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.

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

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

12. The lipopeptide of any preceding claim, wherein the peptide comprises from 3 to 10 amino acid residues.

13. The lipopeptide of any preceding claim, wherein the hydrocarbyl group: a) is branched; b) comprises from 5 to 25 carbon atoms; and / or c) is unsaturated.

14. The lipopeptide of any preceding claim, wherein the hydrocarbyl group is a terpenoid.

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

16. The lipopeptide of claim 15 wherein the sesquiterpene is a famesyl group.

17. The lipopeptide of claim 16, wherein the lipopeptide has the formula:

18. A composition comprising: a) a lipopeptide as defined by any one of claims 1 to 17; and b) at least one additional ingredient.

19. The composition of claim 18, wherein the additional ingredient is a dermatologically acceptable carrier.

20. A cosmetic use of a lipopeptide as defined by any one of claims 1 to 17, or a composition as defined by claim 18 or claim 19.

21. A method of cosmetic prevention and / or treatment, wherein the method comprises: providing the lipopeptide as defined by any one of claims 1 to 17, and applying the lipopeptide to the skin of a user in order to enhance the appearance of the skin.

22. The cosmetic use of claim 20 or the method of claim 21, wherein the use or the method is for regenerating skin, for the treatment of nails, and / or for improving the appearance of hair.

23. A lipopeptide for use in medicine, wherein the lipopeptide is as defined by any one of claims 1 to 17.

24. A lipopeptide for use in a method of prevention, treatment or amelioration of a wound in the skin of a subject and / or in a method of prevention, treatment or amelioration of an inflammation-related condition and / or a collagen-related condition, wherein the lipopeptide is as defined by any one of claims 1 to 17.

25. An article comprising a lipopeptide of any one of claims 1 to 17, wherein the article is configured such that, in use, the lipopeptide can contact a tissue (e.g. the skin) of a subject.37SUBSTITUTE SHEET (RULE 26)

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