PEPTIDE INHIBITORS OF TIGHT JUNCTION PERMEABILITY
20220009965 · 2022-01-13
Inventors
- Sefik Alkan (Baltimore, MD, US)
- Amir Tamiz (Silver Spring, MD, US)
- Kelly Marie Kitchens (Laurel, MD, US)
- Malarvizhi Durai (Ellicott City, MD, US)
- Neil Poloso (Rockville, MD, US)
- Rosa A. Carrasco (Baltimore, MD, US)
Cpc classification
A61P1/04
HUMAN NECESSITIES
C07K5/101
CHEMISTRY; METALLURGY
C07K5/0808
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07K5/1008
CHEMISTRY; METALLURGY
C07K5/0821
CHEMISTRY; METALLURGY
C07K5/0806
CHEMISTRY; METALLURGY
C07K5/081
CHEMISTRY; METALLURGY
International classification
A61K45/06
HUMAN NECESSITIES
Abstract
Novel compounds and methods for the inhibition of biological barrier permeability and for the inhibition of peptide translocation across biological barriers are identified. Assays for determining modulators of biological barrier permeability and for peptide translocation across biological barriers are provided. Methods for treating diseases relating to aberrant biological barrier permeability and peptide translocation across biological barriers are provided. Such diseases include celiac disease, necrotizing enterocolitis, diabetes, cancer, inflammatory bowel diseases, asthma, COPD, excessive or undesirable immune response, gluten sensitivity, gluten allergy, food allergy, rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes mellitus, systemic lupus erythematosus, psoriasis, scleroderma and autoimmune thyroid diseases.
Claims
1. A peptide permeability inhibitor consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-162, wherein said peptide permeability inhibitor inhibits translocation of a gliadin-derived peptide across a biological barrier.
2. The peptide of claim 1, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
3. The peptide of claim 1, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
4. A method of inhibiting gliadin-derived peptide translocation across a biological barrier comprising contacting said barrier with a peptide permeability inhibitor consisting of an amino acid sequence selected from the group consisting of SEQ IDF NOs:1-162.
5. The method of claim 4, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
6. The method of claim 4, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
7. A composition for inhibiting gliadin-derived peptide translocation across a biological barrier, wherein said composition comprises a peptide permeability inhibitor consisting of an amino acid sequence selected from the group consisting of SEQ IDF NOs:1-162.
8. The composition of claim 7, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
9. The composition of claim 7, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-5, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
10. A method for inhibiting gliadin-derived peptide translocation across a biological barrier comprising administering to a subject in need thereof the composition of claim 7 in an amount sufficient to inhibit said gliadin-derived peptide translocation.
11. The method of claim 10, wherein the composition is administered in conjunction with an additional therapeutic agent.
12. The method of claim 11, wherein the composition and the additional therapeutic agent are administered simultaneously.
13. The method of claim 11, wherein the composition and the additional therapeutic agent are not administered simultaneously.
14. The method of claim 11, wherein the composition further comprises the additional therapeutic agent.
15. The method of claim 11, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.
16. A peptide consisting of an amino acid sequence selected from the group consisting of SEQ IDF NOs:1-162, wherein said peptide inhibits a gliadin-induced increase in biological barrier permeability.
17. The peptide of claim 16, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOS: 15, 24 and 25.
18. The peptide of claim 16, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
19. A method of inhibiting a gliadin-induced increase in biological barrier permeability comprising contacting said barrier with a peptide permeability inhibitor consisting of an amino acid sequence selected from the group consisting of SEQ IDF NOs:1-162.
20. The method of claim 19, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
21. The method of claim 19, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
22. A composition for inhibiting a gliadin-induced increase in biological barrier permeability, wherein said composition comprises a peptide permeability inhibitor consisting of an amino acid sequence selected from the group consisting of SEQ IDF NOS:1-162.
23. The composition of claim 22, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
24. The composition of claim 22, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
25. A method for inhibiting a gliadin-induced increase in biological barrier permeability comprising administering to a subject in need thereof the composition of claim 22 in an amount sufficient to inhibit said increase in biological barrier permeability.
26. The method of claim 25, wherein the composition is administered in conjunction with an additional therapeutic agent.
27. The method of claim 26, wherein the composition and the additional therapeutic agent are administered simultaneously.
28. The method of claim 26, wherein the composition and the additional therapeutic agent are not administered simultaneously.
29. The method of claim 26, wherein the composition further comprises the additional therapeutic agent.
30. The method of claim 26, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.
31. A method of treating a patient with celiac disease, comprising administering to the patient a composition comprising a peptide that inhibits translocation of a gliadin-derived peptide across a biological barrier.
32. The method of claim 31, wherein said composition comprises peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-162.
33. The method of claim 32, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
34. The method of claim 32, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
35. The method of claim 31, wherein said composition further comprises an additional therapeutic agent selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.
36. A method of treating a patient with diabetes, comprising administering to the patient a composition comprising a peptide that inhibits a gliadin-induced increase in biological barrier permeability.
37. The method of claim 36, wherein said composition comprises peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-162.
38. The method of claim 37, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
39. The method of claim 37, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
40. The method of claim 36, wherein said composition further comprises an additional therapeutic agent selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.
41. A method of treating a patient with dermatitis herpetiformis, comprising administering to the patient a composition comprising a peptide that inhibits translocation of a gliadin-derived peptide across a biological barrier.
42. The method of claim 41, wherein said composition comprises peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-162.
43. The method of claim 42, wherein the peptide does not consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 24 and 25.
44. The method of claim 42, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
45. the method of claim 41, wherein said composition further comprises an additional therapeutic agent selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0052] The inventors have discovered that peripheral blood mononuclear cells (PBMCs) secrete signals that increase epithelial monolayer permeability on response to stimulation with lipopolysaccharide (PLPS) and pepsin/trypsin treated gliadin (PTG). These secreted signals are present in PBMC culture supernatant, and they increase permeability of CaCO2 cell monolayers to Lucifer yellow when presented to the basolateral aspect of these cells. These permeability changes me inhibited by treatment of the cells with peptide permeability inhibitors of the invention (
[0053] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons. Inc., 1995 (ISBN 0471186341); and other similar technical references.
[0054] As used herein, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0055] As used herein, “biological effect” refers to a biochemical and physiological effect. Biological effect includes, for example, increases or decreases in the activity of the immune system and any of its components (including, for example, complement activation), increases or decreases in receptor binding and increases or decreases in subsequent downstream effector cellular constituents (including, for example, growth factor receptor and downstream effector cellular constituents), increases or decreases in cell signaling, increases or decreases in gene expression, increases or decreased in post-translation modification of proteins (including, for example, phosphorylation), and increases or decreases in protein activity.
[0056] As used herein, “modulate” and all its forms and tenses refer to either increasing or decreasing a particular biochemical or physiological effect.
[0057] As used herein, A “component of the immune system” or an “immune cell” refers to a component or cell of the immune system that is involved in enhancing, eliciting, or maintaining an immune response. The immune system responds to various foreign particles (including, for example, viruses, bacteria, and allergens) and non-foreign particles (including, for example, native endogenous proteins). An immune response includes, for example, antibody production, chemotaxis, phagocytosis, inflammation, complement activation, production of cytotoxic molecules (including, for example, reactive oxygen species and reactive nitrogen species), cell adhesion, cell infiltration, and production and recruitment of mediators of any of the foregoing or other immune responses. A component or cell of the immune system involved in enhancing, eliciting, or maintaining an immune response includes, for example, neutrophils, complement proteins (including, for example, C1q, C1r and C1s), eosinophils, basophils, lymphocytes (including for example, T cells (including, for example, cytotoxic T cells, memory T cells, helper T cells, regulatory T cells, natural killer T cells, and γδ T cells) and B cells (including, for example, plasma B cells, memory B cells, B-1 cells, and B-2 cells)), monocytes, macrophages, dendritic cells (DC), cell adhesion molecules (including, for example, ICAM and VCAM), myeloperoxidase, nitric oxide synthase, cyclooxygenase, and prostaglandin synthase.
[0058] As used herein, “treat” and all its forms and tenses refer to both therapeutic treatment and prophylactic or preventative treatment. Those in need of treatment include those already with the condition or disease as well as those in which the condition or disease is to be prevented.
Present Invention
[0059] The inventors have identified novel methods and compounds that inhibit increased permeability of biological barriers in response to stimuli that are known to induce secretion of pro-inflammatory cytokines. In specific embodiments the inventors have identified methods and compounds that inhibit increased permeability of biological barriers after stimulation by factors secreted by immune cells on exposure to LPS. In further specific embodiments the inventors have identified methods and compounds that inhibit increased permeability of biological barriers after stimulation by factors secreted by immune cells on exposure to PTG. Exemplary compounds of the invention that inhibit increased permeability of biological barriers are presented in Table 20.
[0060] The inventors have also identified novel methods and compounds that inhibit, reduce and/or prevent translocation of PTG-derived peptides across biological barriers. In specific embodiments the inventors have identified methods and compounds that inhibit, reduce and/or prevent translocation of the peptide comprising the amino acid sequence PYPQPQLPY (SEQ ID NO:163). Exemplary compounds of the invention that inhibit, reduce and/or prevent translocation of PTG-derived peptides across biological barriers are presented in Table 20.
[0061] Inhibitors of biological barrier permeability may be used in the practice of the present invention. Such permeability inhibitors may also be antagonists of mammalian tight junction opening. Antagonists of mammalian tight junction opening may also be used in the practice of the present invention. As used herein, permeability inhibitors prevent, inhibit or reduce the permeability of biological barriers to macromolecules including, for example, proteins, peptides and nucleic acids. For example, permeability inhibitors of the invention may comprise peptide permeability inhibitors. Examples of peptide permeability inhibitors that may be used in the practice of the present invention include, but are not limited to, peptides that comprise an amino acid sequence selected from the group consisting of: consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 10-17, 19-23, 27, 32, 34, 36, 48, 49, 55, 58, 67-77, 79-85, 87, 88, 91, 92, 94, 98-104, 106, 110, 111, 113-125, 127, 128, 147, 150, and 160-162.
[0062] Examples of peptide permeability inhibitors include, but are not limited to, peptides that consist of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-162.
[0063] When the permeability inhibitor is a peptide, any length of peptide may be used. Generally, the size of the peptide antagonist will range from about 6 to about 100, from about 6 to about 90, from about 6 to about 80, from about 6 to about 70, from about 6 to about 60, from about 6 to about 50, from about 6 to about 40, from about 6 to about 30, from about 6 to about 25, from about 6 to about 20, from about 6 to about 15, from about 6 to about 14, from about 6 to about 13, from about 6 to about 12, from about 6 to about 11, from about 6 to about 10, from about 6 to about 9, or from about 6 to about 8 amino acids in length. Peptide antagonists of the invention may be from about 8 to about 100, from about 8 to about 90, from about 8 to about 80, from about 8 to about 70, from about 8 to about 60, from about 8 to about 50, from about 8 to about 40, from about 8 to about 30, from about 8 to about 25, from about 8 to about 20, from about 8 to about 15, from about 8 to about 14, from about 8 to about 13, from about 8 to about 12, from about 8 to about 11, or from about 8 to about 10 amino acids in length. Peptide antagonists of the invention may be from about 10 to about 100, from about 10 to about 90, from about 10 to about 80, from about 10 to about 70, from about 10 to about 60, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 25, from about 10 to about 20, from about 10 to about 15, from about 10 to about 14, from about 10 to about 13, or from about 10 to about 12 amino acids in length. Peptide antagonists of the invention may be from about 12 to about 100, from about 12 to about 90, from about 12 to about 80, from about 12 to about 70, from about 12 to about 60, from about 12 to about 50, from about 12 to about 40, from about 12 to about 30, from about 12 to about 25, from about 12 to about 20, from about 12 to about 15, or from about 12 to about 14 amino acids in length. Peptide antagonists of the invention may be from about 15 to about 100, from about 15 to about 90, from about 15 to about 80, from about 15 to about 70, from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 30, from about 15 to about 25, from about 15 to about 20, from about 19 to about 15, from about 15 to about 18, or from about 17 to about 15 amino acids in length.
[0064] The peptide permeability inhibitors 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.
Compositions
[0065] Typically, compositions, such as pharmaceutical compositions, comprise one or more compounds of the invention, and optionally one or more additional active agents. Compounds of the invention may be present in an amount sufficient to inhibit the increased biological barrier permeability in a subject in need thereof. Compounds of the invention may be present in an amount sufficient to inhibit, reduce and/or prevent translocation of a gliadin-derived peptide across a biological barrier in a subject in need thereof. The amount of a compound of the invention employed in may given composition may vary according to factors such as the disease state, age, sex, and weight of the subject. 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.
[0066] Generally, a pharmaceutical composition of the invention will comprise an amount of a compound of the invention in the range of about 1 μg to about 1 g. preferably about 1 mg to about 1000 mg, from about 10 mg to about 100 mg, from about 10 mg to about 50 mg, or from about 10 mg to about 25 mg of the compound. As used herein, “about” used to modify a numerical value means within 10% of the value.
[0067] Compositions of the invention may comprise one or more compounds of the invention 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. As used herein, “about” used to modify a numerical value means within 10% of the value. Compositions of the invention may comprise one or more compounds of the invention 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.
[0068] Compositions of the invention may comprise one or more compounds of the invention 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. As used herein, “about” used to modify a numerical value means within 10% of the value. Compositions of the invention may comprise one or more compounds of the invention 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.
[0069] Compositions of the invention, for example, pharmaceutical compositions comprising one or more compounds of the invention and one or more additional active agents, may be 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).
[0070] In one embodiment, the dosage forms are in the form of a powder aerosol (i.e, comprise particles). These are particularly suitable for use in inhalation delivery systems. Powders may comprise particles of any size suitable for administration to the lung.
[0071] Powder formulations may optionally contain at least one particulate pharmaceutically acceptable carrier known to those of skill in the at. Examples of suitable pharmaceutical carriers include, but are not limited to, saccharides, including monosaccharides, disaccharides, polysaccharides and sugar alcohols such as arabinose, glucose, fructose, ribose, mannose, sucrose, trehalose, lactose, maltose, starches, dextran, mannitol or sorbitol. In one embodiment, a powder formulation may comprise lactose as a carrier.
[0072] Powder formulations may be contained in any container known to those in the art. Containers may be capsules of, for example, gelatin or plastic, or in blisters (e.g. of aluminum or plastic), for use in a dry powder inhalation device. In some embodiments, the total weight of the formulation in the container may be from about 5 mg to about 50 mg. In other embodiments, powder formulations may be contained in a reservoir in a multi-dose dry powder inhalation device adapted to deliver a suitable amount per actuation.
[0073] Powder formulations typically comprise small particles. Suitable particles can be prepared using any means known in the art, for example, by grinding in an airjet mill, ball mill or vibrator mill, sieving, microprecipitation, spray-drying, lyophilisation or controlled crystallisation. Typically, particles will be about 10 microns or less in diameter. Particles for use in the compositions of the invention may have a diameter of from about 0.1 microns to about 10 microns, from about 0.1 microns to about 9 microns, from about 0.1 microns to about 8 microns, from about 0.1 microns to about 7 microns, from about 0.1 microns to about 6 microns, from about 0.1 microns to about 5 microns, from about 0.1 microns to about 4 microns, from about 0.1 microns to about 3 microns, from about 0.1 microns to about 2 microns, from about 0.1 microns to about 1 micron, from about 0.1 microns to about 0.5 microns, from about 1 micron to about 10 microns, from about 1 micron to about 9 microns, from about 1 micron to about 8 microns, from about 1 micron to about 7 microns, from about 1 micron to about 6 microns, from about 1 micron to about 5 microns, from about 1 micron to about 4 microns, from about 1 micron to about 3 microns, from about 1 micron to about 2 microns, from about 2 microns to about 10 microns, from about 2 microns to about 9 microns, from about 2 microns to about 8 microns, from about 2 microns to about 7 microns, from about 2 microns to about 6 microns, from about 2 microns to about 5 microns, from about 2 microns to about 4 microns, or from about 2 microns to about 3 microns. As used herein, “about” used to modify a numerical value means within 10% of the value. In some embodiments, particles for use in the invention may be about 1 micron, about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 microns in diameter.
[0074] In one embodiment, the dosage forms are in the form of a solution aerosol (i.e., comprise droplets). Typically, droplets will be about 10 microns or less in diameter. Droplets for use in the compositions of the invention may have a diameter of from about 0.1 microns to about 10 microns, from about 0.1 microns to about 9 microns, from about 0.1 microns to about 8 microns, from about 0.1 microns to about 7 microns, from about 0.1 microns to about 6 microns, from about 0.1 microns to about 5 microns, from about 0.1 microns to about 4 microns, from about 0.1 microns to about 3 microns, from about 0.1 microns to about 2 microns, from about 0.1 microns to about 1 micron, from about 0.1 microns to about 0.5 microns, from about 1 micron to about 10 microns, from about 1 micron to about 9 microns, from about 1 micron to about 8 microns, from about 1 micron to about 7 microns, from about 1 micron to about 6 microns, from about 1 micron to about 5 microns, from about 1 micron to about 4 microns, from about 1 micron to about 3 microns, from about 1 micron to about 2 microns, from about 2 microns to about 10 microns, from about 2 microns to about 9 microns, from about 2 microns to about 8 microns, from about 2 microns to about 7 microns, from about 2 microns to about 6 microns, from about 2 microns to about 5 microns, from about 2 microns to about 4 microns, or from about 2 microns to about 3 microns. As used herein, “about” used to modify a numerical value means within 10% of the value. In some embodiments, particles and/or droplets for use in the invention may be about 1 micron, about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, or about 10 microns in diameter.
[0075] The compositions of the invention may be formulated for enteric delivery, for example, may comprise one or more coatings including, for example, a 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 compounds of the invention and/or additional active agent from the composition in the duodenum or the jejunum.
[0076] The term “stable in gastric fluid” refers to a composition that releases 30% or less by weight of the total compound of the invention and/or additional active agent in the composition in gastric fluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5 or lea, in approximately sixty minutes. Examples of simulated gastric fluid and simulated intestinal fluid include, but we 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.
[0077] 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%, from about 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 compound of the invention and/or additional active agent 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 used 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 compound of the invention 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.
[0078] The term “unstable in intestinal fluid” refers to a composition that releases 70% or more by weight of the total amount of the compound of the invention and/or additional active agent 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 the compound of the invention and/or additional active agent 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 compound of the invention and/or additional active agent 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, from about 10 minutes to about 90 minutes, from about 15 minutes to about 90 minutes, from about 20 minutes to about 90 minutes, from about 25 minutes to about 90 minutes, from about 30 minutes to about 90 minutes, from about 5 minutes to about 60 minutes, from about 10 minutes to about 60 minutes, from about 15 minutes to about 60 minutes, from about 20 minutes to about 60 minutes, from about 25 minutes to about 60 minutes, or from about 30 minutes to about 60 minutes. As used herein, “about” used to modify a numerical value means within 10% of the value.
[0079] Compositions of the invention may be formulated for transcutaneous delivery (e.g., may be transcutaneous dosage forms). Typically such compositions may be provided as topical solutions and/or gels. Those of skill in the art are aware of many different methods and devices for the formation of topical medications, for example, those disclosed by Block, Medicated Topicals, in Remington: The Science and Practice of Pharmacy, 20th Ed., Chapter 44, Gennaro et al. Eds., Lippincott, Williams and Wilkins Publishing Co. (2000).
[0080] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
[0081] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not adsorb.
[0082] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome.
[0083] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose. Other controlled release systems are well known in the art.
[0084] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[0085] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pam at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0086] The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived f om hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0087] The amount of the compound of the invention that will be effective in the treatment, inhibition and/or prevention of a disease or disorder associated with increased biological barrier permeability can be determined by standard clinical techniques. The amount of the compound of the invention that will be effective in the treatment, inhibition and/or prevention of a disease or disorder associated with translocation of one or more gliadin-derived peptides across a biological barrier can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[0088] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
Additional Active Agents
[0089] In addition to one or more compounds of the invention, compositions of the invention may further comprise one or more additional active agents, e.g., therapeutic agents, immunogenic agents and/or imaging agents.
[0090] Additional therapeutic agents that can be used in the compositions of the invention include agents that act on any organ of the body, such as heart, brain, intestine, or kidneys. Suitable additional therapeutic agents include, but are not limited to, glucose metabolism agents (e.g., insulin), antibiotics, antineoplastics, antihypertensives, antiepileptics, central nervous system agents, anti-inflammatory agents and immune system suppressants.
[0091] Additional therapeutic agents that can be used in the compositions of the invention include immunosuppressive agents. Such immunosuppressants used in the method and composition of the invention can be any agent which tends to attenuate the activity of the humoral or cellular immune systems. In particular, in one aspect the invention comprises compositions wherein the immunosuppressant is selected from the group consisting of cyclosporin A, FK506, prednisone, methylprednisolone, cyclophosphamide, thalidomide, azathioprine, and daclizumab, physalin B, physalin F, physalin G, seco-steroids purified from Physalis angulata L., 15-deoxyspergualin (DSG, 15-dos), MMF, rapamycin and its derivatives, CCI-779, FR 900520, FR 900523, NK86-1086, depsidomycin, kanglemycin-C, spergualin, prodigiosin25-c, cammunomicin, demethomycin, tetranactin, tranilast, stevastelins, myriocin, gliooxin, FR 651814, SDZ214-104, bredinin, WS9482, mycophenolic acid, mimoribine, misoprostol, OKT3, anti-IL-2 receptor antibodies, azaporine, leflunomide, mizoribine, azaspirane (SKF 105685), paclitaxel, altretamine, busulfan, chlorambucil, ifosfamide, mechlorethamine, melphalan, thiotepa, cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine, pentostatin, methotrexate, 6-mercaptopurine, cytarabine, carmustine, lomustine, streptozotocin, carboplatin, cisplatin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, JM216, JM335, fludarabine, aminoglutethimide, flutamide, goserelin, leuprolide, megestrol acetate, cyproterone acetate, tamoxifen, anastrozole, bicalutamide, dexamethasone, diethylstilbestrol, bleomycin, dactinomycin, daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone, mitomycin-c, plicamycin, paclitaxel, docetaxel, topotecan, irinotecan, 9-amino camptothecan, 9-nitro camptothecan, GS-211, etoposide, teniposide, vinblastine, vincristine, vinorelbine, procarbazine, asparaginase, pegaspargase, octreotide, estramustine, and hydroxyurea, and combinations thereof. In one more particular aspect, the immunosuppressant is cyclosporin A.
[0092] Furthermore, the additional therapeutic agent can be selected from the group consisting of a chemotherapeutic, a gene therapy vector, a growth factor, a contrast agent, an angiogenesis factor, a radionuclide, an anti-infection agent, an anti-tumor compound, a receptor-bound agent, a hormone, a steroid, a protein, a complexing agent, a polymer, a thrombin inhibitor, an antithrombogenic agent, a tissue plasminogen activator, a thrombolytic agent, a fibrinolytic agent, a vasospasm inhibitor, a calcium channel blocker, a nitrate, a nitric oxide promoter, a vasodilator, an antihypertensive agent, an antimicrobial agent, an antibiotic, a glycoprotein IIb/IIIa inhibitor, an inhibitor of surface glycoprotein receptors, an antiplatelet agent, an antimitotic, a microtubule inhibitor, a retinoid, an antisecretory agent, an actin inhibitor, a remodeling inhibitor, an antisense nucleotide, an agent for molecular genetic intervention, an antimetabolite, an antiproliferative agent, an anti-cancer agent, a dexamethasone derivative, an anti-inflammatory steroid, a non-steroidal anti-inflammatory agent, an immunosuppressive agent, a PDGF antagonist, a growth hormone antagonist, a growth factor antibody, an anti-growth factor antibody, a growth factor antagonist, a dopamine agonist, a radiotherapeutic agent, an iodine-containing compound, a barium-containing compound, a heavy metal functioning as a radiopaque agent, a peptide, a protein, an enzyme, an extracellular matrix component, a cellular component, an angiotensin converting enzyme inhibitor, a 21-aminosteroid, a free radical scavenger, an iron chelator, an antioxidant, a sex hormone, an antipolymerase, an antiviral agent, an IgG2 Kappa antibody against Pseudomonas aeruginosa exotoxin A and reactive with A431 epidermoid carcinoma cells, monoclonal antibody against the noradrenergic enzyme dopamine beta-hydroxylase conjugated to saporin or other antibody targeted therapy agents, gene therapy agents, a prodrug, a photodynamic therapy agent, and an agent for treating benign prostatic hyperplasia (BHP), a .sup.14C-, .sup.3H-, .sup.131I-, .sup.32P- or .sup.36S-radiolabelled form or other radiolabelled form of any of the foregoing, and combinations thereof.
[0093] More particularly, the additional therapeutic agent can be selected from the group consisting of parathyroid hormone, heparin, human growth hormone, covalent heparin, hirudin, hirulog, argatroban, D-phenylalanyl-L-poly-arginyl chloromethyl ketone, urokinase, streptokinase, nitric oxide, triclopidine, aspirin, colchicine, dimethyl sulfoxide, cytochalasin, deoxyribonucleic acid, methotrexate, tamoxifen citrate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, cyclosporin, trapidal, angiopeptin, angiogenin, dopamine, .sup.60Co, .sup.192Ir, .sup.32P, .sup.111In, .sup.90Y, .sup.99mTc, pergolide mesylate, bromocriptine mesylate, gold, tantalum, platinum, tungsten, captopril, enalapril, ascorbic acid, α-tocopherol, superoxide dismutase, deferoxamine, estrogen, azidothymidine (AZT), acyclovir, famciclovir, rimantadine hydrochloride, ganciclovir sodium, 5-aminolevulinic acid, meta-tetrahydroxyphenylchlorin, hexadecafluoro zinc phthalocyanine, tetramethyl hematoporphyrin, and rhodamine 123, and combinations thereof.
[0094] Compositions of the invention may comprise one or more immunogenic agents, for example, antigens. Examples of antigens that can be used in the compositions of the invention (e.g., immunogenic and/or vaccine compositions) include peptides, proteins, microorganisms (e.g., attenuated and/or recombinant microorganisms), cells (e.g., cancer cells and/or recombinant cells) and viruses (e.g., attenuated and/or recombinant viruses). Examples of peptide antigens include the B subunit of the heat-labile enterotoxin of enterotoxigenic E. coli, the B subunit of cholera toxin, capsular antigens of enteric pathogens, fimbriae or pili of enteric pathogens, HIV surface antigens, cancer antigens (e.g., cancer cells comprising antigens, isolated antigens, etc.), dust allergens, and acari allergens. Other immunogenic compounds as am known in the art can also be used.
[0095] Examples of attenuated microorganisms and viruses that can be used in the compositions of the invention (e.g., vaccine compositions) include those of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, Vibrio cholerae, Shigella flexneri, Salmonella typhi and rotavirus (Fasano et al, In: Le Vaccinazioni in Pediatria, Eds. Vierucci et al, CSH, Milan, pages 109-121 (1991); Guandalini et al, In: Management of Digestive and Liver Disorders in Infants and Children, Elsevior, Eds. Butz et al, Amsterdam, Chapter 25 (1993); Levine et al, Sem. Ped. Infect. Dis., 5.243-250 (1994); and Kaper et al, Clin. Micrbiol. Rev., 8:48-86 (1995), each of which is incorporated by reference herein in its entirety).
[0096] Any antigen capable of inducing a protective immune response may be used in the vaccine compositions of the invention. Examples of suitable antigens include, but are not limited to, measles virus antigens, mumps virus antigens, rubella virus antigens, Corynebacterium diphtheriae antigens, Bordetella pertussis antigens, Clostridium tetani antigens, Bacillus anthracis antigens, Haemophilus influenzae antigens, smallpox virus antigens, and influenza virus antigens.
[0097] Compositions of the invention may further comprise one or more protease inhibitors. Any protease inhibitor can be used, including, but not limited to, a proteinase, peptidase, endopeptidase, or exopeptidase inhibitor. A cocktail of inhibitors can also be used. Alternatively, the protease inhibitors can be selected from the group consisting of bestatin, L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, ethylenediaminetetra-acetic acid (EDTA), phenylmethylsulfonylfluoride (PMSF), aprotinin, amyloid protein precursor (APP), amyloid beta precursor protein, al-proteinase inhibitor, collagen VI, bovine pancreatic trypsin inhibitor (BPTI), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), antipain, benzamidine, chymostatin, ε-aminocaproate, N-ethylmaleimide, leupeptin, pepstatin A, phosphoramidon, and combinations thereof. Novel protease inhibitors can also be used. Indeed, protease inhibitors can be specifically designed or selected to decrease the proteolysis of the tight junction agonist and/or the therapeutic agent.
[0098] 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, sugars, binders, and the like.
Methods of Treatment
[0099] Compounds and pharmaceutical compositions of the invention can be used for treating, ameliorating, and/or preventing a disease. Any disease may be treated using the compositions of the invention by selection of an appropriate active agent, e.g., therapeutic and/or immunogenic agent. In one embodiment, the present invention provides a method of treating diabetes response in a subject (e.g., a mammal such as a human) by administering a composition comprising one or more compounds of the invention together with one or more insulins and/or derivatives thereof. In another embodiment, the invention provides a method of suppressing an excessive or undesirable immune response in a subject (e.g., a mammal such as a human) by administering a composition comprising one or more compounds of the invention together with one or more immune-suppressive drugs that may include, for example, cyclosporin A.
[0100] Examples of diseases that can be treated using the compositions of the invention include, but are not limited to, cancer, autoimmune diseases, vascular disease, bacterial infections, gastritis, gastric cancer, collagenous colitis, inflammatory bowel disease, necrotizing enterocolitis, osteoporosis, systemic lupus erythematosus, food allergy, asthma, celiac disease and irritable bowel syndrome. For example, to treat inflammatory bowel disease, a composition comprising one or more compounds of the invention may be administered to the subject (e.g., a mammal such as a human) in need thereof.
[0101] In another example, to treat cancer of the colon or rectal area, a composition comprising a therapeutically effective amount of Erbitux® (Cetuximab) together with a GM-CSP and/or IL-16 inhibiting amount of one or more compounds of the invention may be administered to the subject (e.g., a mammal such as a human) in need thereof. In another example, to treat breast cancer, a composition comprising a therapeutically effective amount of Herceptin® (Trastuzumab) together with a GM-CSF and/or IL-16 inhibiting amount of one or more compounds of the invention may be administered to the subject (e.g., a mammal such as a human) in need thereof. In another example, to treat various types of cancer, a composition comprising a therapeutically effective amount of Avastin® (Bevacizumab) together with a GM-CSF and/or IL-16 inhibiting amount of one or more compounds of the invention may be administered to the subject (e.g., a mammal such as a human) in need thereof. Another example involves treatment of osteoporosis by administration of a composition comprising one or more compounds of the invention together with a therapeutically effective amount of Fosamax (Alendronate) to the subject in need thereof. Another example involves treatment of transplant rejection by administration of a composition comprising one or compounds of the invention together with a therapeutically effective amount of Cyclosporin A to the subject in need thereof. Another example involves treatment of anemia by administration of a composition comprising one or more compounds of the invention together with a therapeutically effective amount of erythropoietin to the subject in need thereof. Another example involves treatment of hemophilia by administration of a composition comprising one or more compounds of the invention together with a therapeutically effective amount of Factor VIII to the subject in need thereof.
[0102] In some embodiments, compositions of the invention (e.g., pharmaceutical compositions) 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 n attack of the underlying disease condition (e.g., diabetes, cancer, transplant rejection, etc.). Such compositions may be administered chronically, for example, one or more times daily over a plurality of days.
[0103] In some embodiments, compositions of the invention (e.g., pharmaceutical compositions) may be used to treat acute attacks of the underlying disease (e.g., diabetes, cancer, transplant rejection, etc.). 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 administrations may be used.
[0104] In some embodiments, compounds of the invention may be used in the manufacture of compositions and pharmaceutical compositions for use in the methods described above.
[0105] While the invention has been described with reference to certain particular embodiments thereof those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.
Methods of Screening
[0106] Screening for inhibitors of gliadin-derived peptide translocation across biological barriers can be accomplished by a variety of techniques. Likewise, screening for inhibitors of PTG-induced factors that increase biological barrier permeability can be accomplished by a variety of techniques. Gliadin-derived peptide binding to test compounds (inhibitor candidates) can be directly measured, or inhibition of binding of gliadin-derived peptides to a cell preparation can be measured. Gliadin-derived peptides can be labeled to facilitate measurement of binding. Assays may be in cell-free systems or in cell-based systems. Any binding assay format can be used, including formats where the receptor is attached to a solid support, either directly or indirectly.
[0107] Test compounds which can be tested are any compounds. The compounds may be tested as single compounds or in combinations of compounds. The compounds may be structurally identified or of unknown structure. The compounds may be novel or previously known. The compounds may be natural products or synthetic.
[0108] According to one embodiment of the invention the test compounds are fragments of gliadin. Gliadin is a family of proteins which are produced by wheat and other grains. Examples of gliadins are gliadin alpha, gamma, and omega. Gliadins are the aqueous alcohol-soluble storage proteins in the seed. There is great heterogeneity even within a single class of gliadins. At least six, seven, eight, nine, ten, eleven, fifteen, twenty, thirty, thirty-five, fifty, or seventy-five amino acid residues may be used in fragments of gliadin as test compounds. Fragments include any molecule which is less than full length. Fragments may be, e.g., synthesized or the result of proteolytic degradation. The following tables provide the sequences of a representative number of gliadins.
TABLE-US-00001 TABLE 1 Amino acid sequence of alpha-gliadin from Triticum aestivum (NCBI accession no. CAB76964, (SEQ ID NO: 165)) 1 mvrvpvqlqp pqnpsqqqpq eqvplvqqqq fpgqqqpfpp qqpypqpqpf 51 psqqpylqlq pfpqpqlpyp qpqlpypqpq lpypqpqpfr pqqpypqsqp 101 qysqpqqpis qqqqqqqqqq qqkqqqqqqq qilqqilqqq lipcrdvvlq 151 qhsiaygssq vlqqstyqlv qqlccqqlwq ipeqsrcqai hnvvhaiilh 201 qqqqqqqqqq qqplsqvsfq qpqqqypsgq gsfqpsqqnp qaqgsvqpqq 251 lpqfeeirnl aletlpamcn vyippyctia pvgifgtnyr
TABLE-US-00002 TABLE 2 Amino acid sequence of alpha-gliadin precursor from Triticum turgidum subsp. durum (NCBI accession no. CA135909, (SEQ ID NO: 166)) 1 mktflilall aivattatta vrvpvpqlqr qnpsqqqpqe qvplvqqqqf 51 lgqqqpfppq qpypqpqpfp sqqpylqlqp fpqpqlpysq pqpfrpqqpy 101 pqpqprysqp qqpisqqqqq qhqqhqqhhq eqqilqqilq qqlipcmdvv 151 lqqhniahrr sqvlqqstyq llqelccqhl wqipeqsqcq aihnvvhaii 201 phqqqkqqqq pssqfsfqqp lqqyplgqgs frpsqqnpqa qgsvqpqqlp 251 qfeeirnlal qtlpamcnvy ippyctiapf gifgtn
TABLE-US-00003 TABLE 3 Amino acid sequence of alpha/beta-gliadin precursor from Triticum aestivum (NCBI accession no. AAA34280, (SEQ ID NO: 167)) 1 mktflilvll aivattatta vrfpvpqlqp qnpsqqqpqe qvplvqqqqf 51 lgqqqpfppq qpypqpqpfp sqlpylqlqp fpqpqlpysq pqpfrpqqpy 101 pqpqpqysqp qqpisqqqqq qqqqqqqqqq qqqilqqilq qqlipcmdvv 151 lqqhniahgr sqvlqqstyq llqelccqhl wqipeqsqcq aihnvvhaii 201 lhqqqkqqqq pssqvsfqqp lqqyplgqgs frpsqqnpqa qgsvqpqqlp 251 qfeeirnlal qtlpamcnvy ippyctiapf gifgtn
TABLE-US-00004 TABLE 4 Amino acid sequence of Gamma-gliadin precursor from Triticum aestivum (NCBI accession no. P21292, (SEQ ID NO: 168)) 1 mktlliltil amattiatan mqvdpsgqvq wpqqqpfpqp qqpfcqqpqr 51 tipqphqtfh hqpqqtfpqp qqtyphqpqq qfpqtqqpqq pfpqpqqtfp 101 qqpqlpfpqq pqqpfpqpqq pqqpfpqsqq pqqpfpqpqq qfpqpqqpqq 151 sfpqqqqpai qsflqqqmnp cknfllqqcn hvslvsslvs iilprsdcqv 201 mqqqccqqla qipqqlqcaa ihsvahsiim qqeqqqgvpi lrplfqlaqg 251 lgiiqpqqpa qlegirslvl ktlptmcnvy vppdcstinv pyanidagig 301 gq
TABLE-US-00005 TABLE 5 Amino acid sequence of Gamma-gliadin B precursor from Triticum aestivum (NCBI accession no. P06659, (SEQ ID NO: 169)) 1 mktlliltil amaitiatan mqadpsgqvq wpqqqpflqp hqpfsqqpqq 51 ifpqpqqtfp hqpqqqfpqp qqpqqqflqp rqpfpqqpqq pypqqpqqpf 101 pqtqqpqqpf pqskqpqqpf pqpqqpqqsf pqqqpsliqq slqqqlnpck 151 nfllqqckpv slvsslwsii lppsdcqvmr qqccqqlaqi pqqlqcaaih 201 svvhsiimqq eqqeqlqgvq ilvplsqqqq vgqgilvqgq giiqpqqpaq 251 levirslvlq tlptmcnvyv ppycstirap fasivasigg q
TABLE-US-00006 TABLE 6 Amino acid sequence of Gamma-gliadin (Gliadin B-III) from Triticum aestivum (NCB1 accession no. P04730, (SEQ ID NO: 170)) 1 pqqpfplqpq qsflwqsqqp flqqpqqpsp qpqqvvqiis patpttipsa 51 gkptsapfpq qqqqhqqlaq qqipvvqpsi lqqlnpckvf lqqqcspvam 101 pqrlarsqml qqsschvmqq qccqqlpqip qqsryqaira iiysiilqeq 151 qqvqgsiqsq qqqpqqlgqc vsqpqqqsqq qlgqqpqqqq laqgtflqph 201 qiaqlevmts ialrilptmc svnvplyrtt tsvpfgvgtg vgay
TABLE-US-00007 TABLE 7 Amino acid sequence of Gamma-gliadin precursor from Triticum aestivum (NCBI accession no. P08453, (SEQ ID NO: 171)) 1 mktlliltil amaitigtan iqvdpsgqvq wlqqqlvpql qqplsqqpqq 51 tfpqpqqtfp hqpqqqvpqp qqpqqpflqp qqpfpqqpqq pfpqtqqpqq 101 pfpqqpqqpf pqtqqpqqpf pqqpqqpfpq tqqpqqpfpq lqqpqqpfpq 151 pqqqlpqpqq pqqsfpqqqr pfiqpslqqq lnpcknillq qskpaslvss 201 lwsiiwpqsd cqvmrqqccq qlaqipqqlq caaihsvvhs iimqqqqqqq 251 qqqgidiflp lsqheqvgqg slvqgqgiiq pqqpaqleai rslvlqtlps 301 mcnvyvppec simrapfasi vagiggq
TABLE-US-00008 TABLE 8 Amino acid sequence of Gamma-gliadin B-I precursor from Triticum aestivum (NCBI accession no. P04729, (SEQ ID NO: 172)) 1 mktflvfali avvatsaiaq metscisgle rpwqqqplpp qqsfsqqppf 51 sqqqqqplpq qpsfsqqqpp fsqqqpilsq qppfsqqqqp vlpqqspfsq 101 qqqlvlppqq qqqqlvqqqi pivqpsvlqq lnpckvflqq qcspvampqr 151 larsqmwqqs schvmqqqcc qqlqqipeqs ryeairaiiy siilqeqqqg 201 fvqpqqqqpq qsgqgvsqsq qqsqqqlgqc sfqqpqqqlg qqpqqqqqqq 251 vlqgtflqph qiahleavts ialrtlptmc svnvplysat tsvpfgvgtg 301 vgay
TABLE-US-00009 TABLE 9 Amino acid sequence of Gamma-gliadin precursor from Triticum aestivum (NCBI accession no. P08079, (SEQ ID NO: 173)) 1 mktlliltil amaitigtan mqvdpssqvq wpqqqpvpqp hqpfsqqpqq 51 tfpqpqqtfp hqpqqqfpqp qqpqqqflqp qqpfpqqpqq pypqqpqqpf 101 pqtqqpqqlf pqsqqpqqqf sqpqqqfpqp qqpqqsfpqg qppfiqpslg 151 qqvnpcknfl lqqckpvslv sslwsmiwpq sdcqvmrqqc cqqlaqipqq 201 lqcaaihtii hsiimqqeqq eqqqgmhill plyqqqqvgq gtlvqgqgii 251 q
TABLE-US-00010 TABLE 10 Amino acid sequence of Alpha/beta-gliadin MM1 precursor (Prolamin) from Triticum aestivum (NCBI accession no. P18573, (SEQ ID NO: 174)) 1 mktflilall aivattaria vrvpvpqlqp qnpsqqqpqe qvplvqqqqf 51 pgqqqpfppq qpypqpqpfp sqqpylqlqp fpqpqlpypq pqlpypqpql 101 pypqpqpfrp qqpypqsqpq ysqpqqpisq qqqqqqqqqq qkqqqqqqqq 151 ilqqilqqql ipcrdvvlqq hsiaygssqv lqqstyqlvq qlccqqlwqi 201 peqsrcqaih nvvhaiilhq qqqqqqqqqq qplsqvsfqq pqqqypsgqg 251 sfqpsqqnpq aqgsvqpqql pqfeeirnla letlpamcnv yippyctiap 301 vgifgtn
TABLE-US-00011 TABLE 11 Amino acid sequence of Alpha/beta-gliadin clone PTO-A10 (Prolamin) from Triticum aestivum (NCBI accession no. P04728, (SEQ ID NO: 175)) 1 pqpqpqysqp qqpisqqqqq qqqqqqqqqq eqqilqqilq qqlipcmdvv 51 lqqhniahgr sqvlqqstyq llgelccqhl wqipeqsqcq aihnvvhaii 101 lhqqqqkqqq qpssqfsfqq plqqyplgqg sfrpsqqnpq aqgsvqpqql 151 pqfeirnlal qtlpamcnvy ippyctiapf gifgtn
TABLE-US-00012 TABLE 12 Amino acid sequence of Alpha/beta-gliadin clone PW8142 precursor (Prolamin) from Triticum aestivum (NCBI accession no. P04727, (SEQ ID NO: 176)) 1 mktflilalv attattavrv pvpqlqpknp sqqqpqeqvp lvqqqqfpgq 51 qqqfppqqpy pqpqpfpsqq pylqlqpfpq pqpflpqlpy pgpgsfppqg 101 pypqqrpkyl qpqqpisqqq aqqqqqqqqq qqqqqqqqil qqilqqqlip 151 crdvvlqqhn iahassqvlq qstyqllqql ccqqllqipe qsrcqaihnv 201 vhaiimhqqe qqqqlqqqqg qqlqqqqqqq qqqqqpssqv sfqqpqqqyp 251 ssqgsfqpsq qnpqaqgsvq pqqlpqfaei rnlalqtlpa mcnvyipphc 301 sttiapfgif gtn
TABLE-US-00013 TABLE 13 Amino acid sequence of Alpha/beta-gliadin clone PW1215 precursor (Prolamin) from Triticum aestivum (NCBI accession no. P04726, (SEQ ID NO: 177)) 1 mktflilall aivattatta vrvpvpqpqp qnpsqpqpqg qvplvqqqqf 51 pgqqqqfppq qpypqpqpfp sqqpylqlqp fpqpqpfppq lpypqpppfs 101 pqqpypqpqp qypqpqqpis qqqaqqqqqq qqqqqqqqqq qqilqqilqq 151 qlipcrdvvl qqhniahars qvlqqstyqp lqqlccqqlw qipeqsrcqa 201 ihnvvhaiil hqqqrqqqps sqvslqqpqq qypsgqgffq psqqnpqaqg 251 svqpqqlpqf eeirnlalqt lprmcnvyip pycsttiapf gifgtn
TABLE-US-00014 TABLE 14 Amino acid sequence of Alpha/beta-gliadin A-IV precursor (Prolamin) from Triticum aestivum (NCBI accession no. P04724, (SEQ ID NO: 178)) 1 mktflilalr aivattatia vrvpvpqlqp qnpsqqqpqk qvplvqqqqf 51 pgqqqpfppq qpypqqqpfp sqqpymqlqp fpqpqlpypq pqlpypqpqp 101 frpqqsypqp qpqysqpqqp isqqqqqqqq qqqqqqqilq qilqqqlipc 151 rdvvlqqhsi ahgssqvlqq styqlvqqfc cqqlwqipeq srcqaihnvv 201 haiilhqqqq qqqqqqqqqq qplsqvcfqq sqqqypsgqg sfqpsqqnpq 251 aqgsvqpqql pqfeeirnla letlpamcnv yippyctiap vgifgtn
TABLE-US-00015 TABLE 15 Amino acid sequence of Alpha/beta-gliadin A-III precursor (Prolamin) from Triticum aestivum (NCBI accession no. P04723, (SEQ ID NO: 179)) 1 mktflilall aivattatsa vrvpvpqlqp qnpsqqqpqe qvplmqqqqq 51 fpgqqeqfpp qqpyphqqpf psqqpypqpq pfppqlpypq tqpfppqqpy 101 pqpqpqypqp qqpisqqqaq qqqqqqqtlq qilqqqlipc rdvvlqqhni 151 ahassqvlqq ssyqqlqqlc cqqlfqipeq srcqaihnvv haiilhhhqq 201 qqqqpssqvs yqqpqeqyps gqvsfqssqq npqaqgsvqp qqlpqfqeir 251 nlalqtlpam cnvyippycs ttiapfgifg tn
TABLE-US-00016 TABLE 16 Amino acid sequence of Alpha/beta-gliadin A-II precursor (Prolamin) from Triticum aestivum (NCBI accession no. P04722, (SEQ ID NO: 180)) 1 mktfpilall aivattatta vrvpvpqlql qnpsqqqpqe qvplvqeqqf 51 qgqqqpfppq qpypqpqpfp sqqpylqlqp fpqpqlpypq pqpfrpqqpy 101 pqpqpqysqp qqpisqqqqq qqqqqqqqqq ilqqilqqql ipcrdvvlqq 151 hniahgssqv lqestyqlvq qlccqqlwqi peqsrcqaih nvvhaiilhq 201 qhhhhqqqqq qqqqqplsqv sfqqpqqqyp sgqgffqpsq qnpqaqgsfq 251 pqqlpqfeei rnlalqtlpa mcnvyippyc tiapfgifgt n
TABLE-US-00017 TABLE 17 Amino acid sequence of Alpha/beta-gliadin A-I precursor (Prolamin) from Triticum aestivum (NCBI accession no. P04721, (SEQ ID NO: 181)) 1 mktflilall aivattatta vrvpvpqlqp qnpsqqqpqe qvplvqqqqf 51 lgqqqpfppq qpypqpqpfp sqqpylqlqp flqpqlpysq pqpfrpqqpy 101 pqpqpqysqp qqpisqqqqq qqqqqqqqqq qqqqiiqqil qqqlipcmdv 151 vlqqhnivhg ksqvlqqsty qllqelccqh lwqipeqsqc qaihnvvhai 201 ilhqqqkqqq qpssqvsfqq plqqyplgqg sfrpsqqnpq aqgsvqpqql 251 pqfeeirnla rk
TABLE-US-00018 TABLE 18 Amino acid sequence of gamma gliadin from Triticum aestivum (NCBI accession no. AAQ63860, (SEQ ID NO: 182)) 1 mniqvdpssq vpwpqqqpfp qphqpfsqqp qqtfpqpqqt fphqpqqqfs 51 qpqqpqqqfi qpqqpfpqqp qqtypqrpqq pfpqtqqpqq pfpqsqqpqq 101 pfpqpqqqfp qpqqpqqsfp qqqpsliqqs lqqqlnpckn fllqqckpvs 151 lvsslwsmil prsdcqvmrq qccqqlaqip qqlqcaaihs ivhsiimqqe 201 qqeqrqgvqi lvplsqqqqv gqgtlvqgqg iiqpqqpaql evirslvlqt 251 latmcnvyvp pycstirapf asivagiggq yr
TABLE-US-00019 TABLE 19 Amino acid sequence of Omega-gliadin from Triticum monococcum (NCBI accession no. P02865, (SEQ ID NO: 183)) 1 arqlnpsdqe lqspqqlypq qpypqqpy
[0109] Inhibitors of gliadin-derived peptide translocation across biological barriers are useful for treating diseases characterized by inflammation, including autoimmune diseases and particularly including celiac disease. Inhibitors of PTO-induced factors that increase biological barrier permeability are useful for treating diseases characterized by inflammation, including autoimmune diseases and particularly including celiac disease.
[0110] Activity of inhibitors of gliadin-derived peptide translocation and/or inhibitors of PTO-induced permeability can be measured by any means known in the art. Signaling events which can be determined include decrease in TEER, increase in LY permeability, increase in cytokine release, microglial recruitment, tyrosine kinase phosphorylation and chemotaxis, and increase in MMP-2 and MMP-9 gelatinolytic activity in cell-conditioned media.
[0111] The invention provides methods of identifying agents, compounds or lead compounds for agents active in inhibiting PTO-induced alterations in biological barrier permeability and/or peptide translocation. Generally, screening methods of the invention involve assaying for compounds which modulate the interaction of one or more gliadin fragments with one or more cells (e.g., epithelial cells, immune cells). A wide variety of assays for binding agents is provided including labeled in vitro protein-ligand binding assays, cell based assays, immunoassays, etc. A wide variety of formats may be used, including co-immunoprecipitation, 2-hybrid transactivation, fluorescent polarization, NMR, fluorescent resonance energy transfer (FRET), transcriptional activation, etc. For example, a wide variety of NMR-based methods are available to rapidly screen libraries of small compounds for binding to protein targets (Hajduk, P. J., et al. Quarterly Reviews of Biophysics, 1999, 32 (3): 211-40). In some embodiments, methods of the invention may be automated (e.g., high throughput screening) and may be used to screen chemical libraries for lead compounds. Identified compounds may be used to treat diseases involving increased biological barrier permeability including, for example, celiac disease, inflammatory bowel diseases and autoimmune disease& Compounds identified by the methods of the invention may be further optimized to modulate biological barrier modulation, for example, may be derivatized. Multiple iterations of screening and derivatization may be employed to optimize the modulation of biological barrier permeability.
[0112] In vitro ligand binding assays employ a mixture of components including one or more gliadin-derived peptides or fragments and one or more gliadin binding components. Gliadin-derived peptides or fragments may be provided as fusion proteins (e.g., with purification tags such as 6-His). Assay mixtures typically further comprise a compound to be tested for inhibitory activity. Compounds to be tested may be of any kind known to those skilled in the art, for example, may be organic compounds, peptides, proteins, nucleic acids, lipids, carbohydrates and mixtures thereof. A variety of other reagents may also be included in the mixture including, but not limited to, salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc.
[0113] In general, assay mixtures may be incubated under conditions in which, but for the presence of the compound to be tested, gliadin-derived peptides or fragments specifically bind the gliadin binding components with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding. In some embodiments, incubation periods may be minimized to facilitate rapid, high-throughput screening.
[0114] After incubation, the effect of the compound to be tested on the gliadin binding may be detected by any convenient way. For example, the gliadin-derived peptide or fragment or the gliadin binding component may be immobilized, and the other labeled; then in a solid-phase format, any of a variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.
[0115] A difference in the binding affinity of the gliadin-derived peptide or fragment and the gliadin binding component in the absence of the compound to be tested as compared with the binding affinity in the presence of the compound to be tested indicates that the compound modulates the binding of the gliadin-derived peptide or fragment and the gliadin binding component. A difference, as used herein, is statistically significant and preferably represents at least a 50%, 60%, 70%, 80%, or 90% difference.
[0116] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
Example 1
[0117] Measurement of Trans Epithelial Electric Resistance (TEER) and Epithelial Flux of a Fluorescent Marker Lucifer Yellow
[0118] CaCo2 cells form monolayers that exhibit tight junctions between adjacent cells. Agonists of tight junctions can be identified by their ability to enhance the flux of compounds (e.g. ions, Lucifer Yellow) through a cell monolayer that comprises tight junctions; or by their ability to reduce TEER across a cell monolayer that comprises tight junctions. Treatment of CaCo2 monolayers with peptide tight junction agonist compounds leads to enhancement of Lucifer Yellow permeability through CaCo2 monolayers compared to vehicle alone. Treatment of CaCo2 monolayers with peptide tight junction agonist compounds leads to a decrease in TEER across CaCo2 monolayers compared to vehicle alone.
[0119] Tight junction agonists and agonists of the Clorf43 and CCDC78 proteins can be identified using the following method, and this method may be easily modified to identify antagonists and inhibitors of the Clorf43 and CCDC78 proteins:
[0120] Determination of TEER and Lucifer Yellow Flux
[0121] Prepare Modified Hank's Balanced Salt Solution (MHBSS) by obtaining IL bottle of HBSS removing 10 ml of HBSS and replacing it with 10 ml HEPES buffer pH 7.0. Adjust pH to 7.4±0.1 using concentrated NaOH (10N).
[0122] 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.
[0123] Aspirate cell culture medium from both the apical (AP) and basolateral (BL) compartments, replacing with 0.5 ml and 1.5 ml of MHBSS, respectively. Incubate cells at 37° C. for 30 minutes.
[0124] Using the MilliCell®-ERS instrument (Millipore), measure and record the transepithelial electrical resistance (TEER) across each filter and record.
[0125] Aspirate solution from the apical compartment of each filter (n=3 per condition) and replace with 0.5 ml of control and test solutions containing Lucifer Yellow and test compound if appropriate.
[0126] Place all plates into incubator set at 37° C. (±0.2), 50 RPM (±5) for a total of 180 minutes.
[0127] At t=30, 60, 120 and 180 minutes, measure and record the transepithelial electrical resistance (TEER) across each filter using the MilliCell-ERS instrument.
[0128] At t=60, 120 and 180 minutes remove 100 μl from each basolateral compartment and place it in a 96-well plate for Lucifer Yellow analysis, replace with 100 μl of MHBSS.
[0129] Make a Lucifer Yellow standard curve with the following dilutions (7500 μM, 3750 μM, 750 μM, 375 μM, 75 μM, 37.5 μM, 7.5 μM, 3.75 μM, 0.75 μM) and pipette 100 μL 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.
[0130] Harvest the remaining start solutions and what is left in each apical compartment into 1.5 ml vials. Freeze at −20° C. for future analysis.
[0131] Analyze each 96-well plate in a Ten Spectra Fluor Plus using Magellan at 485 and 535 nm.
[0132] Materials:
[0133] Cells: CaCo-2 cells passage 40-60 grown on Transwell® plates for 21-28 days
[0134] Culture Medium: DMEM supplemented with 10% fetal bovine serum, 1% NEAA, 1% Penn/Strep
[0135] Buffers: Hank's Balanced Salt Solution (HBSS) without calcium and magnesium
[0136] Flasks: 100×20 mm Tissue culture dish Falcon.
[0137] Plates: 12 well polycarbonate Transwell® filters; 0.3 uM pore size
Example 2
[0138] Identification of Cytokines Upregulated on Treatment of THP-1 Cells by PT-Gliadin (PTG)
[0139] The monocytic cell line THP-1 was used to characterize the profile of cytokines whose expression was upregulated on exposure to protease treated gliadin (PTG). THP-1 cells were diluted to 5×10.sup.3 cells/ml in RPMI medium supplemented with 10% heat inactivated fetal bovine serum.
[0140] 5×10.sup.5 (1 ml) cells were plated in each well of a 12 well plate, and cells were incubated at 37° C. overnight. Test compounds (PTG 1 mg/ml; LPS 1 μg/ml) were added to the cultures, and incubation was continued a further 18 hours at 37° C.
[0141] Culture supernatants were harvested, and cytokines/chemokines were measured in each sample using a nitrocellulose membrane based proteomic profiler assay (R&D Systems) Assays were performed in triplicate. The cytokines screened in this assay included C5a, C40 ligand, G-CSF, GM-CSF, GRO-α/CXCL1, I-309/CCL1, ICAM-1, IFNγ, IL-1α, IL-1β, L-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32α, IP-10/CXCL10, I-TAC/CXCL11, MCP-1/CCL2, MIF, MIP-1α/CCL3, MIP-1β/CCL3, RANTES/CCL5, SDF-1/CXCL12, Serpin-E1/PAI-1, TNFα, and TREM-1.
[0142] After 6 hours of PTG exposure THP-1 cells demonstrated increased expression of the cytokines IL-8, MIP-1α, MIP-1β, TNF-α and Gro-α. After 24 hours of exposure to PIM increased expression of RANTES and MIF were also observed.
Example 3
[0143] Identification of Cytokines Upregulated on Treatment of PBMCs by PT-Gliadin (PTG)
[0144] Peripheral blood mononuclear cells were isolated from donated human blood samples using methods known in the art, and these PBMCs were used to characterize the profile of cytokines whose expression was upregulated on exposure to protease treated gliadin (PTG). PBMCs were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum, and 2×10.sup.5 cells were plated in each well of a 96 well plate. Cells were incubated at 37° C. with PTG (1 mg/ml) or LPS (1 μg/ml) in the presence or absence of test compounds being examined for the ability to suppress cytokine production. Supernatant samples were harvested following treatment, and cytokines were assayed by ELISA (R&D Systems).
[0145] Expression of IL-6, IL-8, MIP-1α, and Gro-α were induced by treatment with LPS and PTG. Expression of these cytokines was not reduced by treatment with peptide GGVLVQPG (SEQ ID NO:1).
[0146] Increased expression of GM-CSF and IL-16 was induced by exposure to LPS and PTG. This increased expression of these cytokines was inhibited by treatment with peptide GGVLVQPG (SEQ ID NO:1).
[0147] Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. 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.
TABLE-US-00020 TABLE 20 Peptide permeability inhibitors Prevented TEER Reduced LY SEQ ID NO: Sequence Reduction Permeability 1 Gly-Gly-Val-Leu-Val-Gln-Pro-Gly − + 2 Ala-Gly-Val-Leu-Val-Gln-Pro-Gly − + 3 Gly-Ala-Val-Leu-Val-Gln-Pro-Gly − + 4 Gly-Gly-Ala-Leu-Val-Gln-Pro-Gly − + 5 Gly-Gly-Val-Ala-Val-Gln-Pro-Gly − + 6 Gly-Gly-Val-Leu-Ala-Gln-Pro-Gly − − 7 Gly-Gly-Val-Leu-Val-Ala-Pro-Gly − − 8 Gly-Gly-Val-Leu-Val-Gln-Ala-Gly − − 9 Gly-Gly-Val-Leu-Val-Gln-Pro-Ala − − 10 Gly-Asp-Val-Leu-Val-Gln-Pro-Gly + + 11 Gly-Glu-Val-Leu-Val-Gln-Pro-Gly + + 12 Gly-Gln-Val-Leu-Val-Gln-Pro-Gly + + 13 Gly-Phe-Val-Leu-Val-Gln-Pro-Gly + + 14 Gly-His-Val-Leu-Val-Gln-Pro-Gly + + 15 Gly-Arg-Val-Leu-Val-Gln-Pro-Gly + + 16 Gly-Lys-Val-Leu-Val-Gln-Pro-Gly + + 17 Gly-Ile-Val-Leu-Val-Gln-Pro-Gly + + 18 Gly-Trp-Val-Leu-Val-Gln-Pro-Gly − − 19 Gly-Pro-Val-Leu-Val-Gln-Pro-Gly + + 20 Gly-Val-Val-Leu-Val-Gln-Pro-Gly + + 21 Gly-Leu-Val-Leu-Val-Gln-Pro-Gly + + 22 Gly-Asn-Val-Leu-Val-Gln-Pro-Gly + + 23 Gly-Thr-Val-Leu-Val-Gln-Pro-Gly + + 24 Gly-Gly-Gly-Leu-Val-Gln-Pro-Gly − − 25 Gly-Gly-Leu-Leu-Val-Gln-Pro-Gly − − 26 Gly-Gly-Ile-Leu-Val-Gln-Pro-Gly − − 27 Gly-Gly-Phe-Leu-Val-Gln-Pro-Gly + + 28 Gly-Gly-Arg-Leu-Val-Gln-Pro-Gly − − 29 Gly-Gly-Asp-Leu-Val-Gln-Pro-Gly − − 30 Gly-Gly-Gln-Leu-Val-Gln-Pro-Gly − − 31 Gly-Gly-His-Leu-Val-Gln-Pro-Gly − − 32 Gly-Gly-Met-Leu-Val-Gln-Pro-Gly + + 33 Gly-Gly-Ser-Leu-Val-Gln-Pro-Gly − − 34 Gly-Gly-Thr-Leu-Val-Gln-Pro-Gly + + 35 Gly-Gly-Pro-Leu-Val-Gln- − − 36 Gly-Gly-Val-Gly-Val-Gln-Pro-Gly + + 37 Gly-Gly-Val-Val-Val-Gln-Pro-Gly − − 38 Gly-Gly-Val-Ile-Val-Gln-Pro-Gly − − 39 Gly-Gly-Val-Phe-Val-Gln-Pro-Gly − − 40 Gly-Gly-Val-Arg-Val-Gln-Pro-Gly − − 41 Gly-Gly-Val-Asp-Val-Gln-Pro-Gly − − 42 Gly-Gly-Val-Gln-Val-Gln-Pro-Gly − − 43 Gly-Gly-Val-His-Val-Gln-Pro-Gly − − 44 Gly-Gly-Val-Met-Val-Gln-Pro-Gly − − 45 Gly-Gly-Val-Ser-Val-Gln-Pro-Gly − − 46 Gly-Gly-Val-Thr-Val-Gln-Pro-Gly − − 47 Gly-Gly-Val-Pro-Val-Gla-Pro-Gly − − 48 D-Ala-Gly-Val-Leu-Val-Gln-Pro-Gly + + 49 Asp-Gly-Val-Leu-Val-Gln-Pro-Gly + + 50 Glu-Gly-Val-Leu-Val-Gln-Pro-Gly − − 51 Gln-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 52 Phe-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 53 His-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 54 Arg-Gly-Val-Leu-Val-Gln-Pro-Gly − − 55 Lys-Gly-Val-Leu-Val-Gln-Pro-Gly + + 56 Ile-Gly-Val-Leu-Val-Gln-Pro-Gly − − 57 Trp-Gly-Val-Leu-Va1-Gln-Pro-Gly − − 58 Pro-Gly-Val-Leu-Val-Gln-Pro-Gly + + 59 Val-Gly-Val-Leu-Val-Gln-Pro-Gly − − 60 Leu-Gly-Val-Leu-Val-Gln-Pro-Gly − − 61 Thr-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 62 Asn-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 63 D-Phe-Gly-Val-Leu-Val-Gln-Pro-Gly − − 64 Cha-Gly-Val-Leu-Lav-Gln-Pro-Gly NT NT 65 Met(O)2-Gly-Val-Leu-Val-Gln-Pro-Gly NT NT 66 Gly-Val-Leu-Val-Gln-Pro-Gly − − 67 Val-Leu-Val-Gln-Pro-Gly + + 68 Leu-Val-Gln-Pro-Gly + + 69 Val-Gln-Pro-Gly + + 70 Gln-Pro-Gly + + 71 Gly-Gly-Val-Leu-Val-Gln-Pro − + 72 Gly-Gly-Val-Leu-Val-Gln + + 73 Gly-Gly-Val-Leu-Val + + 74 Gly-Gly-Val-Leu + + 75 Gly-Gly-Val + + 76 Gly-Gly-D-Val-Leu-Val-Gln-Pro-Gly + + 77 Gly-Gly-Val-D-Leu-Val-Gln-Pro-Gly + + 78 Gly-Gly-Val-Leu-D-Val-Gln-Pro-Gly − − 79 Gly-Gly-Val-Leu-Val-D-Gln-Pro-Gly + + 80 Gly-Gly-Val-Leu-Val-Gln-D-Pro-Gly + + 81 Gly-D-Pro-D-Gln-D-Val-D-Leu-D-Val- + + Gly-Gly 82 Gly-D-Pro-D-Gln-D-Val-D-Leu-Val-Gly- + + Gly 83 Gly-D-Pro-D-Gln-D-Val-Leu-D-Val-Gly- + + Gly 84 Gly-D-Pro-D-Gln-Val-D-Lcu-D-Val-Gly- + + Gly 85 Gly-D-Pro-Gln-D-Val-D-Leu-D-Val-Gly- + + Gly 86 Gly-Pro-D-Gln-D-Val-D-Leu-D-Val-Gly- − − Gly 87 Gly-Pro-Gln-Val-Leu-Val-Gly-Gly + + 88 Gly-D-Pro-Gln-Val-Leu-Val-Gly-Gly + + 89 Gly-Pro-D-Gln-Val-Leu-Val-Gly-Gly − − 90 Gly-Pro-Gln-D-Val-Leu-Val-Gly-Gly − − 91 Gly-Pro-Gln-Val-D-Leu-Val-Gly-Gly + + 92 Gly-Pro-Gln-Val-Leu-D-Val-Gly-Gly + + 93 Gly-Gly-D-Val-D-Leu-D-Val-D-Gln-D- Pro-Gly 94 Gly-Gly-D-Val-D-Leu-D-Val-D-Gln-Pro- + − Gly 95 Gly-Gly-D-Val-D-Leu-D-Val-Gln-D-Pro- − − Gly 96 Gly-Gly-D-Val-D-Leu-Val-D-Gln-D-Pro- − − Gly 97 Gly-Gly-D-Val-Leu-D-Val-D-Gln-D-Pro- − − Gly 98 Gly-Gly-Val-D-Leu-D-Val-D-Gln-D-Pro- + + Gly 99 Gly-D-Phe-Val-Leu-Val-Gln-Pro-Gly + + 100 Ala-Pro-Gly + + 101 Gln-Ala-Gly + + 102 Gln-Pro-Ala + + 103 (d)Gln-Pro-Gly + + 104 Gln-(d)Pro-Gly + + 105 (d)Gln-(d)Pro-Gly − − 106 Gly-Pro-Gln + + 107 Gly-(d)Pro-Gln − − 108 Gly-Pro-(d)Gln − − 109 Gly-(d)Pro-(d)Gln − − 110 Ala-Pro-Gly + + 111 His-Pro-Gly + + 112 Asp-Pro-Gly − − 113 Arg-Pro-Gly + + 114 Phe-Pro-Gly + + 115 Gly-Pro-Gly + + 116 Glu-Pro-Gly + + 117 Lys-Pro-Gly + + 118 Leu-Pro-Gly + + 119 Met-Pro-Gly + + 120 Asn-Pro-Gly + + 121 Ser-Pro-Gly + + 122 Tyr-Pro-Gly + + 123 Thr-Pro-Gly − + 124 Ile-Pro-Gly + + 125 Trp-Pro-Gly + + 126 Pro-Pro-Gly − − 127 Val-Pro-Gly − + 128 Glp-Pro-Gly + + 129 Glp-Val-Gly − − 130 Glp-Gln-Gly − − 131 Glp-Ser-Gly − − 132 Glp-Lys-Gly − − 133 Glp-Phe-Gly − − 134 Glp-Glu-Gly − − 135 Glp-Thr-Gly − − 136 Glp-Ile-Gly − − 137 Glp-Tyr-Gly − − 138 Glp-His-Gly − − 139 Glp-Asn-Gly − − 140 Glp-Arg-Gly − − 141 Glp-Gly-Gly − − 142 Glp-Trp-Gly − − 143 Glp-Asp-Gly − − 144 Glp-Met-Gly − − 145 Glp-Leu-Gly − − 146 Glp-Pro-Gln − − 147 Glp-Pro-Asn + − 148 Glp-Pro-Gln − − 149 Glp-Pro-Ser − − 150 Glp-Pro-Pro + − 151 Glp-Pro-Trp − − 152 Glp-Pro-Asp − − 153 Glp-Pro-His − − 154 Glp-Pro-Leu − − 155 Glp-Pro-Arg − − 156 Glp-Pro-Val − − 157 Glp-Pro-Lys − − 158 Glp-Pro-Glu − − 159 Glp-Pro-Phe − − 160 Glp-Pro-Ile + − 161 Glp-Pro-Met + − 162 Glp-Pro-Tyr + − Met(O)2 = Methioninedioxide, Cha = cyclohexyl-Ala