peptides
Patent Information
- Application Number
- PCT/GB2026/050234
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-19
- Filing Date
- 2026-02-18
- Publication Date
- 2026-08-27
Smart Images
Figure GB2026050234_27082026_PF_FP_ABST
Abstract
Description
[0001] PEPTIDES
[0002] Technical Field
[0003] The present invention relates to peptides and compositions comprising the peptides. The peptides and their compositions demonstrate a high degree of solubility and film forming ability. The peptides therefore find particular utility in products for the treatment of skin and / or hair and for cosmetic products. The present invention further relates to a genetic construct for expression of the peptides.
[0004] Background to the Invention
[0005] The solubility of proteins oftens limits their utility. The solubility of collagen, for example, is 70 mg / ml. It would be beneficial to have proteins with a higher degree of solubility, for example a solubility at 20°C in an aqueous buffer of at least 100 mg / ml.
[0006] EP1064010 describes the formation of small peptides by digestion of elastin and suggests that these peptides can improve skin appearance when delivered to the skin in the form of a therapeutic or cosmetic composition. Most preferably the peptides have a molecular weight in the range of about 188-585 kDa.
[0007] There is a need for further and improved peptides having a high degree of solubility.
[0008] There is a need for further and improved peptides having an ability to form a film. The film may be able to act as a barrier, for example in skin and haircare products.
[0009] There is a need for further and improved peptides having anti-oxidant properties.
[0010] The present invention seeks to address one or more of the above objectives.
[0011] Summary of the Invention
[0012] The present invention relates to peptides which are useful in one or more of the above fields. The peptides of the present invention comprise 6 short peptide (SP) sequences which are spaced apart by defined linker sequences. Each shortpeptide sequence can be the same or different. The short peptide (SP) sequences are selected to deliver a pre-determined functionality, such as cell binding, binding to the surface of hair, colour, binding to the surface of the skin, antioxidant properties, UV absorbing, increasing skin firmness, or skin elasticity and may be derived from collagen, spider silk, keratin or other proteins that have desirable properties. The peptides of the present invention also include either a specific N-terminal or C-terminal sequence. Optionally, a starter sequence can also be present. The present inventors have found that use of a linker which has the sequence:
[0013] GSGS(A)nGSGS,
[0014] (wherein n is an integer of 6 to 20, for example is 8 to 16, for example is 8 to 12, for example is 10), provides a consistent spacing between the short peptide sequences and structural integrity and surprisingly improves solubility and film forming ability. A preferred linker, Linker 1, has the sequence GSGSAAAAAAAAAAGSGS (SEQ ID No: 39). An alternative preferred linker, Linker 2, has the sequence GSGS (SEQ ID No: 40). Optionally, Linker 2 can be used to link SP2 with SP3 and / or SP3 with SP4 and / or SP4 with SP5. Optionally Linker 2 can link SP3 with SP4. Optionally Linker 2 can link SP3 with SP4, and Linker 1 can link the other SP motifs within the peptide.
[0015] Additionally, the combination of this linker together with a specific N terminal sequence (SEQ ID No: 1) or C terminal sequence (SEQ ID No: 2) leads to the creation of a stable and soluble peptide.
[0016] The present invention relates to peptides which are useful for skin care, hair care and cosmetic compositions.
[0017] The present invention provides therapeutically effective compositions comprising a therapeutically effective concentration of such peptides. The therapeutically effective compositions can function to enhance the appearance of skin, for example by improving the tone, turgor and / or elasticity of the tissue.
[0018] The present invention is further directed to a composition for improving tissue texture, wherein the composition comprises a peptide as described herein.Preferably the composition includes one or more excipients or carriers in combination with the peptide.
[0019] Optionally, the composition is a cosmetic preparation.
[0020] Optionally, the cosmetic preparation is formulated as a topical preparation to be applied to a patient's skin or hair. For example, the topical preparation can be an emulsion, lotion, spray, aerosol, powder, ointment, cream, or foam.
[0021] In some embodiments the peptide is soluble in an aqueous solution at ambient temperatures. For example, the peptide has a solubility at 20ºC in an aqueous buffer of at least 100 mg / ml, for example at least 120 mg / ml, for example at least 140 mg / ml. The peptide may have a solubility at 20ºC in 20 mM HEPES buffer, pH 7.5 of from 100 to 180 mg / ml, for example of around 150 mg / ml. Optionally, the aqueous buffer includes one or more of the following: Tris-HCI (10-50 mM), HEPES (10 - 50 mM), CAPS (10 - 50 mM), carbonate buffer (10 - 50 mM), or phosphate buffers such as sodium and potassium phosphate (10- 50 mM). Optionally, the pH is from 6.0 to 10.0.
[0022] Optionally, the peptide has a molecular weight of less than 42,000 Da, for example less than 40,000 Da, for example less than 30,000 Da. The peptide can have a molecular weight of at least 14,000 Da, for example 14,000 to 42,000 Da, for example 15,000 to 40,000 Da.
[0023] Optionally, the peptide includes up to 600 amino acid residues and a minimum of 250 amino acid residues, for example has from 250 to 580 amino acid residues.
[0024] Brief Description of the Figures
[0025] Figure 1 shows an exemplary film formulated with the peptide of SEQ ID No: 41 (3% w / v) formulated with palmitic acid (1% w / v) and glycerol (1% w / v).
[0026] Figure 2 shows an exemplary film formed with the peptide of SEQ ID No: 41 (3% w / v) formulated with oleic acid (1% w / v) and glycerol (1% w / v).Figure 3 shows an exemplary film formed with the peptide of SEQ ID No: 41 (3% w / v) formulated with PBS and glycerol (1% w / v).
[0027] Figure 4 shows an image of porous protein hydrogel formation of the peptide according to SEQ ID No:42 (1%w / v peptide, formulated with PBS).
[0028] Figure 5 shows is a bar chart showing results of the antioxidant assay using the peptides of SEQ ID Nos: 41 and 42.
[0029] Figure 6 shows the solubility of a 1% solution of the peptide of SEQ ID No: 41 in HEPES buffer at different pH values (ambient temperature).
[0030] Figure 7 shows the solubility of a 1% solution of the peptide of SEQ ID No: 42 in HEPES buffer at different pH values (ambient temperature).
[0031] Detailed Description of the Invention
[0032] The peptide, polynucleotides and vectors encoding the peptide, compositions comprising the peptide, expression system and methods of the present invention are now described in further detail.
[0033] As used herein, the term "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other.
[0034] As used herein, the term "comprising" is to be construed as encompassing both "including" and "consisting of, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention.
[0035] As used herein the term “peptide” refers to a polymer composed of amino acids joined by peptide bonds and does not refer to a specific length of the polymer. A "peptide bond" is a covalent bond between two amino acids in which the a-amino group of one amino acid is bonded to the a-carboxyl group of the other amino acid. The peptide can be modified, for example by glycosylation, amidation, carboxylation, phosphorylation, or the like. The modification can be in vitro or invivo. Amino acid chains with a length of less than approximately 100 amino acids are generally considered within the art to be "peptides", but both "polypeptides", and "proteins" are included within the definition of "peptides" as used herein.
[0036] The terms “amino acid sequence” and “peptide sequence” are used interchangeably. All amino acid or peptide sequences, unless otherwise designated, are written from the amino terminus (N-terminus) to the carboxy terminus (C-terminus).
[0037] For convenience of nomenclature, this application refers to a “peptide”. However, the designation of “peptide” in the term “peptide” is not intended to suggest any information regarding the size or relative size of the polymer concerned.
[0038] As used herein, when applied to an amino acid sequence, “conservative substitution” refers to the substitution of one amino acid residue with another amino acid residue having a side chain with similar physical and chemical properties. For example, conservative substitution may be conducted among amino acid residues having a hydrophobic side chain (e.g., Met, Ala, VaL, Leu, and He), amino acid residues having a neutral hydrophilic side chain (e.g., Cys, Ser, Thr, Asn, and Gin), amino acid residues having an acidic side chain (e.g., Asp and Glu), amino acid residues having a basic side chain (e.g., His, Lys, and Arg), or amino acid residues having an aromatic side chain (e.g., Trp, Tyr and Phe). It is known in the art that a conservative substitution generally does not cause a significant change in the conformational structure of a protein, and thus can retain the biological activity of the protein.
[0039] The term "polynucleotide" refers to a polymer of nucleic acid, for example, DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric polynucleotide molecule comprising one of these polynucleotides alone or in combination. The term “nucleic acid” is used interchangeably with the term “polynucleotide”.
[0040] The term "vector" as used herein refers to a genetic construct to facilitate the handling of a target polynucleotide. The vector may comprise further genes suchas marker genes, which allow for the selection of the vector in a suitable host cell and under suitable conditions. Expression of said polynucleotide or vector comprises transcription of the polynucleotide into a translatable mRNA. Usually, a vector comprises regulatory sequences ensuring initiation of transcription. Other elements which are responsible for the initiation of transcription, such as regulatory elements, may also be present. The vector may also comprise transcription termination signals downstream of the target polynucleotide.
[0041] When applied to an amino acid sequence (or a nucleic acid sequence), “percent sequence identity” refers to a percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those of a reference sequence, relative to the amino acid (or nucleic acid) residues in the candidate sequence during sequence alignment, and if necessary, after introducing gaps to maximize the number of identical amino acids (or nucleic acids). A conservative substitution of amino acid residue may or may not be considered as an identical residue.
[0042] Percent sequence identity of amino acid (or nucleic acid) sequences can be determined by aligning sequences through tools disclosed in the art. A person skilled in the art may use the default parameters of the tools or adjust the parameters appropriately according to the needs of the alignment, for example by choosing an appropriate algorithm. The percentage identity between two polypeptide sequences may be readily determined by programs such as BLASTp which is freely available at http: / / blast.ncbi.nlm.nih.gov.
[0043] An “isolated” material has been artificially altered from its natural state. If an “isolated” substance or component occurs in nature, it has been altered or removed from its original state, or both. For example, a polynucleotide or polypeptide naturally occurring in a living animal is not isolated but may be considered “isolated” if the polynucleotide or peptide is sufficiently isolated from the materials with which it coexists in its native state and exists in a sufficiently pure state. In some embodiments, the polynucleotide or peptide are at least 90%, 93%, 95%, 96%, 97%, 98%, 99% pure as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis), or chromatography (e.g., ion-exchange chromatography or reverse phase HPLC).The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.
[0044] In a first aspect, the present invention provides a peptide having the following formula I or which has 95% sequence identity thereto:
[0045] [M]x-[Starter Sequence]-[SEQ ID No: 1]a-[SP1]-Link-[SP2]- Link-[SP3]-Link- [SP4]-Link-[SP5]- Link-[SP6]-[SEQ ID No: 2]b
[0046] wherein:
[0047] x is 0 or 1;
[0048] a and b are each independently 0 or 1, but at least one of a or b must be 1;
[0049] Link represents a linker sequence selected from GSGS or GSGS(A)nGSGS, where each n is an integer from 6 to 20, for example is 8 to 16;
[0050] wherein each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are each independently selected from SEQ ID Nos: 3 to 26;
[0051] and wherein the Starter Sequence has the sequence:
[0052] [Motif1]-[Motif2]
[0053] wherein Motif 1 is selected from SEQ ID Nos: 33 to 38; and
[0054] wherein Motif 2 is selected from: SEQ ID Nos: 27 to 30.
[0055] SEQ ID No: 1 has the following sequence:
[0056] ALGQANTPWSSKENADAFIGAFMNAASQSGAFSSDQIDDMSVISNTLMAAMDNM GGRITQSKLQALDMAFASSVAEIAVADGQNVGAATNAISDALRSAFYQTTGVVNN QFITGISSLIGMFAQVSGNEV
[0057] SEQ ID No: 2 has the following sequence:
[0058] SVTSGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASA LPSVISNIYSGVVASGVSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNV VQDSVGQYVG
[0059] At least one of SEQ ID Nos 1 and 2 must be present. Optionally, both SEQ ID Nos: 1 and 2 are present in the peptide.When integer x is 0, this indicates that the N-terminal Methionine residue has been cleaved post-translationally. However, depending upon the expression system used, this post-translational cleavage of methionine may not occur, and the present invention extends to such peptide constructs, in which integer x is 1 and the N-terminal residue is methionine (see, for example, SEQ ID Nos: 45 to 48).
[0060] Optionally, the linker is of formula GSGS(A)nGSGS and n is an integer of 6 to 16, for example is 8 to 12. Thus, (A)n can be a chain of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 alanine residues. Optionally, (A)n can be a chain of 10 alanine residues so that the linker sequence has the sequence of SEQ ID No: 39.
[0061] A preferred linker, Linker 1, has the sequence GSGSAAAAAAAAAAGSGS (SEQ ID No: 39). An alternative preferred linker, Linker 2, has the sequence GSGS (SEQ ID No: 40). Optionally, Linker 2 can be used to link SP2 with SP3 and / or SP3 with SP4 and / or SP4 with SP5. Optionally Linker 2 can link SP3 with SP4. Optionally Linker 2 can link SP3 with SP4, and Linker 1 can link the other SP motifs within the peptide.
[0062] For the avoidance of any doubt, the reference to “M” is to a methionine residue, “A” is to an alanine residue, “G” is to a glycine residue, and “S” is to a serine residue.
[0063] The SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 can be the same or different to each other. Optionally two or more of SP1 to SP6 have a first sequence and the other SP sequences have different sequence(s), which may be the same or different to each other. Optionally three or more of SP1 to SP6 have a first sequence and the other SP sequences have different sequence(s), which may be the same or different to each other. Optionally four or more of SP1 to SP6 have a first sequence and the other SP sequences have different sequence(s), which may be the same or different to each other.
[0064] Optionally, each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 have a different sequence to each other.Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 have an identical sequence.
[0065] Optionally, the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 each consist of one of the following sequences shown in Table 1 below.
[0066] Table 1:
[0067] SEQ ID No: Sequence
[0068] 3 GGRPSDSYGAPGGGN
[0069] 4 GGRPCDSYGAPGGGN
[0070] 5 GGRPSDSYGPPGNPGPP
[0071] 6 GGRPCDSYGPPGPPSGGN
[0072] 7 GSCGIGGGIGAGSS
[0073] 8 SGGACGLGGGYGGGF
[0074] 9 GGDVEKRGDREE
[0075] 10 SIKVAVSADRDCIR
[0076] 11 GVLPGVGGAGVLPGVGGA
[0077] 12 GLVPGGPGFGGLVPGGPGFG
[0078] 13 GLGPERGLGPER
[0079] 14 RGDGGRPSDSYGAPGGGN
[0080] 15 GRGDSPGGRPSDSYGAPGGGN
[0081] 16 IKVAVGGRPSDSYGAPGGGN
[0082] 17 CTGRGDSPACGGRPSDSYGAPGGGN
[0083] 18 YGISRGGRPSDSYGAPGGGN
[0084] 19 VGVAPGVGVAPGVGVAPG
[0085] 20 GLPGERGLPGER
[0086] 21 FNDLGAW
[0087] 22 FPDTTTL
[0088] 23 KFGEGK
[0089] 24 LVNELTEFAQ
[0090] 25 LVNEVTEFAQ
[0091] 26 FVDSSATT
[0092]
[0093] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 3, for example two or more of the SP peptides can be SEQ ID No: 3. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 3.
[0094] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 6, for example two or more of the SP peptides can be SEQ ID No: 6. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 6.
[0095] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 7, for example two or more of the SP peptides can be SEQ ID No: 7. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 7.
[0096] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 21, for example two or more of the SP peptides can be SEQ ID No: 21. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 21.
[0097] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 22, for example two or more of the SP peptides can be SEQ ID No: 22. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 22.
[0098] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 23, for example two or more of the SP peptides can be SEQ ID No: 23. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 23.
[0099] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 24, for example two or more of the SP peptides can be SEQ ID No: 24. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 24.Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 25, for example two or more of the SP peptides can be SEQ ID No: 25. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 25.
[0100] Optionally, at least one of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 is SEQ ID No: 26, for example two or more of the SP peptides can be SEQ ID No: 26. Optionally each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are SEQ ID No: 26.
[0101] Optionally, the Starter Sequence may comprise SEQ ID No: 35 formed from the combination of SEQ ID Nos: 33 and 27, namely:
[0102] GSSHHHHHHSSGVDLGTENLYFQSM
[0103] Optionally, the Starter Sequence may comprise SEQ ID No: 36 formed from the combination of SEQ ID Nos: 37 and 27, namely:
[0104] HHHHHHSSGVDLGTENLYFQSM
[0105] Optionally, the present invention provides a peptide having the following formula la:
[0106] [M]x-[Starter Sequence]-[SEQ ID No: 1 ]-[SP1 ]-Link-[SP2]- Link-[SP3]-Link- [SP4]-Link-[SP5]- Link-[SP6]-[SEQ ID No: 2]
[0107] wherein:
[0108] x is 0 or 1;
[0109] Link represents a linker sequence selected from GSGS or GSGS(A)nGSGS, where each n is an integer from 6 to 20, for example is 8 to 16;
[0110] wherein each of SP1, SP2, SP3, SP4, SP5 or SP6 is independently selected from SEQ ID Nos: 3 to 26;
[0111] and wherein the Starter Sequence has the sequence:
[0112] [Motif1]-[Motif2]
[0113] wherein Motif 1 is selected from SEQ ID Nos: 33 to 38; and
[0114] wherein Motif 2 is selected from: SEQ ID Nos: 27 to 30.Optionally, the present invention provides a peptide having the following formula lb:
[0115] [M]x-[Starter Sequence]-[SEQ ID No: 1 ]-[SP1 ]-Link-[SP2]- Link-[SP3]-Link- [SP4]-Link-[SP5]- Link-[SP6]
[0116] wherein:
[0117] x is 0 or 1;
[0118] Link represents a linker sequence selected from GSGS or GSGS(A)nGSGS, where each n is an integer from 6 to 20, for example is 8 to 16;
[0119] wherein each of SP1, SP2, SP3, SP4, SP5 or SP6 is independently selected from SEQ ID Nos: 3 to 26;
[0120] and wherein the Starter Sequence has the sequence:
[0121] [Motif1]-[Motif2]
[0122] wherein Motif 1 is selected from SEQ ID Nos: 33 to 38; and
[0123] wherein Motif 2 is selected from: SEQ ID Nos: 27 to 30.
[0124] Optionally, the present invention provides a peptide having the following formula Ic:
[0125] [M]x-[Starter Sequence]-[SP1 ]-Li n k-[S P2]- Link-[SP3]-Link-[SP4]-Link-[SP5]- Link-[SP6]-[SEQ ID No: 2]
[0126] wherein:
[0127] x is 0 or 1;
[0128] Link represents a linker sequence selected from GSGS or GSGS(A)nGSGS, where each n is an integer from 6 to 20, for example is 8 to 16;
[0129] wherein each of SP1, SP2, SP3, SP4, SP5 or SP6 is independently selected from SEQ ID Nos: 3 to 26;
[0130] and wherein the Starter Sequence has the sequence:
[0131] [Motif1]-[Motif2]
[0132] wherein Motif 1 is selected from SEQ ID Nos: 33 to 38; and
[0133] wherein Motif 2 is selected from: SEQ ID Nos: 27 to 30.
[0134] An exemplary peptide according to formula 1a is shown in SEQ ID No: 41 below in which Motif 1 is SEQ ID No: 33, Motif 2 is SEQ ID No: 27, and SP1, SP2 and SP3are each SEQ ID No: 3 (the sequence motifs SP1, SP2 and SP3 are shown underlined):
[0135] MGSSHHHHHHSSGVDLGTENLYFQSMALGQANTPWSSKENADAFIGAFM NAASQSGAFSSDQIDDMSVISNTLMAAMDNMGGRITQSKLQALDMAFASS VAEIAVADGQNVGAATNAISDALRSAFYQTTGVVNNQFITGISSLIGMFAQV SG N EVGGRPSDSYGAPGGGNGSGSAAAAAAAAAAGSGSGGRPSDSYG APGGGNGSGSAAAAAAAAAAGSGSGGRPSDSYGAPGGGNGSGSGGRP SDSYGAPGGGNGSGSAAAAAAAAAAGSGSGGRPSDSYGAPGGGNGSG SAAAAAAAAAAGSGSGGRPSDSYGAPGGGNSVTSGGYGYGTSAAAGAG VAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVASG VSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYVG
[0136] The equivalent sequence in which the initial Met residue is not cleaved post-translationally is SEQ ID No: 45.
[0137] An alternative exemplary peptide according to formula 1a is shown in SEQ ID No: 42 below in which Motif 1 is SEQ ID No: 33, Motif 2 is SEQ ID No: 27, SP1 is SEQ ID No: 24, SP2 is SEQ ID No: 25, SP3 is SEQ ID No: 26, SP4 is SEQ ID No: 21, SP5 is SEQ ID No: 22, and SP6 is SEQ ID No: 23 (the SP sequence motifs are shown underlined):
[0138] MGSSHHHHHHSSGVDLGTENLYFQSMALGQANTPWSSKENADAFIGAFM NAASQSGAFSSDQIDDMSVISNTLMAAMDNMGGRITQSKLQALDMAFASS VAEIAVADGQNVGAATNAISDALRSAFYQTTGVVNNQFITGISSLIGMFAQV SGNEVLVNELTEFAQGSGSAAAAAAAAAAGSGSLVNEVTEFAQGSGSAA AAAAAAAAGSGSFVDSSATTGSGSAAAAAAAAAAGSGSFNDLGAWGSGS AAAAAAAAAAGSGSFPDTTTLGSGSAAAAAAAAAAGSGSKFGEGKSVTS GGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASA LPSVISNIYSGVVASGVSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDS TLNVVQDSVGQYVG
[0139] The equivalent sequence in which the initial Met residue is not cleaved post-translationally is SEQ ID No: 46.An exemplary peptide according to formula 1a is shown in SEQ ID No: 43 below in which Motif 1 is SEQ ID No: 33, Motif 2 is SEQ ID No: 27, and SP1, SP2, SP3, SP4, SP5 and SP6 are each SEQ ID No: 7 (the SP sequence motifs are shown underlined):
[0140] GSSHHHHHHSSGVDLGTENLYFQSMALGQANTPWSSKENADAFIGAFMN AASQSGAFSSDQIDDMSVISNTLMAAMDNMGGRITQSKLQALDMAFASSV AEIAVADGQNVGAATNAISDALRSAFYQTTGVVNNQFITGISSLIGMFAQVS GNEVGSCGIGGGIGAGSSGSGSAAAAAAAAAAGSGSGSCGIGGGIGAGS SGSGSAAAAAAAAAAGSGSGSCGIGGGIGAGSSGSGSAAAAAAAAAAGS GSGSCG IGGG IGAGSSGSGSAAAAAAAAAAGSGSGSCG IGGG IG AGSSG SGSAAAAAAAAAAGSGSGSCG IGGG IG AGSSSVTSGG YG YGTSAAAG AG VAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVASG VSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYVG
[0141] The equivalent sequence in which the initial Met residue is not cleaved post-translationally is SEQ ID No: 47.
[0142] An exemplary peptide according to formula 1a is shown in SEQ ID No: 44 below in which Motif 1 is SEQ ID No: 33, Motif 2 is SEQ ID No: 27, and SP1, SP2, SP3, SP4, SP5 and SP6 are each SEQ ID No: 6 (the SP sequence motifs are shown underlined):
[0143] GSSHHHHHHSSGVDLGTENLYFQSMALGQANTPWSSKENADAFIGAFMN AASQSGAFSSDQIDDMSVISNTLMAAMDNMGGRITQSKLQALDMAFASSV AEIAVADGQNVGAATNAISDALRSAFYQTTGVVNNQFITGISSLIGMFAQVS GNEVGVLPGVGGAGVLPGVGGAGSGSAAAAAAAAAAGSGSGVLPGVGG AGVLPGVGGAGSGSAAAAAAAAAAGSGSGVLPGVGGAGVLPGVGGAGS GSAAAAAAAAAAGSGSGVLPGVGGAGVLPGVGGAGSGSAAAAAAAAAA GSGSGVLPGVGGAGVLPGVGGAGSGSAAAAAAAAAAGSGSGVLPGVGG AGVLPGVGGASVTSGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASR VSSNIAAIASGGASALPSVISNIYSGVVASGVSSNEALIQALLELLSALVHVL SSASIGNVSSVGVDSTLNVVQDSVGQYVGThe equivalent sequence in which the initial Met residue is not cleaved post-translationally is SEQ ID No: 48.
[0144] Thus, in one aspect, the present invention provides a peptide of SEQ ID Nos: 41 to 48 or which has at least 80% sequence identity thereto, optionally which has at least 85% sequence identity thereto, optionally which has at least 90% sequence identity thereto, optionally which has at least 95% sequence identity thereto, optionally which has at least 98% sequence identity thereto.
[0145] The present invention further provides a composition comprising a peptide as described above together with a pharmaceutically acceptable carrier or excipient. Optionally, the composition is suitable for topical application to skin or hair.
[0146] In a second aspect of the invention, the invention provides a polynucleotide which encodes the peptide described above. Thus, the isolated polynucleotide according to the present invention can be used to encode a peptide which is in accordance with formula 1.
[0147] In addition, the invention also encompasses a polynucleotide which specifically hybridizes under stringent conditions to the polynucleotide encoding the peptide. For the purposes of the present specification, hybridisation under stringent hybridisation conditions means remaining hybridised after washing with 0.1 xSSC, 0.5% SDS at a temperature of at least 68° C, as described by Sambrook et al (Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press).
[0148] It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same peptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the peptides are to be expressed.
[0149] The polynucleotide of the invention may consist of DNA or RNA. The polynucleotide may be single-stranded or double-stranded. The polynucleotidemay include synthetic or modified nucleotides. Several different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the invention as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or life span of polynucleotides of interest.
[0150] Optionally, the polynucleotide of the invention has a nucleotide sequence which expresses a peptide with at least 80% sequence identity to one of SEQ ID Nos: 41 to 48. Optionally, the polynucleotide of the invention encodes a polypeptide having a sequence identity to SEQ ID Nos: 41 to 48 which is more than 80%, for example which is 85%, 90%. 95%, 98% or even more.
[0151] Optionally, the polynucleotide of the invention has a nucleotide sequence which expresses a peptide with at least 85% sequence identity to one of SEQ ID Nos: 41 to 48. Optionally, the polynucleotide of the invention encodes a peptide having a sequence identity to SEQ ID Nos: 41 to 48 which is more than 80%, for example more than 85%, for example which is 90%, 95%, 98% or even more.
[0152] In a third aspect, the present invention provides a vector comprising such a polynucleotide according to the invention as described above, in particular an expression vector expressing, or overexpressing, said polynucleotide.
[0153] The present invention provides a polynucleotide of any of SEQ ID Nos: 49 to 52.
[0154] The present invention also encompasses a polynucleotide which specifically hybridizes under stringent conditions to such a polynucleotide. For the purposes of the present specification, hybridisation under stringent hybridisation conditions means remaining hybridised after washing with 0.1 xSSC, 0.5% SDS at a temperature of at least 68° C, as described by Sambrook et al (Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press).In a fourth aspect, the present invention provides a vector comprising such a polynucleotide according to the invention as described above, in particular an expression vector expressing, or overexpressing, said polynucleotide. The vector can include a polynucleotide having at least 85% sequence identity to a polynucleotide according to the invention, for example having a sequence identity which is 90%, 95%, 98% or even more identity to a polynucleotide according to the invention, such as any of SEQ ID Nos 49 to 52.
[0155] A “vector” in the present invention refers to a vehicle into which a polynucleotide encoding a peptide can be operably inserted for enabling the peptide to be expressed. The vector can be used to transform, transduce, or transfect (which terms are used interchangeably herein) a host cell, such that the genetic elements carried by the vector are expressed in the host cell. A variety of vectors are available. The vector may comprise a variety of elements that control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a signal sequence, one or more marker genes, a selection element, a reporter gene, and a transcription termination sequence. Further, the vector may also comprise an origin of replication. The vector may also comprise a component that facilitates the vector to enter into cells, including, but not limited to, viral particle, liposome, or protein shell.
[0156] For example, the vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or R1 -derived artificial chromosome (PAC), bacteriophages such as 2 bacteriophage or M13 bacteriophage, animal viruses, and the like.
[0157] In some embodiments, the vector systems include mammalian, bacterial, and yeast systems, and will include plasmids such as, but not limited to, ‘pENDO-2’ and other vectors available from the laboratory or commercially available vectors. Suitable eukaryotic vectors include vectors having a 2 micron or centromeric origin of replication. Suitable vectors may include plasmid or viral vectors (e.g., replicationdefective retroviruses, adenoviruses, and adeno-associated viruses). Suitable vectors include pET28 and pET32, for example.The present invention thus provides an expression vector comprising the above polynucleotide according to the invention. The expression vector of the present invention can be prepared by subcloning the polynucleotide as described above into the expression vector by any conventionally known genetic engineering method. The type of expression vector that can be used in the present embodiment is not particularly limited, and examples thereof include any expression vector suitable for heterologous gene expression in eukaryotes and able to drive expression of the target peptide. For example, a eukaryotic vector having a 2 micron or centromeric origin of replication together with a constitutive promoter / terminator cassette can conveniently be used, for example the Tef1 promoter.
[0158] A vector comprising a polynucleotide encoding the peptide may be introduced into a host cell for cloning (amplification of DNA) or gene expression using recombinant techniques well known in the art. In another embodiment, the peptide can be prepared by homologous recombination methods well known in the art.
[0159] In a fifth aspect, the present invention provides a host cell comprising a vector or a polynucleotide according to the invention as described above.
[0160] A “host cell” in the present invention refers to a cell into which an exogenous polynucleotide and / or a vector are introduced. Amino acid sequences of the fusion protein of the present application may be converted to corresponding DNA coding sequences using genetic engineering techniques well known in the art. Due to the degeneracy of genetic code, the transformed DNA sequences may not be completely identical, while the encoded protein sequences remain unchanged.
[0161] Host cells suitable for cloning or expressing the DNA in the vectors of the present invention are prokaryotic, yeast or the above-mentioned advanced eukaryotic cells. Prokaryotic cells suitable for use in the present invention include E. coli (for example E. coli DH5a and BL21de3).
[0162] In one embodiment, eukaryotic host cells are used for cloning or expressing vectors encoding the peptide according to the invention. Saccharomycescerevisiae (S288C) or baker's yeast is the most used lower eukaryotic host microorganism. However, many other genera, species and strains are common and suitable for use in the present invention, such as other members of the Saccharomyces clade (including S. pastorianus. S. eubayanus and S. paradoxus), Komagataella (including K. pastoris), Kluyveromyces (including K. lactis) and Yarrowia (including Y. lipolytica).
[0163] In another aspect of the present invention, the present invention provides a recombinant cell or recombinant microorganism which contains the polynucleotide or vector as described above (such as any of SEQ ID Nos 49 to 52). Thus, the recombinant cells or recombinant microorganisms according to the present invention can express the peptide of the present invention. The invention further relates to a recombinant host cell comprising the polynucleotide, or the vector as described above. The polynucleotide or vector of the present invention, which is present in the host cell, may either be integrated into the genome of the host cell, or it may be maintained extra-chromosomally. Once the polynucleotide or vector has been incorporated into the appropriate “host cell”, the host cell is maintained under conditions suitable for high level expression of the polynucleotide or vector.
[0164] The transformed host cells can be grown according to methodology known in the art to achieve cell growth.
[0165] Optionally, once expressed, the peptide can be purified according to standard procedures of the art. Mention may be made of affinity columns, column chromatography, such as size exclusion chromatography (SEC), gel electrophoresis, ammonium sulphate precipitation and the like. The peptide of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the peptide may be by any conventional means such as, for example, preparative chromatographic separations.
[0166] The host cell is transformed with the above-mentioned expression or cloning vector that can produce the peptide, and then cultured in a conventional nutrient medium,which is suitable for inducing promoters, selecting transformed cells, or amplifying genes encoding target sequences after being modified.
[0167] The host cells used to produce the peptides in the present invention can be cultured in a variety of media known in the art. The media may also comprise any other necessary additives known in the art in a suitable concentration. The conditions of the media, such as temperature, pH and the like are those selected previously for expression of host cells, which are well known to those of ordinary skill.
[0168] The present invention further provides a method for producing a peptide as described above, wherein the method comprises the following steps of suitably culturing a recombinant host cell comprising and expressing a polynucleotide encoding the peptide according to formula I or a vector encoding the peptide according to formula I. The polynucleotide can include a sequence according to any one of SEQ ID Nos: 49 to 52.
[0169] The polynucleotide can express a peptide having a sequence of any one of SEQ ID Nos: 41 to 48.
[0170] The peptide can improve the elasticity of the tissue to which the peptide is applied. Optionally, the administration step be comprised of a number of separate administration steps which are conveniently repeated twice daily over a predetermined time, for example where the predetermined time exceeds one week of daily administration of the peptide, for example where the predetermined time exceeds two weeks, or (in some embodiments) is at least a month of daily topical application (with twice daily of the peptide administration over the month being preferable).
[0171] The peptide of the present invention is suitable for prophylactic or cosmetic use. The peptide can be provided to the relevant tissue, typically to skin or hair. As used herein, the term "providing", when used in conjunction with the peptide, can include, but is not limited to, providing the peptide into or onto the target tissue, for example topically.Mammalian skin consists of a number of overlapping layers of cells. The outer layer of skin is the “stratum corneum” and acts as a physical barrier against physical damage and damage due to the sun. The moisture content of the stratum corneum affects the texture and “softness” of this layer. In lower skin layers a lack of elasticity decreases the tone of the skin and can influence its overall appearance. Use of the inventive peptides within a topical formulation can ameliorate loss of moisture and / or help to maintain hydration of cells.
[0172] As stated above, the present invention is directed to a peptide according to formula I which is useful as a therapeutic and / or cosmetic composition or agent for modifying tissue, especially skin or hair. The term "modify" is used to mean that the peptide of the present invention (or composition containing such peptide) changes either the appearance, form, characteristics and / or the physical attributes of the tissue to which it is being provided, applied or administered. The change in form can be reflected in any of the following alone or in combination: enhanced appearance of the skin or hair (shine or lustre); increased softness of the skin; increased smoothness of hair; decreased static within hair; reduction of split-ends within hair; increased turgor of the skin; increased texture of the skin; increased elasticity of the skin; decreased wrinkle formation and increased endogenous elastin production in the skin.
[0173] Details on techniques for formulation and administration of pharmaceuticals may be found in the latest edition of Remington' s Pharmaceutical Sciences (Mack Publishing Co, Easton Pa.). Although local topical delivery is desirable, there are other means of delivery, for example: oral, parenteral, aerosol, intramuscular, subcutaneous, transcutaneous, intamedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The delivery system of the present invention is preferably a topical delivery system but can alternatively be a subcutaneous, transcutaneous, aerosol, or patch delivery system.
[0174] The term "cosmetic," as used herein, refers to a beautifying substance or preparation which preserves, restores, bestows, simulates, or enhances theappearance of bodily beauty, specifically as it relates to the appearance of skin or hair.
[0175] The present invention can be formulated in a number of carrier vehicles, for example, in a spray; an aerosol; a water and an oil-type emulsion; an oil and watertype emulsion; a face cream or body cream; a sun lotion or after-sun lotion; or other topical administration vehicle. The concentration of the peptide of the present invention in an end-use formulation would typically be 5% (by weight) or less, for example 4% (by weight) or less, for example 3% (by weight) or less, for example 2% (by weight) or less, for example 1 % (by weight) or less, such as 0.5% by weight. Typically, an end-use formulation may contain from 0.5% to 3% by weight of the peptide, for example around 0.8% to 2.5% by weight.
[0176] Optionally, the composition can be a film-forming composition. The film may be able to act as a barrier. The barrier can be a breathable barrier and can be formulated for application to skin or hair. The present invention further provides the film formed using the peptides of the invention. The benefit of forming films in a formulation is that it will leave a protective barrier on the surface of the skin protecting against pollution and potentially sun damage and helping to prevent moisture loss. The film will also smoothen fine lines and wrinkles delivering antiaging effects.
[0177] The composition can be an aqueous composition and can be formulated to include (i) an aqueous phase, that optionally contains glycerol and / or thickening agents (such as salts and / or gums), (ii) an oil phase that optionally contains fatty acids, ether lipids and triglycerides (e.g., as found in coconut milk and oil) and (iii) optionally one or more other excipients such as preservatives (such as phenoxyethanol or ethylhexylglycerin), colourants, aroma or fragrance compounds, or emulsifier compounds.
[0178] Optionally, the composition includes more than one peptide of the invention, for example include two or more peptides of the invention, for example includes three or more peptides of the invention.It is preferable that the topical administration of the composition of the present invention occur repeatedly over a predetermined time period, preferably in the range of about one week to about one month.
[0179] Optionally the peptide of the present invention or a composition comprising a peptide of the present invention can be for application to hair, for example human hair. Optionally the composition can be a shampoo, hair conditioner, hair colourant, hair setting or holding composition or hair spray. Thus, the peptide of the invention or the composition comprising the peptide is useful for treating hair, for example human hair.
[0180] Optionally the peptide of the present invention or a composition comprising a peptide of the present invention can be for application to skin, for example human skin. Optionally the composition can be a skin emollient to the skin, for example can be a soap, cleanser, scrub, cream, lotion, or moisturiser. Optionally the composition can be for improving the appearance of skin, for example can improve the turgor of skin or can be for reduction of wrinkles. Optionally the composition can be a cosmetics (make-up) composition. Optionally the composition can be a face mask. Optionally the composition can be for treatment of scars. Optionally the composition can be a sunscreen. Optionally, the composition can be a deodorant. Thus, the peptide of the invention or the composition comprising the peptide is useful for treating skin, for example human skin.
[0181] Preferred or alternative features of each aspect or embodiment of the invention apply mutatis mutandis to each other aspect or embodiment of the invention (unless the context demands otherwise).
[0182] All documents referred to herein are incorporated by reference. Any modifications and / or variations to described embodiments that would be apparent to one of skill in art are hereby encompassed. Whilst the invention has been described herein with reference to certain specific embodiments and examples, it should be understood that the invention is not intended to be unduly limited to these specific embodiments or examples.The following examples are provided to further illustrate the invention but are not intended to limit the scope of the invention in any manner.
[0183] Examples
[0184] Example 1: Sequence creation
[0185] DNA polynucleotide sequences encoding SEQ ID Nos: 41 to 44 and were created synthetically (see SEQ ID Nos 49 to 52). Each DNA sequence was then loaded into the T7-driven expression plasmid pET32.
[0186] Example 2: Expression and Purification of Peptides
[0187] A pET32 vector containing a relevant coding sequence for each peptide of interest was separately used to transform chemically competent BL21 E. coli, using a standard heat shock approach. Thus, ‘empty’ bacterial cells were caused to take up copies of the expression plasmid encoding the target sequence(s), facilitating downstream protein expression. Cell growth for purification was done in BL21 (DE3) and BL21 STAR (DE3) cells. LB Broth media was used for cell growth.
[0188] Growth Conditions
[0189] All cells were grown at 37oC until an OD of approximately 0.6 is reached and were then induced with 1 mM IPTG. The cells were then left to grow at 20oC overnight and spun down and stored dry in -20oC. Each test is run with cells grown with 1 litre of LB Broth.
[0190] Purification of the Peptides
[0191] The peptides were expressed using IPTG. The cells were harvested and lysed using a sonicator. After centrifugation to remove insoluble proteins and cell lysate, the soluble fraction was run through an immobilised metal affinity column to separate the polypeptide from the other contaminants. Each protein was then concentrated to (100 mg / mL) and a buffer exchange was performed to store the protein in its final storage buffer (20 mM HEPES pH 7.5).Example 3 - Characteristics of the Expressed Peptides
[0192] Colour of Peptides
[0193] Each of the peptides expressed were colourless. At higher concentrations, the protein solutions turn a slight yellow solution and are slightly viscous in consistency.
[0194] Solubility of Peptides
[0195] Each of the peptides expressed were soluble up to 150 mg / mL in 20 mM HEPES pH 7.5 at 23oC.
[0196] pH Stability of Peptides
[0197] Each of the peptides were soluble in aqueous solutions at pH above 5 at 23oC.
[0198] Additionally, solubility across citric acid-Na2HPO4and HEPES buffers with differing pH were conducted for a 1% solution of the peptides of SEQ ID Nos: 41 and 42 at ambient temperature. The peptides were found to be soluble at pH 5 to 6. It was noted that the peptides precipitated out at a pH below 4, but did not precipitate out at pH 5 or above.
[0199] Details of the results for the peptides of SEQ ID Nos: 41 and 42 are shown below:
[0200] Protein Citric Acid Buffer HEPES Buffer
[0201] pH 3 pH 3.5 pH 4.0 pH 5.0 pH 5.5 pH 6.0 SEQ ID poor poor poor soluble soluble soluble No. 41
[0202] SEQ ID poor poor poor soluble soluble soluble No. 42
[0203]
[0204] Figure 6 shows the solubility of a 1% solution of the peptide of SEQ ID No: 41 in HEPES buffer at different pH values (ambient temperature). The protein is soluble at pH 5, pH5.5 and pH 6, but has precipitated at pH3, pH3.5 and pH4.Figure 7 shows the solubility of a 1% solution of the peptide of SEQ ID No: 42 in HEPES buffer at different pH values (ambient temperature). The protein is soluble at pH 5, pH5.5 and pH 6, but has precipitated at pH3, pH3.5 and pH4.
[0205] Thermostability of Peptides
[0206] Each of the peptides expressed were shown to be able to handle up to 3 freeze thaw cycles (-20°C to + 25°C) when stored in 20 mM HEPES pH 7.5. The peptides were also stable for 6 months at room temperature and 4°C. The peptides were also stable for 1 year at -20°C and -80°C.
[0207] Each of the peptides were able to remain in aqueous solution at a temperature of up to 95°C for 30 minutes.
[0208] Example 4 - Film Formation by Peptides - SEQ ID Nos: 41 to 44
[0209] 2 mL of each peptide (1 -3% w / v) was left to dry overnight at 50oC in silicone moulds. Higher concentrations formed more robust films, as did drying the peptides at ambient temperature. Water and oil penetration tests were performed to confirm the permeability of the films. This was done by the addition of these solutions on top of the film and the time taken for the solutes to penetrate through the film was measured. The films showed no penetration of oil. The films also showed that water penetrated through the films, but higher concentrations of proteins had a longer water penetration time.
[0210] Example 5 - Formulations with PEG 20,000.
[0211] PEG 20,000 and glycerol are well known plasticizers. Plasticizers are chemicals that are added than can make the films more flexible increasing its plasticity.
[0212] Solutions of peptides according to the invention were mixed together with plasticizers and the solution was left to form films.
[0213] A 2mL solution containing 1-3% (w / v) peptide and 1-3% (w / v) PEG 20,000 was made and left to form films. The films were then tested for transparency, flexibility, water and oil permeability. It was observed that the films became more stiffer, more opaque and more brittle as the concentration of PEG was increased. Higher concentration of PEG also showed a decrease in water permeability creating morehydrophobic films. Oil did not penetrate through any of the films. Water penetration tests were carried out to test the hydrophobicity of the films formed.
[0214] Example 6 - Formulations with Glycerol
[0215] A 2mL solution containing 1-3% (w / v) protein and 1-3% (w / v) glycerol was made and left to form films. The films were then tested for transparency, flexibility, water and oil permeability. It was observed that the films became more flexible and transparent as the concentration of glycerol was increased. Water penetration tests were carried out to test the hydrophobicity of the films formed.
[0216] Example 7 - Formulations with Palmitate acid
[0217] Palmitates have been used as an emollient in cosmetics. Palmitic acid at varying concentrations (0.5 to 1 % w / v) are to be formulated with SEQ ID Nos: 41 to 44 (at 2% w / v).
[0218] These formulations can be used to form films with varying characteristics. The results for the peptide of SEQ ID No: 41 (2%w / v and 3%w / v) is shown below:
[0219] SEQ ID Palmitic Glycerol Water Oil Film Structure No. Acid (% w / v) (% w / v) Resistance Resistance
[0220] SEQ 41 1 1 Yes Yes flexible, cloudy, opaque film
[0221]
[0222] Figure 1 shows the exemplary film formed with the peptide SEQ ID No: 41 (3% w / v), with palmitic acid (1% w / v) and glycerol (1% w / v).
[0223] Example 8 - Formulations with Oleic acid
[0224] Oleic acid has been used as an emollient in cosmetics. The peptides of the invention have been successfully formulated using oleic acid at varying concentrations. For example, oleic acid at 1% w / v can be formulated with 3% w / v peptides of the invention. These formulations were then used to form films with varying characteristics.
[0225] The results for the peptide of SEQ ID No: 41 is shown below:SEQ ID No: Oleic Glycerol Water Oil Film Structure Acid (% (% w / v) Resistance Resistance
[0226] w / v)
[0227] SEQ ID No: 1 1 Yes Yes flexible, clear, 41 shiny film
[0228]
[0229] Figure 2 shows the exemplary film formed with the peptides of SEQ ID No: 41 (3% w / v), formulated with oleic acid (1% w / v) and glycerol (1% w / v).
[0230] Example 9 - Formulations with Phosphate Buffered Saline
[0231] Phosphate Buffered Saline (PBS) is used as a carrier, solvent and buffer in cosmetics. The peptides of the invention have been successfully formulated using PBS at varying concentrations. The peptide is diluted into a PBS buffer which created different types of films (for example to a buffer such as HEPES). For example, PBS at 1% w / v can be formulated with 3% w / v peptides of the invention. These formulations were then used to form films with varying characteristics. The results for the peptide of SEQ ID No: 41 is shown below:
[0232] SEQ ID Glycerol Water Oil Film Structure
[0233] No: (% w / v) Resistance Resistance
[0234] SEQ ID 1 Yes Yes rigid, opaque, cloudy film No: 41
[0235]
[0236] Figure 3 shows an exemplary film formed with the peptide of SEQ ID No: 41 (3% w / v) formulated with PBS (1% w / v) and glycerol (1% w / v). In this example, a 10% w / v protein (originally in 25 mM HEPES pH 7.5) buffer has been diluted with PBS (+ glycerol) to form a 3% w / v protein solution. This solution (containing both PBS and glycerol) formed the water- and oil-resistant films.
[0237] Example 10 - Hair Binding Activity
[0238] All hair is washed with mild soap solution in water followed by a water rinse and left to dry overnight at room temperature. Dry clean hair is to be soaked for 5 minutes in a solution (20 mM HEPES pH 7.5) containing the peptide (2% w / v). The hair isthen rinsed with water for 0 secs (No Rinse), rinsed by a quick immersion of the soaked hair for 2-3 sec (Dunk Rinse) or rinsed for a period of 5 mins (Soak Rinse). The rinsed hair is then left to air dry at room temperature overnight and imaged using a scanning electron microscope.
[0239] The Control hair is soaked for 5 mins in buffer (20 mM HEPES pH 7.5) and left to air dry overnight at room temperature.
[0240] Example 11 - Antioxidant Activity
[0241] Antioxidant assay was carried out using the ABTS Antioxidant Assay Kit by Zen-Bio.
[0242] Figure 5 shows the results of the anti-oxidant assay conducted for the peptides of SEQ ID Nos: 41 and 42.
[0243] Example 12 - Gel Formation
[0244] The peptides according to the invention can form hydrogels when heated up or when formulated with chemicals such as ethanol and chloroform and plasticizers. The peptides can also form hydrogels when formulated with PBS and heated to 95°C for 5-10 minutes. Figure 4 shows a porous protein hydrogel formed using the peptide of SEQ ID No: 42 at a concentration of 1% w / v protein formulated into PBS and heated to 95°C for 10 minutes. Specifically, 0.1 mL of 10% w / v peptide (in 20mM HEPES) was dissolved in 0.9 mL of PBS and then heated to 95°C for 10 minutes.
[0245] Example 13 - Differential Gene Expression Analysis of Proteins
[0246] A 3D in-vitro skin model containing epidermal keratinocytes and dermal fibroblasts (Mattek EFT-400) was used for skin testing and a 3D in-vitro skin model containing human dermal papilla cell culture was used for hair testing. Each model was exposed to the proteins for 24 hours at 37°C. Differential gene expressions of the skin model and dermal cells were assessed using mRNA-Seq. The results were then curated to a list of target genes with known skin and hair functions. Positive values indicate upregulation and negative values indicate downregulation.The following target genes were assessed:
[0247] Standard Skin Function Genes: ADAM17, AHR, AQP3,, ARNT, BMP4, CALML5, CAPN1, CASP14, CASP3, CAT, CD44, CDSN, CLDN1, CLDN7, COL17A1, COL1A1, COL3A1, COL4A2, COL7A1, CSF2, CTGF, CXCL8 / IL8, DCN, DEFB1, DPT, DSC1, DSG1, DSG3, EDN1, EGFR, ELN, F2RL1, FBN1, FLG, FN1, FOXO3, GBA, GPX1, GRHL3, GSK3B, HAS2, HBEGF, HMOX1,, HNRNPD, HSPG2, ICAM1, IFNA1, IGF1 R, IL10, IL1A, IL1 B, IL1 RN, IL23A, IL6, ITGB1, ITGB4, IVL, KITLG, KLK5, KLK7, KRT1, KRT10, KRT14, KRT5, LCE3D, LOR, MFN1, MFN2, MMP1, MMP10, MMP2, MMP9, MT1A, MT2A, NFE2L2, NMRK1, NQO1, OCLN, PANK4, PCNA, PKP1, POLG1 / MDP1,, PPARD,, PPARG, PTGS1, PTGS2, SERPINB3, SERPINH1, SIRT1, SMPD1, SOD1, SOD2, SPINK5, ST14, TGFB1, TGM1, TIMP1, TIMP2, TLR2, TLR3, TNC, TNF, TP63, TXN, TXNRD1, VCAN, VEGFA,
[0248] Standard Hair Function Genes: AR, ASIP, AXIN2,, BAX,, BCL2, BMP2, BMP4, BMP6,, CCND1,, CDKN2A, CORIN, CTNNB1, DCN,, DKK1, ESR1, ESR2, FGF10, FGF5, FGF7, GLI1, GSK3B,, HES1,, HEY1,, HGF,, IGF1, IL1 B, IL6, KITLG, LEF1, LEP, MC1R,, MKI67, NOG, NOTCH1,, PDGFA, PROM1, PTCH1, SFRP1, SOX2, SRD5A2,, STAT3,, STAT5A,, STAT6, TCF4, TGFB1, TGFB2, VCAN,, VEGFA, WIF1, WNT10B, WNT3A, WNT5A.
[0249] SEQ 41
[0250] SEQ41 showed a balanced inflammatory and immune response, with moderate activation of pro-inflammatory signals that support healing and hair regeneration without causing excessive inflammation. These signals help regulate dermal papilla function and maintain a steady hair growth cycle.
[0251] SEQ 42
[0252] SEQ 42 showed upregulation of skin markers related to skin repair, hydration, and barrier regeneration. These effects were evidenced by improved markers of moisture and lipid synthesis and by significant upregulation of wound-healing markers.Example 14 - UV boosting / protective properties
[0253] Formulations of the peptides at 1% (w / v) and 2% (w / v) are to be used to measure the absorbance across the UV-Vis spectrum and determine UV protective properties.
[0254] To measure UV-A, UV-B and UV-C absorbances accurately, 1:10 dilutions of the peptides were performed, making a working concentration of proteins at 0.1% (w / v) or 0.2% (w / v).
Claims
CLAIMS1. A peptide having the following formula I:[M]x-[Starter Sequence]-[SEQ ID No: 1]a-[SP1]-Link-[SP2]- Link-[SP3]-Link- [SP4]-Link-[SP5]- Link-[SP6]-[SEQ ID No: 2]bwherein:x is 0 or 1;a and b are each independently 0 or 1, but at least one of a or b must be 1; Link represents a linker sequence selected from GSGS or GSGS(A)nGSGS, where each n is an integer from 6 to 20;wherein each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 are each independently selected from SEQ ID Nos: 3 to 26;and wherein the Starter Sequence has the sequence:[Motif1]-[Motif2]wherein Motif 1 is selected from SEQ ID Nos: 33 to 38; andwherein Motif 2 is selected from: SEQ ID Nos: 27 to 30.
2. The peptide as claimed in claim 1, wherein a and b are each 1.
3. The peptide as claimed in either one of claims 1 and 2, wherein each of SP1, SP2, SP3, SP4, SP5 and SP6 independently comprise a different sequence to each other.
4. The peptide as claimed in either one of claims 1 and 2, wherein at least two of SP1, SP2, SP3, SP4, SP5 and SP6 have the same sequence.
5. The peptide as claimed in claim 4, wherein each of the SP peptides SP1, SP2, SP3, SP4, SP5 or SP6 have the same sequence.
6. The peptide as claimed in any one of claims 1 to 5 wherein the Starter Sequence has the sequence of SEQ ID No: 31.
7. The peptide as claimed in any one of claims 1 to 5 where Link represents the linker sequence GSGS(A)nGSGS wherein n is an integer from 8 to 16.
8. The peptide as claimed in claim 7, wherein n is an integer selected from 9, 10, 11 or 12.
9. The peptide as claimed in any one of claims 1 to 8, wherein said peptide has a sequence which has at least 80% sequence identity to one of the sequences of SEQ ID Nos: 41 to 44.
10. The peptide as claimed in claim 9 which has at least 90% sequence identity to one of the sequences of SEQ ID Nos: 41 to 44.
11. The peptide as claimed in any of claims 1 to 9 which includes SEQ ID No: 1.
12. The peptide as claimed in any of claims 1 to 9 which includes SEQ ID No: 2.
13. A composition comprising a peptide as claimed in any one of claims 1 to 12 in combination with a pharmaceutically acceptable carrier or excipient.
14. The composition as claimed in claim 13 which is for cosmetic use.
15. The composition as claimed in claim 14 as a topical preparation for application to skin or hair.
16. The composition as claimed in claim 15, wherein said topical preparation is an emulsion, lotion, spray, aerosol, powder, ointment cream, foam or patch.
17. The composition as claimed in any one of claims 13 to 16 which comprises at least 0.5% by weight of the peptide of claims 1 to 12.
18. The composition as claimed in any one of claims 13 to 17 which is formulated to form a film after application.
19. The composition as claimed in claim 18 wherein said film is a breathablebarrier.
20. A film comprising a peptide of any one of claims 1 to 12.
21. The peptide as claimed in any one of claims 1 to 12 for treatment of hair or skin.
22. The composition as claimed in any one of claims 13 to 19 for the treatment of hair or skin.
23. A polynucleotide which encodes a peptide as claimed in any one of claims 1 to 12.
24. A vector comprising a polynucleotide as claimed in claim 23.
25. A host cell transformed with a vector as claimed in claim 24.