AU2002309842B2 - Treating epidermlyosis bullosa with thymosin beta 4 - Google Patents

Description

WO 02/091969 PCT/US02/15394 "TREATING EPIDERMLYOSIS BULLOSA WITH THYMOSIN BETA 4" BACKGROUND OF THE INVENTION CROSS-REFERENCE TO RELATED APPLICATION The present application claims the benefit of U.S. Provisional Application Serial No. 60/291,326, filed May 17, 2001.

1. FIELD OF THE INVENTION The present invention relates to the field of healing or preventing inflammatory degenerative, immunological and other disorders of the skin and surrounding tissue that occur due to Epidermolysis Bullosa, and all of its subtypes.

2. DESCRIPTION OF THE BACKGROUND ART The phenomenon called Epidermolysis Bullosa (EB) is a rare genetic disorder that afflicts all ethnic and racial groups. EB is a group of diseases characterized by blister formation after minor trauma to the skin. This may lead to open sores, ulcerations and scars. This family of disorders range in severity from mild to the severely disabling, mutilating and life threatening diseases of the skin. Unlike burns, these afflictions sometimes never go away. The most severe cases require great lifestyle adjustments.

Afflicted children should never ride a bike, skate, or participate in sports, because the normal play of children causes chronic sores. Such sores may cover as much as percent of the child's body. Blistering and scarring also occur in the mouth and esophagus. Therefore, frequently a diet of only liquids or soft foods is possible. Scarring also causes the fingers and toes to fuse, leaving deformities which severely limit function.

Much of their life is tied to hospitals for treatment, blood transfusions, biopsies and surgeries. The eyes often blister preventing sight for days. Chronic anemia reduces energy, and growth is retarded. The life span for an individual afflicted with EB is usually not longer than 30 years.

There are three main types of EB: EB Simplex, Dystrophic EB (dominant or recessive) and Junctional EB. The severity of symptoms varies between these types. In general terms, EB causes blisters which may be restricted to specific areas, for example hands or feet, or may affect large areas of the body. In the milder forms the blisters heal normally without leaving permanent damage to the skin. In the other forms, the blisters WO 02/091969 PCT/US02/15394 heal with scarring which can result in permanent change to the skin, for example fingers may fuse and hands contract, reducing movement. Some forms of Junctional EB are life threatening in infancy.

EB results from deleterious changes in the physiological, biochemical and immunological properties of the skin. All forms of EB are genetic in origin and the genes responsible for several different subtypes of the condition are now known. The genetic defects result in the skin layers not adhering properly to each other, causing areas of structural weakness. This fragile skin is particularly vulnerable to damage from mild friction, causing the blisters which are the characteristic feature of the condition. Skin is an important barrier to infection, it is the first line of defense of the immune system. The fragile skin of those afflicted with EB loses this important defense mechanism. Such changes in vasculature decrease capacity to repair damage, increase propensity for skin cancers such as squamous cell carcinoma, and increase risk of infection. In addition, the open sores in the oral and digestive cavity can lead to increased dehydration and malnutrition.

There have been many attempts to treat EB, but none have had a substantial impact on prevention or treatment of EB. Various growth factors, synthetic skins, antibiotics and other therapies have failed to adequately and effectively treat EB. While EB is a genetic disease, treatment that would more rapidly heal or better heal the sores would be extremely important. Further, preventative therapy would clearly confer substantial, perhaps life-saving benefit to the patient.

Numerous pharmaceutical, nutriceutical or cosmeceutical formulations have been proposed to reduce or reverse EB or its affects.

There remains a need in the art for improved methods and compositions for healing or preventing the blisters and sores associated with EB.

SUMMARY OF THE INVENTION In accordance with the present invention, a method of treatment for promoting healing or prevention of blisters, sores or skin degeneration associated with EB involves administration to a subject or patient in need of such treatment an effective amount of a composition comprising an EB-inhibiting polypeptide comprising amino acid sequence LKKTET or a conservative variant thereof having EB-inhibiting activity.

WO 02/091969 PCT/US02/15394 DETAILED DESCRIPTION OF THE INVENTION The present invention is based on a discovery that actin-sequestering peptides such as thymosin p4 (Tp4) and other actin-sequestering peptides or peptide fragments containing amino acid sequence LKKTET or conservative variants thereof, promote healing or prevention of blisters, sores and skin degeneration associated with Epidermolysis Bullosa. Included are N- or C-terminal variants such as KLKKTET and LKKTETQ. Without being bound to any particular theory, these peptides may have the capacity to promote repair, healing and prevention by having the ability to induce terminal deoxynucleotidyl transferase (a non-template directed DNA polymerase), to decrease the levels of one or more inflammatory cytokines or chemokines, and to act as a chemotactic and/or angiogenic factor for endothelial cells and thus heal and prevent degenerative changes in the skin of patients with afflicted EB, even though EB is the result of an inherited defect.

Thymosin p4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin p4 was originally isolated from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Several roles have been ascribed to this protein including a role in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration and vascularization.

In accordance with one embodiment, the invention is a method of treatment for promoting healing and prevention of blisters, sores and skin degradation associated with EB comprising administering to a subject in need of such treatment an effective amount of a composition comprising an EB-inhibiting peptide comprising amino acid sequence LKKTET, or a conservative variant thereof having EB-inhibiting activity, preferably Thymosin p4, an isoform of Thymosin p4, oxidized Thymosin p4, Thymosin p4 sulfoxide, or an antagonist of Thymosin p4.

Compositions which may be used in accordance with the present invention include Thymosin p4 (Tp4), Tp4 isoforms, oxidized TP4, Thymosin p4 sulfoxide, polypeptides or any other actin sequestering or bundling proteins having actin binding domains, or peptide fragments comprising or consisting essentially of the amino acid sequence LKKTET or conservative variants thereof, having EB-inhibiting activity. International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses isoforms of Tp4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET and conservative variants thereof having EB-inhibiting activity, which may be utilized with the present invention. International Application Serial WO 02/091969 PCT/US02/15394 No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Thymosin 04 which may be utilized in accordance with the present invention.

Although the present invention is described primarily hereinafter with respect to Tp4 and Tp4 isoforms, it is to be understood that the following description is intended to be equally applicable to amino acid sequence LKKTET, peptides and fragments comprising or consisting essentially of LKKTET, conservative variants thereof having EB-inhibiting activity, as well as oxidized Thymosin p4.

In one embodiment, the invention provides a method for healing and preventing blisters and sores of skin in a subject by contacting the skin with an EB-inhibiting effective amount of a composition which contains Tp4 or a Tp4 isoform. The contacting may be topically or systemically. Examples of topical administration include, for example, contacting the skin with a lotion, salve, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, or oil comprising Tp4, alone or in combination with at least one agent that enhances Tp4 penetration, or delays or slows release of Tp4 peptides into the area to be treated. Systemic administration includes, for example, intravenous, intraperitoneal, intramuscular or subcutaneous injections, or inhalation, transdermal or oral administration of a composition containing Tp4 or a TB4 isoform, etc. A subject may be a mammal, preferably human.

TP4, or its analogues, isoforms or derivatives, may be administered in any suitable EB-inhibiting amount. For example, Tp4 may be administered in dosages within the range of about 0.1-50 micrograms of Tp4, more preferably in amounts of about 1-25 micrograms.

A composition in accordance with the present invention can be administered daily, every other day, etc., with a single administration or multiple administrations per day of administration, such as applications 2, 3, 4 or more times per day of administration.

TP4 isoforms have been identified and have about 70%, or about 75%, or about or more homology to the known amino acid sequence of Tp4. Such isoforms include, for example, TP4 T39, TP10, T111, Tp12, Tp13, T114 and Tp15. Similar to Tp4, the T110 and T115 isoforms have been shown to sequester actin. Tp4, T310 and T315, as well as these other isoforms share an amino acid sequence, LKKTET, that appears to be involved in mediating actin sequestration or binding. Although not wishing to be bound to any particular theory, the activity of Tp4 isoforms may be due, in part, to the ability to regulate the polymerization of actin. For example, Tp4 can modulate actin polymerization in skin 1-thymosins appear to depolymerize F-actin by sequestering free G-actin). Tp4's ability to modulate actin polymerization may therefore be due to all, WO 02/091969 PCT/US02/15394 or in part, its ability to bind to or sequester actin via the LKKTET sequence. Thus, as with TP4, other proteins which bind or sequester actin, or modulate actin polymerization, including Tp4 isoforms having the amino acid sequence LKKTET, are likely to reduce EB, alone or in a combination with Tp4, as set forth herein.

Thus, it is specifically contemplated that known Tp4 isoforms, such as TP34' a T39, T130, Tp311, Tp312, T13, Tp14 and T315, as well as Tp4 isoforms not yet identified, will be useful in the methods of the invention. As such Tp4 isoforms are useful in the methods of the invention, including the methods practiced in a subject. The invention therefore further provides pharmaceutical compositions comprising Tp4, as well as T34 isoforms TP4a l Tp9, Tp10, T311, Tp12, Tp13, Tp14 and T315, and a pharmaceutically acceptable carrier.

In addition, other proteins having actin sequestering or binding capability, or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET, for example, can similarly be employed in the methods of the invention. Such proteins include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adsevertin, propomyosin, fincilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, P-actinin and acumentin, for example. As such methods include those practiced in a subject, the invention further provides pharmaceutical compositions comprising gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, p-actinin and acumentin as set forth herein. Thus, the invention includes the use of an EBinhibiting polypeptide comprising the amino acid sequence LKKTET (which may be within its primary amino acid sequence) and conservative variants thereof.

As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue.

Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.

TP4 has been localized to a number of tissue and cell types and thus, agents which stimulate the production of Tp4 can be added to or comprise a composition to effect Tp4 production from a tissue and/or a cell. Such agents include members of the family of growth factors, such as insulin-like growth factor (IGF-1), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-P), WO 02/091969 PCT/US02/15394 basic fibroblast growth factor (bFGF), thymosin a 1 (Tal) and vascular endothelial growth factor (VEGF). More preferably, the agent is transforming growth factor beta (TGF-3) or other members of the TGF-P superfamily. Tp4 compositions of the invention may reduce the affects of EB by effectuating growth of the connective tissue through extracellular matrix deposition, cellular migration and vascularization of the skin.

In accordance with one embodiment, subjects are treated with an agent that stimulates production in the subject of an EB-inhibiting peptide as defined above.

Additionally, agents that assist or stimulate EB reduction may be added to a composition along with Tp4 or a Tp4 isoform. Such agents include angiogenic agents, growth factors, agents that direct differentiation of cells, agents that promote migration of cells and agents that stimulate the provision of extracellular matrix material in the skin.

For example, and not by way of limitation, Tp4 or a Tp4 isoform alone or in combination can be added in combination with any one or more of the following agents: VEGF, KGF, FGF, PDGF, TGFP, IGF-1, IGF-2, IL-1, prothymosin a and thymosin al in an effective amount.

The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of Tp4 or a Tp4 isoform in a pharmaceutically acceptable carrier. Such carriers include those listed above with reference to parenteral administration.

The actual dosage or reagent, formulation or composition that heals or prevents blisters, sores and skin degeneration associated with EB may depend on many factors, including the size and health of a subject. However, persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, supra, and the references cited therein, to determine the appropriate dosage to use.

Suitable topical formulations include TP4 or a T4 isoform at a concentration within the range of about 0.001 10% by weight, more preferably within the range of about 0.01 0.1% by weight, most preferably about 0.05% by weight.

The therapeutic approaches described herein involve various routes of administration or delivery of reagents or compositions comprising the Tp4 or other compounds of the invention, including any conventional administration techniques (for example, but not limited to, topical administration, local injection, inhalation, or systemic administration), to a subject. The methods and compositions using or containing Tp4 or other compounds of the invention may be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or carriers.

O The invention includes use of antibodies which interact with Tp4 peptide or

C

functional fragments thereof. Antibodies which consists essentially of pooled monoclonal antibodies with different epitopic specifications, as well as distinct monoclonal antibody Spreparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art as disclosed in PCT/US99/17282, supra. The term antibody as used in this invention is meant to include monoclonal and polyclonal antibodies.

00 O In yet another embodiment, the invention provides a method of treating a subject N by administering an effective amount of an agent which modulates Tp4 gene expression.

The term "modulate" refers to inhibition or suppression of Tp4 expression when Tp4 is over expressed, and induction of expression when Tp4 is under expressed. The term "effective amount" means that amount of Tp4 agent which is effective in modulating T34 gene expression resulting in reducing the symptoms of the Tp4 associated EB. An agent which modulates Tp4 or Tp4 isoform gene expression may be a polynucleotide for example, an antisense directed to the structural gene region or to the promoter region of Tp4 may be utilized.

In another embodiment, the invention provides a method for utilizing compounds that modulate Tp4 activity. Compounds that affect Tp4 activity antagonists agonists) include peptides, peptidomimetics, polypeptides, chemical compounds, minerals such as zincs, and biological agents.

While not be bound to any particular theory, the present invention may promote healing or prevention of blisters, sores and skin degeneration associated with Epidermolysis Bullosa by inducing terminal deoxynucleotidyl transferase (a non-template directed DNA polymerase), to decrease the levels of one or more inflammatory cytokines, or chemokines, and to act as a chemotactic factor for endothelial cells, and thereby promoting healing or preventing degenerative changes in skin brought about by Epidermolysis Bullosa or other degenerative or environmental factors.

Throughout the 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 integer or group of integers but not the exclusion of any other integer or group of integers.

Claims (12)

1. A method of treatment for promoting healing or prevention of blisters, sores or skin degeneration associated with Epidermolysis Bullosa, comprising administering to a subject in need of such treatment an effective amount of a composition comprising an Epidermolysis Bullosa-inhibiting polypeptide comprising amino acid sequence LKKTET, or a conservative variant thereof having Epidermolysis Bullosa-inhibiting activity.

2. The method of claim 1 wherein said polypeptide promotes a skin condition improvement including an increase in skin elasticity of said subject.

3. The method of claim 1 wherein said polypeptide comprises Thymosin p4 (Tp4), an N-terminal variant of Tp4, a C-terminal variant of Tp4, an isoform of Tp4, oxidized TP4 or Tp4 sulfoxide.

4. The method of claim 1 wherein said composition is administered systemically. The method of claim 1 wherein said composition is administered topically.

6. The method of claim 5 wherein said composition is in the form of a gel, creme, paste, lotion, spray, suspension, dispersion, salve, hydrogel or ointment formulation.

7. The method of claim 6, further including at least one agent that delays release or enhances penetration of Tp4 into an area to be treated.

8. The method of claim 1 wherein said polypeptide is recombinant or synthetic.

9. The method of claim 1 wherein said polypeptide is an antibody. The method of claim 9 wherein said antibody is polyclonal or monoclonal.

11. A method of treatment for promoting healing or prevention of blisters, sores or skin degeneration associated with Epidermolysis Bullosa comprising administering to a subject in need of such treatment an effective amount of a composition comprising an agent that stimulates production of an Epidermolysis Bullosa-inhibiting polypeptide comprising amino acid sequence LKKTET, or a conservative variant c thereof having Epidermolysis Bullosa-inhibiting activity.

12. The method of claims 11 wherein said polypeptide is Thymosin p4.

13. The method of claim 11 wherein said agent is an antagonist of Thymosin 5

34. 00 14. A composition when used to promote healing or prevent blisters, sore or (N skin degeneration associated with Epidermolysis Bullosa comprising an 0 effective amount of a composition including an Epidermolysis Bullosa- inhibiting polypeptide comprising amino acid sequence LKKTET or a conservative variant thereof having Epidermolysis Bullosa-inhibiting activity. The composition of claim 14 wherein said composition comprises an N- or C-terminal variant of LKKTET. 16. The composition of claim 14 wherein said composition comprises KLKKTET or LKKTETQ. 17. The composition of claim 14 where in said polypeptide comprises Tp4, and isoform of Tp4 or Tp4 sulfoxide. 18. The composition of claim 14, comprising a gel, creme, paste, lotion, spray, suspension, dispersion salve, hydrogel or ointment formulation. -9-