KR20070019668A - Method of treating or preventing biological or immunological responses to a reactive chemical or biological or toxic agent - Google Patents

Abstract

The present invention provides a reaction-inhibitor comprising the amino acid sequence LKKTET, a conserved variant thereof, or a reagent that stimulates the production of an LKKTET peptide to inhibit a biological or immunological response to a reactive chemical, biological or toxin by a tissue of a subject or Treating, preventing a biological or immunological response to a reactive chemical, biological substance, or toxin by a subject's tissue, the method comprising administering to the subject in need thereof an effective amount of a composition comprising a conserved variant thereof , Method of inhibiting or reducing.

Description

METHOD OF TREATING OR PREVENTING BIOLOGICAL OR IMMUNOLOGICAL RESPONSES TO A REACTIVE CHEMICAL OR BIOLOGICAL OR TOXIC AGENT}

The present invention claims the priority of US Provisional Application No. 60 / 530,893, filed December 22, 2003.

The present invention relates to the field of treating or preventing a biological or immunological response to a reactive chemical or biological substance.

Contact dermatitis and other allergic reactions due to chemical or biological skin sensitizers, environmental toxins or irritants can cause redness, edema, hardening, rash, blisters, burning sensation, inflammation or eczema skin changes in people.

Many therapies have been proposed for such diseases, but erythema, redness, edema, swelling, rash, itching, blisters and / or inflammation due to physiological and immunological reactions to reactive chemicals, biological materials or toxins are suggested. There is still a need in the art for improved methods and compositions for treating or preventing.

According to one aspect, a therapeutic method for treating, preventing, inhibiting or reducing a biological or immunological response to a reactive chemical, biological or toxin by a subject's tissue comprises a reaction comprising the amino acid sequence LKKTET to inhibit the response. Administering to the subject in need of such treatment an effective amount of a composition comprising an inhibitor, a conserved variant thereof, or a reagent that stimulates the production of an LKKTET peptide or a conserved variant thereof.

Although not shown in any particular theory, actin-sequencing polypeptides such as thymosin beta 4 (Tβ4 or TB4) and other including actin-sequencing peptides or peptide fragments comprising the amino acid sequence LKKTET or a conserved variant thereof Response-inhibitors encourage the reversal or prevention of biological or immunological responses from exposure to reactive chemicals, biological materials or toxins. The present invention includes: biological or immunological reactions of surface tissues such as skin or mucous membranes, dermatology and other diseases due to allergic reactions, reactions to chemicals and toxins, contact dermatitis and ivy sumac, toxic oak, toxic lacquer But reaction to plants not limited to these; Contact of various animals with the poison of their skin, such as toxic frogs; And allergic reactions of the lung and gastrointestinal system. The invention may also be applied to skin sensitizers, psoriasis, atopic dermatitis and other symptoms that may be manifested by eczema and scaling patches and plaques or by blister and vesicle changes. The invention may also be applied to allergic contact dermatitis by occupation, including but not limited to nickel-related dermatitis.

Thymosin 4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin 4 is a polypeptide that is native throughout the 4.9 kDa, originally isolated from the thymus and 43 amino acids identified in various tissues. Several roles are believed to belong to this protein, including its role in endothelial cell differentiation and migration, T cell differentiation, actin questration, angiogenesis and wound healing.

According to one embodiment, the present invention provides a biological or immunological response-inhibitor, preferably thymosin β4, which may be a polypeptide comprising an amino acid sequence LKKTET or a conserved variant thereof having biological or immunological response-inhibitory activity, and And / or an effective amount of a composition comprising a Tβ4 isoform, analog or derivative, such as KLKKTET, LKKTETQ, oxidized Tβ4, Tβ4 soxide, N-terminal variant of Tβ4, C-terminal variant of Tβ4 and antagonist of Tβ4 A method of treatment for treating, preventing, inhibiting or reducing a biological or immunological response to a reactive chemical, biological substance or toxin by a subject's tissue, comprising administering to a subject in need of such treatment. Compositions that can be used according to the invention include thymosin β4 (Tβ4) and / or Tβ4 isoforms, analogs or derivatives, such as oxidized Tβ4, Tβ4 sloxides, N-terminal variants of Tβ4, C-terminal variants of Tβ4. And reagents such as antagonists of Tβ4, amino acid sequence LKKTET or a conserved variant thereof, consisting essentially of them, polypeptides having biological or immunological response-inhibiting activity, or peptide fragments. International patent application PCT / US99 / 17282, which is incorporated herein by reference, discloses not only Tβ4 which can be used according to the invention, but also amino acids having biological or immunological response-inhibiting activity which can be used according to the invention. The sequence LKKTET or a conserved variant thereof is disclosed. International patent application PCT / GB99 / 00833 (WO 99/49883), incorporated herein by reference, discloses oxidized thymosin β4 that can be used in accordance with the present invention. The present invention is mainly described below with respect to Tβ4 and Tβ4 isoforms, but the following description comprises the amino acid sequence LKKTET, essentially LKKTET, and peptides or fragments composed thereof, having a biological or immunological response-inhibiting activity thereof It should be understood that it is intended to be applied to conserved variants and / or Tβ4 isoforms, analogs or derivatives such as oxidized Tβ4, Tβ4 sloxides, N-terminal variants of Tβ4, C-terminal variants of Tβ4 and antagonists of Tβ4. .

In one embodiment, the present invention relates to reactive chemicals, biological substances or toxins by a subject's tissue by contacting the tissue with a biological or immunological response-inhibiting effective amount of a composition containing a reaction-inhibitor as disclosed herein. Methods of treatment are provided that treat, prevent, inhibit or reduce biological or immunological responses. By way of non-limiting example, the tissue may be selected from surface tissue such as skin or mucous membranes of the subject, lung tissue of the subject, or gastrointestinal tract tissue of the subject. Contact may be local or systemic. Examples of topical administration include contacting the skin with oils, including lotions, plasters, gels, creams, pastes, sprays, suspensions, dispersants, hydrogels, ointments or reaction-inhibiting agents as disclosed herein. Systemic administration includes, for example, intravenous, intraperitoneal, intramuscular infusion of a composition containing a reaction-inhibitor disclosed herein in a pharmaceutically acceptable carrier such as water for injection.

Response-inhibitors for use in the present invention as disclosed herein may be administered in any suitable biological or immunological response-inhibitory amount. For example, the reaction-inhibitor as disclosed herein may be in dosages in the range of about 0.001-1,000,000 micrograms, more preferably about 0.1-5,000 micrograms, and most preferably about 1-25 micrograms. May be administered.

The composition according to the present invention may be administered daily, every other day, such as a single administration or multiple administrations per administration day such as 2, 3, 4 or more administrations per day.

Many Tβ4 isoforms are identified against known amino acid sequences of Tβ4 and have at least about 70%, or about 75%, or about 80% homology. Such isoforms include, for example, Tβ4 ^ala , Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15. Similar to Tβ4, Tβ10 and Tβ15 isoforms appear to request actin. Tβ4, Tβ10 and Tβ15, as well as other isoforms, share the amino acid sequence LKKTET which appears to be involved in mediating actin sequencer or binding. Without wishing to be bound by any particular theory, the activity of Tβ4 isoforms may be due, in part, to the ability to polymerize actin. For example, T [beta] 4 can modulate actin polymerization reactions in the skin (e.g., [beta] -thymosin appears to depolymerize F-actin by ordering free G-actin). The ability of Tβ4 to modulate the actin polymerization reaction may be due, in whole or in part, to the ability to bind or sequence actin via the LKKTET sequence. Thus, other proteins that bind to or sequence actin, or modulate actin polymerization, including Tβ4 isoforms having the amino acid sequence LKKTET, such as by Tβ4, can be used alone or in combination with Tβ4 as shown herein. Easy to be

Thus, Tβ4 isoforms, such as Tβ4 ^ala , Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15, as well as unidentified Tβ4 isoforms are expected to be used in the methods of the present invention. As such, the Tβ4 isoforms are useful in the methods of the present invention, including methods performed on a subject. Accordingly, the present invention further provides pharmaceutical compositions comprising Tβ4 as well as Tβ4 isoforms Tβ4 ^ala , Tβ9, Tβ10, Tβ11, Tβ12, Tβ13, Tβ14 and Tβ15, and pharmaceutically acceptable carriers.

It also has actin sequencer or binding ability, indicated by appropriate sequencer, binding, transfer or polymerization reaction analysis, or identified in the presence of an amino acid sequence that mediates actin binding such as LKKTET, or moves actin, or Or other reaction-inhibitors or proteins that can control the actin polymerization reaction can be similarly used in the methods of the present invention. Such proteins include, for example, gelzoline, vitamin D binding protein (DBP), propyline, cophylline, dipactin, dennazel, billin, pragmin, severin, capping protein, β-actin and accutin. Since such methods include those conducted in a subject, the present invention provides gelzoline, vitamin D binding protein (DBP), propyline, cophylline, dipactin, dennazel, billin, pragmin, severin, capping as described herein. It further comprises a pharmaceutical composition comprising a protein, β-actin and accutin. Accordingly, the present invention encompasses the use of EB-inhibiting polypeptides comprising the amino acid sequence LKKTET or a conserved variant thereof.

As used herein, the term "conserved variant" or grammatical variants thereof refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservation changes include replacement of hydrophobic residues such as isoleucine, valine, leucine, or methionine with other residues, polar residues with arginine for lysine, glutamic acid for aspartaic acid, glutamine for asparagine, and other residues. It includes replacing with

Tβ4 is localized to many tissue and cell types, and as disclosed herein, reagents that stimulate the production of LKKTET peptides, such as Tβ4 or other reaction-inhibitors, affect the production of reaction-inhibitors from tissues and / or cells. The medicinal composition may include or be added to the composition. Such stimulants include insulin type growth factor (IGF-1), platelet induced growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), T Members of the group of growth factors such as mother kin α1 (Tα1) and vascular endothelial growth factor (VEGF). More specifically, the stimulant is transforming growth factor beta (TGF-β) or other members of the TGF-β superfamily. The compositions of the present invention can reduce the effects of biological or immunological reactions on reactive chemicals or biological materials by effecting growth of connective tissue through extracellular matrix, precipitation, cell migration and angiogenesis.

According to one embodiment, subjects are treated with a stimulant that stimulates intra-subject production of a biological or immunological response-inhibitor as defined herein.

In addition, other reagents that assist in the reduction of biological or immunological responses to reactive chemicals, biological materials, or toxins may be added to the composition with a reaction-inhibitor as disclosed herein. Such other reagents include angiogenic reagents, growth factors, reagents that direct cell differentiation, reagents that stimulate cell migration and reagents that stimulate the provision of extracellular matrix material in tissues. For example, but not limited to, the reaction-inhibitor as disclosed herein, alone or in the following reagents: VEGF, KGF, FGF, PDGF, TGFβ, IGF-1, IGF-2, IL-1, prothymosin It may be added in combination with one or more effective amounts of α and / or thymosin α1.

The invention also includes pharmaceutical compositions comprising a therapeutically effective amount of a reaction-inhibitor as disclosed herein in a pharmaceutically acceptable carrier. Such carriers include those listed herein.

Actual dosages or reagents, formulations or compositions that provide a therapeutic method for treating, preventing, inhibiting or reducing a biological or immunological response to a reactive chemical, biological or toxin by a subject's tissue include the subject's body and health Will depend on many factors. However, those of ordinary skill in the art may use the teachings describing methods and techniques for determining clinical dosages disclosed in PCT / US99 / 17282 supra to determine appropriate dosages, which are incorporated herein by reference. Quote.

Suitable formulations may comprise the reaction-inhibitors disclosed herein at a concentration in the range of about 0.001-50% by weight, more preferably in the range of about 0.1-0.1% by weight, most preferably about 0.05% by weight.

Treatment attempts described herein may be performed by subjecting a subject to a response-inhibitor as disclosed herein, including, but not limited to, any conventional dosing technique (eg, including but not limited to topical administration, local infusion, inhalation, or systemic administration). Include various paths to pass. Methods of using or containing a reaction-inhibitor as disclosed herein and compositions thereof may be formulated into pharmaceutical compositions as a mixture with pharmaceutically acceptable nontoxic excipients or carriers.

The present invention includes the use of antibodies that interact with reaction-inhibitors as disclosed herein. Antibodies are provided that consist essentially of pooled monoclonal antibodies with different epitope specificities as well as unique monoclonal antibody preparations. Monoclonal antibodies are well known to those skilled in the art as well as prepared from antigens containing fragments of the protein by the methods disclosed in PCT / US99 / 17282 supra. The term antibody as used herein is intended to include monoclonal and polyclonal antibodies.

In another embodiment, the present invention provides a method of treating a subject by administering an effective amount of a stimulant that modulates gene expression. The term "modulate" means inhibiting or inhibiting expression when the response-inhibitor as disclosed herein is overexpressed and inducing expression when the response-inhibitor as disclosed herein is underexpressed. The term "effective amount" refers to the amount of stimulant effective to modulate the gene expression of a response-inhibitor as disclosed herein and to result in a reduction in the symptoms of a biological or immunological response to reactive chemicals, biological substances or toxins. . Stimulants that regulate gene expression of response-inhibitors as disclosed herein are, for example, polynucleotides. This polynucleotide may be an antisense triplex reagent, or ribozyme. For example, antisense directed to a structural gene region or to a promoter region of a reaction-inhibitor as disclosed herein can be used. Stimulators that regulate gene expression of response-inhibitors as disclosed herein may also be small interfering RNAs (siRNAs).

In another embodiment, the present invention provides a method of using compounds that modulate the activity of a reaction-inhibitor as disclosed herein. Compounds (eg, antagonists and agonists) that affect the activity of a reaction-inhibitor as disclosed herein include peptides, peptidomimetics, chemical compounds, zinc and other inorganics and biologicals.

The invention further comprises contacting a tissue exhibiting a biological or immunological response with a candidate compound; Measuring the level of reduction of a biological or immunological response in said tissue, wherein said decrease in said level is less than that of said candidate compound treating said biological or immunological response as compared to the level in a corresponding tissue lacking said candidate compound. To a method for screening a reaction-inhibitor as disclosed herein which indicates that it can be prevented, inhibited or reduced.

The invention further comprises contacting the tissue with a candidate compound; Contacting said tissue with a substrate inducing a biological or immunological response in said tissue in the absence of said candidate compound; Determining a level of reduction of a biological or immunological response in the viable tissue, wherein the reduction of said level treats, prevents, or inhibits a biological or immunological response as compared to the level in a corresponding tissue lacking said candidate compound. Or to a method of screening a reaction-inhibitor as disclosed herein which indicates that it can be reduced.

The invention further comprises contacting a tissue exhibiting a biological or immunological response with a candidate compound as disclosed herein; Measuring the intracellular activity of a Tβ4 or other response-inhibitor as disclosed herein, wherein said activity within said tissue as compared to the activity level of said response-inhibitor in a corresponding tissue lacking said candidate compound An increase in the activity of a Tβ4 or other response-inhibitor as disclosed indicates that the compound can induce the stimulant, as disclosed herein, which can encourage the intra-tissue production of a reaction-inhibitor as disclosed herein. A method for screening stimulants of bars.

The invention further comprises contacting the tissue with a candidate compound; Contacting said tissue with a substrate inducing a biological or immunological response in said tissue in the absence of said candidate compound; Measuring the intracellular activity of the response-inhibitor, wherein an increase in the intra-tissue activity of the response-inhibitor as disclosed herein is compared to the level of activity in the corresponding tissue lacking the candidate compound. A stimulant as disclosed herein that can inspire the production of a reaction-inhibitor as disclosed herein, indicating that the candidate compound is capable of stimulating the intra-tissue production of a reaction-inhibitor as disclosed herein. It relates to a method of screening.

Exposure to ivy lacquer causes one area of skin surface with visible redness, swelling, edema and erythema to be treated by topical application of a drug containing 2% by weight of Tβ4, while exposed to ivy lacquer on the same skin surface Other visible redness, sclerosis, edema and erythema areas due to left untreated. After one day, cure and erythema in the treated area were significantly reduced compared to the untreated area, and the itch of the treated area was significantly less than the untreated area.

Claims (29)

Reaction-inhibitors comprising the amino acid sequence LKKTET, conserved variants thereof, or reagents that stimulate the production of LKKTET peptides or conserved variants thereof to inhibit biological or immunological responses to reactive chemicals, biological substances or toxins by the tissue of a subject Treating, preventing, inhibiting, or treating a biological or immunological response to reactive chemicals, biological substances, or toxins by a subject's tissue, the method comprising administering to the subject in need thereof an effective amount of a composition comprising: Reducing treatment method. The method of claim 1, wherein the biological or immunological response comprises redness, sclerosis, edema, itching, rash, blisters, inflammation, erythema, or a combination thereof. The method of claim 1, wherein the response inhibitor has the ability to down-regulate inflammatory cytokines, chemokines or combinations thereof to result in biological or immunological response-inhibition in the tissue. The method of claim 1, wherein the reaction-inhibitor is thymosin beta 4 (Tβ4). The method of claim 1, wherein the reaction-inhibitor is other than Tβ4. The method of claim 1, wherein the reagent is amino acid sequence KLKKTET, amino acid sequence LKKTETQ and an N-terminal variant of Tβ4, a C-terminal variant of Tβ4, an isoform of Tβ4, oxidized Tβ4 or Tβ4 sulfoxide. The method of claim 1, wherein the reaction-inhibiting agent inhibits the reaction directly or indirectly. 8. The method of claim 7, wherein the response-inhibitor inhibits the response indirectly and the response-inhibitor stimulates the production of LKKTET peptide in the tissue of the subject. The method of claim 1, wherein the response-inhibitor is administered to the patient at a dosage within the range of about 1-25 micrograms. The method of claim 1, wherein the response-inhibitor is administered to the subject by direct infusion into the tissue or by intravenous, intraperitoneal, intramuscular, subcutaneous, inhalation transdermal or oral administration. The method of claim 1, wherein the composition is administered systemically. The method of claim 1, wherein the composition is administered topically. The method of claim 12, wherein the composition is in the form of a gel, cream, paste, lotion, spray, suspending agent, dispersant, plaster, hydrogel or ointment formulation. The method of claim 1, wherein said reagent is a recombinant or synthetic peptide. The method of claim 1, wherein said reagent is an antibody. 8. The method of claim 7, wherein said antibody is polyclonal or monoclonal. Administering to a patient in need thereof an effective amount of a composition comprising the amino acid sequence LKKTET or a conserved variant thereof and comprising a stimulant that encourages the production of a biological or immunological response-inhibiting polypeptide with biological or immunological response-inhibiting activity A target method for treating, preventing, inhibiting or reducing a biological or immunological response to a reactive chemical, biological substance or toxin by a subject's tissue. 18. The method of claim 17, wherein said polypeptide is thymosin beta 4. 18. The method of claim 17, wherein said reagent is an antagonist of thymosin beta 4. The method of claim 1, wherein said tissue is a surface tissue selected from skin or mucous membranes of said subject, lung tissue of said subject, or gastrointestinal tract tissue of said subject. 18. The method of claim 17, wherein said tissue comprises surface tissue selected from skin or mucous membranes of said subject, lung tissue of said subject, or gastrointestinal tract tissue of said subject. Contacting the candidate compound with a tissue that exhibits a biological or immunological response; Measuring the level of reduction of a biological or immunological response in said tissue, wherein said decrease in said level is less than that of said candidate compound treating said biological or immunological response as compared to the level in a corresponding tissue lacking said candidate compound. Or a biological or immunological response-inhibitor that indicates that it can be prevented, inhibited or reduced. Contacting the tissue with the candidate compound; Contacting said tissue with a substrate inducing a biological or immunological response in said tissue in the absence of said candidate compound; Determining the level of reduction of a biological or immunological response in the viable tissue, wherein the reduction of said level as compared to the level in a corresponding tissue lacking said candidate compound treats, prevents, A method for screening a biological or immunological response-inhibitor that can be inhibited or reduced. Contacting the candidate compound with a tissue that exhibits a biological or immunological response; Measuring the activity in said tissue of a biological or immunological response-inhibitor, wherein said activity of said response-inhibitor in said tissue as compared to the activity level of said response-inhibitor in the corresponding tissue lacking said candidate compound Wherein the increase in indicates that the candidate compound is capable of inducing a stimulant. The method of screening a stimulant capable of encouraging the production of a biological or immunological response-inhibitor in tissue. The method of claim 24, wherein the reaction-inhibitor is an LKKTET peptide. The method of claim 25, wherein the LKKTET peptide is thymosin beta 4. Contacting the tissue with the candidate compound; Contacting said tissue with a substrate inducing a biological or immunological response in said tissue in the absence of said candidate compound; Measuring the intracellular activity of the response-inhibitor, wherein an increase in the intracellular activity of the response-inhibitor is compared to the level of activity in the corresponding tissue lacking the candidate compound. And a stimulant capable of stimulating the production of a biological or immunological response-inhibitor in a tissue, which is capable of stimulating the production in said tissue of said response-inhibitor. The method of claim 27, wherein the reaction-inhibitor is an LKKTET peptide. The method of claim 28, wherein the LKKTET peptide is thymosin beta 4.