WO2008150981A2

WO2008150981A2 - Novel short peptides that inhibit the opening of mammalian tight junctions

Info

Publication number: WO2008150981A2
Application number: PCT/US2008/065268
Authority: WO
Inventors: Amir Tamiz
Original assignee: Alba Therapeutics Corporation
Priority date: 2007-05-30
Filing date: 2008-05-30
Publication date: 2008-12-11
Also published as: WO2008150981A3

Abstract

The present invention provides novel peptide antagonists of tight junctions that inhibit and/or reduce the opening of mammalian tight junctions. The peptides are preferably 7 or less amino acids in length. The present invention also provides methods for the treatment of excessive or undesirable permeability of a tissue by administering to a subject suffering from such a condition a composition comprising a peptide tight junction antagonist of the invention.

Description

NOVEL SHORT PEPTIDES THAT INHIBIT THE OPENING OF MAMMALIAN TIGHT JUNCTIONS

BACKGROUND

[0001] The tight junctions (tj) or zonula occludens (ZO) are one of the hallmarks of absorptive and secretory epithelia (Madara, J. Clin. Invest., 83:1089-1094 (1989); and Madara, Textbook of Secretory Diarrhea Eds. Lebenthal et al, Chapter 11, pages 125-138 (1990). Tight junctions act as a barrier between apical and basolateral compartments, selectively regulating the passive diffusion of ions and water-soluble solutes through the paracellular (between cells) pathway (Gumbiner, Am. J. Physiol., 253 (Cell Physiol. 22):C749-C758 (1987)). This barrier maintains any gradient generated by the activity of pathways associated with the transcellular route (Diamond, Physiologist, 20:10-18 (1977)).

[0002] Variations in transepithelial conductance can usually be attributed to changes in the permeability of the paracellular pathway, since the resistances of enterocyte plasma membranes are relatively high (Madara, supra). The ZO represents the major barrier in this paracellular pathway, and the electrical resistance of epithelial tissues seems to depend on the number of transmembrane protein strands, and their complexity in the ZO, as observed by freeze-fracture electron microscopy (Madara et al, J. Cell Biol., 101 :2124-2133 (1985)).

[0003] Tight Junction dysfunctions

[0004] Tight junction dysfunction occurs in a variety of clinical conditions, including food allergies, infections of the gastrointestinal tract, autoimmune diseases, celiac disease and inflammatory bowel diseases (Fasano A. 2001. Pathological and therapeutical implications of macromolecule passage through the tight junction. In Tight Junctions, CRC Press, Inc., Boca Raton, FL. 697-722.). Healthy, mature gut mucosa with its intact tight junction serves as the main barrier to the passage of macromolecules. During the healthy state, small quantities of immunologically active antigens cross the gut host barrier. These antigens are absorbed across the mucosa through at least two pathways. Up to 90% of the absorbed proteins cross the intestinal barrier via the transcellular pathway, followed by lysosomal degradation that converts proteins into smaller, non-immunogenic peptides. These residual peptides are transported as intact proteins through the paracellular pathway, which mediates a subtle, but sophisticated, regulation of intercellular tight junction that leads to antigen tolerance. [0005] When the integrity of the tight junction system is compromised, in premature infants or after exposure to radiation, chemotherapy, or toxins, a deleterious immune response to environmental antigens, resulting in autoimmune diseases and food allergies, may be elicited, In normal bowels, the immune reaction is regulated to maintain homeostasis of the gut.

[0006] Celiac disease (CD) is a chronic autoimmune disease that is HLA-DQ2/DQ8 haplotype restricted. Glutens, the major protein fraction of wheat, and related proteins in rye and barley are the triggering agents of the disease. Ingested gluten or its' derivative fractions (gliadin and subunits) elicit a harmful T cell-mediated immune response after crossing the small bowel epithelial barrier, undergoing deamidation by tissue transglutaminase (tTG) and engaging class II MHC molecules. When the integrity of the tight junction system is compromised, as in CD, a paracellular leak ("leaky gut") and an inappropriate immune response to environmental antigens (i.e., gluten) may develop.

[0007] Inflammatory bowel disease (IBD) is a phrase used to describe an inappropriate immune response that occurs in the bowels of affected individuals. Two major types of IBD have been described: Crohn's disease and ulcerative colitis (UC). Both forms of IBD show abnormal profiles of T cell mediated immunity. In the gut of Crohn's disease a strong ThI reaction is induced, while the Th2 response is upregulated in the colon of UC.

[0008] The barrier function of the intestines is impaired in IBD. For example, Crohn's disease is associated with increased permeability of the intestinal barrier even in quiescent patients (Oshitani, et al., hit J MoI Med 15(3):407-10, 2005). A TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions (see Ye et al., American Journal of Physiology- Gastrointestinal and Liver Physiology, 290(3):496-504, 2006). Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex (Willemsen, et al. Clin. Exp. Immunol. 142(2): 275-284, 2005).

[0009] Zonula occludens toxin (ZOT), which is produced by Vibrio cholerae, has been characterized by Fasano et al., (Proc. Natl. Acad. Sci., USA, 8:5242-5246 (1991)) and the sequence has been determined (GenBank accession no. A43864). ZOT increases the intestinal permeability of rabbit ileal mucosa by modulating the structure of intercellular tight junctions. Mammalian proteins that are immunologically and functionally related to ZOT have been identified. See US 5,945,510. These proteins, referred to as "zonulin," function as the physiological effector of mammalian tight junctions. These proteins are useful for enhancing absorption of therapeutic agents across tight junction of intestinal and nasal mucosa, as well as across tight junction of the blood brain barrier.

[0010] Peptide antagonists of tight junction opening were described in U.S. Patent 6,458,925, which is incorporated by reference herein in its entirety, which corresponds to WO 00/07609. Peptide antagonists of tight junction opening may bind to the receptor utilized by the zonnula occludens toxin expressed by Vibrio cholerae, yet not function to physiologically modulate the opening of mammalian tight junctions. The peptide antagonists may competitively inhibit the binding of ZOT and/or zonulin to the ZOT receptor, thereby inhibiting the ability of ZOT and/or zonulin to physiologically modulate the opening of mammalian tight junctions.

[0011] There remains a need in the art for materials and methods to treat diseases characterized by excessive or undesirable permeability of tissues containing tight junctions. This need and others are met by the present invention.

SUMMARY OF THE INVENTION

[0012] The present invention provides peptide antagonists of tight junctions. Peptide antagonists of tight junctions according to the invention are preferably 7 amino acids or shorter in length. In one embodiment, peptide antagonists of the invention may comprise 3-7 amino acids. In one embodiment, a peptide antagonist of the invention may comprise 3 amino acids. In one embodiment, a peptide antagonist of the invention may consist of, or may consist essentially of, 3 amino acids. In one embodiment, a peptide antagonist of the invention may comprise the amino acid sequence Q-P-G (SEQ ID NO:6; corresponding to residues 6-8 of SEQ ID NO:1). In another embodiment, a peptide antagonist of the invention may consist of a peptide having the sequence Q-P-G. In one embodiment, a peptide antagonist of the invention may comprise the amino acid sequence G-G-V (SEQ ID NO: 11 ;corresponding to residues 1 -3 of SEQ ID NO: 1 ). In another embodiment, a peptide antagonist of the invention may consist of a peptide having the sequence G-G-V.

[0013] In some embodiments, the invention provides methods of treating an excessive or undesirable permeability of a tissue containing tight junctions comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. As used herein, a "subject" may be any mammal, for example, a human, dog, cat, horse, cow, etc. In some embodiments, a subject may be a human. In other embodiments, a subject may be a dog. Compositions of the invention may comprise one or more peptide antagonists of tight junctions that may be 7 amino acids or shorter in length. In one embodiment, compositions of the invention may comprise one or more peptide antagonists that may comprise 3-7 amino acids. In one embodiment, compositions of the invention may comprise one or more peptide antagonists of the invention that may comprise 3 amino acids. In one embodiment, compositions of the invention may comprise a peptide antagonist of the invention that may consist of, or may consist essentially of, 3 amino acids. In one embodiment, compositions of the invention may comprise a peptide antagonist comprising the amino acid sequence Q-P-G (SEQ ID NO:6; corresponding to residues 6-8 of SEQ ID NO:1). In another embodiment, compositions of the invention may comprise a peptide antagonist that consists of a peptide having the sequence Q-P-G. In one embodiment, compositions of the invention may comprise a peptide antagonist comprising the amino acid sequence G-G-V (SEQ ID NO: 11 ; corresponding to residues 1 -3 of SEQ ID NO: 1 ). In another embodiment, compositions of the invention may comprise a peptide antagonist that consists of a peptide having the sequence G-G-V. Compositions of the invention may comprise one or more tight junction antagonists and one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.

[0014] In some embodiments, the invention provides methods of treating celiac disease comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. A composition suitable for treating celiac disease may comprise a tight junction antagonist and may be a delayed release composition. Compositions suitable for use in treating celiac disease may comprise one or more peptide antagonists of tight junctions that may be 7 amino acids or shorter in length. In one embodiment, compositions for use in treating celiac disease may comprise one or more peptide antagonists that may comprise 3-7 amino acids. In one embodiment, compositions for use in treating celiac disease may comprise one or more peptide antagonists of the invention that may comprise 3 amino acids. In one embodiment, compositions of the invention may comprise a peptide antagonist of the invention that may consist of, or may consist essentially of, 3 amino acids. In one embodiment, compositions for use in treating celiac disease may comprise a peptide antagonist comprising the amino acid sequence Q-P-G (SEQ ID NO:6;corresponding to residues 6-8 of SEQ ID NO:1). In another embodiment, compositions for use in treating celiac disease may comprise a peptide antagonist that consists of a peptide having the sequence Q-P-G. In one embodiment, compositions for use in treating celiac disease may comprise a peptide antagonist comprising the amino acid sequence G-G-V (SEQ ID NO: 11 ;corresponding to residues 1 -3 of SEQ ID NO: 1 ). In another embodiment, compositions for use in treating celiac disease may comprise a peptide antagonist that consists of a peptide having the sequence G-G-V. Compositions for use in treating celiac disease may comprise one or more tight junction antagonists and one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines, and biologic therapeutics.

[0015] In some embodiments, the invention provides methods of treating inflammatory bowel disease comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. A composition suitable for treating IBD may comprise a tight junction antagonist and may be a delayed release composition. Compositions suitable for use in treating IBD may comprise one or more peptide antagonists of tight junctions that may be 7 amino acids or shorter in length. In one embodiment, compositions for use in treating IBD may comprise one or more peptide antagonists that may comprise 3-7 amino acids. In one embodiment, compositions for use in treating IBD may comprise one or more peptide antagonists of the invention that may comprise 3 amino acids. In one embodiment, compositions of the invention may comprise a peptide antagonist of the invention that may consist of, or may consist essentially of, 3 amino acids, In one embodiment, compositions for use in treating IBD may comprise a peptide antagonist comprising the amino acid sequence Q-P-G (SEQ ID NO:6; corresponding to residues 6-8 of SEQ ID NO: 1). In another embodiment, compositions for use in treating IBD may comprise a peptide antagonist that consists of a peptide having the sequence Q-P-G. In one embodiment, compositions for use in treating IBD may comprise a peptide antagonist comprising the amino acid sequence G-G-V (SEQ ID NO:11; corresponding to residues 1-3 of SEQ ID NO: 1). In another embodiment, compositions for use in treating IBD may comprise a peptide antagonist that consists of a peptide having the sequence G-G-V. Compositions for use in treating IBD may comprise one or more tight junction antagonists and one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines, and biologic therapeutics.

[0016] The present invention provides methods and materials for treating Crohn's disease. In some embodiments, the invention provides methods of treating Crohn's disease comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. A composition suitable for treating Crohn's may comprise a tight junction antagonist and may be a delayed release composition. Compositions suitable for use in treating Crohn's may comprise one or more peptide antagonists of tight junctions that may be 7 amino acids or shorter in length. In one embodiment, compositions for use in treating Crohn's may comprise one or more peptide antagonists that may comprise 3-7 amino acids. In one embodiment, compositions for use in treating Crohn's may comprise one or more peptide antagonists of the invention that may comprise 3 amino acids. In one embodiment, compositions of the invention may comprise a peptide antagonist of the invention that may consist of, or may consist essentially of, 3 amino acids. In one embodiment, compositions for use in treating Crohn's may comprise a peptide antagonist comprising the amino acid sequence Q-P-G (SEQ ID NO:6; corresponding to residues 6-8 of SEQ ID NO:1). In another embodiment, compositions for use in treating Crohn's may comprise a peptide antagonist that consists of a peptide having the sequence Q-P-G. In one embodiment, compositions for use in treating Crohn's may comprise a peptide antagonist comprising the amino acid sequence G-G-V (SEQ ID NO:11; corresponding to residues 1-3 of SEQ ID NO:1). In another embodiment, compositions for use in treating Crohn's may comprise a peptide antagonist that consists of a peptide having the sequence G-G-V. Compositions for use in treating Crohn's may comprise one or more tight junction antagonists and one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, and biologic therapeutics.

[0017] The present invention provides methods and materials for treating ulcerative colitis. In some embodiments, the invention provides methods of treating ulcerative colitis comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. A composition suitable for treating ulcerative colitis may comprise a tight junction antagonist and may be a delayed release composition. Compositions suitable for use in treating ulcerative colitis may comprise one or more peptide antagonists of tight junctions that may be 7 amino acids or shorter in length. In one embodiment, compositions for use in treating ulcerative colitis may comprise one or more peptide antagonists that may comprise 3-7 amino acids. In one embodiment, compositions for use in treating ulcerative colitis may comprise one or more peptide antagonists of the invention that may comprise 3 amino acids. In one embodiment, compositions of the invention may comprise a peptide antagonist of the invention that may consist of, or may consist essentially of, 3 amino acids. In one embodiment, compositions for use in treating ulcerative colitis may comprise a peptide antagonist comprising the amino acid sequence Q-P-G (SEQ ID NO:6; corresponding to residues 6-8 of SEQ ID NO: 1). In another embodiment, compositions for use in treating ulcerative colitis may comprise a peptide antagonist that consists of a peptide having the sequence Q-P-G. In one embodiment, compositions for use in treating ulcerative colitis may comprise a peptide antagonist comprising the amino acid sequence G-G-V (SEQ ID NO:11; corresponding to residues 1-3 of SEQ ID NO:1). In another embodiment, compositions for use in treating ulcerative colitis may comprise a peptide antagonist that consists of a peptide having the sequence G-G-V. Compositions for use in treating ulcerative colitis may comprise one or more tight junction antagonists and one or more therapeutic agents. Suitable therapeutic agents include, but are not limited to, aminosalicylates, corticosteroids, immunomodulators, antibiotics, and biologic therapeutics.

[0018] In specific embodiments, the instant invention includes: (1) A peptide antagonist of tight junctions, wherein the peptide is 7 amino acids or less in length;

(2) A peptide antagonist of tight junctions according to (1), wherein the peptide comprises 3-7 amino acids;

(3) A peptide antagonist of tight junctions according to (1), wherein the peptide consists of 3-7 amino acids;

(4) A peptide antagonist of tight junctions according to (1), wherein the peptide comprises 3 amino acids;

(5) A peptide antagonist of tight junctions according to (1), wherein the peptide consists of 3 amino acids;

(6) A peptide antagonist of tight junctions according to (1), wherein the peptide comprises Q-P-G, residues 6-8 of SEQ ID NO:1 ;

(7) A peptide antagonist of tight junctions according to (1), wherein the peptide consists of Q-P-G;

(8) A peptide antagonist of tight junctions according to (1), wherein the peptide comprises G-G-V, residues 1-3 of SEQ ID NO:1; (9) A peptide antagonist of tight junctions according to (1), wherein the peptide consists of G-G-V;

(10) A method of treating an excessive or undesirable permeability of a tissue containing tight junctions comprising: administering to a subject in need thereof a composition comprising a peptide according to (1);

(11) A method according to ( 10), wherein the subject is a human;

(12) A method according to (10), wherein the peptide comprises 3 amino acids;

(13) A method according to (10), wherein the peptide consists of 3 amino acids;

(14) A method according to (13), wherein the peptide is selected from the group consisting of Q-P-G and G-G-V;

(15) A method according to ( 10), wherein the composition further comprises a therapeutic agent;

(16) A method according to ( 15), wherein the therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics;

(17) A method according to ( 10), wherein the composition is formulated for intestinal delivery;

(18) A method according to (10), wherein the composition is formulated for pulmonary delivery;

(19) A method of treating inflammatory bowel disease comprising: administering to a subject in need thereof a composition comprising a peptide according to (1);

(20) A method according to (19), wherein the subject is a human;

(21) A method according to (19), wherein the peptide comprises 3 amino acids;

(22) A method according to (19), wherein the peptide consists of 3 amino acids;

(23) A method according to (22), wherein the peptide is selected from the group consisting of Q-P-G and G-G-V;

(24) A method according to ( 19), wherein the composition further comprises a therapeutic agent; (25) A method according to (24), wherein the therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics;

(26) A method according to (24), wherein the composition is formulated for intestinal delivery;

(27) A method of treating Crohn's disease comprising: administering to a subject in need thereof a composition comprising a peptide according to (1);

(28) A method according to (27), wherein the subject is a human;

(29) A method according to (27), wherein the peptide comprises 3 amino acids;

(30) A method according to (27), wherein the peptide consists of 3 amino acids;

(31) A method according to (30), wherein the peptide is selected from the group consisting of Q-P-G and G-G-V;

(32) A method according to (27), wherein the composition further comprises a therapeutic agent;

(33) A method according to (32), wherein the therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics;

(34) A method according to (33), wherein the composition is formulated for intestinal delivery;

(35) A method of treating ulcerative colitis comprising: administering to a subject in need thereof a composition comprising a peptide according to (1);

(36) A method according to (35), wherein the subject is a human;

(37) A method according to (35), wherein the peptide comprises 3 amino acids;

(38) A method according to (35), wherein the peptide consists of 3 amino acids;

(39) A method according to (38), wherein the peptide is selected from the group consisting of Q-P-G and G-G-V;

(40) A method according to (38), wherein the composition further comprises a therapeutic agent; (41) A method according to (40), wherein the therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics; and

(42) A method according to (41 ), wherein the composition is formulated for intestinal delivery.

DETAILED DESCRIPTION OF THE INVEDNTION [0019] Antagonists of tight junction opening

[0020] As used herein, tight junction antagonists prevent, inhibit or reduce the opening of tight junctions, for example, the opening of tight junctions induced by a tight junction agonist. A tight junction antagonist may bind to the receptor that mediates tight junction agonist induced opening of tight junctions. For example, a tight junction antagonist may bind to the ZOT receptor and prevent, inhibit, reduce or reverse the tight junction opening triggered by the tight junction agonist ZOT.

[0021] As used herein a subject is any animal, e.g., mammal, upon which methods of the invention may be practiced and/or to which materials of the present invention may be administered. Subjects include, but are not limited to, humans.

[0022] Antagonists of the invention may comprise peptide antagonists. An example of a peptide antagonist of tight junctions is a peptide that comprises the amino acid sequence Gly Gly VaI Leu VaI Gln Pro Gly (SEQ ID NO:1). Additional examples of peptide antagonists of the invention include, but are not limited to, peptides that comprise amino acids 1 -3 of SEQ ID NO: 1 (SEQ ID NO:11), amino acids 1-4 of SEQ ID NO: 1 (SEQ ID NO:10), amino acids 1-5 of SEQ ID NO: 1 (SEQ ID NO:9), amino acids 1-6 of SEQ ID NO: 1 (SEQ ID NO:8), amino acids 1-7 of SEQ ID NO: 1 (SEQ ID NO:7), amino acids 2-8 of SEQ ID NO: 1 (SEQ ID NO:2), amino acids 3-8 of SEQ ID NO: 1 (SEQ ID NO:3), amino acids 4-8 of SEQ ID NO: 1 (SEQ ID NO:4), amino acids 5-8 of SEQ ID NO: 1 (SEQ ID NO:5), and amino acids 6-8 of SEQ ID NO: 1 (SEQ ID NO:6). Additional examples of peptide antagonists of the invention include, but are not limited to, peptides that consist essentially of amino acids 1-3 of SEQ ID NO: 1 (SEQ ID NO: 11), amino acids 1-4 of SEQ ID NO: 1 (SEQ ID NO: 10), amino acids 1-5 of SEQ ID NO: 1 (SEQ ID NO:9), amino acids 1-6 of SEQ ID NO: 1 (SEQ ID NO:8), amino acids 1-7 of SEQ ID NO: 1 (SEQ ID NO:7), amino acids 2-8 of SEQ ID NO: 1 (SEQ ID NO:2), amino acids 3-8 of SEQ ID NO: 1 (SEQ ID NO:3), amino acids 4-8 of SEQ ID NO: 1 (SEQ ID NO:4), amino acids 5-8 of SEQ ID NO: 1 (SEQ ID NO:5), and amino acids 6-8 of SEQ ID NO: 1 (SEQ ID NO:6). Additional examples of peptide antagonists of the invention include, but are not limited to, peptides that consist of amino acids 1-3 of SEQ ID NO: 1 (SEQ ID NO:11), amino acids 1-4 of SEQ ID NO: 1 (SEQ ID NO: 10), amino acids 1-5 of SEQ ID NO: 1 (SEQ ID NO:9), amino acids 1-6 of SEQ ID NO: 1 (SEQ ID NO:8), amino acids 1-7 of SEQ ID NO: 1 (SEQ ID NO:7), amino acids 2-8 of SEQ ID NO: 1 (SEQ ID NO:2), amino acids 3-8 of SEQ ID NO: 1 (SEQ ID NO:3), amino acids 4-8 of SEQ ID NO: 1 (SEQ ID NO:4), amino acids 5-8 of SEQ ID NO: 1 (SEQ ID NO:5), and amino acids 6-8 of SEQ ID NO: 1 (SEQ ID NO:6).

[0023] Peptide antagonists of tight junctions of the invention are preferably, 3, 4, 5, 6, or 7 amino acids in length.

[0024] The peptide antagonists can be chemically synthesized and purified using well- known techniques, such as described in High Performance Liquid Chromatography of Peptides and Proteins: Separation Analysis and Conformation, Eds. Mant et al., C.R.C. Press (1991), and a peptide synthesizer, such as Symphony (Protein Technologies, Inc); or by using recombinant DNA techniques, i.e., where the nucleotide sequence encoding the peptide is inserted in an appropriate expression vector, e.g. , an E. coli or yeast expression vector, expressed in the respective host cell, and purified therefrom using well-known techniques.

[0025] Compositions

[0026] Typically, compositions, such as pharmaceutical compositions, comprising a tight junction antagonist (e.g., peptide antagonist) comprise a pharmaceutically effective amount of the antagonist. The pharmaceutically effective amount of antagonist (e.g., peptide antagonist) employed in any given composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Generally, the amount of antagonist used for preventing, ameliorating and/or treating a disease in a subject will be in the range of about 1.0 μg to 1 g, preferably about 1 mg to about 1000 mg, or from about 10 mg to about 100 mg, or from about 10 mg to about 50 mg, or from about 10 mg to about 25 mg of antagonist.

[0027] Compositions of the invention may comprise one or more tight junction antagonists at a level of from about 0.1 wt% to about 20 wt%, from about 0.1 wt% to about 18 wt%, from about 0.1 wt% to about 16 wt%, from about 0.1 wt% to about 14 wt%, from about 0.1 wt% to about 12 wt%, from about 0.1 wt% to about 10 wt%, from about 0.1 wt% to about 8 wt%, from about 0.1 wt% to about 6 wt%, from about 0.1 wt% to about 4 wt%, from about 0.1 wt% to about 2 wt%, from about 0.1 wt% to about 1 wt%, from about 0.1 wt% to about 0.9 wt%, from about 0.1 wt% to about 0.8 wt%, from about 0.1 wt% to about 0.7 wt%, from about 0.1 wt% to about 0.6 wt%, from about 0.1 wt% to about 0.5 wt%, from about 0.1 wt% to about 0.4 wt%, from about 0.1 wt% to about 0.3 wt%, or from about 0.1 wt% to about 0.2 wt% of the total weight of the composition. Compositions of the invention may comprise one or more tight junction antagonists at a level of about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, or about 0.9 wt% based on the total weight of the composition.

[0028] Compositions of the invention may comprise one or more tight junction antagonists at a level of from about 1 wt% to about 20 wt%, from about 1 wt% to about 18 wt%, from about 1 wt% to about 16 wt%, from about 1 wt% to about 14 wt%, from about 1 wt% to about 12 wt%, from about 1 wt% to about 10 wt%, from about 1 wt% to about 9 wt%, from about 1 wt% to about 8 wt%, from about 1 wt% to about 7 wt%, from about 1 wt% to about 6 wt%, from about 1 wt% to about 5 wt%, from about 1 wt% to about 4 wt%, from about 1 wt% to about 3 wt%, or from about 1 wt% to about 2 wt% of the total weight of the composition. Compositions of the invention may comprise one or more tight junction effectors at a level of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, or about 9 wt% based on the total weight of the composition.

[0029] Compositions of the invention may formulated for pulmonary delivery (e.g., may be pulmonary dosage forms). Typically such compositions may be provided as pharmaceutical aerosols, e.g., solution aerosols or powder aerosols. Those of skill in the art are aware of many different methods and devices for the formation of pharmaceutical aerosols, for example, those disclosed by Sciarra and Sciarra, Aerosols, in Remington: The Science and Practice of Pharmacy, 20th Ed., Chapter 50, Gennaro et al. Eds., Lippincott, Williams and Wilkins Publishing Co., (2000).

[0030] The compositions of the invention may be formulated for enteric delivery, for example, may comprise one or more coatings, for example, delayed release coating containing one or more enteric agents. A delayed release coating is typically substantially stable in gastric fluid and substantially unstable (e.g., dissolves rapidly or is physically unstable) in intestinal fluid, thus providing for substantial release of the tight junction antagonist from the composition in the duodenum or the jejunum.

[0031] The terms "stable in gastric fluid" or "stable in acidic environments" refers to a composition that releases 30% or less by weight of the total tight junction antagonist in the composition in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes.

[0032] Compositions of the of the invention may release from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 15%, from about 0% to about 10%, 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10% by weight of the total tight junction antagonist in the composition in gastric fluid with a pH of 5, or less or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes. As use herein, "about" used to modify a numerical value means within 10% of the value. Compositions of the invention may release about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight of the total tight junction antagonist in the composition in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or less, in approximately sixty minutes.

[0033] The term "unstable in intestinal fluid" refers to a composition that releases 70% or more by weight of the total tight junction antagonist in the composition in intestinal fluid or simulated intestinal fluid in approximately sixty minutes. The term "unstable in near neutral to alkaline environments" refers to a composition that releases 70% or more by weight of the total amount of tight junction antagonist in the composition in intestinal fluid with a pH of 5 or greater, or simulated intestinal fluid with a pH of 5 or greater, in approximately ninety minutes. For example, a composition that is unstable in near neutral or alkaline environments may release 70% or more by weight of a tight junction antagonist peptide in a fluid having a pH greater than about 5 (e.g., a fluid having a pH of from about 5 to about 14, from about 6 to about 14, from about 7 to about 14, from about 8 to about 14, from about 9 to about 14, from about 10 to about 14, or from about 11 to about 14) in from about 5 minutes to about 90 minutes, or from about 10 minutes to about 90 minutes, or from about 15 minutes to about 90 minutes, or from about 20 minutes to about 90 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 90 minutes, or from about 5 minutes to about 60 minutes, or from about 10 minutes to about 60 minutes, or from about 15 minutes to about 60 minutes, or from about 20 minutes to about 60 minutes, or from about 25 minutes to about 90 minutes, or from about 30 minutes to about 60 minutes.

[0034] In addition to a tight junction antagonist, compositions of the invention may further comprise one or more therapeutic agents. Therapeutic agents include, but are not limited to, steroids and other anti-inflammatory compounds. Suitable therapeutic agents may include one or more of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapies. Examples of suitable therapeutic agents that maybe included in the compositions of the invention to treat IBD (e.g., Crohn's disease and/or ulcerative colitis) include, but are not limited to:

5-ASA agents (e.g., Sulfasalazine), Azulfidine®, Asacol,® Dipentum,® Pentasa,® and

Rowasa®;

Antibiotics, for example, metronidazole (Flagyl®) and ciprofloxacin (Cipro®), although there are many others that may be effective in certain individuals;

Steroids, e.g., corticosteroids. Suitable steroids include, but are not limited to, prednisone, hydrocortisone, Medrol®, and budesonide multiple-release capsule MRC (EntocortREC®).

6-mercaptopurine (6-MP, Purinethol®) and azathioprine (Imuran®); and antibodies against inflammatory cytokines, e.g., Infliximab (Remicade™).

[0035] Compositions of the invention may also comprise one or more pharmaceutically acceptable excipients. Suitable excipients include, but are not limited to, buffers, buffer salts, bulking agents, salts, surface active agents, acids, bases, and binders.

[0036] Methods of use

[0037] The compositions of the invention can be used for preventing, slowing the onset of, ameliorating and/or treating any disease associated with an excessive or undesirable permeability of tissues containing tight junctions. Specific examples of disease of this type include, but are not limited to, celiac disease and IBD (e.g., Crohn's disease and/or ulcerative colitis). In one embodiment, the present invention provides a method of treating celiac disease comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. In one embodiment, the present invention provides a method of treating Crohn's disease comprising administering to a subject in need thereof a composition comprising a tight junction antagonist, In one embodiment, the present invention provides a method of treating ulcerative colitis comprising administering to a subject in need thereof a composition comprising a tight junction antagonist. [0038] In some embodiments, compositions of the invention may be given repeatedly over a protracted period, i.e., may be chronically administered. Typically, compositions may be administered one or more times each day in an amount suitable to prevent, reduce the likelihood of an attack of, or reduce the severity of an attack of the underlying disease condition (e.g., celiac disease, IBD etc.). Such compositions may be administered chronically, for example, one or more times daily over a plurality of days.

[0039] In some embodiments, compositions of the invention may be use to treat acute attacks of the underlying disease (e.g., celiac disease, IBD (e.g., Crohn's disease and/or ulcerative colitis)). Typically, embodiments of this type will require administration of the compositions of the invention to a subject undergoing an attack in an amount suitable to reduce the severity of the attack. One or more administration may be used.

EXAMPLES

EXAMPLE 1

[0040] Measurement Of The Inhibition Of The Decrease In Trans Epithelial Electric Resistance (TEER) And Epithelial Flux OfA Fluorescent Marker Lucifer Yellow

[0041] CaCo2 cells form monolayers that exhibit tight junctions between adjacent cells. Treatment of CaCo2 monolayers with peptide FCIGRL enhanced 51 -fold Lucifer Yellow permeability through CaCo2 monolayers compared to vehicle alone. Peptide FCIGRL decreased TEER 16-fold in CaCo2 monolayers compared to vehicle alone. Antagonists of tight junctions can be identified by their ability to prevent or decrease the enhancement of the flux of compounds (e.g. lucifer yellow) through the monolayer induced by agonists of tight junctions (e.g., peptide FCIGRL also known as AT1002). Antagonist of tight junctions can also be identified by their ability to prevent the decrease in TEER induced by agonists of tight junctions (e.g., peptide FCIGRL also known as AT1002).

[0042 ] One method to test the effect of potential tight junction antagonists is by using the following methods.

[0043 ] Prepare Hank's Balanced Salt solution by weighing obtaining 1 L bottle of HBSS removing 10ml of HBSS and replacing it with 10ml HEPES buffer pH 7.0. Adjust pH to 7.4 ±0.1 using concentrated NaOH (10N).

[0044 ] Remove Caco-2 cells from incubator, grown on 12-well, 3.0 μM, polycarbonate Transwell® filters (Corning) and record passage#, date cells seeded and age in days. [0045] Aspirate cell culture medium from both the apical (AP) and basolateral (BL) compartments, replacing with 0.5 ml and 1.5 ml of HBSS, respectively. Incubate cells at 37°C for 30 minutes.

[0046] Using the MilliCell-ERS instrument (Millipore), measure and record the transepithelial electrical resistance (TEER) across each filter and record.

[0047 ] Prepare a stock solution of the Antagonist Pretreatment Solution by dissolving the appropriate amount of antagonist in HBSS. Vortex or sonicate the solution until it is clear then adjust pH to 7.4 ±0.1 using 1N NaOH. Preincubate each filter for 30min with these solutions at 37°C and 50 RPM.

[0048] Prepare stock solutions of Antagonist Treatment Solution by dissolving appropriate amounts of antagonist and agonist (for example, a peptide agonist such as a peptide having the sequence FCIGRL) in 7.5mM Lucifer Yellow solution in HBSS. Vortex or sonicate the solution until it is clear then adjust pH to 7.4 ± 0.1 using 1N NaOH.

[0049] Aspirate Antagonist Pretreatment Solution from the apical compartment of each filter (n=3 per condition) and replace with 0.5ml of control and test solutions.

[0050] Place all plates into incubator set at 37 °C (± 0.2), 50 RPM (±5) for a total of 180 minutes.

[0051] At t = 30, 60, 120 and 180 minutes, measure and record the transepithelial electrical resistance (TEER) across each filter using the MilliCell-ERS instrument.

[0052] At t = 60, 120 and 180 minutes remove 100μl from each basolateral compartment and place it in a 96- well plate for Lucifer Yellow analysis, replace with 100μl of HBSS.

[0053] Make a Lucifer Yellow standard curve with the following dilutions (7500μM, 3750μM, 750μM, 375μM, 75μM, 37.5μM, 7.5μM, 3.75μM, 0.75μM) and pipette 100μL of each into a 96-well plate except for the first three standards mentioned above which require a 1 :10 dilutions prior to transferring to the 96-well plate.

[0054] At t=0 make 1:10 dilutions of each starting Antagonist Treatment Solution mentioned above and pipette 100μL of each into a 96-well plate.

[0055] At t = 180 make 1 :10 dilutions of Antagonist Treatment Solution from the apical compartment of each filter and pipette 100μL of each into the 96-well plate [0056] Harvest the remaining start solutions and what is left in each apical compartment into 1.5ml vials. Freeze at -20°C for future analysis.

[0057] Analyze each 96-well plate in a Tecan Spectra Fluor Plus using Magellan at 485 and 535nm.

[0058] Materials:

[0059] Cells: Caco-2 cells passage 40-60 grown on Transwell plates for 21-28 days

[0060] Culture Medium: DMEM supplemented with 10% fetal bovine serum, 1% NEAA, l% Penn/Strep

[0061] Buffers: Hank's Balanced Salt Solution (HBSS) without calcium and magnesium

[0062] Flasks: 100 X 20 mm Tissue culture dish Falcon.

[0063] Plates: 12 well polycarbonate Transwell filters; 0.3uM pore size

[0064] The results of these assays are provided in table 1. The first column of the table provides the sequences of the peptides tested, the second column indicates the activity of the peptide in attenuating the Lucifer yellow flux induced by AT 1002, and the third column indicates the activity of the peptide in attenuating the reduction of TEER induced by AT1002. AT 1002 is a 6-mer peptide tight junction agonist having the sequence FCIGRL (SEQ ID NO: 12). See US patent publication US 2005/0059593 Al .

Table 1 : Tight junction inhibition by SEQ ID NOs: 1-11

EXAMPLE 2 [0065] Assay of inhibition of cytoskeletal rearrangement induced by PT-Gliadin

[0066] Gliadin treated with the peptidases pepsin and trypsin (termed PT-gliadin or PTG) induces a cytoskeletal arrangement in CaCo2 cells grown in monolayers. The rearrangement can be visualized using a Nikon-TE2000 epifluorescence microscope and a 40x objective and Alexa Fluor 555 conjugated phalloidin (Invitrogen, Carlsbad, CA), which binds specifically to F-actin.. Exposure times were identical for control and agonist/antagonist treated samples. The figures were generated using Adobe Photoshop CS2 v 9.0.2. The concentration of antagonist was 10 mg/mL. Tight junction antagonists can be identified by their ability to inhibit or decrease the cytoskeletal rearrangement induced by the tight junction agonist AT 1002.

[0067] The following table shows the results of cytoskeletal rearrangement assays with tight junction antagonists having the identified sequences.

[0068] Active indicates the peptide protected central actin network (measured by high intensity of fluorescence) compared to control cells treated with agonist alone

[0069] Inactive indicates the peptide provided no protection of central actin network compared to control treated with agonist alone (low total fluorescence intensity)

[0070] The columns entitled Blood, Plasma, Gastric Fluid, and Intestinal Fluid indicate if the shorter peptide having the indicated sequence is formed when incubated in the specified fluid. Gastric fluid and intestinal fluid are simulated fluids, fore example, simulated gastric fluid and simulated intestinal fluid include, but are not limited to, those disclosed in the 2005 Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or other simulated gastric fluids and simulated intestinal fluids known to those of skill in the art, for example, simulated gastric fluid and/or intestinal fluid prepared without enzymes. The blood and plasma used were of human origin.

[0071] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A peptide antagonist of tight junctions, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-11.

2. The peptide antagonist of tight junctions of claim 1 , wherein the peptide is between about three and about seven amino acids in length.

3. The peptide antagonist of tight junctions of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 11.

4. The peptide antagonist of tight junctions of claim 1 , wherein the peptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-

11.

5. The peptide antagonist of tight junctions of claim 1, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-11.

6. The peptide antagonist of tight junctions of claim 1, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 11.

7. The peptide antagonist of tight junctions of claim 6, wherein the peptide consists of the amino acid sequence of SEQ ID NO:6.

8. The peptide antagonist of tight junctions of claim 6, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 11.

9. A method of treating a disease or disorder characterized by excessive or undesirable permeability of a tissue containing tight junctions comprising: administering to a subject in need thereof a composition comprising the peptide antagonist of tight junctions of claim 1.

10. The method of claim 10, wherein the subject is a human.

11. The method of claim 10, wherein the disease or disorder characterized by excessive or undesirable permeability of a tissue containing tight junctions is selected from the group consisting of: inflammatory bowel disease; Crohn's disease; ulcerative colitis; celiac disease; necrotizing enterocolitis; autism; type 1 diabetes; acute lung injury; acute respiratory distress syndrome; chronic obstructive pulmonary disorder; asthma; and irritable bowel syndrome.

11. The method of claim 10, wherein the peptide antagonist consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 11.

13. The method of claim 10, wherein the peptide antagonist consists of the amino acid sequence of SEQ ID NO:6.

14. The method of claim 10, wherein the peptide antagonist consists of the amino acid sequence of SEQ ID NO: 11.

15. The method of claim 10, wherein the composition further comprises an additional therapeutic agent.

16. The method of claim 14, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.

17. The method of claim 10, wherein the composition is formulated for intestinal delivery.

18. The method of claim 10, wherein the composition is formulated for pulmonary delivery.