WO2012126047A1

WO2012126047A1 - Agent and method for treating pain and reducing inflammation

Info

Publication number: WO2012126047A1
Application number: PCT/AU2012/000281
Authority: WO
Inventors: Vasilis Paspaliaris
Original assignee: Adistem Ltd
Priority date: 2011-03-18
Filing date: 2012-03-16
Publication date: 2012-09-27

Abstract

The invention provides a method of treating pain, reducing inflammation and reducing skin irritation particularly itching, the method comprising administering a polypeptide comprising thymosin, or an active fragment or variant thereof or a peptidomimetic thereof. Also provided are compositions for use in the methods.

Description

AGENT AND METHOD FOR TREATING PAIN AND REDUCING INFLAMMATION

The present invention relates to agents and methods for treating pain and reducing inflammation. BACKGROUND

Beta (β) endorphin is an endogenous opioid peptide neurotransmitter found in the neurons of both the central and peripheral nervous system. It is a peptide of 31 amino acids and is created by processing from its precursor proopiomelanocortin (POMC).

β endorphin is an agonist of opioid receptors, with evidence suggesting that it serves as the endogenous ligand of the μ-opioid receptor; the same receptor to which chemicals extracted from opium, such as morphine, have their analgesic effect.

While opioid drugs are used clinically to relieve pain their usefulness is limited by the tolerance and dependence that often develops on chronic treatment. Opioid drugs such as morphine can be addictive and can have centrally mediated side effects such as respiratory and cardiac problems, depression and drowsiness.

It is an aim of an embodiment of the present invention to provide an agent for promoting endogenous β endorphin release for use in treating pain.

β endorphin is also known to have anti-inflammatory and anti-rheumatic effects and β endorphin agonists have been proposed for treating rheumatoid arthritis (Yin H. et al., (2005) Neuroendocrinology 81 : 10-18).

It is an aim of an embodiment of the present invention to provide an agent for promoting endogenous β endorphin release to reduce inflammation and therefore treat rheumatoid arthritis.

"Pruritus" (itching) is alleviated by scratching due to the release of β endorphin. It is an aim of an embodiment of the present invention to provide an agent for promoting endogenous β endorphin release to reduce itching.

SUMMARY

A first aspect provides a method of treating pain in a subject, comprising

administering a polypeptide comprising thymosin, or an active fragment or variant thereof.

Alternative first aspects provides a polypeptide comprising thymosin, or an active fragment or variant thereof for use as a medicament for the treatment of pain or use of a polypeptide comprising thymosin, or an active fragment or variant thereof in the manufacture of a medicament for treating pain. A second aspect provides a method of treating pain comprising administering a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof

Alternative second aspects provides a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof for treating pain, or use of a composition comprising a peptidomimetic of thymosin or an active fragment of variant thereof in the manufacture of a medicament for treating pain.

A third aspect provides a method of reducing inflammation in a subject, comprising administering a polypeptide comprising thymosin, or an active fragment or variant thereof.

Alternative third aspects provides a polypeptide comprising thymosin, or an active fragment or variant thereof for use as a medicament for reducing inflammation or use of a polypeptide comprising thymosin, or an active fragment or variant thereof in the manufacture of a medicament for reducing inflammation.

A fourth aspect provides a method of reducing inflammation comprising

administering a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof

Alternative fourth aspects provides a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof for reducing inflammation, or use of a composition comprising a peptidomimetic of thymosin or an active fragment of variant thereof in the manufacture of a medicament for reducing inflammation.

A fifth aspect provides a method of reducing Pruritus (itching) in a subject, comprising administering a polypeptide comprising thymosin, or an active fragment or variant thereof.

Alternative fifth aspects provide a polypeptide comprising thymosin, or an active fragment or variant thereof for use as a medicament for reducing Pruritus (itching) or use of a polypeptide comprising thymosin, or an active fragment or variant thereof in the manufacture of a medicament for reducing itching.

A sixth aspect provides a method of reducing Pruritus (itching) comprising administering a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof

Alternative sixth aspects provides a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof for reducing Pruritus (itching), or use of a composition comprising a peptidomimetic of thymosin or an active fragment of variant thereof in the manufacture of a medicament for reducing itching. In one embodiment of the first, third or fifth aspects the active fragment of thymosin is LKKTETQ (SEQ ID NO: 1 ). In one embodiment of the first, third or fifth aspects the polypeptide comprises thymosin beta 4 or a fragment thereof comprising or consisting of LKKTETQ (SEQ ID NO: 1 ).

In one embodiment of the second, fourth or sixth aspects the peptidomimetic is based on the polypeptide LKKTETQ (SEQ ID NO:1 ). In one embodiment of the second, fourth or sixth aspects the peptidomimetic is based on thymosin beta 4 or a fragment thereof comprising or consisting of LKKTETQ (SEQ ID NO:1 ).

A seventh aspect provides a composition comprising thymosin, or an active fragment, variant or peptidomimetic thereof as active ingredient, hyaluronic acid/sodium hyaluronate and at least one of sodium chloride, sodium benzoate and potassium sorbate..

In an embodiment of the seventh aspect the active fragment of thymosin comprises or consists essentially of LKKTETQ (SEQ ID NO: 1 ).

An eighth aspect provides a method of treating pain by topical administration of the composition of the seventh aspect.

A ninth aspect provides a method of reducing inflammation by topical administration of the composition of the seventh aspect. DETAILED DESCRIPTION

Thymosin is family of a small polypeptides found in high quantity in thymus and spleen, but widely distributed in many tissues. Members of the thymosin family have been shown to bind to actin monomers and thus to inhibit actin polymerization. The physiological processes that these polypeptides affect include stimulation or suppression of immune responses, regulation of actin dynamics and cell motility, neuroplasticity, repair and remodelling of vessels of the heart and other injured tissues, angiogenesis, and stem cell differentiation.

A number of polypeptides are members of the thymosin family; the most abundant member of the family is thymosin beta 4 (Τβ4). It was first isolated as a thymic hormone that induces terminal deoxynucleotidyltransferase.

Regenerx Biopharmaceuticals Inc. has a number of patents relating to thymosin β4 (TB4) and its fragments containing the amino acid sequence LKKTET (SEQ ID NO: 2). They claim that TB4 and active fragments such as LKKTETQ (SEQ ID NO: 1 ) sequester actin and can inhibit or promote reversal of skin degeneration associated with skin aging; promote healing or prevention of blisters, sores or skin degeneration associated with Epidermolysis Bullosa; inhibit respiratory or pulmonary microbial infection of respiratory or pulmonary tissue; inhibit or promote reversal of eye degeneration associated with dry eye syndrome; treat or reduce tissue deterioration, injury or damage due to a neuro-, muscular- or neuro-muscuiar-clegenerative disease, or restore tissue adversely affected by said disease; treat a biological or immunological response to a reactive chemical agent, biological agent or toxin; treat tissue deterioration, injury or damage due to congestive heart failure disease, or restore tissue adversely affected by said disease; treat tissue deterioration, injury or damage resulting from administration of a quaternary ammonium salt to a subject; treat tissue damage occurring subsequent to affecting an increase in blood flow through a blood vessel which is in communication with said tissue for example when introducing a stent; treat tissue deterioration, injur/ or damage due to a periodontal disease or disease of oral mucosa, or for restoring tissue adversely affected by said disease; reduce extracellular matrix build-up in a body tissue or a bodily fluid transport vessel; conserve or prepare an organ or tissue for transplant; treat microbial infections; treat damage due to radiation exposure; promote regeneration or repair of damaged cardiovascular tissue, or to prevent damage to cardiovascular tissue; and accelerate wound healing, and tissue regeneration and stimulate wound repair.

Until the present invention, it had not been disclosed or suggested that thymosin β4 or its active fragments such as LKKTETQ (SEQ ID NO: 1 ) releases β endorphin and therefore can be used in circumstances where β endorphin release is desirable, for example to treat pain, to reduce inflammation, to treat rheumatoid arthritis and to reduce itching, particularly caused by insect bites.

The inventors have determined that the peptide LKKTETQ (SEQ ID NO: 1 ) is capable of promoting β endorphin release in various cell lines and that it binds the cannabinoid receptor CB₂. β endorphin is a known analgesic. Additionally the CB₂ receptor is known to mediate pain relief. Activation of CB₂ receptors on keratinocytes in the skin has been shown to induce the release of β endorphin. This effector molecule subsequently acts on μ opioid receptors on the terminals of primary sensory neurons, leading to analgesic action (Ibrahim, M.M. et al. Proc. Natl. Acad. Sci. USA (2005) 102, 3093-3098). β endorphin is also known to block the activity and expression of NF-κΒ, reduce the release of proinflammatory cytokines TNF-a, IL-Ι , IL-6, ANTES, MMP-2 and MMP-9 in synovial fluid and is a potent anti-inflammatory and anti-rheumatic agent, β endorphins are released from leukocytes in inflamed tissues. They also alleviate itching.

The inventors have evidence that the peptide LKKTETQ (SEQ ID NO: 1 ), an active fragment of thymosin, is able to reduce pain associated with muscle and joint injuries often associated with sports or aging, such as soft muscle pain, knee and joint pain, osteoarthritis and superficial burns and is able to reduce inflammation in these conditions. They also have evidence that the peptide LKKTETQ (SEQ ID NO: 1 ) is able to reduce itching caused by mosquito bites and pimples or acne. The inventors propose that thymosin would have the same effect.

By "thymosin" we include polypeptides of the thymosin family. Members of the thymosin family include human thymosin beta 4 (X and Y chromosome); human thymosin beta 10; thymosin beta-4-like protein 1 ; thymosin beta-4-like protein 2; thymosin beta-4-like protein 3; thymosin beta-4-like protein 6; thymosin-like protein 8; thymosin like 4; any further members of the family from other species. Information concerning the members of the thymosin protein family may be readily obtained from, for example, UniProt

(http://www.uniprot.org) where a search for 'thymosin' can be performed.

(http://www.uniprot.orq/uniprot/?querv=thymosin&sort=score) The UniProt database directly provides the amino acid sequence of the thymosin proteins. Further members of the thymosin protein family may also be located by database searching.

In one embodiment thymosin does not include thymosin fraction 5. A preferred embodiment of the first aspect of the invention is wherein the thymosin is thymosin beta 4. An example of the amino acid sequence of the thymosin beta 4 polypeptide encoded by the X-chromosomal gene is given below:

Thymosin beta-4 (Swiss-Prot P62328)

MSDKPDMAEI EKFDKSKLKK TETQEKNPLP SKETIEQEKQ AGES (SEQ ID NO:2)

Thymosin polypeptide can be routinely prepared using well known laboratory techniques. Also, there are a number of commercial sources of thymosin beta 4, which can be used in the invention.

By "an active fragment of thymosin" we include where the thymosin used as a medicament comprises less than the total amino acid sequence of the full native

polypeptide; preferably the fragment is therapeutically effective, in that it retains its biological activity: in this case, its ability to promote release of β endorphins. The fragment can be any size and any part of the thymosin polypeptide; examples of representative fragments of thymosin are provided in the specification. For example, the fragment may be between 7 and 30 amino acids; 7 and 25 amino acids; 7 and 16; 7 and 15 amino acid residues.

By "a variant of thymosin" we include a peptide wherein at one or more amino acid positions, there are amino acid mutations selected from insertions, deletions, or substitutions, either conservative or non-conservative; preferably the variant is therapeutically effective in that such changes result in a protein whose properties (for example its ability to promote release of β endorphins) property; protein interaction; thermostability; activity in a certain pH- range (pH-stability)), have not significantly been changed. In one embodiment the variant comprises a mutation at one, two, three, four or five amino acid residues of Τβ4 or an active fragment thereof.

By "conservative substitutions" is intended combinations such as Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using routine methods of protein engineering and site-directed mutagenesis as would be well known to those skilled in the art. Examples of representative variants of thymosin are provided in the specification.

In one embodiment variants are based on the consensus sequence

X₁LKX₂TX₃X4X₅X₆ (SEQ ID NO: 3), wherein X is any amino acid. Preferably X is a conservative substitution of the native amino acid of Τβ4 given in SEQ ID NO: 2.

In one embodiment X₁ is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 amino acid residues or is absent, X₂ is K, H or A, X₃ is E or N, X₄ is T or M, X₅ is Q, N, E or A and X₆ is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 amino acid residues or is absent.

Preferably X₁ comprises (F/L)(D/N)(S/A T/K/N)(K N/G)

Preferably X₆ comprises (E/T)(K/E)(N/E).

The polypeptide may comprise one of amino acid sequences:

FDKSKLKKTETQEKN (SEQ ID NO: 4)

FDKAKLKKTETQEKN (SEQ ID NO: 5)

FDRSKLKKTETNTEE (SEQ ID NO: 6)

FDKTKLKKTETQEKN (SEQ ID NO: 7)

FDKSKLKKTNTEEKN (SEQ ID NO: 8)

FDRSKLKKTNTEEKN (SEQ ID NO: 9)

FDKTKLKKTETAEKN (SEQ ID NO: 10)

FNRAKLKKTETQEKN (SEQ ID NO: 1 1 )

FNKAKLKKTEMQEKN (SEQ ID NO: 12)

FDAKKLKHTETNEKN (SEQ ID NO: 13)

FNQNNLKHTETNEKN (SEQ ID NO:14)

LDKAKLKATEMQEKN (SEQ ID NO:15)

FDKAGLKKTETEEKE (SEQ ID NO: 16). In a preferred embodiment the polypeptide is LKKTETQ (SEQ ID NO: 1 ) or comprises LKKTETQ (SEQ ID NO: 1 ) and up to 15 amino acid residues at the N and C terminus but does not contain a full length thymosin β4 amino acid sequence.

In one embodiment the polypeptide comprises the amino acid sequence provided in any one of SEQ ID NOs: 1 -16 as the only active agent. In another embodiment the polypeptide consists essentially of the amino acid sequence provided in any one of SEQ ID NOs: 1-16.

In another embodiment the polypeptide comprises or consists essentially of oxidized TB4, N-terminal variants of Τβ4, C-terminal variants of Τβ4, polypeptides or peptide fragments comprising or consisting essentially of the amino acid sequence LKKTETQ (SEQ ID NO: 1 ), LKKTNTQ (SEQ ID NO: 17), KLKKTETQ (SEQ ID NO: 18), LKKTETQQ (SEQ ID NO: 19), LKKTET (SEQ ID NO: 20), conservative variants thereof.

International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses LKKTET (SEQ ID NO: 20) and isoforms of Τβ4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTETQ (SEQ ID NO: 1 ) and conservative variants thereof, which may be utilized with the present invention.

International Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Τβ4 which may be utilized in accordance with the present invention.

Although the present invention is described primarily hereinafter with respect to SEQ

ID NO: 1 , it is to be understood that the following description is intended to be equally applicable to amino acid sequences provided as SEQ ID NOs: 2-20 and to Τβ4 and Τβ4 isoforms.

Many Τβ4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of Τβ4. Such isoforms include, for example, Τβ4³'³, Τβ9, Τβ10, Τβ1 1 , Τβ12, Τβ13, Τβ14 and Τβ15. Thus, it is specifically contemplated that known Τβ4 isoforms, such as Τβ4³'³, Τβ9, Τβ10, Τβ1 1 , Τβ12, Τβ13, Τβ14 and Τβ15, as well as Τβ4 isoforms not yet identified, will be useful in the methods of the invention. The invention may utilize Τβ4 or fragments or variants or Τβ4 isoforms Τβ4³'³, Τβ9, Τβ10, Τβ1 1 , Τβ12, Τβ13, Τβ14 and Τβ15 or fragments or variants for treating pain or reducing inflammation and thus accelerating recovery of cells and tissues from injury.

In one embodiment the thymosin, active fragment, variant or peptidomimetic thereof is administered as a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. In another embodiment the thymosin, active fragment, variant or peptidomimetic thereof is administered as a veterinary composition which comprises a carrier suitable for veterinary use.

Laboratory techniques are well known for the preparation of peptides, such methods being readily performed by a skilled person; for example, using recombinant DNA technologies as set out in Sambrook et al (2001 ): Molecular cloning, a laboratory manual, 3^nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York. Hence the skilled person can prepare thymosin polypeptides and fragments using the information provided herein and from common general knowledge.

Peptides and polypeptides used according to the invention may be subject to degradation by a number of means (such as protease activity in biological systems). Such degradation may limit the bioavailability of the polypeptides and hence the ability of the polypeptides to achieve their biological function. There are wide ranges of well-established techniques by which polypeptides that have enhanced stability in biological contexts can be designed and produced. Such polypeptides may have improved bioavailability as a result of increased resistance to protease-mediated degradation. Preferably, a variant suitable for use according to the invention is more protease-resistant than the polypeptide or peptide from which it is derived.

The N and/or C terminal of the peptide/polypeptide may be protected by a protecting group. For example, the N terminal may be protected by an acetyl group, or by an alkyl or aryl group, or an alkyl-CO- or aryl-CO- group, each of which may be optionally substituted. The C terminal may be protected by an amide group or by a substituted amide group.

In one embodiment the polypeptide has an N terminal acetyl group and a C terminal hydroxyl group, for example Ac-SEQ ID NO: 1-OH.

The addition of a protecting group to the N and/or C terminus of the

peptide/polypeptide may enhance the protease resistance. Protease-resistance may be evaluated by means of well-known protein degradation assays. The relative values of protease resistance for the protected polypeptide and polypeptide may then be compared.

Peptoid derivatives of the polypeptides used in the invention may be readily designed from knowledge of the structure of the polypeptide. Commercially available software may be used to develop peptoid derivatives according to well-established protocols.

A further embodiment of a modified form of peptides used according to the invention comprises D-amino acid forms of the peptide. The preparation of peptides using D-amino acids rather than L-amino acids greatly decreases any unwanted breakdown of such an agent by normal metabolic processes, decreasing the amounts of agent which need to be administered, along with the frequency of its administration.

The invention also extends to the use of a retro-inverso form of a peptide having an amino acid sequence provided as SEQ ID NOs: 1 -20 and to Τβ4 and Τβ4 isoforms.

Retro-inverso peptides will be known to persons skilled in the art. Such peptides reverse a given peptide sequence and provide all amino acid residues in D form. The peptides, fragments or variants used in the invention may be expressed by biological cells and the inventions include the use of such agents produced recombinantly.

The term "peptidomimetic" refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent, but that avoids the undesirable features. For example, morphine is a compound which can be orally administered, and which is a peptidomimetic of the peptide endorphin. There are a number of different approaches to the design and synthesis of peptidomimetics, as is well known in the art.

The peptides used in the invention can also contain further amino acid sequences which are not derived from the amino acid sequence thymosin: for example, other amino acid sequences which provide a separate function of the peptide (such as a tag, or a catalytic domain). The use of such peptides is also included in the aspects of the invention.

All peptides, polypeptides, fragments, variants, derivatives or peptidomimetics of thymosin or SEQ ID NO:1 or variants thereof used in the invention are hereinafter referred to as therapeutic agent(s). Such therapeutic agents are all capable of treating pain and reducing inflammation.

As can be appreciated, the medicament may be administered to a variety of different subjects. By "subject" we include any animal that is susceptible to developing pain or who is in pain, preferably a vertebrate, more preferably a mammal such as a domesticated (cat, dog) or farmyard (cow, sheep, goat, horse) animal or a human. Most preferably the subject is a human or a race horse.

The therapeutic agent may be administered orally, topically, or parenterally in medicaments containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

The term parenteral as used herein includes intravenous, intra-arterial,

intraperitoneal, intramuscular, subcutaneous, subconjunctival, intracavity, transdermal and subcutaneous injection, aerosol for administration to lungs or nasal cavity or administration by infusion by, for example, osmotic pump. Various means by which a medicament comprising a therapeutic agent can be formulated are provided below.

Therapeutic agents may be presented 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, micelle, transdermal patch, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the vehicle used to provide the treatment should be one which is well tolerated by the subject to whom it is given, and preferably enables delivery of the therapeutic to the target cell, tissue, or organ.

In one embodiment, the composition is administered with a carrier. In one embodiment the carrier is a liquid and the composition is in the form of a solution. In another embodiment, the carrier is a gel and the composition is in the form of a cream.

Therapeutic agents may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted on or under the skin, and the therapeutic agent may be released over weeks or even months. Such devices may be particularly advantageous when long term treatment with the therapeutic agents is required and which would normally require frequent administration (e.g. at least daily injection).

Therapeutic agents may also be presented in medical devices such as implants and prostheses.

It will be appreciated that the amount of a therapeutic agent that is required is determined by its biological activity and bioavailability which in turn depends on the mode of administration, the physicochemical properties of the therapeutic agent employed, and whether the therapeutic agent is being used as a mono-therapy or in a combined therapy. Also, the amount will be determined by the number and state of target cells to be treated. The frequency of administration will also be influenced by the above-mentioned factors and particularly the half-life of the therapeutic agent 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 therapeutic agent in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. 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.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to establish specific formulations of therapeutic agents and precise therapeutic regimes (such as daily doses of the therapeutic agents and the frequency of administration).

Daily doses may be given as a single administration. Alternatively, the therapeutic agents used may require administration twice or more times during a day. As an example, therapeutic agents may be administered as two (or more depending upon the severity of the condition) daily doses of between 1 mg and 1000 mg (i.e. assuming a body weight of 70kg). A patient receiving treatment may take 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 to a patient without the need to administer repeated doses.

In one embodiment the medicament comprising the therapeutic agent is

administered subcutaneously or is formulated for subcutaneous administration to a subject. In one embodiment the medicament is an injectable composition.

In one embodiment the medicament is formulated to provide between 1 to 1000 mg of the therapeutic agent per administration. In one embodiment, the polypeptide is provided an amount from about 0.01 mg to about 10000 mg. In another embodiment, the amount of the peptide is an amount from about 0.1 mg to about 5000 mg. In another embodiment, the amount of the peptide is an amount from about 1 mg to about 1000 mg.

The topical composition of the fifth aspect comprises thymosin or an active fragment thereof, such as LKKTETQ (SEQ ID NO: 1 ) as active ingredient and sodium hyaluronate.

The invention provides methods of treating pain, particularly chronic pain, although they could also be used to treat acute pain. The term pain as used herein also includes mild pain, irritation or discomfort.

The pain, irritation or discomfort may be that associated with an insect bite.

In one embodiment the insect is a mosquito.

The pain, irritation or discomfort may be associated with burns, rashes or inflammation, particularly of the skin.

Means to diagnosis chronic pain include classical clinical and psychological evaluations, which can be augmented by various laboratory procedures, as described herein. Such means are well-described in the medical/scientific and patent literature; some illustrative examples are provided below.

One criterion to diagnose a "chronic" pain is whether the pain persists for a month beyond the usual course of an acute disease or a reasonable time for an injury to heal. This evaluation is made by the clinician on a case by case basis. Acute diseases or injuries can heal in 2, 3, or, at most, 6 weeks, depending on the nature of the condition or injury, the age and health of the patient, and the like. Clinicians are trained to be very aware of this "acute" versus "chronic" pain distinction, for it is critical to make correct diagnosis and treatment plans. For example, a simple wrist fracture can remain painful for a week to ten days;

however, if pain persists longer than this period, a dystropathy could be developing which will be irreversible if not treated. Accordingly, a chronic pain is diagnosed by the practitioner based on clinical and laboratory results, depending on the particular condition or injury, patient, and the like.

Another means to identify a "chronic" pain is by diagnosis of a pathologic process (which is usually also chronic) known to produce or be associated with chronic pain. Such conditions are well characterized and include, e.g., chronic pain syndrome, arthralgia, arthritis (e.g. , osteoarthritis and rheumatoid arthritis), causalgia, hyperpathia, neuralgia, neuritis, radiculagia, fibromyalgia, orofacial pain and temporomandibular disorders, reflex sympathetic dystrophy, chronic back pain, certain cancers, and the like.

Chronic pain is also associated with particular injuries to the nerves. These include, e.g., nerve transection (traumatic or surgical), chronic abnormal pressure on a nerve, chemical (e.g., formalin) destruction of nerve tissue, and the like.

Chronic pain can also be distinguished from acute pain by its non-responsiveness to pharmacologic therapies known to significantly ameliorate or abate acute pain. When pain is initially diagnosed as acute or of unknown etiology, the clinician typically administers one of several analgesics known in the art to be effective for acute pain, such as, e.g., a nonsteroid anti-inflammatory drug (NSAID), such as, e.g., aspirin, ibuprofen, propoxyphene, tramadol, acetaminophen and the like. If there is no significant amelioration of pain, as assessed by the clinician, over an approximately six week period, then a provisional diagnosis of chronic pain can be made. Ultimately, as discussed above, a diagnosis of chronic pain depends upon determination as to whether pain would be expected, given each individual situation.

Other treatments to which chronic pain is also typically incompletely or totally unresponsive include tricyclic antidepressant administration, psychotherapy, or alternative medicines, such as acupuncture, biofeedback, and the like.

Laboratory, radiographic and other types of imaging procedures can also be used to diagnose chronic pain. In particular, positron emission tomography, or PET, now allows the clinician to objectify such otherwise merely subjective symptoms, including chronic pain.

The methods and compositions find particular utility in the reduction of inflammation and thus can be used to improve recovery in joint, tissue and muscle pain and can be used to treat inflammatory conditions including rheumatoid arthritis, osteoarthritis and asthma. They can also provide relief from swelling caused by insect bites and can reduce spots, pimples, blemishes and rashes on the skin that involve inflammation.

In one embodiment the insect is a mosquito.

The methods and compositions find particular utility in the reduction of itching, particularly cause by insect bites including mosquito bites.

The topical composition of the seventh aspect comprises thymosin or an active fragment thereof, such as LKKTETQ (SEQ ID NO: 1 ) as active ingredient and sodium hyaluronate.

Topical administration of pain-relieving drugs, inflammation relieving drugs or anti- itch drugs to the periphery offers important advantages over systemic or local, non-topical administration. The action of topically administered agents is restricted to the region below the surface of the skin or mucosa where the application has occurred. In using topical routes of administration, the amount of agent absorbed systemically is so minimal that no pharmacologic effect is produced away from the application site. Topical administration of the composition is directed to cutaneous, mucosal, vaginal, rectal, ocular, or nasal surfaces. The composition is topically administered to a subject in an amount and duration sufficient to treat pain and reduce inflammation

The topical composition may be in the form of a cream, gel, serum, lotion, spray, foam, paste, patch, suspension, dispersion, or pad for use with a needle stick, such as a diabetic needle stick or lancet or may be incorporated onto or into disposables such as haemorrhoid wipes, gauze, sponge, bandages, and wraps; mouth guards, dental trays; needles, needle sticks or catheters; adult diapers; gloves, socks or wrist bands, for ease of application.

In a preferred embodiment the topical formulation is a serum, gel, cream or spray.

The composition is applied topically to a site at or adjacent to a painful region. The composition is reapplied as necessary. Relief is typically obtained within minutes and lasts for periods of variable duration ranging from minutes to several hours and even, in some cases, days. The composition is applied such that the dosage of active agent is sufficient to provide an effective dose in the painful area or immediately adjacent areas, to ameliorate or eliminate pain.

The compositions of the present invention can be administered topically by preparing a solution of the active agent and hyaluronic acid/sodium hyaluronate in a solvent such as water, saline, aqueous dextrose, glycerol, ethanol or dimethyl sulfoxide (DMSO), with or without other excipients. The composition can also contain other medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary substances such as wetting agents, emulsifying agents, thickeners, such as xanthan gum, preservatives, such as potassium sorbate and sodium benzoate and pH buffering agents such as citric acid and sodium chloride.

The composition may contain a penetration enhancer, most preferably one with membrane disruptive properties. One long-standing approach for improving transdermal drug delivery uses penetration enhancers (also called sorption promoters or accelerants) which penetrate into skin to reversibly decrease the barrier resistance. Numerous compounds have been evaluated for penetration enhancing activity, including sulphoxides (e.g., dimethylsulfoxide ("DMSO") and decylmethylsulfoxide (C10MSO)), Azones (e.g.

laurocapram), pyrrolidones (for example 2-pyrrolidone, 2P), alcohols and alkanols (ethanol, or decanol), glycols (for example propylene glycol, PG, a common excipient in topically applied dosage forms), surfactants (also common in dosage forms) and terpenes. Many potential sites and modes of action have been identified for skin penetration enhancers, such as the intercellular lipid matrix in which the accelerants may disrupt the packing motif, the intracellular keratin domains, or through increasing drug partitioning into the tissue by acting as a solvent for the permeant within the membrane. Further potential mechanisms of action, for example with the enhancers acting on desmosomal connections between corneocytes or altering metabolic activity within the skin, or exerting an influence on the thermodynamic activity/solubility of the drug in its vehicle are possible.

Preferred penetration enhancers include the sulfoxide decylmethylsulfoxide

(C10MSO); ethers such as diethylene glycol monoethyl ether, dekaoxyethylene-oleylether, and diethylene glycol monomethyl ethers; surfactants, fatty acids such as C8-C22 and other fatty acids, C8-C22 fatty alcohols, and polyols. Other suitable penetration enhancers include, but are not limited to, urea, (carbonyldiamide), imidurea, Ν,Ν-diethylformamide, N-methyl-2- pyrrolidine, 1 -dodecal-azacyclopheptane-2-one, calcium thioglycate, 2-pyyrolidine, N,N- diethyl-m-toluamide, oleic acid and its ester derivatives, such as methyl, ethyl, propyl, isopropyl, butyl, vinyl and glycerylmonooleate, sorbitan esters, such as sorbitan monolaurate and sorbitan monooleate, other fatty acid esters such as isopropyl laurate, isopropyl myristate, isopropyl palmitate, diisopropyl adipate, propylene glycol monolaurate, propylene glycol monooleatea and non-ionic detergents such as Brij.RTM. 76 (stearyl poly(10 oxyethylene ether), Brij.RTM. 78 (stearyl poly(20)oxyethylene ether), Brij.RTM. 96 (oleyl poly(10)oxyethylene ether), and Brij.RTM. 721 (stearyl poly(21 )oxyethylene ether) (ICI Americas Inc. Corp.). Fatty acids such as linoleic acid, capric acid, lauric acid, and neodecanoic acid, which can be in a solvent such as ethanol or propylene glycol, can be used as lipid bilayer disrupting agents. DMSO is not a particularly preferred penetration enhancer due to its strong door and the fact that it is not approved for use in humans by the Food and Drug Administration.

Detergents such as Dawn.RTM. detergent contain sodium lauryl sulfate, sodium pareth-23. Sodium dodecyl sulfate (or sulphate) (SDS or NaDS)

(C.sub.12H.sub.25Na0.sub.4S), also known as sodium lauryl sulfate (SLS), is an ionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to create a lather. The molecule has a tail of 12 carbon atoms, attached to a sulfate group, giving the molecule the amphiphilic properties required of a detergent.

In one embodiment the topical composition comprises 1 % active ingredient or less.

In a further embodiment the topical composition comprises 0.5% active ingredient or less. In a further embodiment the topical composition comprises 0.25% active ingredient or less. In a further embodiment the topical composition comprises 0.1 % active ingredient or less. In a further embodiment the topical composition comprises 0.05% active ingredient or less. In a further embodiment the topical composition comprises 0.025% active ingredient or less. In a further embodiment the topical composition comprises 0.02% active ingredient or less.

In one embodiment the topical composition is made up as follows:

It is also appreciated by the inventors that the medicaments disclosed herein in all aspects of the invention can be used alone or in combination with one or more additional medicament for treating pain or inflammation. Examples of further medicaments include NSAIDS and opioid analgesics, while further medicaments would be well known to the skilled person.

A tenth aspect provides a method of treating pain in which the thymosin, or an active fragment, variant or peptidomimetic thereof is provided by topical administration of the composition of the seventh aspect. An eleventh aspect provides a method for reducing inflammation in which the thymosin, or an active fragment, variant or peptidomimetic thereof is provided by topical administration of the composition of the seventh aspect.

A twelfth aspect of the invention provides a method of treating pain or reducing inflammation or reducing itching comprising administering a population of stem cells that secrete thymosin.

The inventors have surprisingly identified that, when administered to a subject, stem cells which secrete thymosin can be effective therapeutic agents for the treatment of pain. Until the present disclosure, it had not been disclosed or suggested that such stem cells would have this utility.

Stem cells are cells that have the potential to differentiate into a number of cell types in the body. Theoretically, stem cells may divide without limit to replenish other cells for as long as the organism is alive. Upon differentiation, the daughter cell has the potential to remain a stem cell or become another cell type, for example lung cell and display its characteristics, thus holding promise for many diseases by replacing damaged tissues. These phenomena may be induced under specific physiological and experimental conditions.

In general, stem cell therapy represents a therapeutic method by which degenerative and/progressive diseases (such as those caused by premature death or malfunction of cell types that the body is unable to replace) may be treated. It is hoped that addition of stem cells may help nucleate and promote the development of functional cells and/or tissues to replace those lost, thereby restoring normal healthy activity/function.

For the purposes of the present invention, "stem cells" are taken to comprise nullipotent, totipotent or pluripotent cells, and progenitor cells (or precursor cells) to comprise multipotent cells. For the avoidance of doubt, the medicament and methods of the invention can comprise a therapeutically effective quantity of either stem or progenitor cells, or both stem and progenitor cells. Preferred culture conditions for use in accordance with the present invention may be determined with reference to the type of biological cell to be cultured. Consideration should be given both to the nature of the cell (e.g. stem or progenitor cell), to the source of the cell, and also to the manner in which the cell is to be utilised. Suitable culture conditions are well known to those skilled in the art.

A suitable source of stem cells that may be used in accordance with the present invention are cells derived from the inner cell mass/epiblast of pre-implantation embryos. Such embryonic stem (ES) cells are readily obtainable and are capable of giving rise to all possible embryonic and adult cell lineages. In particular, the undifferentiated human ESC (H1 line from WiCell Research Institute, Inc, Madison, Wl: www.wicell.org) could be used in the invention; this cell line is commercially available. A further source of stem cells that can be used in the present invention are umbilical cord-derived cells. A still further source of stem cells are those isolated from adult tissues, including adipose tissues.

Where the medicament of the invention involves the use of biological cells, preferably the formulation for comprises biological cells in a suitable liquid carrier. Such a liquid carrier is preferably non-immunogenic, and may comprise a saline solution, cell culture medium, or distilled water. Formulations for injection may be as described above, or may also be provided in the form of a gel, which may preferably be capable of resolution by the body of the subject treated. Formulations suitable for implantation may take the forms described for injection or inhalation, and may also comprise biological cells provided in a scaffold or matrix capable of providing a foundation for new tissue development.

By "thymosin, or a fragment, variant or derivative thereof, and/or a population of stem cells" we include all those particular embodiments discussed above in relation to other aspects of the invention.

The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms (prophylaxis) and/or their underlying cause, and

improvement or remediation of damage. Thus, for example, the present method of "treating" a disorder encompasses both prevention of the disorder in a predisposed individual and treatment of the disorder in a clinically symptomatic individual.

"Treating" as used herein covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human, and includes: inhibiting the condition, i.e., arresting its development; or relieving or ameliorating the effects of the condition, i.e. , cause regression of the effects of the condition.

"Prophylaxis" or "prophylactic" or "preventative" therapy or "prevent" or "prevention" as used herein includes preventing the condition from occurring or ameliorating the subsequent progression of the condition in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it.

In one embodiment the method or composition prevents or slows progression of pain, alleviates, prevents or dulls the sensation of pain.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. It must also be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

The invention is now further described in detail by reference to the following example. The example is provided for purposes of illustration only, and is not intended to be limiting unless otherwise specified. Thus, the invention encompasses any and all variations which become evident as a result of the teaching provided herein.

Example 1 : Evaluation of β-endorphin secretion with LKKTETQ

In this study, we evaluated the expression level of β-endorphin secretion stimulated by administering the test peptide Ac- LKKTETQ-OH (SEQ ID NO: 1 ) with 6 titration (30, 3, 0.3, 0.03, 0.003, and 0.0003μΜ) under 3 different time periods (15, 30, 60min) on white blood cells as well as keratinocyte cells.

Protocol Cell lines:

White blood cells: IM-9 (ATCC, #CCL-159) and Jurkat (ATCC, #TIB-152)

Keratinocyte: HaCaT (XiangYa Central Experiment Laboratory)

96 well plates were seeded at optimal cell density in 150pL/well CM.

The cells were Incubated for 24 hours at 37°C with 5% C0₂. The supernatants were discarded by spinning plates at 1200rpm for 5min, fresh culture medium was added and the cells incubated for 1 hour at 37°C with 5% C0₂ prior to adding test samples, VEH (vehicle), & AM1241 (A CONTROL CB2 RECEPTOR AGONIST (Ibrahim, M.M. et al. Proc. Natl. Acad. Sci. USA (2005) 102, 3093-3098).).

Master and Daughter dilution plates were prepared. The test peptide was titrated with top concentration at 30μΜ for samples requiring EC50 for full-log 6-pt curve.

16.5μΙ_ of test samples from Daughter plate was added to 150μΙ_ CM + cells wells, add VEH and AM1241 samples.

Controls:

Cell negative control: VEH (1 %₀ DMSO)

Reference control: AM1241 (1 uM)

The plates were incubated at 37°C in 5% C0₂ for different time periods (15min, 30min, 1 hour). The supernatants were harvested by spinning plates at 1200rpm for 10m in. 150μΙ_ supernatants were collected for the following assay.

Assay for β-endorphin secretion followed the manufacture's guide (Peninsula Laboratories, #S-1 134).

β-endorphin levels and EC50 values were determined using a "Nonlinear Regression (Curve fit), Sigmoidal dose-response (variable slope)" by GraphPad Prism software.

The results are shown in Figures 1 to 9 and Tables 1 to 9.

Table 1 :

Table 2:

AM 1241/ β-Endorphin / HaCaT cell

15min 30min 60min

AM1241 446.5 416 537

Table 3:

TB4-7/ β-Endorphin / HaCaT cell

Table 4:

Table 5:

AM 1241/ β-Endorp in / IM-9 cell

15min 30min 60min

AM 1241 520 406.5 409 Table 6:

Table 7:

Table 8:

AM 1241/ β-Endorphin / Jurkat cell

15min 30min 60min

AM 1241 570 618.5 806.5 Table 9:

The results of ELISA assay showed that β-endorphin secretion was lower than 1000 pg/mL in these 3 cell lines with or without peptide (TB4-7) treatment. In HaCaT and IM-9 cells, the expression of β-endorphin was up-regulated with increasing concentration of the peptide, the highest expression level in both cell lines was around 125% compared with vehicle samples under the conditions of 30μΜ treated for 60min. EC50 value of the tested peptide was about 0.25μΜ in HaCaT cells when treated for 60min, while which of that was more than 30μΜ in HaCaT and IM-9 cells or non detected in Jurkat cells under other experiment conditions. Interestingly, the secretion of β-endorphin was decreased in Jurkat cells with the peptide treatment under our experiment conditions. Example 2: Evaluation of the ability of Ac-LKKTETQ- OH to bind CB₂ in a Radio-ligand Binding assay

The ability of the peptide Ac-LKKTETQ-OH (SEQ ID NO: 1 ) to bind the cannabinoid receptor CB₂ was assayed according to the method described in Munro, S. et al, (1993) Nature 365, 61 -65.

Human recombinant CHO-K1 cells were incubated with 1 nm, 0.1 μιη or 10 μιη of peptide with peptide with 10 μιη R(+) WIN-55,212-2 (non-specific ligand) in incubation buffer consisting of 20mM HEPES, pH 7.0, 0.5 mg/ml BSA for 90 minutes at 37°C. TB4-7 peptide (Ac-LKKTETQ-OH) displaced the radiolabeled ligand by 9% at 10uM and 7% at 1 μΜ.

Example 3: Clinical evaluation of the ability to reduce pain

A serum comprising SEQ ID NO: 1 was made with the following ingredients:

A serum comprising the same ingredients as above in which SEQ ID NO: 1 is replaced by any one of SEQ ID NO: 2 to 20 or peptidomimetics thereof can also be made.

Female patient, 67 years old, presented with hands hurting and sore, particularly in the joints at the end of her thumbs. The patient commented that there was also a weakness in both hands due to arthritis.

Patient demonstrated the problem by trying to lift a large jug of water. Her right hand could raise it only about 3 inches off a table and her left hand could lift it only about an inch and a half high.

After topical application of the serum as made above on her hands by rubbing all over, in 15 minutes patient noticed the pain in her hands was mostly gone and she had more flexibility opening and closing her fist. After 45 minutes, she was curious and tried to lift the jug of water again. She could easily lift it high off the table a foot or more with both hands with little effort or discomfort. Patient slept with the serum still on her hands that night and felt much better the next couple of days without another application.

Similar results are expected with a serum in which SEQ ID NO: 1 is substituted by any one of SEQ ID NO: 2 to 20 or peptidomimetics thereof.

Example 4 Clinical evaluation of the ability to reduce pain, irritation and itching caused by mosquito bites

The serum produced in Example 3 was applied topically to the site of irritation in patients with mosquito bites. The pain, irritation and itching associated with the bites was found to cease within a minute or so of the application of the serum.

Example 5 Clinical evaluation of the ability to reduce acne and pimples

The serum produced in Example 3 was applied topically to the pimples and acne sores. The serum was able to reduce inflammation and thereby reduce pimples and acne sores.

Claims

1 . A method of treating pain in a subject, comprising administering a polypeptide comprising thymosin, or an active fragment or variant thereof.

2. A method of treating pain in a subject, comprising administering a peptidomimetic of thymosin, or an active fragment or variant thereof.

3. A method of reducing inflammation in a subject, comprising administering a polypeptide comprising thymosin, or an active fragment or variant thereof.

4. A method of reducing inflammation comprising administering a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof.

5. A method of reducing Pruritus (itching) in a subject, comprising administering a polypeptide comprising thymosin, or an active fragment or variant thereof.

6. A method of reducing Pruritus (itching) comprising administering a composition comprising a peptidomimetic of thymosin, or an active fragment or variant thereof

7. The method of claim 1 , claim 3 or claim 5 in which the polypeptide comprises X₁LKX₂TX₃X₄X5X6 (SEQ ID NO: 3), wherein X is any amino acid.

8. The method of claim 7 in which:

Xi is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 amino acid residues or is absent, X₂ is K, H or A,

X₃ is E or N,

X₄ is T or M,

X₅ is Q, N, E or A, and

X₆ is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 amino acid residues or is absent.

9. The method of claim 7 in which X₁ comprises (F/L)(D/N)(S/A/T/K/N)(K/N/G)

10. The method of claim 7 in which X₆ comprises (E/T)(K E)(N/E).

1 1 . The method of claim 7 in which the polypeptide comprises or consists essentially of:

LKKTETQ (SEQ ID NO: 1 )

FDKSKLKKTETQEKN (SEQ ID NO: 4)

FDKAKLKKTETQEKN (SEQ ID NO: 5)

FDRSKLKKTETNTEE (SEQ ID NO: 6)

FDKTKLKKTETQEKN (SEQ ID NO: 7)

FDKSKLKKTNTEEKN (SEQ ID NO: 8)

FDRSKLKKTNTEEKN (SEQ ID NO: 9) FDKTKLKKTETAEKN (SEQ ID NO: 10)

FNRAKLKKTETQEKN (SEQ ID NO: 1 1 )

FNKAKLKKTEMQEKN (SEQ ID NO: 12)

FDAKKLKHTETNEKN (SEQ ID NO: 13)

FNQNNLKHTETNEKN (SEQ ID NO:14)

LDKAKLKATEMQEKN (SEQ ID NO:15)

FDKAGLKKTETEEKE (SEQ ID NO: 16).

12. The method of claim 1 , claim 3 or claim 5 in which composition comprises thymosin beta 4 or a fragment thereof comprising LKKTETQ (SEQ ID NO: 1 ).

13. The method of claim 2, claim 4 or claim 6 in which the peptidomimetic is based on X₁LKX₂TX₃X₄X₅X₆ (SEQ ID NO: 3), wherein X is any amino acid.

14. The method of claim 13 in which:

Xs is E or N,

X₄ is T or M,

X₅ is Q, N, E or A, and

15. The method of claim 13 in which X₁ comprises (F/L)(D/N)(S/A/T/K/N)(K N/G).

16. The method of claim 13 in which X₆ comprises (E/T)(K/E)(N/E).

17. The method of claim 13 in which the peptidomimetic is based on:

LKKTETQ (SEQ ID NO: 1 )

FDKSKLKKTETQEKN (SEQ ID NO: 4)

FDKAKLKKTETQEKN (SEQ ID NO: 5)

FDRSKLKKTETNTEE (SEQ ID NO: 6)

FDKTKLKKTETQEKN (SEQ ID NO: 7)

FDKSKLKKTNTEEKN (SEQ ID NO: 8)

FDRSKLKKTNTEEKN (SEQ ID NO: 9)

FDKTKLKKTETAEKN (SEQ ID NO: 10)

FNRAKLKKTETQEKN (SEQ ID NO: 1 1 )

FNKAKLKKTEMQEKN (SEQ ID NO: 12)

FDAKKLKHTETNEKN (SEQ ID NO: 13) FNQNNLKHTETNEKN (SEQ ID NO:14)

LDKAKLKATEMQEKN (SEQ ID N0:15)

FDKAGLKKTETEEKE (SEQ ID NO: 16).

18. The method of claim 2, claim 4 or claim 6 in which the peptidomimetic is based on thymosin beta 4 or a fragment thereof comprising LKKTETQ (SEQ ID NO: 1 ).

19. The method of any preceding claim in which the pain or inflammation is caused by an insect bite.

20. The method of claim 5 or claim 6 in which the itching is caused by an insect bite.

21 . The method of claim 19 or claim 20 in which the insect is a mosquito.

22. A composition comprising thymosin, or an active fragment, variant or peptidomimetic thereof and hyaluronic acid or sodium hyaluronate and at least one of sodium chloride, sodium benzoate and potassium sorbate.

23. The composition of claim 22 in which the active fragment of thymosin is Ac- LKKTETQ-OH.

24. The method of any one of claims 1 to 21 in which the polypeptide comprising thymosin, or an active fragment or variant thereof is provided by topical administration of the composition of claim 22 or claim 23.