AU2021248625A1

AU2021248625A1 - Polypeptides derived from the C-terminus of acetylcholinesterase for use in skin conditions

Info

Publication number: AU2021248625A1
Application number: AU2021248625A
Authority: AU
Inventors: Susan Greenfield
Original assignee: Neuro Bio Ltd
Current assignee: Neuro Bio Ltd
Priority date: 2020-04-01
Filing date: 2021-03-31
Publication date: 2022-09-29
Also published as: JP2023519935A; GB202004804D0; CN115515622A; EP4126006A1; WO2021198684A1

Abstract

The invention relates to skin, and to novel compositions, therapies and methods for treating, preventing or ameliorating a variety of skin conditions, such as skin wrinkling, discolouration, and wound treatment. The invention also extends to cosmetics and pharmaceutical compositions, and methods of using them on skin to treat various conditions. For example, the compositions can be used to treat hair loss, baldness, and as a skin whitening agent.

Description

POLYPEPTIDES DERIVED FROM THE C-TERMINUS OF ACETYLCHOLINESTERASE FOR USE IN SKIN CONDITIONS

The enzyme acetylcholinesterase (AChE) is expressed at different stages of development in various forms, all of which have the same catalytic enzymatic activity, but which have different molecular compositions. The ‘tailed’ (T-AChE - SEQ ID No: l) is expressed at synapses and the inventors have previously identified two peptides that could be cleaved from the C-terminus of T-AChE, one being a 14 amino acid long peptide referred to as “T14” (SEQ ID No: 3), within the other which is a 30 amino acid long peptide known as “T30” (SEQ ID No: 2). The AChE C-terminal peptide “T14”’ has been identified as being the salient part of the AChE molecule responsible for its range of non-hydrolytic actions.

The synthetic analogue (i.e. “T14”), and subsequently the larger, more empirically tractable , and more potent amino acid sequence in which it is embedded (i.e. “T30”) display actions comparable to those reported for ‘non-cholinergic’ AChE, whereas the inert 15 amino acid long peptide within the T30 sequence (i.e. “T15” - SEQ ID No: 4) is without effect. The T14 peptide binds to an allosteric site on the a 7 nicotinic-receptor, where, on its own, it has no effect. However, in the presence of a primary ligand, such as acetylcholine or dietary choline, T14 enhances the calcium influx induced by these primary agents. Excessive calcium can be taken up into the mitochondria where it compromises oxidative phosphorylation, and causes a leakage of electrons. Free radicals are consequently formed that then destabilize the cell membrane, and the cell then dies.

The epidermal layer of the skin is one of the few examples of a continuously renewing process in the mature adult. The cell cycle process is driven by activation of the alpha-7 receptor, (Arreondo et al. 2002 J Cell Biol. ΐ59(2):325-3ό) which is the target of T14 (Greenfield et al., 2004). The inventors, therefore, investigated the effects of various peptides derived from the C-terminus of acetylcholinesterase on a keratinocyte skin cell line, and observed that the T30 peptide (SEQ ID No: 2), which comprises the T14 sequence, surprisingly not only stimulates intracellular calcium influx into the skin cells, but also induces cell proliferation. Without wishing to be bound to any particular theory, the inventors propose that T14, and biologically active variants and fragments thereof binds to an allosteric site on the alpha 7 receptor and thereby modulates calcium entry, which in turn promotes cell growth and proliferation.

Accordingly, the inventors believe that peptides derived from the C-terminus of acetylcholinesterase will have application in various skin conditions related to skin cell renewal, including anti-aging and wound healing. The inventors hypothesise that the proliferative effect of the peptides derived from acetylcholinesterase on keratinocytes will have applicability in the treatment of skin conditions that are associated with aging. Proliferation of keratinocytes and associated attenuation of keratinocyte differentiation is acknowledged to improve skin conditions associated with aging (Gilhar et al, 2004, aged human skin exhibits decreased epidermal thickness, flattening of the dermal- epidermal junction, and decreased keratinocyte proliferation).

Keratinocyte growth factor has been shown to regulate proliferation and differentiation in epithelial tissues and may even regulate the clonogenic stem cells of the hair follicle (J. Invest Dermatol. 2000, April, 114(4): 667-730). Thus, the inventors believe that the polypeptides of the invention can also be used to treat hair loss and/ or balding.

Thus, in a first aspect of the invention, there is provided a polypeptide, or a biologically active variant or fragment thereof, derived from the C-terminus of acetylcholinesterase, for use in preventing, treating or ameliorating a skin condition related to aging.

In a second aspect of the invention, there is provided a method of preventing, treating or ameliorating a skin condition related to aging, the method comprising, administering, or having administered to a subject in need of such treatment, a therapeutically effective amount of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof.

In a third aspect of the invention, there is provided the use of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, for preventing or treating a skin condition related to aging. In a fourth aspect of the invention, there is provided a method for cosmetic treatment of skin, the method comprising applying, to skin, a polypeptide derived from the C- terminus of acetylcholinesterase, or a biologically active variant or fragment thereof. In a fifth aspect, therefore, there is provided a polypeptide, or a biologically active variant or fragment thereof, derived from the C-terminus of acetylcholinesterase, for use in preventing, treating or ameliorating hair loss or baldness.

In a sixth aspect of the invention, there is provided a method of preventing, treating or ameliorating hair loss or baldness, the method comprising, administering, or having administered to a subject in need of such treatment, a therapeutically effective amount of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof. In a seventh aspect of the invention, there is provided the use of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, for preventing or treating hair loss or baldness.

Furthermore, the inventors believe that the polypeptides of the invention may be used as a skin whitening agent.

Accordingly, in an eighth aspect, there is provided a polypeptide, or a biologically active variant or fragment thereof, derived from the C-terminus of acetylcholinesterase, for use as a skin whitening agent.

In a ninth aspect of the invention, there is provided a method of whitening skin, the method comprising, administering, or having administered to a subject in need of such treatment, a therapeutically effective amount of a polypeptide derived from the C- terminus of acetylcholinesterase, or a biologically active variant or fragment thereof.

In a tenth aspect of the invention, there is provided the use of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, for skin whitening. Acetylcholinesterase is a serine protease that hydrolyses acetylcholine, and will be well- known to the skilled person. The major form of acetylcholinesterase, which is found in the brain, is known as tailed acetylcholinesterase (T-AChE). The protein sequence of one embodiment of human tailed acetylcholinesterase (Gen Bank: AAA68151.1) is 614 amino acids in length, and is provided herein as SEQ ID No: 1, as follows:

1 mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv 61 saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn

121 relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm 181 nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv

241 gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel

301 vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg

361 vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe

421 dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy 481 eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq

541 yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn

601 qfdhyskqdr csdl

[SEQ ID No:l] It will be appreciated that the first 31 amino acid residues of SEQ ID No:i are removed while the protein is released, thereby leaving a 583 amino acid sequence. Thus, preferably acetylcholinesterase comprises or consists of an amino acid sequence substantially as set out in SEQ ID NO: 1, or a biologically active variant or fragment thereof, more preferably excluding the 31 amino acids at the N-terminal.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, comprises an amino acid sequence derived from the last 300, 200, too or 50 amino acids forming the C-terminus of acetylcholinesterase, or a truncation thereof, preferably wherein the acetylcholinesterase comprises an amino acid sequence substantially as set out in SEQ ID No:i.

The polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, preferably comprises an amino acid sequence derived from the last 50 amino acids forming the C-terminus of acetylcholinesterase, or a truncation thereof. The polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, preferably comprises an amino acid sequence derived from the last 40 amino acids forming the C- terminus of acetylcholinesterase, or a truncation thereof. The polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, preferably comprises an amino acid sequence derived from the last 30 amino acids forming the C-terminus of acetylcholinesterase, or a truncation thereof.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 3 and 50 amino acid residues, between 3 and 40 amino acid residues, between 3 and 35 amino acid residues, or between 3 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 3 and 25 amino acid residues, between 3 and 20 amino acid residues, between 3 and 15 amino acid residues, or between 3 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 4 and 50 amino acid residues, between 4 and 40 amino acid residues, between 4 and 35 amino acid residues, or between 4 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 4 and 25 amino acid residues, between 4 and 20 amino acid residues, between 4 and 15 amino acid residues, or between 4 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 5 and 50 amino acid residues, between 5 and 40 amino acid residues, between 5 and 35 amino acid residues, or between 5 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 5 and 25 amino acid residues, between 5 and 20 amino acid residues, between 5 and 15 amino acid residues, or between 5 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 6 and 50 amino acid residues, between 6 and 40 amino acid residues, between 6 and 35 amino acid residues, or between 6 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 6 and 25 amino acid residues, between 6 and 20 amino acid residues, between 6 and 15 amino acid residues, or between 6 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 7 and 50 amino acid residues, between 7 and 40 amino acid residues, between 7 and 35 amino acid residues, or between 7 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 7 and 25 amino acid residues, between 7 and 20 amino acid residues, between 7 and 15 amino acid residues, or between 7 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 8 and 50 amino acid residues, between 8 and 40 amino acid residues, between 8 and 35 amino acid residues, or between 8 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 8 and 25 amino acid residues, between 8 and 20 amino acid residues, between 8 and 15 amino acid residues, or between 8 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 9 and 50 amino acid residues, between 9 and 40 amino acid residues, between 9 and 35 amino acid residues, or between 9 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 9 and 25 amino acid residues, between 9 and 20 amino acid residues, between 9 and 15 amino acid residues, or between 9 and 10 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 10 and 50 amino acid residues, between 10 and 40 amino acid residues, between 10 and 35 amino acid residues, or between 10 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 10 and 25 amino acid residues, between 10 and 20 amino acid residues, between 10 and 15 amino acid residues, or between 10 and 12 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 11 and 50 amino acid residues, between 11 and 40 amino acid residues, between 11 and 35 amino acid residues, or between 11 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 11 and 25 amino acid residues, between 11 and 20 amino acid residues, between 11 and 15 amino acid residues, or between 11 and 13 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 12 and 50 amino acid residues, between 12 and 40 amino acid residues, between 12 and 35 amino acid residues, or between 12 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 12 and 25 amino acid residues, between 12 and 20 amino acid residues, between 12 and 15 amino acid residues, or between 12 and 14 amino acid residues.

Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 13 and 50 amino acid residues, between 13 and 40 amino acid residues, between 13 and 35 amino acid residues, or between 13 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 13 and 25 amino acid residues, between 13 and 20 amino acid residues, between 13 and 15 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 14 and 50 amino acid residues, between 14 and 40 amino acid residues, between 14 and 35 amino acid residues, or between 14 and 30 amino acid residues. Preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises between 14 and 25 amino acid residues, between 14 and 20 amino acid residues, between 14 and 17 amino acid residues. More preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof comprises between 10 and 35 amino acids. Most preferably, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof comprises between 14 and 30 amino acids.

Even more preferably, the polypeptide, or a biologically active variant or fragment thereof comprises between 3 and 12 amino acids, between 3 and 10 amino acids, between 3 and 8 amino acids. Even more preferably, the polypeptide, or a biologically active variant or fragment thereof comprises 4, 5 or 6 amino acids. Most preferably, the polypeptide, or a biologically active variant or fragment thereof comprises 4 or 6 amino acids. The amino acid sequence of T30 (which corresponds to the last 30 amino acid residues of SEQ ID No:i) is provided herein as SEQ ID No:2, as follows:-

KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

[SEQ ID No: 2]

Thus, in one embodiment, the polypeptide derived from the C-terminus of acetylcholinesterase, or biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 2, or a biologically active variant or fragment thereof.

The amino acid sequence of the T14 peptide (which corresponds to the 14 amino acid residues located towards the end of SEQ ID No:i, and lacks the final 15 amino acids found in T30) is provided herein as SEQ ID No:3, as follows:- AEFHRWSSYMVHWK

[SEQ ID NO:3]

Thus, in one embodiment, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No:3, or a biologically active variant or fragment thereof. It will be appreciated that any of the above fragment lengths will be active if they comprise T14 (SEQ ID No: 3) or a biologically active variant or fragment thereof, as shown in Figures 1 and 3, for example, which demonstrate that T30, a polypeptide derived from the C-terminus of acetylcholinesterase and comprising the T14 sequence, induces a calcium influx in keratinocytes and stimulates proliferation.

Thus, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof may have more or less amino acid residues that T14 (SEQ ID No:3), but nonetheless may comprise T14 (SEQ ID No: 3), or a biologically active variant or fragment thereof. Hence, in some embodiments, it is preferred that the polypeptides, and biologically active variants or fragments thereof, according to the invention, are derived from the C-terminus of acetylcholinesterase, and comprise or consist of T14 (SEQ ID No:3), or a biologically active variant or fragment thereof.

For example, in one embodiment, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, may be between 8 and 50 amino acid residues in length, and comprise T14 (SEQ ID No: 3), or a biologically active variant or fragment thereof. In another embodiment, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof maybe up to 50, 40 or 30 amino acids in length and comprise T14 (SEQ ID No: 3), or a biologically active variant or fragment thereof. As described in the Examples, however, in other embodiments, peptides shorter than T14 (for example, Peptides NBP-402, 403, 610, 611, 806, 807, 808 and 1012) have been shown to be surprisingly active.

Accordingly, in a preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of three amino acids.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of four amino acids. Preferably, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 5 or 6. The amino acid sequence of SEQ ID No: 5 (i.e. “NBP-402”) is: WKAE. Advantageously, this peptide is not naturally occurring and so exhibits resistance to proteases, which might otherwise degrade it when used on a subject’s skin. As such, SEQ ID No:5 is preferred.

The amino acid sequence of SEQ ID No: 6 (i.e. “NBP-403”) is: KAEF.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of five amino acids.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of six amino acids. Preferably, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 7 or 8.

The amino acid sequence of SEQ ID No: 7 (i.e. “NBP-610”) is: SSYMVH.

The amino acid sequence of SEQ ID No: 8 (i.e. “NBP-611”) is: SYMVHW.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of seven amino acids.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of eight amino acids. Preferably, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 9, 10 or 11.

The amino acid sequence of SEQ ID No: 9 (i.e. “NBP-806”) is: FHRWSSYM.

The amino acid sequence of SEQ ID No: 10 (i.e. “NBP-807”) is: HRWSSYMV.

The amino acid sequence of SEQ ID No: 11 (i.e. “NBP-808”) is: RWSSYMVH.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of nine amino acids.

In one preferred embodiment, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of ten amino acids. Preferably, the polypeptide, or a biologically active variant or fragment thereof comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 12.

The amino acid sequence of SEQ ID No: 12 (i.e. “NBP-1012”) is: YMVHWKAEFH. Advantageously, this peptide is also not naturally occurring and so exhibits resistance to proteases, which might otherwise degrade it when used on a subject’s skin. As such, SEQ ID No:i2 is preferred.

As described in the examples, these linear peptides bind to an allosteric site on the alpha 7 receptor and thereby modulate calcium ion entry, which in turn promotes cell growth and proliferation. Furthermore, these peptides do not negatively affect cell viability.

The amino acid sequence of T15 (which corresponds to the last 15 amino acid residues of SEQ ID No:i) is provided herein as SEQ ID No:4, as follows: -

NQFDHYSKQDRCSDL

[SEQ ID NO:4] Thus, in one embodiment, the polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, does not consist of an amino acid sequence substantially as set out in SEQ ID No: 4.

The polypeptide, variant or fragment thereof defined herein may be cyclized. Cyclic polypeptides are peptide chains whose N- and C-termini are themselves linked together with a peptide bond that forms a circular chain of amino acids. For example, the polypeptide derived from the C-terminus of acetylcholinesterase may be a cyclized form of T14 (SEQ ID No: 3)· It will be appreciated that any of the polypeptides or peptides described herein may be synthesised de novo using standard peptide synthesis methods commonly known to the skilled person, and, as such, may then be used in any of the cosmetic/therapeutic applications described herein. Accordingly, any of the peptides can be produced by forming a peptide bond between adjacent amino acids of the sequences provided herein to build up to the full sequence length, i.e. the first amino acid is provided, to which a second amino acid is attached, and so on up to the desired length of peptide. Therefore, it is not necessary to start with the full acetyl cholinesterase sequence, or a shorter truncation thereof, and reduce the length of the polypeptide by removing amino acids from the N- and/or C- terminal until the desired peptide length is reached. Indeed, for the sake of speed, convenience and cost, it is preferred that the polypeptide, or a biologically active variant or fragment thereof, is created using a de novo peptide synthesis method.

The skin condition related to aging may be selected from a group of conditions consisting of: fine lines; wrinkles; discoloration; uneven pigmentation; sagging; enlarged pores; rough skin; dry skin; stretch marks; uneven tone; blemishes; skin thickening or thinning; and any combination thereof.

Preferably, the polypeptides of the invention are for slowing down signs of aging. Preferably, the skin condition related to aging is wrinkling or wrinkle formation. Thus, slowing down the signs of aging may mean preventing, retarding, arresting, or reversing the process of wrinkle formation in mammalian skin.

It will be appreciated, therefore, that the peptides described herein reduce the “effects of aging” rather than “age” per se. In other words, administration of the peptides reduces the apparent age of the subject, and can make skin look younger than it actually is.

Currently, detection of DNA methylation (or epigenetics) is used as a read-out of skin aging (Clinical Epigenetics, 12, 105 (2020)), and retinol (also known as vitamin A - alcohol) is used as the “gold standard” benchmark against which other anti-aging skin treatment products are compared. However, there is clearly a need to provide improved methods by which the appearance of skin age can be improved.

The term "wrinkle", as used herein, can refer to a fold or crease in the skin. Wrinkle formation may be assessed by measuring wrinkle length.

The severity of wrinkle formation may be determined using a wrinkle severity scale, for example the Wrinkle Severity Rating Scale (WSRS) and the Glogau classification. Wrinkles can vary in size and intensity, from fine lines to deep furrows. Wrinkles in skin maybe classified into three different types: dynamic wrinkles, static wrinkles and wrinkle folds. Dynamic wrinkles are caused by repeated contractions of muscles underlying the skin. For example, frowning or furrowing causes wrinkles between the eyebrows (i.e., glabellar lines), while smiling and/or squinting causes wrinkles at the distal corners of the eyes (i.e., lateral canthal lines). Static wrinkles, or wrinkles at rest, when the face is in a neutral or natural position, result from a loss of elasticity in skin, which may arise from a variety of factors, including sun damage, poor nutrition, smoking, and genetic factors, or from spasms or tones of muscles. Wrinkle folds, which may appear as deep grooves between the nose and mouth, for example, arise from the sagging of underlying facial structure.

The polypeptides of the invention may also be used as a whitening agent.

As described in the examples, the inventors performed assays of intracellular calcium influx and cell proliferation assays on the keratinocyte cell line, HaCaTs, a widely used and characterized model for human keratinocytes. The inventors have surprisingly shown that that the linear peptide, T30, is able to induce an intracellular calcium influx in HaCaTs, which correlates with stimulation of cell proliferation. The inventors have also shown that an inert component of T30, “T15” does not induce intracellular calcium influx and does not stimulate cell proliferation, suggesting that the active component of T30 is T14.

The inventors also believe that, in addition to being effective in preventing, treating or ameliorating a skin condition related to aging, such as wrinkles etc., the polypeptides of the invention may also be used to treat wounds.

Thus, in a eleventh aspect of the invention, there is provided a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, for use in treating a wound. In a twelfth aspect of the invention, there is provided a method of treating wound, the method comprising, administering, or having administered to a subject in need of such treatment, a therapeutically effective amount of a polypeptide derived from the C- terminus of acetylcholinesterase, or a biologically active variant or fragment thereof. The polypeptide or a biologically active variant or fragment thereof may be as defined in the first aspect. “Wound treatment” according to the invention preferably comprises re-epithelisation of epithelial tissue. Re-epithelialization of a wound requires migration and proliferation of keratinocytes. Advantageously, the polypeptides of the invention stimulate keratinocyte proliferation and thus aid in the proliferative phase of wound healing.

Re-epithelialisation is defined as the restoration of an intact epithelium through migration of epithelial cells to close a wound. Epithelia coat all surfaces of the body, both inside and out. Therefore, the treatment comprises re-epithelisation and can be used in any epithelial wound, i.e. internal or external of the body.

Preferably, the rate of wound healing is increased.

Rate of wound healing Preferably, the polypeptides of the invention increase the rate of wound healing. The rate of wound healing may relate to the absolute area healed per day, percentage of initial area healed per day and advance of the wound margin towards the wound centre per day, time to complete wound closure, or any other method known in the art, including those described herein. An increase in the rate of wound healing refers to that achieved compared to the level of healing occurring on healing of a control-treated or untreated wound.

Wound sites

The wound treated with polypeptides of the invention may be present at any body site, and in any tissue or organ, where a wound may occur. The skin represents the preferred site at which the rate of wound healing is increased. The inventors believe that the polypeptides of the present invention may beneficially increase wound healing in all types of epithelial wounds. Examples of specific wounds in which the effects of the invention may be seen include wounds selected from the group consisting of wounds of the skin (such as burns, incision wounds, pressure ulcers), the lungs, the eye (including the inhibition of scarring resulting from eye surgery such as LASIK surgery, LASER surgery, PRK surgery, glaucoma filtration surgeiy, cataract surgery, or surgery in which the lens capsule may be subject to scarring) such as those giving rise to corneal cicatrisation; wounds subject to capsular contraction (which is common surrounding breast implants); wounds of the oral cavity, including the lips and palate (for example, to inhibit scarring resulting from treatment of cleft lip or palate or to promote closure or oral ulcers); wounds of the internal organs such as the digestive tissues and reproductive tissues; wounds of body cavities such as the abdominal cavity, pelvic cavity and thoracic cavity (where inhibition of scarring may reduce the number of incidences of adhesion formation and/or the size of adhesions formed); and surgical wounds (in particular wounds associated with cosmetic procedures, such as scar revision or isolation of strip grafts for hair transplant surgery). It is particularly preferred that the polypeptides of the present invention be used to increase the rate of re-epithelisation, wound healing and/or prevent, reduce or inhibit scarring associated with wounds of the skin.

Incisional wounds may be a preferred group of wounds which may be treated with polypeptides of the invention. Surgical incisional wounds may constitute a particularly preferred group of wounds in respect of which wound healing may be increased, utilising the medicaments and methods of the invention.

Polypeptides of the present invention may be used to heal wounds associated with plastic or cosmetic surgery. Since a large number of plastic or cosmetic surgeries consist of elective surgical procedures it is readily possible to administer a polypeptide of the present invention, prior to surgery, and/ or around the time of closure of the wound (for instance, before or after the application of sutures), and this use represents a particularly preferred embodiment of the invention.

In surgical procedures in general, a preferred route by which a polypeptide of the present invention maybe administered is via localised injection (such as intradermal injection). Such injections may form raised blebs, which may then be incised as part of the surgical procedure, or alternatively the bleb maybe raised by injecting the wound margins after the wound has been closed e.g. by sutures. Alternatively, the polypeptide may be administered in a cream formulation or in a bandage, or may be coated on the sutures used for incision closure.

Scar revisions are surgical procedures in which existing scars are “revised” (for example through excision or realignment) in order to reduce the cosmetic and/ or mechanical disruption caused by the existing scar. Probably the best known of these is "Z-plasty" in which two V-shaped flaps of skin are transposed to allow rotation of a line of tension. The use of the polypeptides of the invention in procedures associated with scar revision represents a preferred use in accordance with the present invention. It is recognised that wounds resulting from burns injuries (which for the purposes of the present invention may be taken to encompass exposure to heated gasses or solids, as well as scalding injuries involving hot liquids; "freezer burn" injuries caused by exposure to extreme low temperatures; radiation burns; and chemical burns, such as those caused by caustic agents) may extend over great areas of an individual so afflicted. Accordingly, burns may give rise to scar formation covering a large proportion of a patient's body. This great extent of coverage increases the risk that the scar formed will cover areas of elevated cosmetic importance (such as the face, neck, arms or hands) or of mechanical importance (particularly the regions covering or surrounding joints). Burns injuries caused by hot liquids are frequently suffered by children (for example as a result of upsetting pans, kettles or the like) and, due to the relatively smaller body size of children, are particularly likely to cause extensive damage over a high proportion of the body area. Thus there is an elevated risk of both cosmetic and mechanical impairment associated with scarring after burns. After large burns, skin grafts are used as a treatment. This invention can be used in combination with a skin graft, to promote migration of epithelial cells from the graft to the uncovered wound, to quickly establish a barrier in non-grafted areas of skin. The inventors have demonstrated that a number linear peptides of differing lengths, as described herein, have a range of therapeutic and cosmetic applications, relating to the skin or keratinocytes. These peptides can be synthesised and applied exogenously to the skin (or a hair follicle) to exert their cosmetic or therapeutic effects. Thus, in a thirteenth aspect of the invention, there is provided one or more peptide, derivative or analogue thereof comprising or consisting of an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12, for use in preventing, treating or ameliorating: -

(i) a skin condition related to aging; (ii) a wound;

(iii) hair loss or baldness; or

(iv) as a skin whitening agent.

In a fourteenth aspect of the invention, there is provided a method of preventing, treating or ameliorating:-

(i) a skin condition related to aging; (ii) a wound;

(iii) hair loss or baldness; or

(iv) as a skin whitening agent, the method comprising, administering, or having administered to a subject in need of such treatment, a therapeutically effective amount of one or more peptide, derivative or analogue thereof comprising or consisting of an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12.

In a fifteenth aspect of the invention, there is provided the use of one or more peptide, derivative or analogue thereof comprising or consisting of an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12, for use in preventing, treating or ameliorating: -

(i) a skin condition related to aging;

(ii) a wound; (iii) hair loss or baldness; or as a skin whitening agent.

In a sixteenth aspect of the invention, there is provided a method for cosmetic treatment of skin, the method comprising applying, to skin, one or more peptide, derivative or analogue thereof comprising or consisting of an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12.

The term “derivative or analogue thereof’ can mean a peptide within which amino acid residues are replaced by residues (whether natural amino acids, non-natural amino acids or amino acid mimics) with similar side chains or peptide backbone properties. Additionally, the terminals of such peptides may be protected by N- and/or C-terminal protecting groups with similar properties to acetyl or amide groups.

Derivatives and analogues of peptides according to the invention may also include those that increase the peptide’s half-life in vivo. For example, a derivative or analogue of the peptides of the invention may include peptoid and retropeptoid derivatives of the peptides, peptide-peptoid hybrids and D-amino acid derivatives of the peptides.

Peptoids, or poly-N-substituted glycines, are a class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the alpha-carbons, as they are in amino acids. Peptoid derivatives of the peptides of the invention may be readily designed from knowledge of the structure of the peptide. Retropeptoids (in which all amino acids are replaced by peptoid residues in reversed order) are also suitable derivatives in accordance with the invention. A retropeptoid is expected to bind in the opposite direction in the ligand-binding groove, as compared to a peptide or peptoid-peptide hybrid containing one peptoid residue. As a result, the side chains of the peptoid residues are able to point in the same direction as the side chains in the original peptide.

The term “derived from” can mean an amino acid sequence which is a derivative or a modification of an amino acid sequence that is present in, or forms, the C-terminus of AChE, and portions thereof.

The term “truncation thereof’ can mean the peptide derived from AChE is reduced in size by the removal of amino acids. The reduction of amino acids may be achieved by removal of residues from the C- and/ or N-terminal of the peptide, or may be achieved by deletion of one or more amino acids from within the core of the peptide.

It will be appreciated that the polypeptides according to the invention may be used in a medicament and/or a cosmetic, which may be used as a monotherapy (i.e. use of the polypeptides according to the invention), for treating a wound, in particular increasing the rate of wound healing and/or treating, preventing or ameliorating a skin condition related to aging, hair loss, balding or as a skin whitening agent. Alternatively, the polypeptides according to the invention may be used as an adjunct to, or in combination with, known therapies for treating a wound, in particular increasing the rate of wound healing and/or preventing, treating or ameliorating a skin condition related to aging, hair loss, balding or as a skin whitening agent.

The polypeptide according to the invention maybe combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments or cosmetics according to the invention should be one which is well -tolerated by the subject to whom it is given and preferably enables delivery of the polypeptide to the skin. The polypeptides according to the invention may also be incorporated within a slow- or delayed-release device, such as a layer-by-layer assembled bandage. Such devices may, for example, be inserted on or under the skin, and the medicament or cosmetic may be released over weeks or even months. The device may be located at least adjacent to the treatment site. Such devices maybe particularly advantageous when long-term treatment with the polypeptide is required and which would normally require frequent administration (e.g. at least daily injection). In a preferred embodiment, medicaments or cosmetics according to the invention may be administered topically to the skin, preferably directly at a site requiring treatment.

It will be appreciated that the amount of the polypeptide that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the polypeptide and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the polypeptide (linear or cyclic) within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular polypeptide in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement or stage of the disorder. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration. Generally, a daily dose of between o.ooipg/kg of body weight and lomg/kg of body weight, or between o.oipg/kg of body weight and lmg/kg ofbody weight, of the polypeptide according to the invention may be used for treating a wound or skin condition associated with ageing, depending upon the polypeptide used. The polypeptides may be administered before, during or after onset of the injury causing the wound. Daily doses maybe given as a single administration (e.g. a topical cream or spray). Alternatively, the polypeptide may require administration twice or more times during a day. As an example, the polypeptide may be administered as two (or more depending upon the severity of the disorder being treated) daily doses of between 0.07 pg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may administer a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of the polypeptide according to the invention to a patient without the need to administer repeated doses. Known procedures, such as those conventionally employed by the pharmaceutical or cosmetic industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the polypeptide according to the invention and precise therapeutic or cosmetic regimes (such as daily doses of the agents and the frequency of administration).

The inventors realise, therefore, that the novel pharmaceutical compositions comprising the polypeptides of the invention may be produced.

Thus, in a seventeenth aspect of the invention, there is provided a skin condition treatment pharmaceutical composition comprising a therapeutically effective amount of the polypeptide, or biologically active variant or fragment thereof, according to the first or thirteenth aspect, and optionally a pharmaceutically acceptable vehicle.

The invention also provides in an eighteenth aspect, a process for making the skin condition treatment composition according to the seventeenth aspect, the process comprising combining a therapeutically effective amount of the polypeptide, or a biologically active variant or fragment thereof, according to the first or thirteenth aspect, with a pharmaceutically acceptable vehicle. The pharmaceutical composition is preferably used for preventing, treating or ameliorating a skin condition related to aging, or for treating a wound.

Preferably, the pharmaceutical composition comprises one or more peptide having an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12, or a derivative or analogue thereof.

However, the inventors also appreciate that the novel cosmetic compositions comprising the polypeptides of the invention may be produced. Hence, in a ninteenth aspect of the invention, there is provided a cosmetic composition comprising an effective amount of the polypeptide, or a biologically active variant or fragment thereof, according to the first or thirteenth aspect, and optionally a cosmetically acceptable vehicle.

The invention also provides in a twenteeith aspect, a process for making cosmetic composition according to the ninteenth aspect, the process comprising combining a therapeutically effective amount of the polypeptide, or biologically active variant or fragment thereof, to the first or thirteenth aspect, with a cosmetically acceptable vehicle. Preferably, the cosmetic composition comprises one or more peptide having an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12, or a derivative or analogue thereof.

In some embodiments, the composition described herein may be a suntan cream, oil or lotion.

In other embodiments, the composition may be a moisturiser or moisturising formulation. In other embodiments, the composition may be an anti-aging or anti-wrinkle formulation.

In other embodiments, the composition maybe hair growth stimulating or promoting formulation.

In other embodiments, the composition may be a skin whitening formulation.

The cosmetic composition is preferably used for preventing, treating or ameliorating: - (i) a skin condition related to aging; (ii) a wound;

(iii) hair loss or baldness; or

(iv) as a skin whitening agent.

The skin condition related to aging may be selected from a group of conditions consisting of: fine lines; wrinkles; discoloration; uneven pigmentation; sagging; enlarged pores; rough skin; dry skin; stretch marks; uneven tone; blemishes; skin thickening or thinning; and any combination thereof. Preferably, the cosmetic composition slows down signs of aging, such as reducing wrinkling or wrinkle formation. A “subject” may be a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary applications. Most preferably, however, the subject is a human being. A “therapeutically effective amount” of the polypeptide, the cosmetic composition or pharmaceutical composition is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to treat a wound or to treat a skin condition associated with ageing. For example, the therapeutically effective amount of the polypeptide, the cosmetic composition or the pharmaceutical composition used may be from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 mg. It is preferred that the amount of the polypeptide, the cosmetic composition or the pharmaceutical composition is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.

A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

A “cosmetically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating cosmetic compositions. In one embodiment, the cosmetically or pharmaceutically acceptable vehicle may be a solid, and the composition maybe in the form of a powder or tablet. In another embodiment, the cosmetic or pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like. However, the cosmetic or pharmaceutical vehicle may be a liquid, and the cosmetic or pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The polypeptide according to the invention may be dissolved or suspended in a cosmetically or pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both, or cosmetically or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable cosmetic or pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other cosmetically or pharmaceutically acceptable propellant.

The polypeptides, the cosmetic composition and the pharmaceutical composition of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The polypeptide, the cosmetic composition or the pharmaceutical composition according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. In the situation in which it is desired to administer a polypeptide of the present invention by means of oral ingestion, it will be appreciated that the chosen agonist will preferably be one having an elevated degree of resistance to degradation. For example, the chosen agonist maybe protected (using the techniques well known to those skilled in the art) so that its rate of degradation in the digestive tract is reduced. Preferably, the polypeptide of the invention maybe administered topically in the form of a cream, gel, lotion, ointment, cutaneous solution, suspension, spray, foam, bath additive, collodion, impregnated dressing or medicated plaster. The cream may be either oil-in-water or water-in-oil type. The polypeptide and compositions of the invention may be administered topically with emulgators such as alkyl sulphates, alkyl amines, alkyl pyrimidin compounds, etc. Acceptable oils for cream formulation include: white petrolatum, paraffin, cetearyl alcohol, cocoglycerides, cetyl alcohol, isopropyl miristate, cetyl palmitate, butyrum cacao, oleum helianthi, cera alba, lanolin, isopropyl palmitate, stearic acid, magnesium stearate. For preparation of a gel, the following gel forming additives maybe used: cellulose gum (carboxymethyl cellulose), hydroxypropyl cellulose, methylcellulose, hydroxyethyl cellulose, ethylhydroxy cellulose, or laponite.

Preferably, the polypeptide of the invention may be administered topically with preserving agents, antioxidants, complexing agents, solvents, fragrances, bactericides, odor absorbers, vitamins, moisturizers, self-tanning compounds and anti-wrinkle active agents.

The compositions of the invention may contain cosmetically acceptable additives or adjuvants as well as cosmetic or dermatologic active agents. Representative additives and adjuvants include, for example, oil-soluble or oil-miscible solvents or co-solvents. Suitable examples of additives and adjuvants include, but are not limited to, fatty alcohols, fatty amides, alkylene carbonates, glycols, lower alcohols (e.g. ethanol, propanediol), dispersion enhancing agents, polymers, thickening agents, stabilizers, moisturizers, humectants, colorants, fillers, chelating agents, antioxidants (e.g. BHT, tocopherol), essential oils, fragrances, dyes, neutralizing or pH-adjusting agents (e.g., citric acid, triethylamine (TEA) and sodium hydroxide), preservatives, bactericides, conditioning or softening agents (e.g., panthenol and allantoin), extracts, such as botanical extracts, or any other ingredient commonly used in cosmetics for this type of application. Additives and adjuvants maybe present in the compositions in amounts generally ranging from about 0.01% to about 10%, by weight. Examples of cosmetic active agents or dermatological active agents include free-radical scavengers, vitamins (e.g., Vitamin E and derivatives thereof), anti-elastase and anti-collagenase agents, peptides, fatty acid derivatives, steroids, trace elements, extracts of algae and of planktons, enzymes and coenzymes, flavonoids and ceramides, hydroxy acids and mixtures thereof, and enhancing agents. These ingredients may be soluble or dispersible in oil phase(s) that is/are present in the composition. Medicaments and compositions comprising a polypeptide of the present invention that are for use in treating wounds in the lungs or other respiratory tissues may be formulated for inhalation.

Any suitable route capable of achieving the desired effect of the invention can be used to administer a therapeutically effective amount of a polypeptide of the present invention. However, it may generally be preferred that the polypeptide of the invention is provided to a tissue by local administration.

Suitable methods by which such local administration maybe achieved will depend on the identity of the tissue or organ in question. The selection of preferred routes of administration may also depend on whether or not a tissue or organ to be treated is permeable to the chosen medicament or cosmetic. Suitable routes of administration may be selected from the group consisting of: injections; application of sprays, ointments, gels or creams; inhalation of medicaments; release from biomaterials or other solid medicaments or cosmetics, including sutures or wound dressings.

Suitable delivery systems may include particulate systems, scaffolds or hydrogels. Particulate particles include micro particles or nanoparticles. Such particulate particles may be lipid based or polymer based. Preferably, polymer based particles are biodegradable. Scaffolds may include those biomaterials derived from native ECM, such as HA, collagen, and chitosan. Scaffolds may also comprise biomimetic materials fabricated to mimic ECM, including micro/nanofibers scaffolds produced by electrospinning. The polypeptides of the invention may preferably be provided in the form of one of more dosage units providing a therapeutically effective amount (or a known fraction or multiple of a therapeutically effective amount) of polypeptides of the invention. Methods of preparing such dosage units will be well known to the skilled person; for example see Remington's Pharmaceutical Sciences 18th Ed. (1990).

Suitable polypeptides maybe provided on a sterile dressing or patch, which maybe used to cover a wound where a wound is to be treated.

A polypeptide of the invention may be released from a device or implant, or may be used to coat such a device, e.g. a stent, or a controlled release device, or a wound dressing, or sutures for use in wound closure. It will be appreciated that the vehicle of a composition comprising a polypeptide of the invention should be one that is well tolerated by the patient and allows release of the polypeptide to the wound to be treated. Such a vehicle is preferably relatively "mild", i.e. non-inflammatory, biodegradeable, bioresolveable, or bioresorbable.

A dose of a composition comprising a polypeptide of the present invention may preferably be sufficient to provide a therapeutically or cosmetically effective amount of a suitable agonist in a single administration. However, it will be appreciated that each dose need not in itself provide a therapeutically or cosmetically effective amount of a polypeptide of the present invention, but that a therapeutically or cosmetically effective amount may instead be built up through repeated administration of suitable doses.

In a further embodiment, polypeptide of the invention maybe formulated as a part of a pharmaceutically or cosmetically acceptable trans-epidermal delivery system, e.g. a patch/dressing. A solid vehicle can include one or more substances that may also act as flavouring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also comprise an encapsulating material.

Medicaments in accordance with the invention for use in treating wounds in the body cavities, e.g. abdomen or pelvis, maybe formulated as an irrigation fluid, lavage, gel or instillate. Polypeptides for use in the medicaments or cosmetics or methods of the invention may be incorporated in a biomaterial, from which it may be released to treat a wound, in particular. Biomaterials incorporating polypeptide of the present invention are suitable for use in many contexts, and at many body sites but may be of particular utility in providing a suitable polypeptides of the invention to the eye (for example after retina surgery or glaucoma filtration surgery), or to sites where it is wished to inhibit restenosis or adhesions.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms “substantially the amino acid/nucleotide/peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID NO:I-12.

Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/ polynucleotide/ polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.

The skilled technician will appreciate howto calculate the percentage identity between two amino acid/polynucleoti de/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleoti de/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et ah, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW maybe as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty =

10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino acid/polynucleoti de/polypeptide sequences may then be calculated from such an alignment as (N /T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:- Sequence Identity = (N/T)^*ioo.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence, which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/0.1% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or too amino acids from the sequences shown in SEQ ID No: 1-12.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.

Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which: - Figure 1 shows the ability of the linear peptides T30 and T15 to induce intracellular calcium influx into a keratinocyte cell line under low calcium conditions.

Figure 2 shows the ability of the linear peptides T30 and T15 to induce intracellular calcium influx into a keratinocyte cell line under high calcium conditions.

Figure 3 shows A) the degree of cell viability and B) cell proliferation of a keratinocyte cell line treated with the linear peptides T30 and T15 under low calcium conditions.

Figure 4 shows A) the degree of cell viability and B) cell proliferation of a keratinocyte cell line treated with the linear peptides T30 and T15 under high calcium conditions. Figure 5 shows the results of experiments using further embodiments of the linear peptide according to the invention of different lengths (referred to herein as Peptide 402, 403, 610, 611, 806, 807, 808 and 1012) compared to T30/NBP-14 (control) and their ability to influence calcium ion influx into PC12 cells (“Calcium”), acetylcholinesterase activity (“AChE”) and cell “Viability”. The right hand column shows the value of “Calcium” + “AChE” / “Viability”.

Figure 6 shows the effects of T14, T30, 402 and 611 linear peptides on calcium influx of PC12 cells. Top panels: Dose response of T14 (left) and T30 (right) effects at 0.5, 1, 2.5 and 5mM. Bottom panels: Dose response of 402 (left) and 611 (right) effects at 10,

50, too and 5qqmM. All treatments were analysed in the same manner with Baseline subtraction and normalisation to their respective controls. The first time point (2 sec) of acetylcholine (Ach) injection was excluded from normalisation due to possible injections artefacts and/or acute side effects of Ach (too mM).

Figure 7 shows a comparison of the effects on calcium ion influx from T30 and linear peptide 402.

Figure 8 shows the effects of linear peptides 402 and 611 on calcium influx of PC12 cells. Left panel: Time course of calcium influx since acetylcholine (Ach) injection. Values were expressed as a percentage (mean ± SEM) of untreated control cells after baseline subtraction. The baseline before Ach injection was averaged between the values from o (start of the recording) to 1.8 seconds. Right panel: Peak of calcium influx expressed as control percentage (mean ± SEM). The peak was determined for each n of “column of cells” (consisting of 6 replicates/wells per column) within the first second following Ach injection (2.2-3. o sec). Statistical analysis was performed on the peaks. One-sample t tests (two tailed P values) was used to compare mean peak calcium influx against the value of too (which indicates relative calcium influx of untreated cells). Significant differences were indicated by one small triangle (P<0.05) and two squares (P<o.oi) above the histograms. The star (*) showed significant difference between peptides 402 and 611 after an unpaired t test.

Figure 9 shows the effects of AchE-peptides (T14 and T30; left panel) and shorter peptides (402 and 611; right panel) on the concentration-response curve of calcium influx peaks from nicotinic acetylcholine receptor (nAChR) in PC12 cells. Peak of calcium influx were expressed as control percentage as in Figure 8. The peak was determined within the first second following Ach injection (2.2-3. o sec).

Figure 10 shows the results of acetylcholinesterase (AChE) release in the extracellular matrix as a compensatory effect of the extant cells.

Examples

Rationale The inventors have generated a number of linear peptides based on the C-terminus of acetylcholinesterase, known as T14, T15 and T30 peptides, and evaluated their effects in a keratinocyte cell line. The inventors have produced a series of other, shorter linear peptides derived from acetylcholinesterase, known as Peptide: 402, 403, 610, 611, 806, 807, 808 and 1012, and also a cyclised version of linear T14 (known as cyclic NBP-14), and these peptides were then tested in a series of assays, including calcium ion influx, acetylcholinesterase activity and cell viability. The experiments show how the linear peptides can be used in a variety of skin-related applications, as skin wrinkling, discolouration, wound treatment, to treat hair loss, baldness, and as a skin whitening agent.

Example 1 - T14. NBP-14. and T20.

The ‘tailed’ acetylcholinesterase (T-AChE) is expressed at synapses and the inventors have previously identified two peptides that could be cleaved from its C-terminus, one referred to as “T14” (14 amino acids long), within the other which is known as “T30” (30 amino acids long). The amino acid sequence of the linear peptide, T14, is

AEFHRWSSYMVHWK [SEQ ID No:3]. This peptide has also been cyclised via the terminal alanine (A) and lysine (K) residues to form NBP-14. Cyclisation can be achieved by several different means. For example, Genosphere Biotechnologies (France) performed the cyclisation of T14 by transforming the linear peptide into an N- terminal to C-terminal lactam. Cyclisation of T14 to create cyclic NBP-14 brings together both ends, i.e. HWK-AEF.

The amino acid sequence of the linear peptide, T30, is

KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL [SEQ ID No:2] T30 is endogenous, and the active part of T30 is T14, both of which are found in the C-terminus of AChE. Another peptide referred to as “T15” corresponds to the last 15 amino acid residues of SEQ ID No:i, i.e. NQFDHYSKQDRCSDL [SEQ ID No: 4].

The AChE C-terminal peptide “T14”’ has been identified as being the salient part of the AChE molecule responsible for its range of non-hydrolytic actions. The synthetic 14 amino acids peptide analogue (i.e. “T14”), and subsequently the larger, more stable, and more potent amino acid sequence in which it is embedded (i.e. “T30”) display actions comparable to those reported for ‘non-cholinergic’ AChE. Example 2 - The effect of acetylcholinesterase-derived peptides on intracellular calcium in the keratinocvte HaCaTs cell line

The inventors examined the ability of the acetylcholinesterase-derived peptides, T30 and T15, to induce intracellular calcium influx into the keratinocyte (HaCaTs) cell line. As shown in Figure 1, the application of T30 surprisingly induced an increase in intracellular calcium in the keratinocyte cell line, while application of the inert T15 peptide did not induce a raise of intracellular calcium in the cell line. T30 is used experimentally as a substitute for endogenously produced T14, primarily because T30 is more stable than T14 in solution, which has a tendency to aggregate. However, as mentioned above, T30 contains the active T14.

Example 3 - The effect of acetylcholinesterase-derived peptides on proliferation of the keratinocvte HaCaTs cell line

The inventors next examined the ability of the acetylcholinesterase-derived peptides, T30 and T15, to induce proliferation of the HaCaT cell line and also tested cell viability to determine cell toxicity of T15 and T30 to the HaCaT cell line. The results are shown in Figures 3 and 4. Referring to Figures 3B and 4B, T30 significantly induced proliferation of HaCaT cells when compared to untreated controls (no peptide). In contrast, T15 did not alter cell proliferation. Neither T15 nor T30 induced significant cytotoxic effects in the cell line, as shown in Figures 3A and 4A.

Conclusions

Keratinocytes share much in common with key brain cells, including embryological provenance and high levels of free T14 (as indicated by dominance of the AChE monomer) and, as brain cells decline with age, it follows that a similar trend will occur in the epidermis, i.e. T14 levels will decline with age. If, as shown here, T14 and polypeptides (such as T30) comprising T14 or biologically active variants or fragments thereof enhance keratinocyte proliferation, then addition of T14 would therefore supplement the dwindling endogenous levels and restore a rate of cell proliferation commensurate with younger age, thus providing a new and improve anti-aging treatment.

Example 4 - The effect of other acetylcholinesterase-derived peptides on calcium influx, acetylcholinesterase and cell viability

The inventors prepared a series of linear peptides which are all smaller than T14, T15 and T30. These were 4-mers, 6-mers, 8-mers and 10-mers and are known as Neuro-Bio Peptide (NBP): 402, 403, 610, 611, 806, 807, 808 and 1012, as summarised in Table 1 below.

Table 1 - Linear peptides and their sequence

Each of these linear peptides were tested for their ability to influence calcium ion influx into PC12 cells (“Calcium”), acetylcholinesterase activity (“AChE”) and cell “Viability”, and the initial results are shown in Figure 5. The three values for these tests were introduced into the following formula:

Value score = (Calcium + AchE) / Viability

As shown in Figure 5, T30 (reflecting T14) has a combined score of 171.05 (Calcium) + 169.46 (AChE) / 74.31 (Viability), which gives a Value score of: 4.58. The inventors also investigated the effects of a cyclic NBP-14 on a keratinocyte cell line and found that it inhibits the T30-induced intracellular calcium influx into keratinocytes. Accordingly, when T30 is combined with cyclic NBP14 (at 0.5 uM), it gave a Value score of only 1.93 due to the inhibitor effect of NBP-14.

Each of Peptide 402, 403, 610, 611, 806, 807, 808 and 1012 were then assessed and, as can be seen in Figure 5, these candidate peptides all perform in a comparable way to T30 (i.e. T14) with Score values all significantly higher than the T30/NBP14 control sample. Surprisingly, when all three parameters are taken into consideration, Peptides 402, 611 and 1012 behave very similar to T30, with Value scores of 4.32, 4.39 and 1012, respectively.

However, when only calcium influx is taken into consideration, Peptide 402 and 1012 behave better than T30, with Calcium values of 212 and 179, respectively, with Peptide 402 significantly outperforming the benchmark of T30.

The inventors also note that peptides 402 and 1012 are not naturally occurring in T14. Thus, they should exhibit resistance to proteases, which might otherwise degrade them when used in vivo on a subject’s skin.

Example 5 - The effect of linear T14. T20. 402 and 611 linear peptides on calcium influx of PC12 cells

Calcium fluorometry PC12 cells were plated in 96 well plates with 0.1 ml (per well: 40000-80000 cells) of complete medium (DMEM including lomM Hepes, 2 mM glutamine, 10% Heat inactivated Horse serum, 5% Foetal bovine serum, and 0.25% Penicillin/Streptomycin) at 37°C and 5% CO2 the day before the experiment. On the day of the experiment, the Fluo-8 solution (Abeam) was prepared as described by the manufacturer by adding 20 mΐ of Fluo-8 in the assay buffer that contains 9 ml of Hank's Balanced Salt Solution

(HBSS) and 1 ml of pluronic F127 Plus. Subsequently, 0.100 ml of growth medium was removed and 70 mΐ of Fluo-8 solution were added. Treatments with the peptides 402, 611, T14 or T30, were added within the Fluo-8 at different concentrations (402 and 611: 10, 50, 100 and 5qqmM; T14 and T30: 0.5, 1, 2.5 and 5mM) and incubated for 30 min at 37°C (and 5% CO2) and 30 min at room temperature. After 1 h, the plate was placed in the fluorescence plate reader (Fluostar, Optima, BMG Labtech, Ortenberg, Germany). Before reading the fluorescence, acetylcholine (ACh) 100 mM, an agonist of the nicotinic receptors, was prepared and placed in the Fluostar injector. For each well, the reading was formed by a basal fluorescence reading followed by acetylcholine injection that induced an increase of calcium via nicotinic receptors.

Results

Referring to Figure 6, there are shown the effects of T14, T30, 402 and 611 peptides on calcium influx in PC12 cells. The top panels illustrate the dose response of T14 (left) and T30 (right) effects at 0.5, 1, 2.5 and 5mM, and the bottom panels show the dose response of 402 (left) and 611 (right) effects at 10, 50, 100 and 5qqmM.

Referring to Figure 6, there are shown dose responses for T14 and T30 (top panels) as well as the short Amino Acid (AA) peptides (<7 AA) (Bottom panels). A more comprehensive range was included for T14 and T30 on the concentration-response curves. T14 and T30 are endogenous (naturally occurring) as compared to the synthesised shorter peptides. The inventors therefore expect T14 and T30 to have a higher affinity for nAChR (as allosteric modulators), explaining their effects at lower doses (<icr⁶ M). The inhibitory effects of higher dose (>icr⁶ M) of AChE-peptide can block nAChR responses via different mechanisms as described initially by Greenfield et al. 2004. These inhibitory mechanisms of nAChR can be explained by rapid desensitization of the receptor in presence of Acetylcholine. Indeed, phosphorylation of the nAChR by cAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, which become inactive in presence of ACh (Huganir 1982). In addition, these higher doses (>icr⁶M) can also increase basal intracellular Calcium to high levels that would in turn inactivate some nACh receptors.

In summary, the 402 and 611 linear peptides act as positive allosteric modulators similar to T14, and so could serve as novel treatments for the various skin conditions described herein. Furthermore, peptide 402 is not part of the natural sequence and thus could be more resistant to in vivo degradation.

Example 6 - The effect of linear peptide 402 on calcium influx of PC12 cells The inventors repeated the calcium influx test using peptide 402, and the results are shown in Figure 7. As can be seen, 402 triggered a higher calcium ion influx than T30, which confirms the results shown in Figure 6. Example 7 - The effect of linear peptides 402 and 611 on calcium influx of PC12 cells Analysis method

Data were obtained over three separate experiments/plates (of 96 wells), consisting of 6 (611) or 5 (402) independents columns. Each “treatment column” contained 6 wells replicates which were averaged first to determine an individual value “n”. The first time point (2 sec) of acetylcholine (Ach) injection was excluded from normalisation due to possible injections artefacts and/or acute side effects of Ach (100 mM). All treatments were analysed in the same manner with Baseline subtraction and normalisation to their respective controls.

Results

Referring to Figure 8, there are shown the effects of linear peptides 402 and 611 on calcium influx of PC12 cells. The left panel shows the time course of calcium influx since acetylcholine (Ach) injection, and the right panel shows the peak of calcium influx expressed as control percentage. ± SEM).

As can be seen, these peptides could serve as novel treatments for skin conditions, and peptide 402 is not part of the natural sequence of T14, and thus will likely be more resistant to protease degradation.

Example 8 - Concentration-response experiments with T14. T20. peptide 402 and 611 Referring to Figure 9, there is shown the effects of AchE-peptides (T14 and T30; left panel) and shorter peptides (402 and 611; right panel) on the concentration-response curve of calcium influx peaks from nicotinic acetylcholine receptor (nAChR) in PC12 cells.

Example Q - The effect of linear peptides 402 and 611 on acetyl cholinesterase activity in supernatant AChE Activity assay

AChE activity was measured using the Ellman reagent that measures the presence of thiol groups as a result of AChE activity. The PC12 cells were plated the day before the experiment as for the cell viability assay. Cells were treated with T30, T14, 402 or 611 peptides (1, 5, 10, too or 500 uM) alone or combined with NBP14 (1 uM). After treatment, supernatant (perfusate) of each treatment was collected and 65 ul from each condition were added to a new flat bottomed 96 well plate followed by the addition of 50 ul of Ellman reagent (Phosphate Buffer (0.1 M), pH 7.0; substrate: Acetylthiocholine Iodide 0.15 M; Reagent: 5, 5’-dithiobis (2-nitrobenzoic acid (DTNB)) 0.01 M and NaHC03 17.86 mM using the following ratio: i8(Phosphate Buffer): (Acetylthiocholine Iodide): (DTNB). The plate was then incubated at room temperature for 30 minutes and the absorbance values read for 10 minutes at 412 nm using BMG CLARIOstar Plus. Results were processed using MARS data analysis software.

Results

Referring to Figure 10, there are shown the results of acetylcholinesterase (AChE) release in the extracellular matrix as a compensatory effect of the extant cells. As can be seen, the 402 peptide is more potent at 1 uM than the 611 peptide. However, at 500 uM, the 611 peptide has an even higher value than that of 402 at 1 uM. Peptide 402 is more potent and so, at low doses, it potentiates the release of AchE after activation of the nicotinic receptor. At high doses, as with T14/T30, the peptide will be inhibitory. Indeed, at higher doses, peptide 402 let’s too much calcium in and so the receptor phosphorylates and shuts down, and so there is then no release of AChE. From 5 uM, peptide 402 has an inhibitory effect on the calcium ion channels which leads to a decrease in activity. For peptide 611, this effect is shifted to higher doses as it is not as potent. This shift is reflected by the lines crossing over.

Claims

1. A polypeptide, or a biologically active variant or fragment thereof, derived from the C-terminus of acetylcholinesterase, for use in preventing, treating or ameliorating a skin condition related to aging.

2. The polypeptide, or a biologically active variant or fragment thereof, for use according to claim l, wherein the acetylcholinesterase comprises an amino acid sequence substantially as set out in SEQ ID NO:i, or a biologically active variant or fragment thereof.

3. The polypeptide, or a biologically active variant or fragment thereof, for use according to either claim l or claim 2, wherein the polypeptide or a biologically active variant or fragment thereof, comprises: (i) between 3 and 50 amino acid residues, between 3 and 40 amino acid residues, between 3 and 35 amino acid residues, or between 3 and 30 amino acid residues; or

(ii) between 3 and 25 amino acid residues, between 3 and 20 amino acid residues, between 3 and 15 amino acid residues, or between 3 and 10 amino acid residues; or

(hi) between 3 and 8 amino acid residues, or between 3 and 7 amino acid residues.

4. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 2, or a biologically active variant or fragment thereof.

5. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide, or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No:3, or a biologically active variant or fragment thereof.

6. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide, or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 5 or 6.

7. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide, or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 7 or 8.

8. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide, or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 9, 10 or 11.

9. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide, or a biologically active variant or fragment thereof, comprises or consists of an amino acid sequence substantially as set out in SEQ ID No: 12.

10. The polypeptide, or a biologically active variant or fragment thereof, for use according any preceding claim, wherein the polypeptide, or a biologically active variant or fragment thereof, is created using a de novo peptide synthesis method.

11. Use of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, for preventing or treating a skin condition related to aging.

12. The use according to claim 11, wherein the polypeptide, or a biologically active variant or fragment thereof, is as defined in any one of claims 1-10.

13. The polypeptide, or biologically active variant or fragment thereof, for use according to any one of claims 1 to 10, or the use according to either claim 11 or claim 12, wherein the skin condition related to aging may be selected from a group of conditions consisting of: fine lines; wrinkles; discoloration; uneven pigmentation; sagging; enlarged pores; rough skin; dry skin; stretch marks; uneven tone; blemishes; skin thickening or thinning; and any combination thereof.

14. The polypeptide, or biologically active variant or fragment thereof, for use according to any one of claims 1 to 10, for use in preventing, retarding, arresting, or reversing the process of wrinkle formation in mammalian skin.

15. A method for cosmetic treatment of skin, the method comprising applying, to skin, a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof.

16. The method according to claim 15, wherein the polypeptide or a biologically active variant or fragment thereof, is as defined in any one of claims 1 to 10.

17. A polypeptide, or a biologically active variant or fragment thereof, derived from the C-terminus of acetylcholinesterase, for use in (i) preventing, treating or ameliorating hair loss or baldness, or (ii) as a skin whitening agent.

18. A polypeptide, or a biologically active variant or fragment thereof for use according to claim 17, wherein the polypeptide or a biologically active variant or fragment thereof, is as defined in any one of claims 1 to 10.

19. A method of preventing, treating or ameliorating hair loss or baldness, or for whitening skin, the method comprising, administering, or having administered to a subject in need of such treatment, a therapeutically effective amount of a polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof.

20. A polypeptide derived from the C-terminus of acetylcholinesterase, or a biologically active variant or fragment thereof, for use in treating a wound.

21. The polypeptide, for use according to claim 20, wherein the polypeptide, or a biologically active variant or fragment thereof is as defined in any one of claims 1 to 10.

22. A skin condition treatment pharmaceutical composition comprising a therapeutically effective amount of the polypeptide, or a biologically active variant or fragment thereof, according to any one of claims 1 to 10, and optionally a pharmaceutically acceptable vehicle.

23. A process for making the skin condition treatment composition according to claim 22, the process comprising combining a therapeutically effective amount of the polypeptide, or biologically active variant or fragment thereof according to any one of claims 1 to 10, with a pharmaceutically acceptable vehicle.

24. A cosmetic composition comprising an effective amount of the polypeptide, or a biologically active variant or fragment thereof, according to any one of claims 1 to 10, and optionally a cosmetically acceptable vehicle.

25. A process for making cosmetic composition according to claim 24, the process comprising combining a therapeutically effective amount of the polypeptide, or a biologically active variant or fragment thereof, according to any one of claims 1 to 10, with a cosmetically acceptable vehicle.

26. The composition according to either claim 22 or 24, wherein the composition is:

(a) a suntan cream, oil or lotion;

(b) a moisturiser or moisturising formulation;

(c) an anti-aging or anti-wrinkle formulation;

(d) a hair growth stimulating or promoting formulation; or (e) a skin whitening formulation.

27. One or more peptide, derivative or analogue thereof comprising or consisting of an amino acid sequence substantially as set out in SEQ ID No: 2, 3 or 5-12, for use in preventing, treating or ameliorating: - (i) a skin condition related to aging;

(ii) a wound;

(iii) hair loss or baldness; or

(iv) as a skin whitening agent.