MXPA03010446A - Treating epidermlyosis bullosa with thymosin beta 4. - Google Patents

Abstract

Blister, sores or skin degradation associated with Epidermolysis Bullosa is treated or prevented by administration of a actin-sequestering peptide such as Thymosin beta4, an isoform of Thymosin beta4 or oxidized Thymosin beta4.

Description

PHARMACEUTICAL COMPOSITIONS BASED ON TIMOSIN ß4, ANALOGUES, ISORPHORMS AND OTHER DERIVATIVES FOR THE TREATMENT OF THE VESICULAR EPIDERMOLYSIS AND ASSOCIATED DERMATOLOGICAL INDICATIONS, AND METHODS FOR THIS FIELD OF THE INVENTION The present invention is related to the field of 1 stroke or the prevention of degenerative inflammatory disorders, immunological disorders and other disorders of the skin and surrounding tissue that occur due to hereditary vesicular epidermolysis, and to the totality of its subtypes.

Background of the Invention The present application claims the benefit of the provisional patent application of the United States of America number 60 / 291,326, filed on May 17, 2001. The phenomenon called Hereditary Vesicular Epidermolysis (EB, Epidermolysis Bullosa) is a disorder rare genetic that affects all ethnic and racial groups. EB is a group of diseases characterized by the formation of blisters after a minor skin trauma. This can lead to open sores, ulcers and scars. This family of disorders varies in severity from mild skin diseases to diseases that severely incapacitate, maim and threaten life. Unlike burns, these conditions sometimes never appear. The most severe crashes require major adjustments in lifestyle. Affected children should never ride a bicycle, skate or participate in sports, because children's normal play causes them chronic sores. These sores can cover as much as 75 percent of the child's body. Blistering and scarring also occurs in the mouth and esophagus. Therefore, often only a diet of fluids and soft foods is possible. The scarring also causes the fingers and toes to melt, leaving deformities that severely limit their function. Much of his life is tied to hospitals for treatment, blood transfusions, biopsies and surgeries. The eyes are often filled with blisters that prevent it from being possible to see for days. Chronic anemia reduces energy and slows growth. The life expectancy for an individual affected by EB is normally not greater than 30 years. There are three main types of EB: Simple EB, dystrophic EB (dominant or recessive) and Conective EB. The severity of symptoms varies among these types. In general terms, EB causes blisters that can be restricted to specific areas, for example, hands or feet, or can affect large areas of the body. In soft forms the blisters heal normally without leaving permanent damage to the skin. In the other forms, the blisters heal with scars that can result in a permanent change of the skin, for example, the fingers can fuse and the hands can contract, reducing their movement. Some forms of Connective EB threaten life in childhood. The EB results from the harmful changes in the physiological, biochemical and immunological properties of the skin. All forms of EB are of genetic origin and the genes responsible for several different subtypes of this condition are now known. Genetic defects result in the layers of skin not adhering properly between them, causing areas with structural weakness. This easy skin is particularly vulnerable to damage from a soft friction, causing the blisters that are a characteristic feature of this condition. The skin is an important barrier to infection, it is the first line of defense of the immune system. The fragile skin of those affected with BD loses this important defense mechanism. Such changes in the vasculature decrease the ability to repair damage, increase the propensity for skin cancers such as squamous cell carcinoma, and increase the risk of infection. further, open sores in the oral cavity and digestive can lead to increased dehydration and malnutrition. There have been many attempts to treat EB but none has had a major impact on the prevention or treatment of EB. Several growth factors, synthetic skins, antibiotics and other therapies have failed to adequately and effectively treat EB. While EB is a genetic disease, treatment that was able to heal faster or improve the healing of the sores would be extremely important. In addition, preventive therapy would clearly confer an important benefit to the patient, perhaps saving his life.

Numerous pharmaceutical, nutraceutical or cosmeceutical formulations have been proposed to reduce or reverse the EB or its effects. There is still a need in the art for improved methods and compositions for the cure or prevention of blisters and sores associated with EB.

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

Detailed Description of Preferred Modes The present invention is based on the discovery that actin-sequestering peptides, such as thymosin β4 (β4) and other peptides sequestering actin or fragments of peptide containing the amino acid sequence LKKTET or Conservative variants of this, promote the healing or prevention of blisters, sores and skin degeneration, associated with Hereditary Vesicular Epidermolysis. Variants with N or C termination, such as EXKKTET and LKKTETQ are included. Without being bound to any particular theory, these peptides may have the ability to promote repair, healing and prevention by having the ability to induce terminal deoxynucleotidyl transferase (a DNA polymerase not driven by model), to reduce levels of one or more cytosine or inflammatory chemosins, and to act as a chemotactic and / or angiogenic factor for endothelial cells and thus cure and prevent degenerative changes in the skin of patients affected by EB, even when the EB is the result of a hereditary defect. Thymosin ß4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin ß4 was originally isolated from the thymus and is a ubiquitous 43 amino acid polypeptide, 4.9 kDa, which has been identified in a variety of tissues. Several roles have been attributed to this protein, including a role in the migration and differentiation of endothelial cells, the differentiation of T cells, and the vascularization and sequestration of actin.

According to one embodiment, the invention consists of a method of treatment for promoting the healing and prevention of blistering, sores and skin degradation associated with EB, which comprises administration to a subject in need of such treatment. , of an effective amount of a composition comprising an EB-inhibiting polypeptide, which comprises the amino acid sequence LKKTET, or a conservative variant thereof, which has EB-inhibiting activity, preferably Thymosin-4, an isoform of the Thymosin ß4, Oxidized thymosin ß4, Thymosin sulfoxide ß4 or a thymosin β4 antagonist. Compositions which may be used in accordance with the present invention include thymosin β4 (β4), isoforms of β4, oxidized β4, thymosin β4 sulphoxide, polypeptides or any other protein or protein that is so 1 aa and which have actin binding domains, or peptide fragments that comprise or consist essentially of the LKKTET amino acid sequence or conservative variants thereof, which have EB inhibitory activity. International patent application number PCT US99 / 17282, which is incorporated herein by reference, discloses ß4 isoforms which may be useful in accordance with the present invention, as well as an amino acid sequence LKKTET and conservative variants of it having EB inhibiting activity, which can be used with the present invention. International patent application number PCT / GB99 / 00833 (WO 99/49883), which is incorporated herein by reference, describes oxidized thymosin β4 which can be used in accordance with the present invention. Although the present invention is described hereinafter primarily with respect to the ß4 and isoforms of the ß4, it should be understood that the following description is intended to be equally applicable to an amino acid sequence LKKTET, peptides and fragments comprising or consist essentially of LKKTET, conservative variants thereof having EB inhibitory activity, as well as oxidized ß4 thymosin. In one embodiment, the invention provides a method of treatment for curing and preventing the formation of blisters and sores on the skin of a subject by contacting said skin with an effective EB-inhibiting amount of a composition containing? ß4 or an isoform of? ß4. The contact can be topically or systemically. Examples of topical administration include, for example, contact of the skin with a lotion, balm, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment, or. oil comprising ß4, alone or in combination with at least one agent that improves the penetration of ß4, or slows down or slows down the release of the ß4 peptides within the area to be treated. Systemic administration includes, for example, intravenous, intraperitoneal, intramuscular or subcutaneous injections, or inhalation, or transdermal or oral administration of a composition containing the? 4 or an isoform of the? 4, etc. A subject can be any mammal, preferably a human being. The? ß4 or its analogs, isoforms or derivatives can be administered in any suitable inhibitory amount of EB. For example, it can be administered in doses within the range of about 0.1 to 50 micrograms of ß4, more preferably of about 1 to 25 micrograms. A composition according to the present invention can be administered daily, every third day, etc., with a single application or with multiple applications per day of administration, such as applications 2, 3, 4 or more times per day of administration.

Isoforms of ß4 have been identified that are approximately 70%, or approximately 75%, or approximately 80% or more in homology to the known amino acid sequence of ß4. Such isoforms include, for example, ß43?!?? 9,? ß?,? ß?,? ß412,? ß13,? ß14 and ß15. In the same way as? ß4, it has been shown that isoforms? ß ?? and? ß15 sequester actin. The? ß4,? ß ?? and? ß15, as well as these other isoforms share the amino acid sequence LKKTET, which seems to be involved in the mediation of the sequestration or binding of actin. While not wishing to be bound by any particular theory, the activity of the ß4 isoforms may be due, in part, to the ability to regulate the polymerization of actin. For example,? ß4 can modulate the polymerization of actin in the skin (for example, ß-thymosins appear to depolymerize F-actin by sequestering free G-actin). The ability of? ß4 to modulate the polymerization of actin may, therefore, be wholly, or in part, due to its ability to bind to or sequester actin through the LKKTET sequence. Thus, as with? ß4, other proteins that bind or sequester to actin, or that modulate the polymerization of actin, including isoforms of? ß4 having the amino acid sequence LKKTET, are likely to reduce the EB, alone or in combination with the? ß4, as stated here. Therefore, it is specifically contemplated that the known isoforms of the ? ß4, such as? ß4a? 3,? ß9,? ß ??,? ß? ?,? ß412,? ß13,? ß14? ? ß15, as well as isoforms of? ß4 that have not yet been identified, will be useful in the methods of the invention. As such, isoforms of ß4β are useful in the methods of the invention, including methods carried out in a subject. The invention therefore further provides pharmaceutical compositions comprising the? 4, as well as the? -4 isoforms, such as?? 4 ?,? 9,? Β, β? ?,? ß412,? ß13,? ß14 and ß15, and a pharmaceutically acceptable carrier. In addition, other proteins that have the ability to sequester or bind to actin, or that can mobilize actin or modulate actin polymerization, as demonstrated in a suitable assay for sequestration, binding, mobilization or polymerization, or as identified by the presence of an amino acid sequence that mediates the binding of actin, such as LKKTET, for example, can similarly be employed in the methods of the invention. P rotein tials include 1 g alsolin, vitamin D de nion (DBP), profilin, cofilin, adverse, propomycin, fincilin, depactin, Dnasel, vilin, fragmin, severin, coronal coating protein, β- actinin, and acumetin, for example. As such methods include those that are practiced in a subject, the invention further provides pharmaceutical compositions comprising gelsolin, vitamin D binding protein (DBP), profilin, cofilin, depactin, Dnasel, vilin, fragmin, severin, coating protein coronal, ß -actinin, and acumetin as established here. In this manner, the invention includes the use of a polypeptide that inhibits EB, which comprises 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 variants include the replacement of a hydrophilic residue such as isoleucine, valine, leucine or methionine, on the other hand, the replacement of one polar residue by another, such as the substitution of arginine with lysine, glutamic acid with aspartic acid, or glutamine for asparagine, and the like. The? ß4 has been located in a number of tissues and types of cells and, in this way, agents that stimulate the production of? ß4 can be added to the composition, or which comprise a composition for effecting the production of the ß4 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), epidemial growth factor (EGF), growth factor beta. transformation (TGF-ß), basic fibroblast growth factor (bFGF), thymosin al (Tal) and vascular endothelial growth factor (VEGF). More preferably, the agent is the transforming growth beta factor (TGF-β) or other members of the TGF-β super family. The compositions of the ß4 of the invention can reduce the effects of EB by effecting the growth of connective tissue through extracellular matrix deposition, cell migration and vascularization of the skin. According to one embodiment, the subjects are treated with an agent that stimulates the production in the subject of a peptide inhibitor of EB as defined above. Additionally, agents that help or that stimulate the reduction of EB, together with the ß4 or an isoform of the ß4, can be added to the composition. Such agents include angiogenic agents, growth factors, agents that direct cell differentiation, agents that promote the migration of cells and agents that stimulate the provision of extracellular matrix material in the skin. For example, and not by way of limitation, either ß4 or an isoform of ß4 alone or in combination with any or more of the following agents may be added: V EGF, K GF, FGF, P DGF, T GF-β, IGF-1, IGF-2, IL-1, protymosin ay thymosin al in an effective amount. The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of ß4 or a ß4 isoform in a pharmaceutically acceptable carrier. Such carriers include those of the above list with reference to parenteral administration.

The actual dose of reagent, formulation or composition that heals or prevents the blisters, sores and skin degeneration that is associated with EB may depend on many factors, including the size and health of the subject. However, persons with ordinary skill in the art can use the teachings described in the methods and techniques for the determination of clinical doses as described in PCT US99 / 17282, supra, and the references cited herein, for determine the appropriate dose of use. Suitable topical formulations include ß4 or a ß4 isoform in a concentration within the range of about 0.001 to 10% by weight, more preferably within the range of about 0.01 to 0.1% by weight, more preferably about 0.05% by weight. The therapeutic approaches described herein involve various routes of administration or release of the reagents or compositions comprising the ß4 or other compounds of the invention, including any conventional administration techniques (for example), but not limited to, topical administration, injection local, inhalation or systemic administration), to a subject. The methods and compositions which use or contain the ß4 or other compounds of the invention can be formulated into pharmaceutical compositions by mixing them with non-toxic and pharmaceutically acceptable carriers or excipients. The invention includes the use of antibodies that interact with the? -4 peptide or functional proteins thereof. Antibodies are provided which consist essentially of accumulated monoclonal antibodies with different epitopic specificities, as well as different preparations of monoclonal antibodies. Monoclonal antibodies are prepared from fragments of the antigen-containing protein, by methods that are well known to those skilled in the art as described in PCT US99 / 17282, supra. The term "antibody" as used in this invention means that it includes monoclonal and polyclonal antibodies. In still another embodiment, the invention provides a method for the treatment of a subject by administering an effective amount of an agent that regulates the expression of the? ß4 gene. The term "regular" refers to the inhibition or suppression of ß4 expression when ß4 is over-expressed, and the induction of ß4 expression when ß4 is poorly expressed. The term "effective amount" means that amount of? ß4 agent that is effective in regulating the expression of the? ß4 gene resulting in the reduction of? ß4 symptoms associated with EB. An agent that regulates the expression of the ß4 gene or the isoforms of the ß4 can be a polynucleotide for example. The polynucleotide may be an antisense, a triplex agent, or a ribosome. For example, an antisense directed to the region of the structural gene or to the region of the? -4 promoter can be used. In another embodiment, the invention provides a method for the use of compounds that regulate the activity of β4. Compounds that affect the activity of β4 (for example, antagonists and agonists) include peptides, peptimetimetics, polypeptides, chemical compounds, minerals such as zinc and biological agents. While not bound by a particular theory, it is believed that the present invention can promote the healing or prevention of blisters, sores and skin degeneration associated with Hereditary Vesicular Epidermolysis by inducing terminal deoxynucleotidyl transferase (a DNA polymerase). not directed by model), to decrease the levels of one or more of the inflammatory cytokines or chemokines, and to act as a chemotactic factor for endothelial cells, and in this way promote the healing or prevention of degenerative changes in the skin that are Caused by Hereditary Vesicular Epidermolysis or other degenerative or environmental factors.

Claims (18)

1. Novelty of the Invention 1. A method of treatment for promoting healing or preventing the formation of blisters, sores or skin degeneration that is associated with Hereditary Vesicular Epidermolysis, which comprises administration to a subject in need of such treatment. , of an effective amount of a composition comprising a polypeptide inhibitor of Hereditary Vesicular Epidermolysis, which comprises the amino acid sequence LKKTET, or a conservative variant thereof, which has inhibitory activity of Hereditary Vesicular Epidermolysis. The method of claim 1, wherein said polypeptide promotes the improvement of skin condition, including an increase in skin elasticity of said subject. 3. The method of claim 1, wherein said polypeptide comprises thymosin ß4 (ß4), a variant with N-terminus of ß4, a variant with C-terminus of ß4, an isoform of ß4, ß4 oxidized or ß4-sulfoxide. 4. The method of claim 1, wherein said composition is administered systemically. The method of claim 1, wherein said composition is topically administered. The method of claim 5, wherein said composition is in the form of a gel, cream, paste, lotion, spray, suspension, dispersion, balsam, hydrogel, or ointment formulation. The method of claim 6, which further comprises at least one agent that delays the release or that improves the penetration of the? ß4 within an area to be treated. 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 8, wherein said antibody is polyclonal or monoclonal. 11. A method of treatment for promoting healing or preventing the formation of blisters, sores or skin degeneration, associated with Hereditary Vesicular Epidermolysis, which comprises the administration to a subject in need of such treatment, of an effective amount of a composition comprising an agent that stimulates the production of a polypeptide inhibitor of Hereditary Vesicular Epidermolysis, which comprises the amino acid sequence LKKTET, or a conservative variant thereof, which has inhibitory activity of Hereditary Vesicular Epidermolysis. 12. The method of claim 11, wherein said polypeptide is thymosin β4. The method of claim 11, wherein said agent is a thymosin β4 antagonist. 14. A composition for use in promoting healing or preventing the formation of blisters, sores or skin degeneration, associated with Hereditary Vesicular Epidermolysis, which comprises an effective amount of a composition that includes a polypeptide inhibitor of the Vesicular Hereditary Epidermolysis, which comprises the amino acid sequence LKKTET, or a conservative variant thereof, which has activity of inhibition of Hereditary Vesicular Epidermolysis. 15. The composition of claim 14, wherein said composition comprises a variant of LKKTET with N or C terminus. 16. The composition of claim 14, wherein said composition comprises KLKKTET or LKKTETQ. 17. The composition of claim 14, wherein said polypeptide comprises ß4, an isoform of γ4, γ4 oxidized or ß4 sulfoxide. The composition of claim 14, which comprises a gel, cream, paste, lotion, spray, suspension, dispersion, balsam, hydrogel, or ointment formulation.