MXPA04007579A - Treatment of infections and other disorders. - Google Patents

Abstract

Microbial infections including anthrax infection, and gastrointestinal disorders, are treated or prevented by administration of an actin-sequestering peptide including amino acid sequence LKKTET, such as Thymosin beta4, an isoform of Thymosin beta4, oxidized Thymosin beta4, or Tbeta4 sulfoxide.

Description

PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF INFECTIONS AND OTHER DISORDERS Field of the Invention The present invention relates to the field of treatment of microbial infections and other gastrointestinal disorders.

Background of the Invention The present patent application claims the benefit of the provisional patent applications of the United States of America numbers 60 / 421,038, filed on October 25, 2002, and 60 / 354,250, filed on February 6, 2002. The treatment of microbial, bacterial, viral and fungal infections can be difficult by conventional methods. These infections can include gastrointestinal infections (E. Coli, H. Pylori, VRE, etc.), abdominal infections (peritonitis, pancreatitis, gallbladder infections, etc.), infections due to surgical operations and osteomyelitis (bone infection). Other examples of microbial infection is anthrax. Anthrax is an infectious agent caused by Bacillus anthracis, a gram-positive organism. It is primarily a disease of herbivores. Anthrax can affect many different vertebrates, including humans. The symptoms of anthrax vary widely depending on the route of infection. Three forms of disease commonly occur, which include the cutaneous form, the gastrointestinal form and the pulmonary form (inhalation). The pulmonary form of the disease is typically caused by the inhalation of anthrax spores. The symptoms of systemic anthrax can be reproduced in a large number of animal models by administering virulence factors (endotoxins) that are responsible for the main pathologies, morbidity and mortality observed with anthrax. Once large quantities of anthrax toxins are produced within the body by the bacteria, the administration of antibiotics is usually ineffective. The pathologies induced by anthrax produce septic shock and sudden death that is observed with other gram-positive and gram-negative bacterial infections, such as multi-organ failure, edema and A DS. In both animals and humans anthrax-induced pathologies also include the marked elevation of TNF-α, IL-1β, PAF and a large number of other inflammatory cytokines. The overproduction of reactive oxygen intermediates and the increase in aracidonic acid metabolites such as PGE and thromboxane β2, as well as the destruction of the actin cytoskeleton are also observed in anthrax-induced septic shock. A large number of approaches that delay, prevent and / or treat exposure to anthrax have been reported. In the area of prevention, a vaccine for humans is available, but the effectiveness of the vaccine is unclear. The best treatment currently available consists of treatment with specific antibiotics such as ciprofloxaxin or doxycycline. Antibiotics are effective if they are provided in the very early stages of infection and are basically ineffective once the bacteria have had the opportunity to multiply rapidly by producing lethal amounts of the deadly anthrax toxins. Of the three forms of anthrax, the deadliest form is pulmonary anthrax (inhalation), which has a mortality rate of more than 75% (even with the treatment of appropriate antibiotics). Anthrax produces a multi-component toxin that mounts on the surface of host cells after infection. The lethal action of anthrax toxins occurs in the cytoplasm of the host cells. Anthrax toxin is just one of the many multi-unit toxins that causes severe disease in humans. A major concern when treating bacterial infections with antibiotics is the appearance of an increasing number of strains resistant to antibiotics. further, once the anthrax bacillus has produced large amounts of exotoxins antibiotics are basically ineffective. Millions of Americans suffer from other gastrointestinal (GI) disorders such as colitis, ileitis, Crohn's disease, ulcerative colitis, colic, gingivitis, regional enteritis, ulcers, sac swelling, sclerosis, cholangitis, fistulas. The cause of many of these diseases is not known. However, these may have genetic roots or may result from exposure to certain chemicals, pathogens, immune dysfunctions, or food during the lifetime, or may result from normal aging of the human body. GI disorders occur in both men and women and can be acute or chronic, debilitating and life threatening, and can occur anywhere within the GI tract including, but not limited to, the mouth, throat, esophagus, stomach, large intestines and thin, colon and anus. People suffering from GI disorders can have a greatly diminished quality of life and may suffer premature death. A large number of therapeutic approaches have been reported for the treatment of gastrointestinal disorders and diseases, depending on the location and nature of the GI pathology. Treatments vary from surgical intervention, food manipulations and the use of a variety of drugs and biological agents. These biological agents include antibiotics, anti-viral drugs, anti-inflammatory drugs, glucocorticoids, immunosuppressive drugs, monoclonal antibodies, antacids, anti-secretory drugs, anti-spasmodics, as well as a large number of other agents. Numerous pharmaceutical, nutraceutical or cosmeceutical formulations have been proposed for the treatment of damage caused by microbial infections and gastrointestinal disorders. There is still a need in the art for improved methods and compositions for the cure or prevention of damage caused by microbial infections and gastrointestinal disorders.

SUMMARY OF THE INVENTION In accordance with the present invention, the treatment of infections and gastrointestinal disorders (GI) comprises the administration to a subject or patient in need of such treatment, of an effective amount of a composition comprising the amino acid sequence LKKTET, or a conservative variant of it.

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 treatment of microbial infections and gastrointestinal disorders. 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 the levels of one or more inflammatory cytokines or chemokines, and to act as a chemotactic and / or angiogenic factor for endothelial cells and in this way treat the damage caused by microbial infections and gastrointestinal disorders. Initially Thymosin ß4 was identified as a protein that is up-regulated during endothelial cell migration and in vitro differentiation. 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 treating the damage associated with microbial infections, which comprises the administration to a subject in need of said treatment, of an effective amount of a composition comprising an infection-inhibiting polypeptide. microbial, which comprises the amino acid sequence LKKTET, or a conservative variant thereof, which has microbial infection inhibiting activity, preferably thymosin β4, an isoform of thymosin β4, thymosin β4 oxidized, thymosin sulphoxide β4 or an antagonist of Thimosin ß4. Compositions which may be used according to the present invention include thymosin ß4 (ß4), isoforms of ß4, γ4 oxidized, thymosin sulphoxide ß4, polypeptides or any other sequestering or binding proteins to actin and having actin binding domains, or peptide fragments comprising or consisting essentially of the LKKTET amino acid sequence or conservative variants thereof, which have microbial infection inhibitory activity. International patent application number PCT / US99 / 17282, which is incorporated herein by reference, discloses isoforms of ß4 which may be useful in accordance with the present invention, as well as an amino acid sequence LKKTET and conservative variants of this which have microbial infection 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, discloses oxidized thymosin β4 which can be used in accordance with the present invention. While the present invention is described hereinafter primarily with respect to the? 4 and isoforms of the? 4, it should be understood that the following description is intended to be equally applicable to the amino acid sequence LKKTET, peptides and proteins comprising or consist essentially of LKKTET, conservative variants thereof having microbial infection inhibitory activity, as well as oxidized thymosin β4. In a modality, the invention provides a method for curing and preventing damage caused by microbial infection in a subject by contacting the area to be treated with an effective amount of a microbial infection inhibiting composition, which contains ß4 or an isoform of? ß4. The contact can be topically, systemically or enterically. Examples of topical administration include, for example, contact of the skin with a lotion, ointment, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment or oil comprising the? 4 alone or in combination with when less an agent that improves the penetration of the? ß4, or that delays 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. Enteral administration may include oral or rectal administration. A subject can be any mammal, preferably a human being. The? ß4 or its analogs, isoforms or derivatives can be administered in any effective amount. For example,?? 4 can be administered in doses within the range of about 0.1 to 50 micrograms of?? 4, more preferably in amounts in the range 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?! 1,? ß9,? ß ??,? ß? ?,? ß412,? ß 13,? 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 abduct 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 microbial infection alone or in combination with? ß4, as stated here. Therefore, it is specifically contemplated that the known isoforms of? Β4, such as? Β43'3, ββ9, ββ, ββ? ?,? ß412,? ß13,? ß14 and ß15, as well as the 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 ß4, as well as the isoforms of ß4, such as Tp4ala, ß9, ß3, ß2. ?,? 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. Such proteins include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adsevertin, propomyosin, fincilin, depactin, Dnasel, vilin, fragmin, severin, coronal coating protein, β-actinin, and acumetin, by 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, β-actinin, and acumetin as established here. In this manner, the invention includes the use of a polypeptide inhibitor of microbial infection, 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 hydrophobic residue such as isoleucine, valine, leucine or methionine, on the other, the replacement of one polar residue by another, such as the substitution of arginine by Usina, glutamic acid by aspartic acid, or glutamine for asparagine, and the like. The? ß4 has been located in a number of tissues and cell types and, in this way,can be added to the composition, or have it contain agents that stimulate the production of? ß4, to effect the production of? 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. 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 microbial infections by effecting the growth of the connective tissue through extracellular matrix deposition, cell migration and vascularization of the skin. Additionally, other agents may be added to the composition in conjunction with the ß4 or an ß4 isoform. Such agents include angiogenic agents, growth factors, agents that direct 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 one or more of the following agents may be added: VEGF, KGF, FGF, PDGF, TGF-β, IGF-1, IGF-2, IL-1, protimosin and thymosin al in an effective amount. The actual or reactive dose, formulation or composition that heals the damage associated with the microbial infection 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 here, to 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? Β4 or other compounds of the invention, including any conventional administration techniques to a subject (eg, but not limited to, topical administration). , local injection, inhalation, systemic or enteric administration). The methods and compositions that use or contain the ß4 or other compounds of the invention can be formulated into pharmaceutical compositions by mixing them with pharmaceutically acceptable non-toxic carriers or excipients. The invention includes the use of antibodies that interact with the? -4 peptide or functional fragments thereof. Antibodies are included that include accumulated monoclonal antibodies with different epitopic specificities, as well as different preparations of monoclonal antibodies. The 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 one embodiment, the invention provides a method for the treatment of bacterial infections, which comprises the administration to a subject in need of said treatment, of an effective amount of a composition comprising a polypeptide inhibitor of microbial infection, which comprises the amino acid sequence LKKTET, or a conservative variant thereof and having inhibitory activity of the microbial infection. In another embodiment, the invention provides a method for the treatment of gastrointestinal infections. Common gastrointestinal infections include, but are not limited to, Helicobacter pylori (H. pylori), Escherichia coli (E. coli), Vancomycin-resistant Enterococcus faecalis (VRE), and methicillin-resistant Staphylococcus aureus (MRSA). systemically released by injection, orally, nasally, through suppositories or enemas, transdermally or in any other way that is suitable In another embodiment, the invention provides a method for the treatment of anthrax infections. Damage caused by infection with anthrax includes, septic shock, sudden death, multiple organ failure, edema, ARDS and inflammatory, degenerative and immune damage.The composition can be applied alone or in combination with an antibiotic such as ciprofloxocin, doxycycline or penicillin A therapeutically effective amount of the composition is applied to the site or systemically on a periodic basis during the course of therapy to reduce the effects of mortality and morbidity from exposure to biological agents such as anthrax or to prevent such effects. The composition can also be delivered systemically by injection, orally, nasally, through any other means to reduce the toxicity of pulmonary or gastrointestinal anthrax. Another aspect of the invention is the treatment of other gastrointestinal disorders. According to one embodiment, the invention consists in a method for the treatment of damage associated with gastrointestinal disorders, which comprises the administration to a subject requiring said treatment, of an effective amount of a composition comprising a polypeptide inhibitor of disorders. gastrointestinal, which comprises LKKTET, or a conservative variant thereof and which has inhibitory activity of gastrointestinal disorders. This invention is applicable to inflammatory, ulcerative, degenerative, immunological and other damages and disorders of the gastrointestinal tract (from the mouth to the anus). These disorders may be due to genetic abnormalities, food intolerance, exposure to chemicals, aging and microbial infections. Gastrointestinal disorders to which the invention is applicable include, but are not limited to, gastrointestinal infections that include bacterial, viral and fungal infections, disorders associated with environmental or iatrogenic abrasions, inflammations and other inflammatory disorders, immunological diseases, allergies. which include food allergies, Crohn's disease, ulcerative colitis, recurrent aged stomatitis (recurrent chancre sores), ileitis, colic, gingivitis, regional enteritis, ulcers, sac inflammation, sclerosis, cholangitis, fistulas and genetic abnormalities. These may result from exposure to certain chemicals, pathogens, immune dysfunction, or food during the lifetime, or may result from normal aging of the human body. In one embodiment, the invention provides a method for curing the damage caused by gastrointestinal disorders in a subject by contacting the skin with gastrointestinal disorder, with an effective amount of a composition containing ß4 or an xsoform of Tp4. The contact can be topically, systemically or enterically. Examples of topical administration include, for example, contact of the skin with a lotion, ointment, gel, cream, paste, spray, suspension, dispersion, hydrogel, ointment or oil comprising the? 4 alone or in combination with when less an agent that improves the penetration of the? ß4, or that delays 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 (oral or nasal), or transdermal, suppository 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 invention is also directed to a substance for use in the manufacture of a medicament for the treatment of microbial infections including anthrax, and gastrointestinal disorders, which comprises the amino acid sequence LKKTET, or a conservative variant thereof.

Example The? ß4 showed antimicrobial activity against Staphylococcus aureus and Escherichia coli at concentrations of 5-20 nmol / ml, and increased dose dependent activity against those microorganisms at concentrations of 50-200 n mol / ml.

Claims (15)

1. Novelty of the Invention 1. A pharmaceutical composition for the treatment of microbial infections, which comprises a polypeptide inhibitor of microbial infections, which includes the amino acid sequence LKKTET, or a conservative variant thereof, which has inhibitory activity of microbial infections. The composition of claim 1, wherein said polypeptide comprises ß4 thymosin (ß4), a variant of ß4 with N-terminus, a variant of ß4 with C-terminus, an isoform of ß4, ß4 oxidized, or ß4-sulfoxide. 3. The composition of claim 1, wherein said composition is administered systemically. 4. The composition of claim 1, wherein said composition is administered topically. The composition of claim 1, wherein said composition is administered enterically. 6. The composition of claim 1, wherein said microbial infection is a bacterial infection. The composition of claim 1, wherein said microbial infection is an anthrax infection. The composition of claim 1, wherein said microbial infection is a gastrointestinal infection. 9. A pharmaceutical composition for the treatment of gastrointestinal disorders, which comprises a polypeptide inhibitor of gastrointestinal disorders, which includes the amino acid sequence LKKTET, or a conservative variant thereof, which has gastrointestinal disorders inhibitory activity. The composition of claim 9, wherein said gastrointestinal disorders are associated with infection, abrasion, inflammation, allergy, immune disorder, or genetic abnormality. The composition of claim 9, wherein said gastrointestinal disorder is Crohn's disease. 12. The composition of claim 9, wherein said gastrointestinal disorder is ulcerative colitis. The composition of claim 9, wherein said gastrointestinal disorder is recurrent aged stomatitis. 14. The use of a polypeptide comprising the amino acid sequence LKKTET or a conservative variant thereof, for the manufacture of a drug useful for the treatment of microbial infections. 15. The use of a polypeptide comprising the amino acid sequence LKKTET or a conservative variant thereof, for the manufacture of a medicament useful for the treatment of gastrointestinal disorders.