Novel peptide conjugates

Abstract

The present invention relates to peptides that bind to hair. Furthermore, the invention relates to peptide conjugates wherein the peptides of the invention are linked to a payload. In addition, the invention relates to the use of the peptides or peptide conjugates of the invention for the treatment of hair.

Description

Field of the Invention

[0001] The present invention relates to peptides that bind to hair. In addition, the present invention relates to peptide conjugates, wherein the peptides of the present invention are linked to a payload. Further, the present invention relates to the use of the peptides or peptide conjugates of the present invention for treating hair. Background of the Invention

[0003] Hair dyes can be classified into three categories, namely, permanent, semi-permanent, or direct and temporary hair dyes. Permanent hair dyes are usually oxidative dyes. These dyes consist of oxidative dyes and an oxidizing agent (such as hydrogen peroxide). The oxidizing agent oxidizes the dye precursors, and the dye precursors combine to form large color molecules. Although oxidative hair dyes provide long-lasting color, the oxidizing agents they contain can cause severe hair damage. Semi-permanent hair dyes are pre-formed dye molecules that are applied to the hair and provide short-term color. As described in WO2001 / 045652, the durability of these dyes can be improved by using nanoparticle hair dye materials with a particle size of 10 to 500 nm. Temporary hair dyes are colorants applied to the hair surface and can be removed after one wash. Semi-permanent and temporary dyes cause less damage to the hair, but their effects are short-term.

[0004] Therefore, there has been an ongoing effort to develop technologies that can deliver hair dye molecules to the hair permanently and safely.

[0005] Peptides or polypeptides that can bind to the body surface can promote the targeted and concentrated delivery of beauty or therapeutic agents. For example, JP08104614 and US5,597,386 describe hair dyes composed of anti-keratin antibodies covalently linked to dyes or pigments. The antibody enhances the binding of short-term hair dyes to the hair, thereby extending the effect while maintaining safety. The use of antibodies, antibody fragments, or alternative immunoglobulin structures can effectively enhance the binding of hair care products and extend the effect, but these antibodies or immunoglobulin structures are difficult to produce and costly. Therefore, JP2002363026 describes the use of conjugates for skin and hair care compositions, the conjugates being composed of smaller single-chain antibodies (preferably anti-keratin antibodies) conjugated to dyes, ligands, and beauty substances. These single-chain antibodies are effective and can be prepared using genetic engineering techniques, but due to their large size, they are still difficult and expensive to prepare.

[0006] In view of the above, it is clearly necessary to develop small peptide molecules that are inexpensive to produce and bind to hair with high affinity. The peptides described must be able to conjugate with payload molecules (i.e., hair care products such as hair dye molecules) to promote the targeted delivery of such hair care products, thereby producing a long-lasting and safe effect.

[0007] The inventors of the present application utilized phage display screening technology to identify novel peptides that bind to hair with high affinity. Such peptides can be conjugated with payload molecules, such as dyes, to achieve safe and long-lasting hair treatment. Summary of the Invention

[0009] In a first aspect of the present invention, there is provided a peptide that binds to hair and comprises the following sequence:

[0010] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8

[0011] Wherein,

[0012] The amino acid of X1 is absent or is K;

[0013] The amino acid of X2 is absent or is G;

[0014] The amino acid of X3 is absent or is T;

[0015] The amino acid of X4 is absent or is A;

[0016] The amino acid of X5 is absent or is P;

[0017] The amino acid of X6 is absent or is G;

[0018] The amino acid of X7 is absent or is selected from L and R;

[0019] The amino acid of X8 is absent or is A;

[0020] Wherein if X6 is absent, then X3, X4, and X5 are present and are T, A, and P respectively. Brief Description of the Drawings

[0022] Figure 1 Shows the detection by infrared spectrometry of the production of a peptide-zinc oxide nanoparticle conjugate (product: light gray line) from a peptide (SEQ ID NO.3) and a nanoparticle-spacer moiety (reagent: dark gray line) (see Conjugate Example 1).

[0023] Figure 2 Shows that the peptide-zinc oxide nanoparticle conjugate is capable of binding to hair (Figure A), that the zinc oxide nanoparticles alone are unable to bind to hair (Figure B), and the resistance of the peptide-zinc oxide nanoparticle conjugate binding to shampooing. Detailed Description of the Invention

[0025] Binding to hair can be quantified as described in the hair binding assay (see "Examples" section below). Suitably, the peptide-5-carboxytetramethylrhodamine (TAMRA) conjugate of the invention will bind to hair and produce a fluorescence intensity equivalent to 25% or more, such as 30% or more, such as 35% or more, such as 40% or more, such as 45% or more, such as 50% or more, or such as 60% or more of the fluorescence intensity produced when the peptide-TAMRA conjugate comprises the peptide of SEQ ID NO. 3.

[0026] Peptide

[0027] In a first aspect of the invention, there is provided a peptide that binds to hair and comprises the following sequence:

[0028] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8

[0029] wherein,

[0030] the amino acid of X1 is absent or is K;

[0031] the amino acid of X2 is absent or is G;

[0032] the amino acid of X3 is absent or is T;

[0033] the amino acid of X4 is absent or is A;

[0034] the amino acid of X5 is absent or is P;

[0035] the amino acid of X6 is absent or is G;

[0036] the amino acid of X7 is absent or is selected from L and R;

[0037] the amino acid of X8 is absent or is A;

[0038] wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively.

[0039] In another aspect, the invention provides a peptide that binds to hair and consists of the following sequence:

[0040] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8

[0041] wherein,

[0042] the amino acid of X1 is absent or is K;

[0043] the amino acid of X2 is absent or is G;

[0044] the amino acid of X3 is absent or is T;

[0045] The amino acid of X4 is absent or is A;

[0046] The amino acid of X5 is absent or is P;

[0047] The amino acid of X6 is absent or is G;

[0048] The amino acid of X7 is absent or is selected from L and R;

[0049] The amino acid of X8 is absent or is A;

[0050] Wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively.

[0051] In a suitable aspect, X6 is present and is G.

[0052] In a suitable aspect, X7 is present and is selected from L and R. In a particularly suitable embodiment, X7 is present and is L.

[0053] In a suitable aspect, X5 is present and is P.

[0054] In a suitable aspect, X4 is present and is A.

[0055] In a suitable aspect, X3 is present and is T.

[0056] In a suitable aspect, X2 is present and is G.

[0057] In a suitable aspect, X8 is present and is A.

[0058] In a suitable aspect, X1 is present and is K.

[0059] In one aspect of the present invention, the peptide further comprises one or more (e.g., one or two, particularly one) cysteine residues, such as cysteine residues contained at the N-terminal position, at the C-terminal position and / or at any other position outside the motifs described above. In a suitable aspect, the peptide comprises a cysteine residue at the N-terminal position.

[0060] For example, the peptide that binds to hair comprises the following sequence or consists of the following sequence:

[0061] Y a -X1-X2-X3-X4-X5-R-R-K-X6-X7-X8-Y b

[0062] Wherein X1-X8 are as defined elsewhere herein, and Y a and Y bis independently C or absent, and Y a and Y b one or both of which are C.

[0063] Preferably, Y a and Y b one of which is C and the other is absent. Preferably, Y a is C, and Y b is absent.

[0064] In one aspect of the invention, the peptide comprises 6 or more amino acids, i.e., comprises, for example, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, etc. amino acids. In a suitable aspect, the peptide comprises 7 or more amino acids.

[0065] In one aspect of the invention, the peptide is composed of 30 or fewer amino acids, i.e., composed of 25 or fewer, 20 or fewer, 18 or fewer, 15 or fewer amino acids.

[0066] For example, in one aspect of the invention, the peptide is composed of 6 to 30 amino acids, particularly 6 to 25 amino acids, 6 to 20 amino acids, 6 to 18 amino acids or 6 to 15 amino acids. For example, the peptide can be composed of 7 to 20, 7 to 18, 7 to 15, 8 to 20, 8 to 18 or 8 to 15 amino acids.

[0067] In one aspect of the invention, there is provided a peptide comprising a sequence selected from the group consisting of: CKGTAPRRKGLA, CTAPRRKGL, CGTAPRRKGR, CRRKGLA, KGTAPRRKGL, KGTAPRRKG, GTAPRRKGL, TAPRRKGL, APRRKGL, RRKGL, GTAPRRKG, TAPRRK, CGTAPRRKGL and GTAPRRKGR. In a suitable aspect of the invention, the peptide comprises a sequence selected from the group consisting of: CKGTAPRRKGLA, CTAPRRKGL, CGTAPRRKGR, CRRKGLA, GTAPRRKGL, TAPRRK, CGTAPRRKGL and GTAPRRKGR. In a particularly suitable aspect of the invention, the peptide comprises a sequence selected from the group consisting of: CKGTAPRRKGLA, CTAPRRKGL, CGTAPRRKGR and CRRKGLA.

[0068] In one aspect of the present invention, there is provided a peptide consisting of a sequence selected from the group: CKGTAPRRKGLA, CTAPRRKGL, CGTAPRRKGR, CRRKGLA, KGTAPRRKGL, KGTAPRRKG, GTAPRRKGL, TAPRRKGL, APRRKGL, RRKGL, GTAPRRKG, TAPRRK, CGTAPRRKGL, and GTAPRRKGR. In a suitable aspect of the present invention, the peptide consists of a sequence selected from the group: CKGTAPRRKGLA, CTAPRRKGL, CGTAPRRKGR, CRRKGLA, GTAPRRKGL, TAPRRK, CGTAPRRKGL, and GTAPRRKGR. In a particularly suitable aspect of the present invention, the peptide consists of a sequence selected from the group: CKGTAPRRKGLA, CTAPRRKGL, CGTAPRRKGR, and CRRKGLA.

[0069] It should be understood that the peptides of the present invention can be used in the form of their salts, such as their physiologically acceptable salts. Such salts can be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary, and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium, and magnesium salts).

[0070] Peptide Conjugates

[0071] In one aspect of the present invention, there is provided a peptide conjugate of the following formula that binds to hair

[0072] P-S-Py

[0073] wherein P is the peptide of the present invention, S is an optional spacer moiety, and Py is a payload.

[0074] Payload

[0075] In a suitable aspect of the present invention, the payload is a colorant, such as a dye, such as a fluorescent dye, a nanoparticle, an ultraviolet absorbing substance, a conditioning agent, a beneficial polypeptide, or a vitamin.

[0076] Colorants include any dyes, pigments, etc. that can be used to change the color of hair. Suitable colorants are known in the art and include, but are not limited to, dyes such as 4-hydroxypropylamino-3-nitrophenol, 4-amino-3-nitrophenol, 2-amino-6-chloro-4-nitrophenol, 2-nitro-p-phenylenediamine, N,N-hydroxyethyl-2-nitro-phenylenediamine, 4-nitroindole, henna, HC Blue 1, HC Blue 2, HC Yellow 4, HC Red 3, HC Red 5, Disperse Violet 4, Disperse Black 9, HC Blue 7, HC Blue 12, HC Yellow 2, HC Yellow 6, HC Yellow 8, HC Yellow 12, HC Brown 2, D&C Yellow 1, D&C Yellow 3, D&C Blue 1, Disperse Blue 3, Disperse Violet 1, eosin derivatives such as D&C Red No. 21 and halogenated fluorescein derivatives such as D&C Red No. 27, the combination of D&C Red Orange No. 5 with D&C Red No. 21 and D&C Orange No. 10; and pigments such as D&C Red No. 36 and D&C Orange No. 17, calcium lakes of D&C Red Nos. 7, 11, 31 and 34, barium lake of D&C Red No. 12, strontium lake of D&C Red No. 13, FD&C Yellow No. 5, FD&C Yellow No. 6, aluminium lakes of D&C Red No. 27, D&C Red No. 21 and FD&C Blue No. 1, metal oxides such as iron oxide, chromium oxide, titanium dioxide, zinc oxide and barium oxide, manganese violet, ultramarine blue, bismuth citrate, and carbon black particles.

[0077] Nanoparticles include any metal or semiconductor particles having an average particle size between 1 and 100 nm, for example between 1 and 40 nm. As used herein, "particle size" and "particle diameter" have the same meaning. Metal nanoparticles include, but are not limited to, gold, silver, platinum, palladium, iridium, rhodium, osmium, iron, copper, cobalt, and alloys composed of these metals. "Alloy" is defined herein as a homogeneous mixture of two or more metals. "Semiconductor nanoparticles" include, but are not limited to, cadmium selenide, cadmium sulfide, silver sulfide, cadmium sulfide, zinc sulfide, zinc selenide, lead sulfide, gallium arsenide, silicon, and particles of metal oxides (such as zinc oxide, tin oxide and iron oxide). Nanoparticles can be used for dyeing hair and / or protecting hair from ultraviolet radiation and / or improving hair texture and / or increasing hair volume.

[0078] Nanoparticles can be coated with substances such as (3-aminopropyl)triethoxysilane (APTES) or azobisisobutyronitrile (AIBN) to functionalize the nanoparticles with reactive groups that can promote the direct or indirect ligation of the payload to the peptide.

[0079] A UV absorbing substance is any substance that protects the hair by absorbing or reflecting UV radiation. When such a substance is attached to the hair, it reduces the amount of potentially harmful UV radiation received by the hair, in a similar manner to the substances used in sunscreens. UV absorbing substances include, but are not limited to, zinc oxide and titanium oxide nanoparticles.

[0080] Conditioning agents include any material that improves the appearance, texture and shine of hair and increases volume or smoothness. Suitable hair conditioning agents are known in the art and include, but are not limited to, cationic polymers such as cationic guar gum, diallylquaternium / acrylamide copolymers, quaternized polyvinylpyrrolidones and their derivatives, and polyquaternium compounds; cationic surfactants such as stearyl ammonium chloride, cetrimonium chloride, and sapamin hydrochloride; fatty alcohols such as behenyl alcohol; fatty amines such as stearylamine; waxes; esters; nonionic polymers such as polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol; silicones; siloxanes such as decamethylcyclopentasiloxane; polymer emulsions such as amodimethicone; and compatibilizers such as chitosan.

[0081] Beneficial polypeptides are those that have beneficial properties when the peptide conjugate is combined with hair. The beneficial properties include one or more of the following: improving and / or stimulating hair growth, increasing hair volume, reducing hair thinning, increasing hair strength, and reducing or increasing hair charge. Suitable beneficial polypeptides include, but are not limited to, insulin-like growth factor-1 (IGF-1), acetyl tetrapeptide-3, pea peptides, collagen peptides, copper tripeptide (GHK-Cu), sh-polypeptide 3 (keratin growth factor), sh-oligopeptide-4 (thymosin beta 4), and sh-polypeptide 9 (VEGF).

[0082] Vitamins include any organic molecule, or chemically closely related molecules, i.e., vitamers, that are essential nutrients required in trace amounts for the proper health, function, and growth of an organism. A suitable list of vitamins includes, but is not limited to, vitamin A, vitamin B3, vitamin B7, vitamin B12, vitamin C, vitamin D, and vitamin E.

[0083] Linkage of P and Py

[0084] In one aspect of the present invention, Py is functionalized to facilitate direct or indirect attachment to P. In one aspect of the present invention, Py is functionalized to facilitate direct attachment to P. In another aspect of the present invention, Py is functionalized to facilitate indirect attachment to P (i.e., via an optional spacer moiety S), that is, Py is functionalized to facilitate attachment to an optional spacer moiety, and the optional spacer moiety is functionalized to facilitate attachment to P. Similarly, in one aspect of the present invention, P is functionalized to facilitate direct or indirect attachment to Py. In one aspect of the present invention, P is functionalized to facilitate direct attachment to Py. In another aspect of the present invention, P is functionalized to facilitate indirect attachment to Py, that is, P is functionalized to facilitate attachment to an optional spacer moiety, and the optional spacer moiety is functionalized to facilitate attachment to Py.

[0085] In one embodiment, the optional spacer moiety S is absent. In one embodiment, the optional spacer moiety S is present.

[0086] The optional spacer moiety S isolates the payload from the peptide and may help ensure that the payload does not interfere with the binding of the peptide to the hair. The spacer can be a variety of molecules, such as an alkyl chain, a phenyl compound, ethylene glycol, an amide, an ester, etc. Preferred spacers are hydrophilic and have a chain length of from 1 to about 100 atoms, such as from 2 to about 30 atoms, such as from 2 to about 10 atoms. Examples of suitable spacers include, but are not limited to, ethanolamine, ethylene glycol, polyethylene with a chain length of 6 carbon atoms, polyethylene glycol with 3 to 6 repeating units, phenoxyethanol, propanolamide, butanediol, butanediolamide, propylphenyl, and ethyl, propyl, hexyl, stearyl, acetyl, and palmitoyl alkyl chains.

[0087] In one aspect of the present invention, Py or the optional spacer moiety is attached to P via a cysteine residue in P, i.e., Py is attached to P directly or indirectly (i.e., via the optional spacer moiety S) via a cysteine residue in P.

[0088] In another aspect of the present invention, Py or the optional spacer moiety is attached to P via an amino group in P, i.e., Py is attached to P directly or indirectly (i.e., via the optional spacer moiety S) via an amino group in P. In a suitable aspect of the present invention, Py or the optional spacer moiety is attached to P via the N-terminal amino group in P, i.e., Py is attached to P directly or indirectly (i.e., via the optional spacer moiety S) via the N-terminal amino group in P.

[0089] In one aspect of the present invention, Py and / or an optional spacer moiety comprises a residue of a reactive amino, phenyl, hydroxyl, carboxyl, acyl halide, sulfonyl chloride, dichlorotriazine or sulfoxyethylene group, i.e., is functionalized, which reacts with an optional spacer moiety and / or P to form P-S-Py. In a particularly suitable aspect of the present invention, Py and / or an optional spacer moiety comprises a residue of a reactive benzenesulfonyl chloride, dichlorotriazine or sulfoxyethylsulfonyl, i.e., is functionalized, which reacts with an optional spacer moiety and / or P to form P-S-Py. In a suitable aspect of the present invention, Py and / or an optional spacer moiety comprises a residue of a reactive maleimide group, i.e., is functionalized, which reacts with an optional spacer moiety and / or a nucleophilic group (such as -SH or NH2) on P to form P-S-Py.

[0090] For example, the linkage between P and Py optionally involving the spacer moiety S (if present) is or comprises an amide or thioether group or a triazole ring. For example, the linkage between P and Py or the linkage between P and one end of the spacer moiety S and / or the linkage between the other end of the spacer moiety S and Py is or comprises an amide or thioether group or a triazole ring. The amide linkage can be formed by the reaction of an acid functional group on one reactant with an amine functional group on another reactant. The thioether linkage can be formed by the reaction of a mercapto functional group on one reactant with an alkenyl group on another reactant. The triazole ring linkage can be formed by appropriately using copper catalysis to react an azide functional group on one reactant with an acetylene functional group on another reactant.

[0091] Examples of the binding between Py and P through functional groups are described below:

[0092]

[0093] It should be noted that in the above examples, Py is represented as a dye and is directly linked via an amino group in P. These illustrations are non-limiting.

[0094] It may be suitable for Py to be directly or indirectly linked to multiple copies of P via an optional spacer moiety S. Multiple copies of P of the same sequence or a combination of P of different sequences can be used. In one aspect of the present invention, Py can be directly or indirectly linked to 1 to about 10,000 copies of P. Suitably, Py can be directly or indirectly linked to 1 to about 5000, such as 1 to about 2500, such as 1 to about 1000, such as 1 to about 500 copies of P.

[0095] Methods

[0096] In one aspect of the present invention, the peptides of the present invention are produced synthetically, such as biosynthetically.

[0097] Specifically, the peptides of the present invention can be produced using standard peptide synthesis methods known in the art, for example, those described in the following: Stewart et al., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL., 1984; Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, N.Y., 1984; and Pennington et al., Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994.

[0098] Alternatively, the peptides of the present invention can be obtained and processed using publicly available techniques, such as those disclosed in the following references: Green and Sambrook 2012 Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbour Laboratory Press. In particular, artificial gene synthesis can be used to generate polynucleotides (Nambiar et al., 1984, Science, 223: 1299-1301, Sakamar and Khorana, 1988, Nucl. Acids Res., 14: 6361-6372, Wells et al., 1985, Gene, 34: 315-323, and Grundstrom et al., 1985, Nucl. Acids Res., 13: 3305-3316), which can then be expressed in a suitable organism to produce the peptide. Genes encoding the peptides of the present invention can be synthesized, for example, by solid-phase DNA synthesis. The complete gene can be synthesized de novo without a precursor template DNA. To obtain the desired oligonucleotides, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the product sequence. After chain assembly is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can be separated by high-performance liquid chromatography (HPLC) to obtain the desired high-purity oligonucleotides (Verma and Eckstein, 1998, Annu. Rev. Biochem. 67: 99-134). By using various gene amplification methods (Liang et al., Methods Mol Biol., 2012; 834: 93-109), these relatively short fragments can easily be assembled into longer DNA molecules suitable for use in many recombinant DNA-based expression systems. DNA-based expression vectors combined with appropriate regulatory and control sequences (including promoters, termination signals, etc.) and sequences that promote peptide secretion are suitable for introduction (e.g., transfection, transduction, etc.) into heterologous host cells responsible for peptide production.

[0099] The genes encoding the peptides of the present invention can be produced in heterologous host cells, particularly in cells of microbial hosts. Preferred heterologous host cells include fungal or bacterial cells. Examples of host organisms include, but are not limited to, fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcali-genes, Synechocystis, Anabaena, Thiobacillus, Methanobacterium, and Klebsiella. Alternatively, the peptides of the present invention can be produced by transducing eukaryotic cells (e.g., Chinese hamster ovary cells or Drosophila S2 cells).

[0100] The improved isolation of the peptides of the present invention produced recombinantly can be optionally facilitated by adding a stretch of histidine residues (commonly referred to as His-tag) to one end of the peptide.

[0101] In one aspect of the present invention, there is provided a method for preparing a peptide conjugate of the present invention, the method comprising reacting a functionalized payload molecule Py with a peptide P of the present invention. Suitably, the functionalized payload molecule can react with a cysteine residue in the peptide. Alternatively, the functionalized payload molecule can react with an amino group in the peptide. The amino group can be an N-terminal amino group or a side-chain amino group (e.g., on a lysine residue). In a suitable aspect of the present invention, the functionalized payload molecule reacts with the N-terminal amino group in the peptide.

[0102] In another aspect of the present invention, there is provided a method for preparing the peptide conjugate of the present invention, the method comprising reacting a functionalized payload molecule with a spacer moiety which has been previously functionalized and reacted with a peptide, or will be subsequently functionalized and reacted with a peptide. For example, in one aspect, there is provided a method for preparing a peptide conjugate, the method comprising reacting a functionalized payload molecule Py with an S-P moiety, where S is a functionalized spacer moiety and P is a peptide of the present invention. In an alternative aspect, there is provided a method for preparing a peptide conjugate, the method comprising reacting an S-Py moiety (where S is a functionalized spacer moiety and Py is a payload molecule) with a peptide P of the present invention. Suitably, the functionalized spacer moiety can react with a cysteine residue in the peptide. Alternatively, the functionalized spacer moiety can react with an amino group in the peptide. In a suitable aspect of the present invention, the functionalized spacer moiety reacts with the N-terminal amino group in the peptide.

[0103] Conditions suitable for the above methods are known to those skilled in the art.

[0104] Formulations / Administration

[0105] In one aspect of the present invention, there is provided a composition comprising a peptide or peptide conjugate of the present invention and one or more physiologically acceptable diluents or carriers.

[0106] Such physiologically acceptable diluents or carriers include water and solvents such as C1-C 20 monohydric or polyhydric alcohols and their ethers, acetonitrile, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and glycerol. Suitable solvents include C1-C 10 monohydric and dihydric alcohols and their ethers, such as ethanol, isopropanol, n-propanol, butanol, propylene glycol, ethylene glycol, monoethyl ether and mixtures thereof. In a particularly suitable aspect of the present invention, the diluent is water or comprises water as its main component.

[0107] The compositions of the present invention may optionally include a number of additional optional cosmetically acceptable excipients, such as chelating agents, stabilizers, thickening agents, buffering agents, carriers, surfactants, solvents, antioxidants, polymers, non-oxidizing dyes, and conditioning agents. Common examples of such substances are known to those skilled in the art. Examples of such cosmetically acceptable excipients include water-soluble or soluble preservatives such as DMDM hydantoin, Germall 115, methyl, ethyl, propyl, and butyl esters of hydroxybenzoic acid, EDTA, Euxyl(RTM) K400; natural preservatives such as benzyl alcohol, potassium sorbate, and bisabalol, benzoic acid, sodium benzoate, and 2-phenoxyethanol; antioxidants such as sodium sulfite, hydroquinone, sodium bisulfite, sodium metabisulfite, and thioglycolic acid, sodium dithionite, erythrobic acid, and other mercaptans; humectants such as hyaluronic acid, chitin, starch-grafted sodium polyacrylates, methylcellulose, starch, higher fatty alcohols, natural oils, paraffin oil, and fatty acids; antibacterial agents such as Oxeco (phenoxyisopropanol); viscosity control agents such as magnesium sulfate, and other electrolytes; hair conditioners such as silicones, higher alcohols, cationic polymers, etc.; thickening agents such as oleic acid, cetyl alcohol, oleyl alcohol, sodium chloride, cetearyl alcohol, stearyl alcohol, synthetic thickening agents such as Carbopol, Aculyn, and Acrosyl and their mixtures; and fragrances and fragrance solubilizers.

[0108] Suitable surfactants in the compositions of the present invention generally have a lipophilic chain length of from about 8 to about 22 carbon atoms and may be selected from anionic, cationic, nonionic, amphoteric (zwitterionic) surfactants, and mixtures thereof.

[0109] The optional excipients may be added to the compositions of the present invention at a concentration of from 0.001% to 25% by weight, such as from 0.001% to 15% by weight, such as from 0.01% to 10% by weight, such as from 0.01% to 5% by weight, wherein the percentages by weight are relative to the weight of the total formulation.

[0110] The composition suitably contains a peptide or peptide conjugate at a concentration of from 0.001% to 25% by weight, such as from 0.001% to 10% by weight, such as from 0.1% to 5% by weight, such as from 0.1% to 2.5% by weight, wherein the percentages by weight are relative to the weight of the total formulation.

[0111] The compositions of the present invention can be acidic, neutral or alkaline. Suitably, the compositions of the present invention can have a pH value within the following ranges: 2.0 to 12.0, such as 4.0 to 10.0, such as 6.0 to 8.0, such as approximately neutral pH. The pH value of the composition can be adjusted by a pH regulator, which includes acidifying agents such as hydrochloric acid, tartaric acid, citric acid, succinic acid, phosphoric acid, ascorbic acid, acetic acid, lactic acid, sulfuric acid, formic acid and mixtures thereof, or basic buffers such as ammonium hydroxide, ethylamine, dipropylamine, triethylamine, alkanediamine, ethanolamine, polyalkylenepolyamine, heterocyclic amine, hydroxides of alkali metals (e.g., sodium hydroxide and potassium hydroxide), hydroxides of alkaline earth metals (e.g., magnesium hydroxide and calcium hydroxide), and basic amino acids (e.g., L-arginine, lysine, alanine, leucine, isoleucine, oxylysine and histidine), and mixtures thereof.

[0112] In one aspect of the present invention, the peptide, peptide conjugate or composition thereof is formulated for topical administration. Suitable formulations for topical administration include but are not limited to foams, creams, shampoos, sprays, solutions, gels, serums, dry shampoos and aerosols.

[0113] The present invention also provides the use of the peptide or peptide conjugate of the present invention for treating hair. In one aspect of the present invention, there is provided the use of the peptide of the present invention for binding to hair (i.e., treating hair). In another aspect of the present invention, there is provided the use of the peptide conjugate for binding a payload to hair (i.e., treating hair with the payload).

[0114] In other aspects of the present invention, there are provided compositions for treating hair, which comprise the peptide or peptide conjugate of the present invention and a physiologically acceptable diluent or carrier.

[0115] The present invention also provides a method for treating hair, which comprises administering the peptide or peptide conjugate of the present invention to the hair. In one aspect of the present invention, there is provided a method for binding to hair (treating hair), which comprises administering the peptide of the present invention to the hair. In another aspect of the present invention, there is provided a method for binding a payload to hair (i.e., treating hair with the payload), which comprises administering the peptide conjugate of the present invention to the hair.

[0116] In other aspects of the present invention, there are provided methods for treating hair, which comprise administering to the hair a composition comprising the peptide or peptide conjugate of the present invention and a physiologically acceptable diluent or carrier.

[0117] In one aspect of the present invention, the peptide, peptide conjugate or composition of the present invention is not used for medical uses or methods, i.e., it is used for non-medical uses or methods. For example, the peptide, peptide conjugate or composition of the present invention is suitably used for cosmetic uses or methods.

[0118] Other aspects of the present invention are defined by the following clauses:

[0119] 1. A peptide that binds to hair, comprising the following sequence:

[0120] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8 (SEQ ID NO.1)

[0121] Wherein,

[0122] The amino acid of X1 is absent or is K;

[0123] The amino acid of X2 is absent or is G;

[0124] The amino acid of X3 is absent or is T;

[0125] The amino acid of X4 is absent or is A;

[0126] The amino acid of X5 is absent or is P;

[0127] The amino acid of X6 is absent or is G;

[0128] The amino acid of X7 is absent or is selected from L and R;

[0129] The amino acid of X8 is absent or is A;

[0130] Wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively.

[0131] 2. The peptide according to clause 1, which consists of:

[0132] (i) The following sequence

[0133] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8 (SEQ ID NO.1) Wherein,

[0134] The amino acid of X1 is absent or is K;

[0135] The amino acid of X2 is absent or is G;

[0136] The amino acid of X3 is absent or is T;

[0137] The amino acid of X4 is absent or is A;

[0138] The amino acid of X5 is absent or is P;

[0139] The amino acid of X6 is absent or is G;

[0140] The amino acid of X7 is absent or is selected from L and R;

[0141] The amino acid of X8 is absent or is A;

[0142] wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively; or

[0143] (ii) the corresponding sequence having a cysteine residue at either terminus.

[0144] 3. The peptide according to clause 1 or clause 2, wherein X6 is G.

[0145] 4. The peptide according to any one of clauses 1 to 3, wherein X7 is selected from L and R.

[0146] 5. The peptide according to clause 4, wherein X7 is L.

[0147] 6. The peptide according to any one of clauses 1 to 5, wherein X5 is P.

[0148] 7. The peptide according to any one of clauses 1 to 6, wherein X4 is A.

[0149] 8. The peptide according to any one of clauses 1 to 7, wherein X3 is T.

[0150] 9. The peptide according to any one of clauses 1 to 8, wherein X2 is G.

[0151] 10. The peptide according to any one of clauses 1 to 9, wherein X8 is A.

[0152] 11. The peptide according to any one of clauses 1 to 10, wherein X1 is K.

[0153] 12. The peptide according to any one of clauses 1 to 11, wherein the peptide contains a cysteine residue.

[0154] 13. The peptide according to clause 12, wherein the peptide contains a cysteine residue at the N-terminal position.

[0155] 14. The peptide according to any one of clauses 1 to 13, wherein the peptide contains 6 or more amino acids.

[0156] 15. The peptide according to clause 14, wherein the peptide contains 7 or more amino acids.

[0157] 16. The peptide according to clause 2, which is composed of a sequence selected from the group consisting of:

[0158] CKGTAPRRKGLA (SEQ ID NO.3),

[0159] CTAPRRKGL (SEQ ID NO.5),

[0160] CGTAPRRKGR (SEQ ID NO.6),

[0161] CRRKGLA (SEQ ID NO.4),

[0162] KGTAPRRKGL (SEQ ID NO.7),

[0163] KGTAPRRKG (SEQ ID NO.8),

[0164] GTAPRRKGL (SEQ ID NO.9),

[0165] TAPRRKGL (SEQ ID NO.10),

[0166] APRRKGL (SEQ ID NO.11),

[0167] RRKGL (SEQ ID NO.12),

[0168] GTAPRRKG (SEQ ID NO.13),

[0169] TAPRRK (SEQ ID NO.14),

[0170] CGTAPRRKGL (SEQ ID NO.15), and

[0171] GTAPRRKGR (SEQ ID NO.16).

[0172] 17. A peptide according to clause 16, which consists of a sequence selected from the group consisting of:

[0173] CKGTAPRRKGLA (SEQ ID NO.3),

[0174] CTAPRRKGL (SEQ ID NO.5),

[0175] CGTAPRRKGR (SEQ ID NO.6),

[0176] CRRKGLA (SEQ ID NO.4),

[0177] GTAPRRKGL (SEQ ID NO.9),

[0178] TAPRRK (SEQ ID NO.14),

[0179] CGTAPRRKGL (SEQ ID NO.15), and

[0180] GTAPRRKGR (SEQ ID NO.16).

[0181] 18. A peptide according to clause 17, which is composed of a sequence selected from the following group:

[0182] CKGTAPRRKGLA (SEQ ID NO.3),

[0183] CTAPRRKGL (SEQ ID NO.5),

[0184] CGTAPRRKGR (SEQ ID NO.6), and

[0185] CRRKGLA (SEQ ID NO.4).

[0186] 19. A peptide conjugate of the following formula that binds to hair:

[0187] P - S - Py

[0188] wherein P is a peptide according to any one of clauses 1 to 18, S is an optional spacer moiety S, and Py is a payload.

[0189] 20. The peptide conjugate according to clause 19, wherein the payload is a colorant, nanoparticle, ultraviolet absorbing substance, conditioning agent, metal oxide, metal colloidal particle, beneficial polypeptide, or vitamin.

[0190] 21. The peptide conjugate according to clause 20, wherein the payload is selected from zinc oxide nanoparticles, graphene nanoparticles, and graphene oxide nanoparticles.

[0191] 22. The peptide conjugate according to any one of clauses 19 to 21, wherein Py is functionalized to facilitate direct or indirect (i.e., via an optional spacer moiety S) attachment to P.

[0192] 23. The peptide conjugate according to any one of clauses 19 to 22, wherein Py is attached to P directly or indirectly (i.e., via an optional spacer moiety S) through a cysteine residue in P.

[0193] 24. The peptide conjugate according to any one of clauses 19 to 22, wherein Py is attached to P directly or indirectly (i.e., via an optional spacer moiety S) through an amino group in P.

[0194] 25. The peptide conjugate according to clause 24, wherein Py is attached to P directly or indirectly (i.e., via an optional spacer moiety S) through the N - terminal amino group in P.

[0195] 26. The peptide conjugate according to clause 22, wherein Py contains a residue of a reactive amino group, phenyl, hydroxyl, carboxyl, acyl halide, sulfonyl chloride, dichlorotriazine, or sulfonyloxyethylene group, which reacts with an optional spacer moiety S or P to form P - S - Py.

[0196] 27. A peptide conjugate according to clause 26, wherein Py comprises a residue of a reactive amino group, phenyl, sulfonyl chloride, dichlorotriazine or sulfonyloxyethylsulfonyl, which reacts with an optional spacer moiety S or P to form P-S-Py.

[0197] 28. A peptide conjugate according to any one of clauses 22 to 27, wherein Py comprises a residue of a reactive maleimide group, which reacts with an optional spacer moiety S or P to form P-S-Py.

[0198] 29. Use of a peptide conjugate according to any one of clauses 19 to 28 for binding a payload to hair.

[0199] 30. A method of treating hair, which comprises applying a peptide conjugate according to any one of clauses 19 to 28 to the hair.

[0200] 31. A composition for treating hair, which comprises a peptide conjugate according to any one of clauses 19 to 28 and a physiologically acceptable diluent or carrier.

[0201] 32. A method for preparing a peptide conjugate according to any one of clauses 19 to 28, which comprises;

[0202] (i) reacting a functionalized payload molecule Py with a peptide P according to any one of clauses 1 to 18; or

[0203] (ii) reacting a functionalized payload molecule Py with an S-P moiety, wherein S is a functionalized spacer moiety and P is a peptide according to any one of clauses 1 to 18; or

[0204] (ii) reacting an S-Py moiety with a peptide according to any one of clauses 1 to 18, wherein S is a functionalized spacer moiety and Py is a payload molecule.

[0205] 33. A peptide that binds to hair, which comprises the following sequence:

[0206] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8 (SEQ ID NO.1)

[0207] wherein,

[0208] the amino acid of X1 is absent or is K;

[0209] the amino acid of X2 is absent or is G;

[0210] the amino acid of X3 is absent or is T;

[0211] the amino acid of X4 is absent or is A;

[0212] The amino acid of X5 is absent or is P;

[0213] The amino acid of X6 is absent or is G;

[0214] The amino acid of X7 is absent or is selected from L and R;

[0215] The amino acid of X8 is absent or is A;

[0216] Wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively; and / or

[0217] The peptide comprises a sequence selected from the group consisting of:

[0218] CKGTAPRRKGLA (SEQ ID NO.3),

[0219] CTAPRRKGL (SEQ ID NO.5),

[0220] CGTAPRRKGR (SEQ ID NO.6),

[0221] CRRKGLA (SEQ ID NO.4),

[0222] KGTAPRRKGL (SEQ ID NO.7),

[0223] KGTAPRRKG (SEQ ID NO.8),

[0224] GTAPRRKGL (SEQ ID NO.9),

[0225] TAPRRKGL (SEQ ID NO.10),

[0226] APRRKGL (SEQ ID NO.11),

[0227] RRKGL (SEQ ID NO.12),

[0228] GTAPRRKG (SEQ ID NO.13),

[0229] TAPRRK (SEQ ID NO.14),

[0230] CGTAPRRKGL (SEQ ID NO.15), and

[0231] GTAPRRKGR (SEQ ID NO.16).

[0232] 34. A peptide that binds to hair, comprising the following sequence:

[0233] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8(SEQ ID NO.1)

[0234] Among them,

[0235] the amino acid of X1 does not exist or is K;

[0236] the amino acid of X2 does not exist or is G;

[0237] the amino acid of X3 does not exist or is T;

[0238] the amino acid of X4 does not exist or is A;

[0239] the amino acid of X5 does not exist or is P;

[0240] the amino acid of X6 does not exist or is G;

[0241] the amino acid of X7 does not exist or is selected from L and R;

[0242] the amino acid of X8 does not exist or is A;

[0243] wherein if X6 does not exist, then X3, X4 and X5 exist and are T, A and P respectively; and / or

[0244] wherein the peptide is composed of sequences selected from the following group:

[0245] CKGTAPRRKGLA(SEQ ID NO.3),

[0246] CTAPRRKGL(SEQ ID NO.5),

[0247] CGTAPRRKGR(SEQ ID NO.6),

[0248] CRRKGLA(SEQ ID NO.4),

[0249] KGTAPRRKGL(SEQ ID NO.7),

[0250] KGTAPRRKG(SEQ ID NO.8),

[0251] GTAPRRKGL(SEQ ID NO.9),

[0252] TAPRRKGL(SEQ ID NO.10),

[0253] APRRKGL(SEQ ID NO.11),

[0254] RRKGL(SEQ ID NO.12),

[0255] GTAPRRKG (SEQ ID NO.13),

[0256] TAPRRK (SEQ ID NO.14),

[0257] CGTAPRRKGL (SEQ ID NO.15), and

[0258] GTAPRRKGR (SEQ ID NO.16).

[0259] 35. The peptide according to Clause 1, which consists of:

[0260] (i) the following sequence

[0261] X1-X2-X3-X4-X5-R-R-K-X6-X7-X8 (SEQ ID NO.1)

[0262] wherein,

[0263] the amino acid of X1 is absent or is K;

[0264] the amino acid of X2 is absent or is G;

[0265] the amino acid of X3 is absent or is T;

[0266] the amino acid of X4 is absent or is A;

[0267] the amino acid of X5 is absent or is P;

[0268] the amino acid of X6 is absent or is G;

[0269] the amino acid of X7 is absent or is selected from L and R;

[0270] the amino acid of X8 is absent or is A;

[0271] wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively; or

[0272] (ii) the corresponding sequence having a cysteine residue at either terminus; and / or

[0273] wherein the peptide consists of a sequence selected from the group consisting of:

[0274] CKGTAPRRKGLA (SEQ ID NO.3),

[0275] CTAPRRKGL (SEQ ID NO.5),

[0276] CGTAPRRKGR (SEQ ID NO.6),

[0277] CRRKGLA (SEQ ID NO.4),

[0278] KGTAPRRKGL (SEQ ID NO.7),

[0279] KGTAPRRKG (SEQ ID NO.8),

[0280] GTAPRRKGL (SEQ ID NO.9),

[0281] TAPRRKGL (SEQ ID NO.10),

[0282] APRRKGL (SEQ ID NO.11),

[0283] RRKGL (SEQ ID NO.12),

[0284] GTAPRRKG (SEQ ID NO.13),

[0285] TAPRRK (SEQ ID NO.14),

[0286] CGTAPRRKGL (SEQ ID NO.15), and

[0287] GTAPRRKGR (SEQ ID NO.16). Examples

[0288] Assays

[0289] Hair Binding Assay

[0290] The peptides of the present invention were conjugated with the dye 5-carboxytetramethylrhodamine (TAMRA) via a 6-aminohexanoic acid group to produce peptide-TAMRA conjugates. 150 μL of a peptide-TAMRA conjugate solution (100 μM, 10 μM, or 1 μM) was incubated with a lock of hair for 30 - 40 minutes. Subsequently, the hair was washed 5 times with a 0.1% (w / v) Tween solution for about 1 minute each time, and then washed 2 times with Milli-Q water for about 1 minute each time.

[0291] The fluorescence intensity of stained samples was measured using a multimode imaging plate reader, Cytation 5 (BioTek). Nonspecific binding was estimated using glycine-TAMRA conjugate (NEG) and subtracted from the peptide-TAMRA fluorescence intensity values. All fluorescence intensities were within the dynamic range of the microscope camera, enabling the measurement of baseline fluorescence and peak intensity. The fluorescence intensity of the tested peptide-TAMRA conjugates was measured as a percentage (%) relative to the peptide-TAMRA conjugate in which the peptide was the peptide of SEQ ID NO.3 (100%) (see Table 1). The results of the 100 μM experiment (showing the strongest binding) are shown in Table 1.

[0292] Table 1: Fluorescence intensity of peptide-TAMRA conjugate (100 μM) after binding to a single hair strand

[0293]

[0294] Suitable peptides were identified as those that produced a fluorescence intensity as a peptide-TAMRA conjugate that was 25% or more relative to the peptide-TAMRA conjugate in which the peptide was the peptide of SEQ ID NO.3. The peptides and their variants were incorporated into subsequent hair binding assays. Here, the hair was cut into short segments (1 - 3 mm) and placed horizontally at the bottom of the wells in a 96-well plate. The shredded hair was then stained with the peptide-TAMRA conjugate solution (100 μM, 10 μM, or 1 μM) as described above. Using the above microscope equipment, the fluorescence intensity was measured as a percentage (%) relative to the peptide-TAMRA conjugate in which the peptide was the peptide of SEQ ID NO.3 (100%). The measurements were repeated three times and the average was calculated. The results of the 100 μM experiment (showing the strongest binding) are shown in Table 2.

[0295] Table 2: Average fluorescence intensity of peptide-TAMRA conjugate (100 μM) after binding to shredded hair (n = 3)

[0296]

[0297] As can be seen from Tables 1 and 2, the TAMRA conjugate forms of the peptides of the present invention (SEQ ID NO.3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16) showed good binding to hair, while the control peptides (SEQ ID NO.17 - 25) did not.

[0298] Peptide Examples

[0299] Peptides were synthesized sequentially using conventional methods known in the art.

[0300] The following hair-binding peptides were prepared:

[0301] Examples of the peptides of the present invention:

[0302] SEQ ID NO.3: CKGTAPRRKGLA

[0303] SEQ ID NO.4: CRRKGLA

[0304] SEQ ID NO.5: CTAPRRKGL

[0305] SEQ ID NO.6: CGTAPRRKGR

[0306] SEQ ID NO.7: KGTAPRRKGL

[0307] SEQ ID NO.8: KGTAPRRKG

[0308] SEQ ID NO.9: GTAPRRKGL

[0309] SEQ ID NO.10: TAPRRKGL

[0310] SEQ ID NO.11: APRRKGL

[0311] SEQ ID NO.12: RRKGL

[0312] SEQ ID NO.13: GTAPRRKG

[0313] SEQ ID NO.14: TAPRRK

[0314] SEQ ID NO.15: CGTAPRRKGL

[0315] SEQ ID NO.16: GTAPRRKGR

[0316] Comparative peptides:

[0317] SEQ ID NO.17: KGTAPRRK

[0318] SEQ ID NO.18: KGTAPRR

[0319] SEQ ID NO.19: KGTAPR

[0320] SEQ ID NO.20: KGTAP

[0321] SEQ ID NO.21: PRRKGL

[0322] SEQ ID NO.22: APRKK

[0323] SEQ ID NO.23: TAPRR

[0324] SEQ ID NO.24: LGKRR

[0325] SEQ ID NO.25: STNSHGEDS

[0326] Conjugate Examples

[0327] Conjugate Example 1 - Preparation of Peptide-Zinc Oxide Nanoparticle Conjugate

[0328] First, 2.3 ml of phosphate buffer (pH 7.4) was added to 0.1056 g of APTES-coated zinc oxide nanoparticles, and the dispersion was sonicated in an ultrasonic bath for 10 minutes. 5 mg of spacer (NHS-PEG 12 -maleimide, Thermo Scientific TM ) was dissolved in 0.2 ml of DMSO. The spacer solution was added to the nanoparticle dispersion, and the resulting mixture was stirred with a magnetic stirrer at room temperature for 1 hour. Then the mixture was centrifuged at 11,000 g for 15 minutes at 4 °C and decanted. 2.5 ml of HEPES buffer (pH 7.2) was added to the precipitate, and then it was sonicated for 10 minutes. Then the centrifugation and sonication steps were repeated.

[0329] 11 mg of the peptide of SEQ ID NO.3 was dissolved in 1 ml of deionized water and subsequently added to the nanoparticle-spacer conjugate prepared in the previous paragraph. The mixture was stirred with a magnetic stirrer at room temperature for 1 h and then kept overnight at 4 °C until separation was achieved. The product was separated by centrifuging at 7,000 g for 10 minutes at 4 °C and then decanting. The precipitate was washed 3 times with phosphate buffer (pH 7.4). Specifically, 2.5 ml of phosphate buffer (pH 7.4) was added to the precipitate, the mixture was sonicated for 10 minutes, centrifuged at 7000 g for 10 minutes at 4 °C and decanted. The resulting product was dried at 20 °C under vacuum for 4 h. Yield: 0.0898 g (74%).

[0330] Conjugate Example 2 - Synthesis of Maleimide-Functionalized Methyl Red

[0331] Step 1: As described by Krapcho et al., Boc anhydride was added dropwise to tetramethylenediamine in 1,4-dioxane at room temperature to produce a tetramethylenediamine compound in which one amino group was protected by a Boc group. The product was extracted to remove unreacted diamine and bis-Boc compounds.

[0332]

[0333] Step 2: Activate methyl red by N,N'-dicyclohexylcarbodiimide (DCC), and then react it with the diamine compound generated in Step 1. A product mixture is formed, and the desired amide compound is extracted using flash chromatography technique.

[0334]

[0335] Step 3: Deprotect the amide compound of Step 2 by treatment with trifluoroacetic acid (TFA), and extract the pure product.

[0336]

[0337] Step 4: React the deprotected compound of Step 3 with maleic anhydride to form an amide product, which precipitates out from the reaction mixture. After completion of the reaction, the precipitate is filtered, washed, and dried. Subsequently, the amide product is heated in acetic anhydride in the presence of sodium acetate. The desired maleimide-functionalized methyl red product is separated from the by-products using flash chromatography technique.

[0338]

[0339] Step 5: Dissolve 965 mg of the peptide of SEQ ID NO.3 in 18 mL of 0.2 M potassium phosphate buffer (pH 7.4). Dissolve maleimide-functionalized methyl red (322 mg; see Step 4) in 27 mL of acetonitrile and add it to the peptide solution. Stir the resulting mixture with a magnetic stirrer. Next, generate an inert gas (Ar) atmosphere in the reaction vessel and stir the mixture at room temperature for 24 hours. Evaporate the mixture, dissolve the resulting precipitate in methanol and filter to remove insoluble phosphates. Evaporate the filtrate and purify it by reverse-phase flash chromatography (eluent: water - acetonitrile + 0.1% (w / v) TFA). Collect and combine the desired fractions and remove the solvent on a rotary evaporator. Further dry the product under vacuum overnight. Identify the peptide-methyl red conjugate (1020 mg; yield 79%) by HPLC-MS (column: silica gel; mobile phase: water - acetonitrile 1:1 + 0.1% (w / v) TFA). The retention time of the product is 8.9 min, and its mass-to-charge (m / z) ratio is 837 and 1685.

[0340]

[0341] Test Examples

[0342] Test Example 1

[0343] The peptide-zinc oxide nanoparticle conjugate was produced as described in Conjugate Example 1. The peptide-zinc oxide nanoparticle conjugate and zinc oxide nanoparticles alone (both at a concentration of 0.1% (w / v)) were incubated with hair at room temperature for 15 minutes and then washed with water. The samples were evaluated using a scanning electron microscope. The results are as Figure 2 shown.

[0344] Figure 2 The results in Figure 2 show that zinc oxide nanoparticles alone do not bind to hair with any significant affinity (see Figure 2 , Figure B). However, the peptide-zinc oxide nanoparticle conjugates of the present invention that were tested were able to bind to hair in large amounts and this binding was resistant to shampooing (see

[0345] Test Example 2

[0346] , Figure A).

[0347] Table 3: The peptide-methyl red conjugate was prepared as described in Conjugate Example 2. The conjugate (0.1% aqueous solution) was used to dye blonde hair. Specifically, the hair was incubated with the conjugate (Sample 1) or pure methyl red alone (Sample 2) at room temperature for 15 minutes, then washed with water (30 seconds), and then washed with a Tween 20 (1%) solution (30 seconds). The degree of staining (producing an orange color) of the blonde hair was scored on a scale of 1 to 5. A score of "1" corresponded to no staining (i.e., the hair remained blonde). A score of "2" corresponded to slight staining (i.e., the hair was a light orange). A score of "3" corresponded to moderate staining (i.e., the hair was a medium orange). A score of "4" corresponded to relatively heavy staining (i.e., the hair was a brighter orange). A score of "5" corresponded to heavy staining (i.e., the hair was a bright orange). The results in Table 3 show that compared to untreated blonde hair (Sample 3), the peptide-methyl red conjugate dyed the hair a bright orange, while pure methyl red alone dyed the hair a very light orange-brown. The hair samples were then washed with water 5 times (see Table 3). The binding of the peptide-methyl red conjugate persisted and the hair samples remained bright orange (Sample 1). In contrast, during this process, the pure methyl red was washed out (Sample 2), causing the hair samples to return to the blonde color of the untreated hair samples (Sample 3).

[0347] Table 3: Visual scoring of hair samples dyed with the peptide-methyl red conjugate (Sample 1) or pure methyl red (Sample 2). Visual score "1": no staining; Visual score "2": slight staining (light orange); Visual score "3": moderate staining (medium orange); Visual score "4": relatively heavy staining (brighter orange); Visual score "5": heavy staining (bright orange).

[0348]

[0349] Test Example 3

[0350] A peptide-methyl red conjugate was prepared as described in Conjugate Example 2. The conjugate (0.15% aqueous solution) was incubated with blond hair at room temperature for 15 minutes to stain the hair samples. A commercially available Joico orange hair dye (1.5% aqueous solution) was used to stain alternative samples. The samples were then washed up to 15 times, each wash consisting of washing with water for 30 seconds followed by washing with Tween 20 (1%) for 30 seconds. The degree of staining (resulting in orange) of the blond hair was scored on a scale of 1 to 5. A score of "1" corresponded to no staining (i.e., the hair remained blond). A score of "2" corresponded to light staining (i.e., the hair was pale orange). A score of "3" corresponded to moderate staining (i.e., the hair was moderately orange). A score of "4" corresponded to heavy staining (i.e., the hair was a brighter orange). A score of "5" corresponded to very heavy staining (i.e., the hair was bright orange). The results in Table 4 show that, although 10 times lower in concentration, the peptide-methyl red conjugate stained the hair a more intense orange than the Joico dye. After 15 washes, it could be seen that the peptide-methyl red conjugate retained a rich orange color, while the appearance of the Joico dye was duller, although the concentration of the peptide-methyl red conjugate was 10 times lower than that of the Joico dye.

[0351] Table 4: Visual scoring of hair samples stained with the peptide-methyl red conjugate or Joico orange. Visual score "1": no staining; Visual score "2": light staining (pale orange); Visual score "3": moderate staining (moderately orange); Visual score "4": heavy staining (brighter orange); Visual score "5": very heavy staining (bright orange).

[0352]

[0353] Summary

[0354] The ability of the peptides of the present invention to bind to hair with high affinity has been demonstrated.

[0355] In addition, the inventors of the present application have described the production of the peptide conjugates of the present invention. First, a method for producing a peptide-zinc oxide nanoparticle conjugate was provided. Second, a method for producing a peptide-methyl red conjugate was provided, in which the methyl red dye was linked to an optional spacer moiety and functionalized with a maleimide group. The maleimide group can facilitate the linking of the methyl red-spacer conjugate to the peptide of SEQ ID NO.3.

[0356] Finally, the inventors of the present application have demonstrated that the peptide conjugates of the present invention retain hair-binding affinity. Specifically, the peptide-zinc oxide nanoparticle conjugates are capable of binding to hair with high affinity, such that the binding is resistant to hair washing. However, the zinc oxide nanoparticles alone are unable to bind to hair. In addition, the peptide conjugates of the present invention are capable of effectively treating hair. Specifically, the peptide-methyl red conjugates are capable of dyeing hair a rich orange color, which remains after washing the hair five times, while pure methyl red alone colors the hair very faintly and the effect disappears after five washes. In addition, compared to commercially available products, the peptide-methyl red conjugates are still capable of coloring the hair more richly despite being used at a concentration ten times lower.

[0357] References

[0358] Birr C. Principles of Peptide Synthesis. Von M. Bodanszky. Springer-Verlag, Berlin. 1984. XVI, 308S., geb. DM 148.00.–ISBN 3-540-12395-4.

[0359] Grundstrom T, Zenke WM, Wintzerith M, Matthes HW, Staub A, Chambon P. Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ gene synthesis. Nucl. Acids Res. 1985. 13:3305-3316.

[0360] Krapcho A, Kuell C. Mono-Protected Diamines. N-tert-Butoxycarbonyl-α,ω-Alkanediamines from α,ω-Alkanediamines. Synthetic Communications. 1990. 20:16, 2559-2564.

[0361] Liang X, Peng L, Tsvetanova B et al., Recombination-based DNA assembly and mutagenesis methods for metabolic engineering. Methods Mol Biol. 2012. 834:93-109

[0362] Millner, PA. Peptide synthesis protocols: Methods in molecular biology volume 35: Edited by M W Pennington and B M Dunn. pp 321. Human Press, Totowa, NJ. 1994. ISBN 0-896-0362736. Biochem. Educ., 23:116-116.

[0363] Nambiar KP, Stackhouse J, Stauffer DM, Kennedy WP, Eldredge JK, Benner SA. Total Synthesis and Cloning of a Gene Coding for the Ribonuclease S Protein. Science. 1984. 223:1299-1301.

[0364] Sakamar, Khorana. Total synthesis and expression of a gene for the α-subunit of bovine rod outer segment guanine nucleotide-binding protein. Nucl. Acids Res. 1988. 14:6361-6372.

[0365] Sambrook J, Green MR. Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press. 2012.

[0366] Stewart JM, Young JD. Solid phase peptide synthesis. Pierce Chemical Company; 1984.

[0367] Verma S, Eckstein F. Modified oligonucleotides: synthesis and strategy for users. Annu Rev Biochem. 1998. 67:99-134.

[0368] Wells JA, Vasser M, Powers DB. Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene. 1985. 34(2-3):315-323.

[0369] Other

[0370] All reference documents mentioned in this application, including patents and patent applications, are incorporated herein by reference to the fullest extent possible.

[0371] Throughout the specification and the following claims, unless the context requires otherwise, the word "comprising" and variations such as "comprises" and "comprising" will be understood to mean including the stated integer, step, group of integers or group of steps, but not excluding any other integer, step, group of integers or group of steps.

[0372] As used herein, when referring to a peptide of the present invention, the term "consisting of" means that the peptide does not contain other amino acids except those identified amino acids.

[0373] The application to which this specification and claims pertain may be used as the basis for priority for any subsequent application. The claims of such subsequent applications may relate to any feature or combination of features described herein. They may take the form of product, composition, method or use claims and may include, but are not limited to, the following claims.

[0374] Sequence Listing

[0375] SEQ ID NO.1:

[0376] X1X2X3X4X5RRKX6X7X8

[0377] SEQ ID NO.2:

[0378] Y a X1X2X3X4X5RRKX6X7X8 Y b

[0379] SEQ ID NO.3:

[0380] CKGTAPRRKGLA

[0381] SEQ ID NO.4:

[0382] CRRKGLA

[0383] SEQ ID NO.5:

[0384] CTAPRRKGL

[0385] SEQ ID NO.6:

[0386] CGTAPRRKGR

[0387] SEQ ID NO.7:

[0388] KGTAPRRKGL

[0389] SEQ ID NO.8:

[0390] KGTAPRRKG

[0391] SEQ ID NO.9:

[0392] GTAPRRKGL

[0393] SEQ ID NO.10:

[0394] TAPRRKGL

[0395] SEQ ID NO.11:

[0396] APRRKGL

[0397] SEQ ID NO.12:

[0398] RRKGL

[0399] SEQ ID NO.13:

[0400] GTAPRRKG

[0401] SEQ ID NO.14:

[0402] TAPRRK

[0403] SEQ ID NO.15:

[0404] CGTAPRRKGL

[0405] SEQ ID NO.16:

[0406] GTAPRRKGR

[0407] SEQ ID NO.17:

[0408] KGTAPRRK

[0409] SEQ ID NO.18:

[0410] KGTAPRR

[0411] SEQ ID NO.19:

[0412] KGTAPR

[0413] SEQ ID NO.20:

[0414] KGTAP

[0415] SEQ ID NO.21:

[0416] PRRKGL

[0417] SEQ ID NO.22:

[0418] APRKK

[0419] SEQ ID NO.23:

[0420] TAPRR

[0421] SEQ ID NO.24:

[0422] LGKRR

[0423] SEQ ID NO.25:

[0424] STNSHGEDS

Claims

1. A peptide that binds to hair, comprising the following sequence: X1-X2-X3-X4-X5-R-R-K-X6-X7-X8 (SEQ ID NO.1) wherein, the amino acid of X1 is absent or is K; the amino acid of X2 is absent or is G; the amino acid of X3 is absent or is T; the amino acid of X4 is absent or is A; the amino acid of X5 is absent or is P; the amino acid of X6 is absent or is G; the amino acid of X7 is absent or is selected from L and R; the amino acid of X8 is absent or is A; wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively.

2. The peptide according to claim 1, which consists of: (i) the following sequence X1-X2-X3-X4-X5-R-R-K-X6-X7-X8 (SEQ ID NO.1) wherein, the amino acid of X1 is absent or is K; the amino acid of X2 is absent or is G; the amino acid of X3 is absent or is T; the amino acid of X4 is absent or is A; the amino acid of X5 is absent or is P; the amino acid of X6 is absent or is G; the amino acid of X7 is absent or is selected from L and R; the amino acid of X8 is absent or is A; wherein if X6 is absent, then X3, X4 and X5 are present and are T, A and P respectively; or (ii) the corresponding sequence having a cysteine residue at either terminus.

3. The peptide according to claim 1 or claim 2, wherein one or more of the following conditions apply: (i) X6 is G; (ii) X7 is selected from L and R; (iii) X5 is P; (iv) X4 is A; (v) X3 is T; (vi) X2 is G; (vii) X8 is A; and / or (viii) X1 is K.

4. The peptide according to any one of claims 1 to 3, wherein the peptide contains a cysteine residue at the N-terminal position.

5. The peptide according to any one of claims 1 to 4, wherein the peptide contains 6 or more amino acids.

6. The peptide according to claim 2, which consists of sequences selected from the following group: CKGTAPRRKGLA (SEQ ID NO.3), CTAPRRKGL (SEQ ID NO.5), CGTAPRRKGR (SEQ ID NO.6), CRRKGLA (SEQ ID NO.4), KGTAPRRKGL (SEQ ID NO.7), KGTAPRRKG (SEQ ID NO.8), GTAPRRKGL (SEQ ID NO.9), TAPRRKGL (SEQ ID NO.10), APRRKGL (SEQ ID NO.11), RRKGL (SEQ ID NO.12), GTAPRRKG (SEQ ID NO.13), TAPRRK (SEQ ID NO.14), CGTAPRRKGL (SEQ ID NO.15), and GTAPRRKGR (SEQ ID NO.16).

7. A peptide conjugate of the formula below that binds to hair: P-S-Py wherein P is a peptide according to any one of claims 1 to 6, S is an optional spacer moiety S, and Py is a payload.

8. The peptide conjugate according to claim 7, wherein the payload is a colorant, nanoparticle, ultraviolet-absorbing substance, conditioning agent, metal oxide, metal colloidal particle, beneficial polypeptide, or vitamin.

9. The peptide conjugate according to claim 7 or claim 8, wherein Py is functionalized to facilitate attachment to P, either directly or indirectly, i.e., via an optional spacer moiety S.

10. The peptide conjugate according to any one of claims 7 to 9, wherein Py is attached to P either directly or indirectly, i.e., via an optional spacer moiety S, through a cysteine residue in P or through an amino group in P.

11. The peptide conjugate according to claim 10, wherein Py is attached to P either directly or indirectly, i.e., via an optional spacer moiety S, through the N-terminal amino group in P.

12. The peptide conjugate according to any one of claims 7 to 11, wherein Py contains a residue of a reactive amino group, phenyl, hydroxyl, carboxyl, acyl halide, sulfonyl chloride, dichlorotriazine, or sulfonyloxyethylene group that reacts with an optional spacer moiety S or P to form P-S-Py.

13. The peptide conjugate according to any one of claims 9 to 12, wherein Py contains a residue of a reactive maleimide group that reacts with an optional spacer moiety or P to form P-S-Py.

14. Use of the peptide conjugate according to any one of claims 7 to 13 for binding a payload to hair.

15. A composition for treating hair, comprising a peptide conjugate according to any one of claims 7 to 13 and a physiologically acceptable diluent or carrier.

Images