WO2009110947A2 - Compositions and methods for inhibiting cytokine production - Google Patents

Compositions and methods for inhibiting cytokine production Download PDF

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Publication number
WO2009110947A2
WO2009110947A2 PCT/US2008/087666 US2008087666W WO2009110947A2 WO 2009110947 A2 WO2009110947 A2 WO 2009110947A2 US 2008087666 W US2008087666 W US 2008087666W WO 2009110947 A2 WO2009110947 A2 WO 2009110947A2
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tnfα
biological
microns
present
compounds
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PCT/US2008/087666
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French (fr)
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WO2009110947A3 (en
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Neil Poloso
Amir Tamiz
Edward Oliver
Sefik Alkan
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Alba Therapeutics Corporation
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Publication of WO2009110947A3 publication Critical patent/WO2009110947A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids

Definitions

  • the present invention in the field of immunology and medicine relates to inhibition of cytokine production including tumor necrosis factor alpha (TNF ⁇ ), and to pharmaceutical and diagnostic compositions and production, diagnostic and therapeutic methods thereof, and to methods for treating human TNF-mediated pathologies.
  • TNF ⁇ tumor necrosis factor alpha
  • Inflammatory events play a central role in the pathology of disease conditions that adversely affect a considerable proportion of the population in developed countries. This process is mediated by cytokines, a system of polypeptides that enable one cell to signal to initiate events in another cell that initiate inflammatory sequelae. Normally, the system acts as part of a defensive reaction against infectious agents, harmful environmental agents, or malignantly transformed cells. But when inflammation exceeds the requirements of its defensive role, it can initiate adverse clinical effects, such as arthritis, septic shock, inflammatory bowel disease, and a range of other human disease conditions.
  • Small-molecule antirheumatic drugs such as methotrexate and sulfasalazine are insufficient to control inflammation in about two-thirds of arthritis patients.
  • New biological agents developed in the last decade have proved to be effective for a majority of patients unresponsive to traditional drugs.
  • the target for such agents is often one of the cytokine pathways— either capturing the ligand conveying the signal from one cell to another, or blocking the receptor at the surface of the effector cell, preventing transduction of the cytokine signal, thereby forestalling the inflammatory events.
  • a leading biological agent for treating inflammatory conditions is EnbrelTM (Etanercept), marketed by Amgen Corp.
  • the compound interferes with the binding of TNF to cell-surface TNF receptors—showing the importance of modulating the TNF pathway for clinical therapy of inflammatory conditions.
  • TNF ⁇ modulating agents currently licensed in the U.S. for treating inflammatory conditions include RemicadeTM (Infliximab), a chimeric antibody that binds TNF ⁇ ; and HumiraTM, a humanized anti-TNF ⁇ antibody.
  • RemicadeTM Infliximab
  • HumiraTM a humanized anti-TNF ⁇ antibody
  • TNF ⁇ tumor necrosis factor- ⁇
  • TNF ⁇ tumor necrosis factor- ⁇
  • TNF ⁇ tumor necrosis factor- ⁇
  • TNF ⁇ Cells other than monocytes or macrophages also make TNF ⁇ .
  • human non-monocytic tumor cell lines produce TNF.
  • CD4 + and CD8 + peripheral blood T lymphocytes and some cultured T and B cell lines also produce TNF ⁇ .
  • TNF ⁇ causes pro-inflammatory actions which result in tissue injury, such as inducing procoagulant activity on vascular endothelial cells, increasing the adherence of neutrophils and lymphocytes, and stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells.
  • a large body of evidence associates TNF ⁇ with infections, immune disorders, neoplastic pathologies, autoimmune pathologies and graft-versus host pathologies.
  • Environmental stimuli including for example, microorganisms and gluten, can increase permeability of biological barriers as measured by a decrease in trans-epithelial electrical resistance (TEER) (ex vivo) or the Lactulose/mannitol test (in vivo).
  • TEER trans-epithelial electrical resistance
  • Lactulose/mannitol test in vivo
  • Such increases in barrier permeability are believed to underly many diseases including a large number of inflammatory conditions.
  • inflammatory diseases and disorders examples include, for example, rheumatoid arthritis, multiple sclerosis, immune- mediated or type 1 diabetes mellitus, inflammatory bowel diseases (Crohn's disease and ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, and autoimmune thyroid diseases. Prolonged inflammation is often associated with these diseases, although the inflammation is thought to be a sequela rather than a primary pathological insult.
  • Other diseases and disorders associated with biological barrier dysfunction and which may be treated using the instant inventions include, for example, celiac disease, asthma, acute lung injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease, inflammation (e.g., psoriasis and other inflammatory dermatoses), asthma, allergy, cell proliferative disorders (e.g., hyperproliferative skin disorders including skin cancer), metastasis of cancer cells, ion transport disorders such as magnesium transport defects in the kidney, and exposure to Clostridium perfringens enterotoxin (CPE).
  • CPE Clostridium perfringens enterotoxin
  • autoimmune encephalomyelitis optic neuritis, progressive multifocal leukoencephalopathy (PML), primary biliary cirrhosis, IgA nephropathy, Wegener's granulomatosis, multiple sclerosis, scleroderma, systemic sclerosis, Hashimoto's thyroiditis (underactive thyroid), Graves' disease (overactive thyroid), autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Devic's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome (LEMS), autoimmune lymphproliferative syndrome (ALPS), paraneoplastic syndromes, polyglandular autoimmune syndromes (PGA), alopecia areata, gastrointestinal inflammation that gives rise to increased intestinal permeability, intestinal conditions that cause protein losing enteropathy, C.
  • PML progressive multifocal leukoencephalopathy
  • IgA nephropathy IgA nephropathy
  • One object of the present invention is to identify compounds that inhibit production of TNF ⁇ .
  • Another object of the present invention is to identify compounds that inhibit release of TNF ⁇ .
  • Another object of the present invention is to identify compounds that inhibit the biological effects of TNF ⁇ .
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the production and/or release of cytokines.
  • the biological effect of TNF ⁇ is the production and/or release of TNF ⁇ .
  • the biological effect of TNF ⁇ is the production and/or release of IL-6.
  • the biological effect of TNF ⁇ is the production and/or release of IL- 16.
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the induction of expression of TNF-induced proteins.
  • TNF-induced proteins whose production and/or release is inhibited by the present invention include, for example, CXCLl, CXCL2, CXCL5, CCL2, CCL5, 4- IBBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA, FasL, GITRL, LIGHT, LT-Beta, NGF-Beta, NGFR, OX40L, RANKL, LCN2/22p3, EREG, OSMR, COX2, C/EBP ⁇ , C/EBP ⁇ , MAIL/IKB ⁇ , MMPl 3, TIMP2, RANKL, OPG, SOCS3, PSGD3, 14-3-3- ⁇ , CTTN, COTLl , FAS, CASPl , 4-1BB, BAFFR, BCMA, c-Fos,
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is to regulate biological barrier function.
  • the biological effect of TNF ⁇ is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of TNF ⁇ is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • Another object of the present invention is to identify compounds that inhibit production of IL-4.
  • Another object of the present invention is to identify compounds that inhibit release of IL-4.
  • Another object of the present invention is to identify compounds that inhibit the biological effects of IL-4.
  • the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
  • the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function.
  • the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • Another object of the present invention is to identify compounds that inhibit production of IL-6.
  • Another object of the present invention is to identify compounds that inhibit release of IL-6.
  • Another object of the present invention is to identify compounds that inhibit the biological effects of IL-6.
  • the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
  • the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function.
  • the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • Another object of the present invention is to identify compounds that inhibit production of IL-16.
  • Another object of the present invention is to identify compounds that inhibit release of IL-16.
  • Another object of the present invention is to identify compounds that inhibit the biological effects of IL-16.
  • the present invention provides a compound that inhibits the biological effects of IL-16, wherein the biological effect of IL-16 is the production and/or release of cytokines.
  • the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-16 is to regulate biological barrier function.
  • the biological effect of IL-16 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of TNF ⁇ .
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of TNF ⁇ .
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of TNF ⁇ .
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the production and/or release of cytokines.
  • the biological effect of TNF ⁇ is the production and/or release of TNF ⁇ .
  • the biological effect of TNF ⁇ is the production and/or release of IL-6.
  • the biological effect of TNF ⁇ is the production and/or release of IL-16.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is to regulate biological barrier function.
  • the biological effect of TNF ⁇ is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of TNF ⁇ is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of IL-4.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of IL-4.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-4.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function.
  • the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of IL-6.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of IL-6.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-6.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function.
  • the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of IL- 16.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of IL-16.
  • Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 16.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is to regulate biological barrier function.
  • the biological effect of IL- 16 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL- 16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the invention provides a method of treating a patient with an autoimmune or inflammation-associated disease.
  • the disease is selected from the group consisting of inflammatory bowel disease, including Crohn's disease and ulcerative colitis, type 1 diabetes, celiac disease, autoimmune hepatitis, multiple sclerosis, autism, dermatitis herpetiformis, IgA nephropathy, primary biliary chirrosis, rheumatoid arthritis, systemic lupus erythematosus, Grave's disease, Hashimoto's disease, and depression.
  • a compound that inhibits the production, release and/or the biological effects of TNF ⁇ is administered to the patient.
  • FIG. 1 PTG exposure induces expression of cytokines in the monocytic cell line THP-I .
  • PTG (lmg/ml) and LPS (l ⁇ g/ml) exposure for 24 hours induced expression of characteristic profiles of cytokines and chemokines by THP-I cells. Profiles were determined in triplicate using a nitrocellulose membrane based proteomic profiler assay.
  • FIG. 3 AT-1001 inhibits PTG and LPS induced expression of TNF- ⁇ in human PBMCs.
  • TNF ⁇ levels were measured by ELISA.
  • FIG. 4 AT-1001 inhibits PTG and LPS induced expression of TNF- ⁇ in human PBMCs.
  • TNF ⁇ levels were measured by ELISA.
  • TNF ⁇ induction in human PBMcs is PTG-specific. PTG exposure induced TNF ⁇ expression by human PBMCs while exposure of human PBMCs to PT-treated casein did not.
  • FIG. 6 IL-6 induction in human PBMcs is not PTG-specific. PTG exposure induced IL-6 expression by human PBMCs, and exposure of human PBMCs to PT-treated casein also induced IL-6 expression in these cells.
  • FIG. 7 PTG-induced expression of TNF ⁇ by monocytes in human PBMC samples. Control and PTG stimulated PBMCs were treated with brefeldin A to inhibit protein secretion. Treated cells were stained for intracellular TNF ⁇ and for CD 14, and stained cells were separated by fluorescence flow cytometry.
  • FIG. 8 LPS-induced transcription of TNF ⁇ in monocytes is not inhibited by AT-1001. Monocytes were exposed to LPS in the presence or absence of AT-1001. Total cellular RNA was isolated from the treated cells, and TNF ⁇ mRNA was measured by RT- PCR.
  • FIG. 9 LPS-induced synthesis of TNF ⁇ protein in monocytes is not inhibited by AT-1001. Monocytes were exposed to LPS in the presence or absence of AT-1001 or known translation (actinomycin D) or protein synthesis (cycloheximide) inhibitors. Treated cells were stained for intracellular accumulation of TNF ⁇ and separated by flow cytometry.
  • AT-1001 does not inhibit TACE-mediated release of TNF ⁇ from LPS or PTG treated monocytes. Monocytes were exposed to LPS (l ⁇ g/ml) or PTG (lmg/ml) in the presence or absence of AT-1001. Treated cells were stained for accumulation of TNF ⁇ on the cell surface, and cell populations were separated by flow cytometry.
  • AT-1001 inhibits TNF ⁇ -induced permeability.
  • Caco-2 cells were grown as described for 21 days and treated with TNF- ⁇ (100ng/ml) in the presence or absence of AT-1001 (12.5mM).
  • Monolayer permeability to Lucifer Yellow (LY) was measured by analysis in a Tecan Spectrofluor fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
  • AT-1001 inhibits TNF ⁇ -induced TEER.
  • Caco-2 cells were grown as described for 21 days and treated with TNF- ⁇ (100ng/ml) in the presence or absence of AT- 1001 (12.5mM). TEER measurements were made, and values are expressed relative to untreated control cells.
  • FIG. 14 IL-4 treatment increases permeability in a dose-dependent manner.
  • T- 84 cells were grown as described and treated with IL-4 (I Ong/ml or 100ng/ml).
  • Monolayer permeability to Lucifer Yellow (LY) was measured by analysis in a Tecan Spectrofluor fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
  • FIG. 15 AT-1001 inhibits IL-4-induced permeability.
  • T-84 cells were grown as described and treated with IL-4 (10ng/ml or 100ng/ml) in the presence or absence of AT- 1001.
  • Monolayer permeability to Lucifer Yellow (LY) was measured by analysis in a Tecan Spectrofluor fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
  • AT-1001 inhibits TNF ⁇ -induced secretion of IL-6 from peripheral blood mononuclear cells (PBMCs): Human PBMCs purified from healthy donors (1 & 2) were stimulated with purified TNF- ⁇ in the presence or absence of AT-1001 (6.8mM) for 24 hours. IL-6 levels were measured by standard sandwich ELISA (R&D Systems Duoset).
  • AT-1001 inhibits TNF ⁇ -induced secretion of IL-16 from peripheral blood mononuclear cells (PBMCs): Human PBMCs purified from healthy donors (1 & 2) were stimulated with purified TNF- ⁇ in the presence or absence of AT-1001 (6.8mM) for 24 hours. IL-16 levels were measured by standard sandwich ELISA (R&D Systems Duoset).
  • 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.
  • modulate and all its forms and tenses refer to either increasing or decreasing a particular biochemical or physiological effect.
  • 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, CIq, CIr and CIs), 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 ⁇ 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.
  • neutrophils including, for example, complement proteins (including, for example, CIq, CIr and CIs), eosinophils, basophils, lymphocyte
  • 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.
  • TNF ⁇ inhibitors may also act as inhibitors of mammalian tight junction opening.
  • Exemplary compounds of the invention that inhibit TNF ⁇ are presented in Table 1. Table 1. Inhibition of TNF ⁇ secretion by compounds of the invention
  • Antagonist of mammalian tight junction opening may also be used in the practice of the present invention.
  • tight junction antagonists prevent, inhibit or reduce the opening of tight junctions.
  • antagonists of the invention may comprise peptide antagonists.
  • peptide antagonists 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: GIy Arg VaI Cys VaI GIn Pro GIy (SEQ ID NO:1), GIy Arg VaI Cys VaI GIn Asp GIy (SEQ ID NO:2), GIy Arg VaI Leu VaI GIn Pro GIy (SEQ ID NO:3), GIy Arg VaI Leu VaI GIn Asp GIy (SEQ ID NO:4), GIy Arg Leu Cys VaI GIn Pro GIy (SEQ ID NO:5), GIy Arg Leu Cys VaI GIn Asp GIy (SEQ ID NO:5), GI
  • peptide antagonists include, but are not limited to, peptides that consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24.
  • the peptide antagonist may have the amino acid sequence of SEQ ID NO: 15.
  • the peptide antagonist may have the amino acid sequence GGVLVQPG, also known as AT-1001.
  • 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 1 1, 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 1 1 , 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.
  • 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 ah, 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.
  • an appropriate expression vector e.g., an E. coli or yeast expression vector
  • the present invention provides compounds that inhibit production of TNF ⁇ .
  • the present invention provides compounds that inhibit release of TNF ⁇ .
  • the present invention provides compounds that inhibit the biological effects of TNF ⁇ .
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the production and/or release of cytokines.
  • the biological effect of TNF ⁇ is the production and/or release of TNF ⁇ .
  • the biological effect of TNF ⁇ is the production and/or release of IL-6.
  • the biological effect of TNF ⁇ is the production and/or release of IL- 16.
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the induction of expression of TNF-induced proteins.
  • TNF-induced proteins whose production and/or release is inhibited by the present invention include, for example, CXCLl , CXCL2, CXCL5, CCL2, CCL5, 4-1 BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA, FasL, GITRL, LIGHT, LT-Beta, NGF-Beta, NGFR, OX40L, RANKL, LCN2/22p3, EREG, OSMR, COX2, C/EBP ⁇ , C/EBP ⁇ , MAIL/IKB ⁇ , MMPl 3, TIMP2, RANKL, OPG, SOCS3, PSGD3, 14-3-3- ⁇ , CTTN, COTLl, FAS, CASPl, 4- IBB, BAFFR, BCMA, c-Fos,
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is to regulate biological barrier function.
  • the biological effect of TNF ⁇ is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of TNF ⁇ is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides compounds that inhibit production of IL-4.
  • the present invention provides compounds that inhibit release of IL-4.
  • the present invention provides compounds that inhibit the biological effects of IL-4.
  • the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
  • the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function.
  • the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides compounds that inhibit production of IL-6.
  • the present invention provides compounds that inhibit release of IL-6.
  • the present invention provides compounds that inhibit the biological effects of IL-6.
  • the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
  • the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function.
  • the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides compounds that inhibit production of IL- 16.
  • the present invention provides compounds that inhibit release of IL- 16.
  • the present invention provides compounds that inhibit the biological effects of IL- 16.
  • the present invention provides a compound that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
  • the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL- 16 is to regulate biological barrier function.
  • the biological effect of IL- 16 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit production of TNF ⁇ .
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit release of TNF ⁇ .
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of TNF ⁇ .
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the production and/or release of cytokines.
  • the biological effect of TNF ⁇ is the production and/or release of TNF ⁇ .
  • the biological effect of TNF ⁇ is the production and/or release of IL-6.
  • the biological effect of TNF ⁇ is the production and/or release of IL- 16.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is to regulate biological barrier function.
  • the biological effect of TNF ⁇ is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of TNF ⁇ is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit production of IL-4.
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit release of IL-4.
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-4.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function.
  • the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit production of IL-6.
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit release of IL-6.
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-6.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function.
  • the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit production of IL- 16.
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit release of IL- 16.
  • the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 16.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
  • the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is to regulate biological barrier function.
  • the biological effect of IL- 16 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL- 16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • 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 facilitate the inhibition of TNF ⁇ production in a subject in need thereof.
  • the amount of a compound of the invention employed in any 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.
  • a pharmaceutical composition of the invention will comprise an amount of a compound of the invention in the range of about 1 ⁇ g to about 1 g, preferably about lmg to about lOOOmg, from about lOmg to about lOOmg, from about l Omg to about 50mg, or from about l Omg to about 25mg of the compound.
  • "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 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
  • 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.
  • 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.
  • 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.
  • compositions of the invention may be formulated for pulmonary delivery (e.g., may be pulmonary dosage forms).
  • compositions may be provided as pharmaceutical aerosols, e.g., solution aerosols or powder aerosols.
  • pharmaceutical aerosols e.g., solution aerosols or powder aerosols.
  • 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.
  • Powder formulations may optionally contain at least one particulate pharmaceutically acceptable carrier known to those of skill in the art.
  • 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.
  • a powder formulation may comprise lactose as a carrier.
  • 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.
  • the total weight of the formulation in the container may be from about 5 mg to about 50 mg.
  • powder formulations may be contained in a reservoir in a multi-dose dry powder inhalation device adapted to deliver a suitable amount per actuation.
  • 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 micron
  • 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.
  • the dosage forms are in the form of a solution aerosol (i.e., comprise droplets).
  • 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,
  • 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.
  • 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.
  • 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 less, in approximately sixty minutes.
  • 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.
  • 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.
  • "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.
  • stable 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.
  • stable 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.
  • 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.
  • "about" used to modify a numerical value means within 10% of the value.
  • 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.
  • topical solutions and/or gels 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).
  • 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.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the pharmaceutical compounds or compositions of the invention 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.
  • a protein including an antibody
  • care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome.
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used.
  • polymeric materials can be used.
  • 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.
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • 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.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • 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 from 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.
  • the amount of the compound of the invention that will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant TNF ⁇ production can be determined by standard clinical techniques.
  • 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.
  • 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.
  • compositions of the invention may further comprise one or more additional active agents, e.g., therapeutic agents, immunogenic agents and/or imaging agents.
  • additional active agents e.g., therapeutic agents, immunogenic agents and/or imaging agents.
  • 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.
  • Additional therapeutic agents that can be used in the compositions of the invention include immunosuppressive agents.
  • 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.
  • 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 65
  • 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 Ilb/IIIa inhibitor, an inhibitor of surface glycoprotein receptors, an antiplatelet agent, an antimitotic, a microtubule inhibitor, a retinoid, an antisecretid, a
  • 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-L-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, 60 Co, 192 Ir , 32 P , 1 11 In , 90 Y, 99m Tc, pergolide mesylate, bromocriptine mesylate
  • compositions of the invention may comprise one or more immunogenic agents, for example, antigens.
  • 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).
  • 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 are known in the art can also be used.
  • 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.
  • any antigen capable of inducing a protective immune response may be used in the vaccine compositions of the invention.
  • suitable antigens include, but are not limited to, measles virus antigens, mumps virus antigens, rubella virus antigens, C ⁇ rynebacterium diphtheriae antigens, Bordetella pertussis antigens, Clostridium tetani antigens, Bacillus anthracis antigens, Haemophilus influenzae antigens, smallpox virus antigens, and influenza virus antigens.
  • 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.
  • the protease inhibitors can be selected from the group consisting of bestatin, L- trans-S-carboxyoxiran ⁇ -carbonyl-L-leucylagmatine, ethylenediaminetetra-acetic acid (EDTA), phenylmethylsulfonylfluoride (PMSF), aprotinin, amyloid protein precursor (APP), amyloid beta precursor protein, ⁇ l -proteinase inhibitor, collagen VI, bovine pancreatic trypsin inhibitor (BPTI), 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), antipain, benzamidine, chymostatin, ⁇ -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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, osteoporosis, systemic lupus erythematosus, food allergy, asthma, celiac disease and irritable bowel syndrome.
  • 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.
  • a composition comprising a therapeutically effective amount of Erbitux® (Cetuximab) together with a TNF ⁇ 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.
  • a composition comprising a therapeutically effective amount of Herceptin® (Trastuzumab) together with a TNF ⁇ 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.
  • a composition comprising a therapeutically effective amount of Avastin® (Bevacizumab) together with a TNF ⁇ 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.
  • the subject e.g., a mammal such as a human
  • 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.
  • compositions of the invention may be given repeatedly over a protracted period, i.e., may be chronically administered.
  • 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., diabetes, cancer, transplant rejection, etc).
  • Such compositions may be administered chronically, for example, one or more times daily over a plurality of days.
  • compositions of the invention may be used to treat acute attacks of the underlying disease (e.g., diabetes, cancer, transplant rejection, etc).
  • the underlying disease e.g., diabetes, cancer, transplant rejection, etc.
  • 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.
  • compounds of the invention may be used in the manufacture of compositions and pharmaceutical compositions for use in the methods described above.
  • the present invention provides methods to inhibit production of TNF ⁇ .
  • the present invention provides methods to inhibit release ofTNF ⁇ .
  • the present invention provides methods to inhibit the biological effects ofTNF ⁇ .
  • the present invention provides methods to inhibit the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the production and/or release of cytokines.
  • the biological effect of TNF ⁇ is the production and/or release of TNF ⁇ .
  • the biological effect of TNF ⁇ is the production and/or release of IL-6.
  • the biological effect ofTNF ⁇ is the production and/or release of IL- 16.
  • the present invention provides a compound that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the induction of expression of TNF-induced proteins.
  • TNF-induced proteins whose production and/or release is inhibited by the present invention include, for example, CXCLl, CXCL2, CXCL5, CCL2, CCL5, 4- IBBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA, FasL, GITRL, LIGHT, LT-Beta, NGF-Beta, NGFR, OX40L, RANKL, LCN2/22p3, EREG, OSMR, COX2, C/EBP ⁇ , C/EBP ⁇ , MAIL/IKB ⁇ , MMPl 3, TIMP2, RANKL, OPG, SOCS3, PSGD3, 14-3-3- ⁇ , CTTN, COTLl, FAS, CASPl, 4-1BB, BAFFR, BCMA, c-Fos, c-Jun, c- ReI, Caspase 1, Caspase 10, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspas
  • the present invention provides methods to inhibit the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is to regulate biological barrier function.
  • the biological effect of TNF ⁇ is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of TNF ⁇ is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides methods to inhibit production of IL-4. [0173] In another embodiment the present invention provides methods to inhibit release ofIL-4.
  • the present invention provides methods to inhibit the biological effects of IL-4.
  • the present invention provides methods to inhibit the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
  • the present invention provides methods to inhibit the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function.
  • the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides methods to inhibit production of IL-6.
  • the present invention provides methods to inhibit release of IL-6.
  • the present invention provides methods to inhibit the biological effects of IL-6.
  • the present invention provides methods to inhibit the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
  • the present invention provides methods to inhibit the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function.
  • the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides methods to inhibit production of IL- 16. [0183] In another embodiment the present invention provides methods to inhibit release of IL- 16.
  • the present invention provides methods to inhibit the biological effects of IL- 16.
  • the present invention provides methods to inhibit the biological effects of IL-16, wherein the biological effect of IL-16 is the production and/or release of cytokines.
  • the present invention provides methods to inhibit the biological effects of IL-4, wherein the biological effect of IL-16 is to regulate biological barrier function.
  • the biological effect of IL-16 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans-epithelial electric resistance
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of TNF ⁇ .
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of TNF ⁇ .
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is the production and/or release of cytokines.
  • the biological effect of TNF ⁇ is the production and/or release of TNF ⁇ .
  • the biological effect of TNF ⁇ is the production and/or release of IL-6.
  • the biological effect of TNF ⁇ is the production and/or release of IL-16.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of TNF ⁇ , wherein the biological effect of TNF ⁇ is to regulate biological barrier function.
  • the biological effect of TNF ⁇ is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of TNF ⁇ is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans- epithelial electric resistance
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of IL-4.
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of IL-4.
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 4.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function.
  • the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-4 is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans- epithelial electric resistance
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of IL-6.
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of IL-6.
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 6.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function.
  • the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-6 is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans- epithelial electric resistance
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of IL- 16.
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of IL- 16.
  • the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 16.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
  • the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL-16 is to regulate biological barrier function.
  • the biological effect of IL-16 is to increase the macromolecular permeability of a biological barrier.
  • the biological effect of IL-16 is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
  • TEER trans- epithelial electric resistance
  • 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.
  • compounds e.g. ions, Lucifer Yellow
  • 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.
  • 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:
  • Culture Medium DMEM supplemented with 10% fetal bovine serum, 1% NEAA, l % Penn/Strep
  • Buffers Hank's Balanced Salt Solution (HBSS) without calcium and magnesium
  • Flasks 100 X 20 mm Tissue culture dish Falcon.
  • Plates 12 well polycarbonate Transwell ® filters; 0.3uM pore size
  • THP-I PT- Gliadin
  • the monocytic cell line THP-I was used to characterize the profile of cytokines whose expression was upregulated on exposure to protease treated gliadin (PTG).
  • THP-I cells were diluted to 5x 10 5 cells/ml in RPMI medium supplemented with 10% heat inactivated fetal bovine serum.
  • 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, CD40 ligand, G-CSF, GM-CSF, GRO- ⁇ /CXCLl, I-309/CCL1, ICAM-I, IFN ⁇ , IL-l ⁇ , IL-l ⁇ , IL-lra, 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-32 ⁇ , IP-10/CXCLlO, I-TAC/CXCL1 1 , MCP-1/CCL2, MIF, MIP-l ⁇ /CCL3, MIP- l ⁇ /CCL3, RANTES/CC
  • THP-I cells demonstrated increased expression of the cytokines IL-8, MIP-Ia, MIP-I ⁇ , TNF- ⁇ and Gro- ⁇ . After 24 hours of exposure to PTG increased expression of RANTES and MIF were also observed.
  • 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 2x 10 5 cells were plated in each well of a 96 well plate. Cells were incubated at 37 0 C with PTG (lmg/ml) or LPS (l ⁇ g/ml) in the presence or absence of test compounds being examined for the ability to suppress cytokine production.
  • Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNF ⁇ transcription after exposure to PTG or LPS in the presence or absence of AT-1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum, and incubated for 4 hours at 37°C with PTG (lmg/ml) or LPS (l ⁇ g/ml) in the presence or absence of test compounds being examined for the ability to suppress cytokine production. Following treatment total cellular RNA was isolated from each sample, and the level of TNF ⁇ mRNA was measured by RT-PCR (Applied Biosciences) using methods known in the art. AT-1001 treatment did not reduce the amount of TNFa mRNA transcribed in response to LPS ( Figure 8) or PTG (not shown) exposure.
  • Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNF ⁇ protein synthesis after exposure to PTG or LPS in the presence or absence of AT- 1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum and containing Brefeldin A. Monocytes were incubated for 4 hours at 37 0 C with PTG (lmg/ml) or LPS (l ⁇ g/ml) in the presence or absence of protein synthesis inhibitors or test compounds being examined for the ability to suppress cytokine production.
  • Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNF ⁇ protein synthesis after exposure to PTG or LPS in the presence or absence of AT- 1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human
  • TNF ⁇ release form the cell surface is mediated by the proteolytic activity of the enzyme TNF ⁇ converting enzyme (TACE), which is also known as ADAM- 17.
  • TACE TNF ⁇ converting enzyme
  • ADAM- 17 ADAM- 17.
  • This enzyme is a metalloprotease-disintegrin, and in the absence of its activity TNF ⁇ accumulates at the surface of an expressing cell.
  • Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNF ⁇ protein secretion after exposure to PTG or LPS in the presence or absence of AT-1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum. Monocytes were incubated for 24 hours at 37°C with PTG (l mg/ml) or LPS (l ⁇ g/ml) in the presence or absence of test compounds being examined for the ability to suppress cytokine production. Following treatment cells were stained for accumulation of TNF ⁇ at the cell surface and separated by fluorescence flow cytometry using methods known in the art. AT-1001 treatment did not reduce the amount of TNFa protein present at the cell surface in response to LPS or PTG exposure ( Figure 1 1).

Abstract

Novel inhibitors of TNFα secretion are identified. Assays for determining modulators of TNFα secretion are provided. Methods for treating diseases relating to aberrant TNFα secretion are provided. Such diseases include celiac disease, diabetes, cancer, inflammatory bowel diseases, 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.

Description

COMPOSITIONS AND METHODS FOR INHIBITING CYTOKINE
PRODUCTION
PRIORITY
[0001] This Application claims priority to US Provisional Application No. 61/015,075 filed December 19, 2007, which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention in the field of immunology and medicine relates to inhibition of cytokine production including tumor necrosis factor alpha (TNFα), and to pharmaceutical and diagnostic compositions and production, diagnostic and therapeutic methods thereof, and to methods for treating human TNF-mediated pathologies.
BACKGROUND OF THE INVENTION Inflammation
[0003] Inflammatory events play a central role in the pathology of disease conditions that adversely affect a considerable proportion of the population in developed countries. This process is mediated by cytokines, a system of polypeptides that enable one cell to signal to initiate events in another cell that initiate inflammatory sequelae. Normally, the system acts as part of a defensive reaction against infectious agents, harmful environmental agents, or malignantly transformed cells. But when inflammation exceeds the requirements of its defensive role, it can initiate adverse clinical effects, such as arthritis, septic shock, inflammatory bowel disease, and a range of other human disease conditions.
[0004] Small-molecule antirheumatic drugs such as methotrexate and sulfasalazine are insufficient to control inflammation in about two-thirds of arthritis patients. New biological agents developed in the last decade have proved to be effective for a majority of patients unresponsive to traditional drugs. The target for such agents is often one of the cytokine pathways— either capturing the ligand conveying the signal from one cell to another, or blocking the receptor at the surface of the effector cell, preventing transduction of the cytokine signal, thereby forestalling the inflammatory events. [0005] A leading biological agent for treating inflammatory conditions is Enbrel™ (Etanercept), marketed by Amgen Corp. It is a chimeric molecule comprising the extracellular portion of the human TNF receptor linked as a dimer to the IgG Fc region. The compound interferes with the binding of TNF to cell-surface TNF receptors—showing the importance of modulating the TNF pathway for clinical therapy of inflammatory conditions.
[0006] Other TNFα modulating agents currently licensed in the U.S. for treating inflammatory conditions include Remicade™ (Infliximab), a chimeric antibody that binds TNFα; and Humira™, a humanized anti-TNFα antibody.
Tumor Necrosis Factor
[0007] Monocytes and macrophages secrete cytokines known as tumor necrosis factor-α (TNFα) and tumor necrosis factor-β (TNFβ) in response to endotoxin or other stimuli. TNFα is a soluble homotrimer of 17 kD protein subunits. A membrane-bound 26 kD precursor form of TNF also exists.
[0008] Cells other than monocytes or macrophages also make TNFα. For example, human non-monocytic tumor cell lines produce TNF. CD4+ and CD8+ peripheral blood T lymphocytes and some cultured T and B cell lines also produce TNFα.
[0009] TNFα causes pro-inflammatory actions which result in tissue injury, such as inducing procoagulant activity on vascular endothelial cells, increasing the adherence of neutrophils and lymphocytes, and stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells.
[0010] A large body of evidence associates TNFα with infections, immune disorders, neoplastic pathologies, autoimmune pathologies and graft-versus host pathologies.
Biological barrier dysfunction
[0011] Environmental stimuli, including for example, microorganisms and gluten, can increase permeability of biological barriers as measured by a decrease in trans-epithelial electrical resistance (TEER) (ex vivo) or the Lactulose/mannitol test (in vivo). Such increases in barrier permeability are believed to underly many diseases including a large number of inflammatory conditions. [0012] Examples of such inflammatory diseases and disorders that may be treated using the instant invention include, for example, rheumatoid arthritis, multiple sclerosis, immune- mediated or type 1 diabetes mellitus, inflammatory bowel diseases (Crohn's disease and ulcerative colitis), systemic lupus erythematosus, psoriasis, scleroderma, and autoimmune thyroid diseases. Prolonged inflammation is often associated with these diseases, although the inflammation is thought to be a sequela rather than a primary pathological insult.
[0013] Other diseases and disorders associated with biological barrier dysfunction and which may be treated using the instant inventions include, for example, celiac disease, asthma, acute lung injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease, inflammation (e.g., psoriasis and other inflammatory dermatoses), asthma, allergy, cell proliferative disorders (e.g., hyperproliferative skin disorders including skin cancer), metastasis of cancer cells, ion transport disorders such as magnesium transport defects in the kidney, and exposure to Clostridium perfringens enterotoxin (CPE). autoimmune encephalomyelitis, optic neuritis, progressive multifocal leukoencephalopathy (PML), primary biliary cirrhosis, IgA nephropathy, Wegener's granulomatosis, multiple sclerosis, scleroderma, systemic sclerosis, Hashimoto's thyroiditis (underactive thyroid), Graves' disease (overactive thyroid), autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Devic's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome (LEMS), autoimmune lymphproliferative syndrome (ALPS), paraneoplastic syndromes, polyglandular autoimmune syndromes (PGA), alopecia areata, gastrointestinal inflammation that gives rise to increased intestinal permeability, intestinal conditions that cause protein losing enteropathy, C. difficile infection, enterocolitis, shigellosis, viral gastroenteritis, parasite infestation, bacterial overgrowth, Whipple's disease, diseases with mucosal erosion or ulcerations, gastritis, gastric cancer, collagenous colitis, and mucosal diseases without ulceration, Menetrier's disease, eosinophilic gastroenteritis, diseases marked by lymphatic obstruction, congenital intestinal lymphangiectasia, sarcoidosis lymphoma, mesenteric tuberculosis, after surgical correction of congenital heart disease, and food allergies, primarily to milk.
[0014] There is a continuing need in the art for methods to treat inflammatory and autoimmune diseases as well as diseases associated with biological barrier dysfunction more effectively and to discover or identify drugs which are suitable for treating inflammatory and autoimmune diseases as well as diseases associated with biological barrier dysfunction. Accordingly, there is a need to provide novel methods of inhibiting TNFα, and to pharmaceutical and diagnostic compositions and methods for treating human TNF-mediated pathologies
SUMMARY OF THE INVENTION
[0015] One object of the present invention is to identify compounds that inhibit production of TNFα.
[0016] Another object of the present invention is to identify compounds that inhibit release of TNFα.
[0017] Another object of the present invention is to identify compounds that inhibit the biological effects of TNFα.
[0018] In particular embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the production and/or release of cytokines. In a specific embodiment the biological effect of TNFα is the production and/or release of TNFα. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-6. In another specific embodiment the biological effect of TNFα is the production and/or release of IL- 16.
[0019] In further specific embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the induction of expression of TNF-induced proteins. TNF-induced proteins whose production and/or release is inhibited by the present invention include, for example, CXCLl, CXCL2, CXCL5, CCL2, CCL5, 4- IBBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA, FasL, GITRL, LIGHT, LT-Beta, NGF-Beta, NGFR, OX40L, RANKL, LCN2/22p3, EREG, OSMR, COX2, C/EBPδ, C/EBPβ, MAIL/IKBξ, MMPl 3, TIMP2, RANKL, OPG, SOCS3, PSGD3, 14-3-3-γ, CTTN, COTLl , FAS, CASPl , 4-1BB, BAFFR, BCMA, c-Fos, c-Jun, c- ReI, Caspase 1, Caspase 10, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, CD27, CD30, CD40, DCRl, DCR2, DR3, DR4, DR5, DR6, EDAR, FADD, Fas, Fn 14, GITR, HVEM, I-KappaB, Alpha, I-KappaB-Beta, I-KappaB-Epsilon, IKK-Alpha, IKK-Beta, IKK-Epsilon, IKK-Gamma, JNKl, JNK2, JNK3, LT-BetaR, MEKl , MEK2, MEK3, MEK4, MEK5, MEK6, NF-KappaBl, NF-KappaB2, OX40, p38-Alpha, p38-Beta, p38-Delta, p38-Gamma, PIK3C2Alpha, PIK3C2Beta, PIK3C2Gamma, PIK3C3, PIK3CAlpha, PIK3CBeta, PIK3CDelta, PIK3CGamma, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5, RANK, ReIA, ReIB, RELT, Src, TABl, TAB2, GRO-I, Interleukin-8 (IL-8), A20 (A20), GADD45 (GADD45), Cellular inhibitor of apoptosis protein 1/2 (c-IAPl/2), Interleukin-1 alpha (IL-Ia), v-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), Tumor protein p53 (Li-Fraumeni syndrome) (p53), TGFB-inducible early growth response, Syndecan-4a, MAP kinase phosphatase 1, Small inducible cytokine A2, TNFα-induced protein 2, Natural killer cell transcript 4, TNFα-induced protein 3, Early growth response 1, Superoxide dismutase 2, Human transcription factor ETRlOl, Prostaglandin E synthase, and JunB proto-oncogene.
[0020] In further specific embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is to regulate biological barrier function. In a specific embodiment the biological effect of TNFα is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of TNFα is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0021] Another object of the present invention is to identify compounds that inhibit production of IL-4.
[0022] Another object of the present invention is to identify compounds that inhibit release of IL-4.
[0023] Another object of the present invention is to identify compounds that inhibit the biological effects of IL-4.
[0024] In particular embodiments the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
[0025] In further specific embodiments the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier. [0026] Another object of the present invention is to identify compounds that inhibit production of IL-6.
[0027] Another object of the present invention is to identify compounds that inhibit release of IL-6.
[0028] Another object of the present invention is to identify compounds that inhibit the biological effects of IL-6.
[0029] In particular embodiments the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
[0030] In further specific embodiments the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0031] Another object of the present invention is to identify compounds that inhibit production of IL-16.
[0032] Another object of the present invention is to identify compounds that inhibit release of IL-16.
[0033] Another object of the present invention is to identify compounds that inhibit the biological effects of IL-16.
[0034] In particular embodiments the present invention provides a compound that inhibits the biological effects of IL-16, wherein the biological effect of IL-16 is the production and/or release of cytokines.
[0035] In further specific embodiments the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-16 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-16 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier. [0036] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of TNFα.
[0037] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of TNFα.
[0038] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of TNFα.
[0039] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the production and/or release of cytokines. In a specific embodiment the biological effect of TNFα is the production and/or release of TNFα. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-6. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-16.
[0040] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is to regulate biological barrier function. In a specific embodiment the biological effect of TNFα is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of TNFα is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0041] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of IL-4.
[0042] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of IL-4.
[0043] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-4.
[0044] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
[0045] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0046] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of IL-6.
[0047] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of IL-6.
[0048] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-6.
[0049] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
[0050] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0051] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit production of IL- 16.
[0052] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit release of IL-16.
[0053] Another object of the present invention is to provide pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 16.
[0054] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
[0055] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is to regulate biological barrier function. In a specific embodiment the biological effect of IL- 16 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL- 16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0056] In certain embodiments, the invention provides a method of treating a patient with an autoimmune or inflammation-associated disease. The disease is selected from the group consisting of inflammatory bowel disease, including Crohn's disease and ulcerative colitis, type 1 diabetes, celiac disease, autoimmune hepatitis, multiple sclerosis, autism, dermatitis herpetiformis, IgA nephropathy, primary biliary chirrosis, rheumatoid arthritis, systemic lupus erythematosus, Grave's disease, Hashimoto's disease, and depression. A compound that inhibits the production, release and/or the biological effects of TNFα is administered to the patient.
[0057] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] Figure 1. PTG exposure induces expression of cytokines in the monocytic cell line THP-I . PTG (lmg/ml) and LPS (l μg/ml) exposure for 24 hours induced expression of characteristic profiles of cytokines and chemokines by THP-I cells. Profiles were determined in triplicate using a nitrocellulose membrane based proteomic profiler assay. [0059] Figure 2. Cytokines expressed in the monocytic cell line THP-I on exposure to PTG (lmg/ml).
[0060] Figure 3. AT-1001 inhibits PTG and LPS induced expression of TNF-α in human PBMCs. PTG (lmg/ml; lOOμg/ml) and LPS (10ng/ml) exposure induced expression of TNFα by human PBMCs, and this expression was inhibited in a dose-dependent fasion by AT-1001. TNFα levels were measured by ELISA.
[0061] Figure 4. AT-1001 inhibits PTG and LPS induced expression of TNF-α in human PBMCs. PTG (lmg/ml; n=9) and LPS (10ng/ml; n=10) exposure induced expression of TNFα by human PBMCs, and this expression was inhibited in a dose-dependent fasion by AT-1001. TNFα levels were measured by ELISA.
[0062] Figure 5. TNFα induction in human PBMcs is PTG-specific. PTG exposure induced TNFα expression by human PBMCs while exposure of human PBMCs to PT-treated casein did not.
[0063] Figure 6. IL-6 induction in human PBMcs is not PTG-specific. PTG exposure induced IL-6 expression by human PBMCs, and exposure of human PBMCs to PT-treated casein also induced IL-6 expression in these cells.
[0064] Figure 7. PTG-induced expression of TNFα by monocytes in human PBMC samples. Control and PTG stimulated PBMCs were treated with brefeldin A to inhibit protein secretion. Treated cells were stained for intracellular TNFα and for CD 14, and stained cells were separated by fluorescence flow cytometry.
[0065] Figure 8. LPS-induced transcription of TNFα in monocytes is not inhibited by AT-1001. Monocytes were exposed to LPS in the presence or absence of AT-1001. Total cellular RNA was isolated from the treated cells, and TNFα mRNA was measured by RT- PCR.
[0066] Figure 9. LPS-induced synthesis of TNFα protein in monocytes is not inhibited by AT-1001. Monocytes were exposed to LPS in the presence or absence of AT-1001 or known translation (actinomycin D) or protein synthesis (cycloheximide) inhibitors. Treated cells were stained for intracellular accumulation of TNFα and separated by flow cytometry. [0067] Figure 10. PTG-induced synthesis of TNFα protein in monocytes is not inhibited by AT-1001. Monocytes were exposed to PTG in the presence or absence of AT-1001 or known translation (actinomycin D) or protein synthesis (cycloheximide) inhibitors. Treated cells were stained for intracellular accumulation of TNFα and separated by flow cytometry.
[0068] Figure 11. AT-1001 does not inhibit TACE-mediated release of TNFα from LPS or PTG treated monocytes. Monocytes were exposed to LPS (lμg/ml) or PTG (lmg/ml) in the presence or absence of AT-1001. Treated cells were stained for accumulation of TNFα on the cell surface, and cell populations were separated by flow cytometry.
[0069] Figure 12. AT-1001 inhibits TNFα-induced permeability. Caco-2 cells were grown as described for 21 days and treated with TNF-α (100ng/ml) in the presence or absence of AT-1001 (12.5mM). Monolayer permeability to Lucifer Yellow (LY) was measured by analysis in a Tecan Spectrofluor fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
[0070] Figure 13. AT-1001 inhibits TNFα-induced TEER. Caco-2 cells were grown as described for 21 days and treated with TNF-α (100ng/ml) in the presence or absence of AT- 1001 (12.5mM). TEER measurements were made, and values are expressed relative to untreated control cells.
[0071] Figure 14. IL-4 treatment increases permeability in a dose-dependent manner. T- 84 cells were grown as described and treated with IL-4 (I Ong/ml or 100ng/ml). Monolayer permeability to Lucifer Yellow (LY) was measured by analysis in a Tecan Spectrofluor fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
[0072] Figure 15. AT-1001 inhibits IL-4-induced permeability. T-84 cells were grown as described and treated with IL-4 (10ng/ml or 100ng/ml) in the presence or absence of AT- 1001. Monolayer permeability to Lucifer Yellow (LY) was measured by analysis in a Tecan Spectrofluor fluorescence plate reader at excitation and emission wavelengths of 485 nm and 535 nm, respectively.
[0073] Figure 16. AT-1001 inhibits TNFα-induced secretion of IL-6 from peripheral blood mononuclear cells (PBMCs): Human PBMCs purified from healthy donors (1 & 2) were stimulated with purified TNF-α in the presence or absence of AT-1001 (6.8mM) for 24 hours. IL-6 levels were measured by standard sandwich ELISA (R&D Systems Duoset).
[0074] Figure 17. AT-1001 inhibits TNFα-induced secretion of IL-16 from peripheral blood mononuclear cells (PBMCs): Human PBMCs purified from healthy donors (1 & 2) were stimulated with purified TNF-α in the presence or absence of AT-1001 (6.8mM) for 24 hours. IL-16 levels were measured by standard sandwich ELISA (R&D Systems Duoset).
DETAILED DESCRIPTION OF THE INVENTION Definitions
[0075] 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 0471 186341); and other similar technical references.
[0076] 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.
[0077] 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. [0078] As used herein, "modulate" and all its forms and tenses refer to either increasing or decreasing a particular biochemical or physiological effect.
[0079] 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, CIq, CIr and CIs), 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.
[0080] 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
[0081] The inventors have identified novel compounds that selectively inhibit TNFα secretion in response to stimuli that are known to induce secretion of TNFα and other proinflammatory cytokines. The novel compounds identified as TNFα inhibitors may also act as inhibitors of mammalian tight junction opening. Exemplary compounds of the invention that inhibit TNFα are presented in Table 1. Table 1. Inhibition of TNFα secretion by compounds of the invention
Figure imgf000015_0001
Cbz - Benzylcarbamate OBn - o-Benzyl Pyr - Pyroglutamic acid Boc - Tetrabutylcarbamate
[0082] Antagonist of mammalian tight junction opening may also be used in the practice of the present invention. As used herein, tight junction antagonists prevent, inhibit or reduce the opening of tight junctions. For example, antagonists of the invention may comprise peptide antagonists. Examples of peptide antagonists 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: GIy Arg VaI Cys VaI GIn Pro GIy (SEQ ID NO:1), GIy Arg VaI Cys VaI GIn Asp GIy (SEQ ID NO:2), GIy Arg VaI Leu VaI GIn Pro GIy (SEQ ID NO:3), GIy Arg VaI Leu VaI GIn Asp GIy (SEQ ID NO:4), GIy Arg Leu Cys VaI GIn Pro GIy (SEQ ID NO:5), GIy Arg Leu Cys VaI GIn Asp GIy (SEQ ID NO:6), GIy Arg Leu Leu VaI GIn Pro GIy (SEQ ID NO:7), GIy Arg Leu Leu VaI GIn Asp GIy (SEQ ID NO:8), GIy Arg GIy Cys VaI GIn Pro GIy (SEQ ID NO:9), GIy Arg GIy Cys VaI GIn Asp GIy (SEQ IDNOrI O), GIy Arg GIy Leu VaI GIn Pro GIy (SEQ IDNOrI 1), GIy Arg GIy Leu VaI GIn Asp GIy (SEQ IDNO: 12), GIy GIy VaI Cys VaI GIn Pro GIy (SEQ ID NO: 13), GIy GIy VaI Cys VaI GIn Asp GIy (SEQ ID NO: 14), GIy GIy VaI Leu VaI GIn Pro GIy (SEQ ID NO: 15), GIy GIy VaI Leu VaI GIn Asp GIy (SEQ ID NO: 16), GIy GIy Leu Cys VaI GIn Pro GIy (SEQ ID NO: 17), GIy GIy Leu Cys VaI GIn Asp GIy (SEQ ID NO: 18), GIy GIy Leu Leu VaI GIn Pro GIy (SEQ ID NO: 19), GIy GIy Leu Leu VaI GIn Asp GIy (SEQ ID NO:20), GIy GIy GIy Cys VaI GIn Pro GIy (SEQ ID NO:21), GIy GIy GIy Cys VaI GIn Asp GIy (SEQ ID NO:22), GIy GIy GIy Leu VaI GIn Pro GIy (SEQ ID NO:23), and GIy GIy GIy Leu VaI GIn Asp GIy (SEQ ID NO:24)
[0083] Examples of peptide antagonists include, but are not limited to, peptides that consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-24. In a particular embodiment the peptide antagonist may have the amino acid sequence of SEQ ID NO: 15. In a particular embodiment the peptide antagonist may have the amino acid sequence GGVLVQPG, also known as AT-1001.
[0084] When the antagonist 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 1 1, 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 1 1 , 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.
[0085] 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 ah, 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.
[0086] In one embodiment the present invention provides compounds that inhibit production of TNFα.
[0087] In another embodiment the present invention provides compounds that inhibit release of TNFα.
[0088] In another embodiment the present invention provides compounds that inhibit the biological effects of TNFα.
[0089] In particular embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the production and/or release of cytokines. In a specific embodiment the biological effect of TNFα is the production and/or release of TNFα. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-6. In another specific embodiment the biological effect of TNFα is the production and/or release of IL- 16.
[0090] In further specific embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the induction of expression of TNF-induced proteins. TNF-induced proteins whose production and/or release is inhibited by the present invention include, for example, CXCLl , CXCL2, CXCL5, CCL2, CCL5, 4-1 BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA, FasL, GITRL, LIGHT, LT-Beta, NGF-Beta, NGFR, OX40L, RANKL, LCN2/22p3, EREG, OSMR, COX2, C/EBPδ, C/EBPβ, MAIL/IKBξ, MMPl 3, TIMP2, RANKL, OPG, SOCS3, PSGD3, 14-3-3-γ, CTTN, COTLl, FAS, CASPl, 4- IBB, BAFFR, BCMA, c-Fos, c-Jun, c- ReI, Caspase 1, Caspase 10, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, CD27, CD30, CD40, DCRl , DCR2, DR3, DR4, DR5, DR6, EDAR, FADD, Fas, Fn 14, GITR, HVEM, I-KappaB, Alpha, I-KappaB-Beta, I-KappaB-Epsilon, IKK-Alpha, IKK-Beta, IKK-Epsilon, IKK-Gamma, JNKl, JNK2, JNK3, LT-BetaR, MEKl, MEK2, MEK3, MEK4, MEK5, MEK6, NF-KappaBl, NF-KappaB2, OX40, p38-Alpha, p38-Beta, p38-Delta, p38-Gamma, PIK3C2Alpha, PIK3C2Beta, PIK3C2Gamma, PIK3C3, PIK3CAlpha, PIK3CBeta, PIK3CDelta, PIK3CGamma, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5, RANK, ReIA, ReIB, RELT, Src, TABl, TAB2, GRO-I, Interleukin-8 (IL-8), A20 (A20), GADD45 (GADD45), Cellular inhibitor of apoptosis protein 1/2 (c-IAPl/2), Interleukin-1 alpha (IL-Ia), v-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), Tumor protein p53 (Li-Fraumeni syndrome) (p53), TGFB-inducible early growth response, Syndecan-4a, MAP kinase phosphatase 1 , Small inducible cytokine A2, TNFα-induced protein 2, Natural killer cell transcript 4, TNFα-induced protein 3, Early growth response 1, Superoxide dismutase 2, Human transcription factor ETRlOl , Prostaglandin E synthase, and JunB proto-oncogene.
[0091] In further specific embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is to regulate biological barrier function. In a specific embodiment the biological effect of TNFα is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of TNFα is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0092] In another embodiment the present invention provides compounds that inhibit production of IL-4.
[0093] In another embodiment the present invention provides compounds that inhibit release of IL-4.
[0094] In another embodiment the present invention provides compounds that inhibit the biological effects of IL-4. [0095] In particular embodiments the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
[0096] In further specific embodiments the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0097] In another embodiment the present invention provides compounds that inhibit production of IL-6.
[0098] In another embodiment the present invention provides compounds that inhibit release of IL-6.
[0099] In another embodiment the present invention provides compounds that inhibit the biological effects of IL-6.
[00100] In particular embodiments the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
[0100] In further specific embodiments the present invention provides a compound that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0101] In another embodiment the present invention provides compounds that inhibit production of IL- 16.
[0102] In another embodiment the present invention provides compounds that inhibit release of IL- 16.
[0103] In another embodiment the present invention provides compounds that inhibit the biological effects of IL- 16. [0104] In particular embodiments the present invention provides a compound that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
[0105] In further specific embodiments the present invention provides a compound that inhibits the biological effects of IL-4, wherein the biological effect of IL- 16 is to regulate biological barrier function. In a specific embodiment the biological effect of IL- 16 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0106] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit production of TNFα.
[0107] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit release of TNFα.
[0108] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of TNFα.
[0109] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the production and/or release of cytokines. In a specific embodiment the biological effect of TNFα is the production and/or release of TNFα. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-6. In another specific embodiment the biological effect of TNFα is the production and/or release of IL- 16.
[0110] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is to regulate biological barrier function. In a specific embodiment the biological effect of TNFα is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of TNFα is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0111] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit production of IL-4. [0112] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit release of IL-4.
[0113] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-4.
[0114] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
[0115] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0116] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit production of IL-6.
[0117] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit release of IL-6.
[0118] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of IL-6.
[0119] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
[0120] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0121] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit production of IL- 16. [0122] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit release of IL- 16.
[0123] In another embodiment the present invention provides pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 16.
[0124] In particular embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
[0125] In further specific embodiments the present invention provides a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is to regulate biological barrier function. In a specific embodiment the biological effect of IL- 16 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL- 16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
Compositions
[0126] 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 facilitate the inhibition of TNFα production in a subject in need thereof. The amount of a compound of the invention employed in any 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.
[0127] 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 lmg to about lOOOmg, from about lOmg to about lOOmg, from about l Omg to about 50mg, or from about l Omg to about 25mg of the compound. As used herein, "about" used to modify a numerical value means within 10% of the value.
[0128] 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.
[0129] 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.
[0130] 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).
[0131] 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.
[0132] Powder formulations may optionally contain at least one particulate pharmaceutically acceptable carrier known to those of skill in the art. 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.
[0133] 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.
[0134] 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, frυm 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 frυm 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.
[0135] 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.
[0136] 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.
[0137] 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 less, in approximately sixty minutes. Examples of 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.
[0138] 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.
[0139] 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.
[0140] 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).
[0141] 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. [0142] 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 absorb.
[0143] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome.
[0144] 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.
[0145] 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.
[0146] 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 pain 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.
[0147] 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 from 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.
[0148] The amount of the compound of the invention that will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant TNFα production 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.
[0149] 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
[0150] 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.
[0151] 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.
[0152] 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, azasporine, 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, diethylstllbestrol, bleomycin, dactinomycin, daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone, mitomycin-c, plicamycin, paclitaxel, docetaxel, topotecan, irinotecan, 9-amino camptothecan, 9-nitro camptothecan, GS-21 1, 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.
[0153] 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 Ilb/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 14C-, 3H-, 131I-, 32P- or 36S-radiolabelled form or other radiolabeled form of any of the foregoing, and combinations thereof.
[0154] 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-L-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, 60Co, 192Ir , 32P , 1 11In , 90Y, 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.
[0155] 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 are known in the art can also be used.
[0156] 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).
[0157] 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, Cυrynebacterium diphtheriae antigens, Bordetella pertussis antigens, Clostridium tetani antigens, Bacillus anthracis antigens, Haemophilus influenzae antigens, smallpox virus antigens, and influenza virus antigens.
[0158] 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-S-carboxyoxiran^-carbonyl-L-leucylagmatine, ethylenediaminetetra-acetic acid (EDTA), phenylmethylsulfonylfluoride (PMSF), aprotinin, amyloid protein precursor (APP), amyloid beta precursor protein, αl -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.
[0159] 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
[0160] 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.
[0161] 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, 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.
[0162] 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 TNFα 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 TNFα 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 TNFα 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.
[0163] 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 an 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.
[0164] 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.
[0165] 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.
[0166] In one embodiment the present invention provides methods to inhibit production of TNFα.
[0167] In another embodiment the present invention provides methods to inhibit release ofTNFα.
[0168] In another embodiment the present invention provides methods to inhibit the biological effects ofTNFα.
[0169] In particular embodiments the present invention provides methods to inhibit the biological effects of TNFα, wherein the biological effect of TNFα is the production and/or release of cytokines. In a specific embodiment the biological effect of TNFα is the production and/or release of TNFα. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-6. In another specific embodiment the biological effect ofTNFα is the production and/or release of IL- 16. [0170] In further specific embodiments the present invention provides a compound that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the induction of expression of TNF-induced proteins. TNF-induced proteins whose production and/or release is inhibited by the present invention include, for example, CXCLl, CXCL2, CXCL5, CCL2, CCL5, 4- IBBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA, FasL, GITRL, LIGHT, LT-Beta, NGF-Beta, NGFR, OX40L, RANKL, LCN2/22p3, EREG, OSMR, COX2, C/EBPδ, C/EBPβ, MAIL/IKBξ, MMPl 3, TIMP2, RANKL, OPG, SOCS3, PSGD3, 14-3-3-γ, CTTN, COTLl, FAS, CASPl, 4-1BB, BAFFR, BCMA, c-Fos, c-Jun, c- ReI, Caspase 1, Caspase 10, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, CD27, CD30, CD40, DCRl, DCR2, DR3, DR4, DR5, DR6, EDAR, FADD, Fas, Fn 14, GITR, HVEM, I-KappaB, Alpha, I-KappaB-Beta, I-KappaB-Epsilon, IKK-Alpha, IKK-Beta, IKK-Epsilon, IKK-Gamma, JNKl , JNK2, JNK3, LT-BetaR, MEKl, MEK2, MEK3, MEK4, MEK5, MEK6, NF-KappaBl, NF-KappaB2, OX40, p38-Alpha, p38-Beta, p38-Delta, p38-Gamma, PIK3C2Alpha, PIK3C2Beta, PIK3C2Gamma, PIK3C3, PIK3CAlpha, PIK3CBeta, PIK3CDelta, PIK3CGamma, PIK3R1, PIK3R2, PIK3R3, PIK3R4, PIK3R5, RANK, ReIA, ReIB, RELT, Src, TABl , TAB2, GRO-I , Interleukin-8 (IL-8), A20 (A20), GADD45 (GADD45), Cellular inhibitor of apoptosis protein 1/2 (c-IAPl/2), Interleukin-1 alpha (IL-Ia), v-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), Tumor protein p53 (Li-Fraumeni syndrome) (p53), TGFB-inducible early growth response, Syndecan-4a, MAP kinase phosphatase 1 , Small inducible cytokine A2, TNFα-induced protein 2, Natural killer cell transcript 4, TNFα-induced protein 3, Early growth response 1, Superoxide dismutase 2, Human transcription factor ETRl Ol , Prostaglandin E synthase, and JunB proto-oncogene.
[0171] In further specific embodiments the present invention provides methods to inhibit the biological effects of TNFα, wherein the biological effect of TNFα is to regulate biological barrier function. In a specific embodiment the biological effect of TNFα is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of TNFα is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0172] In another embodiment the present invention provides methods to inhibit production of IL-4. [0173] In another embodiment the present invention provides methods to inhibit release ofIL-4.
[0174] In another embodiment the present invention provides methods to inhibit the biological effects of IL-4.
[0175] In particular embodiments the present invention provides methods to inhibit the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
[0176] In further specific embodiments the present invention provides methods to inhibit the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-4 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0177] In another embodiment the present invention provides methods to inhibit production of IL-6.
[0178] In another embodiment the present invention provides methods to inhibit release of IL-6.
[0179] In another embodiment the present invention provides methods to inhibit the biological effects of IL-6.
[0180] In particular embodiments the present invention provides methods to inhibit the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines.
[0181] In further specific embodiments the present invention provides methods to inhibit the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-6 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0182] In another embodiment the present invention provides methods to inhibit production of IL- 16. [0183] In another embodiment the present invention provides methods to inhibit release of IL- 16.
[0184] In another embodiment the present invention provides methods to inhibit the biological effects of IL- 16.
[0185] In particular embodiments the present invention provides methods to inhibit the biological effects of IL-16, wherein the biological effect of IL-16 is the production and/or release of cytokines.
[0186] In further specific embodiments the present invention provides methods to inhibit the biological effects of IL-4, wherein the biological effect of IL-16 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-16 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-16 is to reduce the trans-epithelial electric resistance (TEER) across a biological barrier.
[0187] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of TNFα.
[0188] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of TNFα.
[0189] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of
TNFα.
[0190] In particular embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is the production and/or release of cytokines. In a specific embodiment the biological effect of TNFα is the production and/or release of TNFα. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-6. In another specific embodiment the biological effect of TNFα is the production and/or release of IL-16.
[0191] In further specific embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of TNFα, wherein the biological effect of TNFα is to regulate biological barrier function. In a specific embodiment the biological effect of TNFα is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of TNFα is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
[0192] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of IL-4.
[0193] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of IL-4.
[0194] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 4.
[0195] In particular embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is the production and/or release of cytokines.
[0196] In further specific embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-4, wherein the biological effect of IL-4 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-4 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-4 is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
[0197] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of IL-6.
[0198] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of IL-6.
[0199] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 6.
[0200] In particular embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is the production and/or release of cytokines. [0201] In further specific embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL-6, wherein the biological effect of IL-6 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-6 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-6 is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
[0202] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit production of IL- 16.
[0203] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit release of IL- 16.
[0204] In another embodiment the present invention provides methods of using pharmaceutical compositions comprising compounds that inhibit the biological effects of IL- 16.
[0205] In particular embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL- 16 is the production and/or release of cytokines.
[0206] In further specific embodiments the present invention provides methods of using a pharmaceutical composition that inhibits the biological effects of IL- 16, wherein the biological effect of IL-16 is to regulate biological barrier function. In a specific embodiment the biological effect of IL-16 is to increase the macromolecular permeability of a biological barrier. In another specific embodiment the biological effect of IL-16 is to reduce the trans- epithelial electric resistance (TEER) across a biological barrier.
[0207] 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.
REFERENCES
[0208] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as having been incorporated by reference in its entirety.
EXAMPLE 1
[0209] Measurement Of Trans Epithelial Electric Resistance (TEER) And Epithelial Flux OfA Fluorescent Marker Lucifer Yellow
[0210] 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.
[0211] 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:
[0212] Determination of TEER and Lucifer Yellow flux
[0213] Prepare Modified Hank's Balanced Salt Solution (MHBSS) by 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).
[0214] 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.
[0215] 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.
[0216] Using the MilliCell®-ERS instrument (Millipore), measure and record the transepithelial electrical resistance (TEER) across each filter and record. [0217] Aspirate solution from the apical compartment of each filter (n=3 per condition) and replace with 0.5ml of control and test solutions containing Lucifer Yellow and test compound if appropriate.
[0218] Place all plates into incubator set at 37°C (± 0.2), 50 RPM (±5) for a total of 180 minutes.
[0219] At t = 30, 60, 120 and 180 minutes, measure and record the transepithelial electrical resistance (TEER) across each filter using the MilliCell-ERS instrument.
[0220] At t = 60, 120 and 180 minutes remove lOOμl from each basolateral compartment and place it in a 96-well plate for Lucifer Yellow analysis, replace with lOOμl of MHBSS.
[0221] 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 lOOμL 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.
[0222] Harvest the remaining start solutions and what is left in each apical compartment into 1.5ml vials. Freeze at -200C for future analysis.
[0223] Analyze each 96-well plate in a Tecan Spectra Fluor Plus using Magellan at 485 and 535nm.
[0224] Materials:
[0225] Cells: CaCo-2 cells passage 40-60 grown on Transwell® plates for 21-28 days
[0226] Culture Medium: DMEM supplemented with 10% fetal bovine serum, 1% NEAA, l % Penn/Strep
[0227] Buffers: Hank's Balanced Salt Solution (HBSS) without calcium and magnesium
[0228] Flasks: 100 X 20 mm Tissue culture dish Falcon.
[0229] Plates: 12 well polycarbonate Transwell® filters; 0.3uM pore size
EXAMPLE 2
[0230] Identification of Cytokines Upregulated on Treatment of THP-I cells by PT- Gliadin (PTG) [0231] The monocytic cell line THP-I was used to characterize the profile of cytokines whose expression was upregulated on exposure to protease treated gliadin (PTG). THP-I cells were diluted to 5x 105 cells/ml in RPMI medium supplemented with 10% heat inactivated fetal bovine serum.
[0232] 5x 105 (ImI) cells were plated in each well of a 12 well plate, and cells were incubated at 370C overnight. Test compounds (PTG lmg/ml; LPS lμg/ml) were added to the cultures, and incubation was continued a further 18 hours at 37°C.
[0233] 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, CD40 ligand, G-CSF, GM-CSF, GRO-α/CXCLl, I-309/CCL1, ICAM-I, IFNγ, IL-l α, IL-l β, IL-lra, 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/CXCLlO, I-TAC/CXCL1 1 , MCP-1/CCL2, MIF, MIP-lα/CCL3, MIP- l β/CCL3, RANTES/CCL5, SDF-1/CXCL12, Serpin-El /PAI-I, TNFα, and TREM-I .
[0234] After 6 hours of PTG exposure THP-I cells demonstrated increased expression of the cytokines IL-8, MIP-Ia, MIP-I β, TNF-α and Gro-α. After 24 hours of exposure to PTG increased expression of RANTES and MIF were also observed.
EXAMPLE 3
[0235] Identification of Cytokines Upregulated on Treatment of PBMCs by PT-Gl iadin (PTG)
[0236] 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 2x 105 cells were plated in each well of a 96 well plate. Cells were incubated at 370C with PTG (lmg/ml) or LPS (l 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). [0237] Expression of IL-6, IL-8, MIP- lα, 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: 15).
[0238] Expression of TNF-α was induced by treatment with LPS and PTG. Expression of this cytokines was inhibited in a dose-dependent manner by treatment with peptide GGVLVQPG (SEQ ID NO: 15; Figures 3 and 4).
EXAMPLE 4
[0239] TNFα Transcription in Monocytes after PTG or LPS exposure is not inhibited by AT-1001
[0240] Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNFα transcription after exposure to PTG or LPS in the presence or absence of AT-1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum, and incubated for 4 hours at 37°C with PTG (lmg/ml) or LPS (l μg/ml) in the presence or absence of test compounds being examined for the ability to suppress cytokine production. Following treatment total cellular RNA was isolated from each sample, and the level of TNFα mRNA was measured by RT-PCR (Applied Biosciences) using methods known in the art. AT-1001 treatment did not reduce the amount of TNFa mRNA transcribed in response to LPS (Figure 8) or PTG (not shown) exposure.
EXAMPLE 5
[0241] TNFα protein synthesis in Monocytes after PTG or LPS exposure is not inhibited by AT-1001
[0242] Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNFα protein synthesis after exposure to PTG or LPS in the presence or absence of AT- 1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum and containing Brefeldin A. Monocytes were incubated for 4 hours at 370C with PTG (lmg/ml) or LPS (l μg/ml) in the presence or absence of protein synthesis inhibitors or test compounds being examined for the ability to suppress cytokine production. Following treatment cells were stained for intracellular accumulation of TNFα and separated by fluorescence flow cytometry using methods known in the art. AT-1001 treatment did not reduce the amount of TNFa protein synthesized in response to LPS (Figure 9) or PTG (Figure 10) exposure.
EXAMPLE 6
[0243] TNFα protein release from Monocytes after PTG or LPS exposure is not inhibited by AT-1001
[0244] TNFα release form the cell surface is mediated by the proteolytic activity of the enzyme TNFα converting enzyme (TACE), which is also known as ADAM- 17. This enzyme is a metalloprotease-disintegrin, and in the absence of its activity TNFα accumulates at the surface of an expressing cell.
[0245] Monocytes were isolated from human PBMCs by magnetic bead negative selection using a Monocyte Isolation Kit II (Miltenyi Biotech), and they were used to study TNFα protein secretion after exposure to PTG or LPS in the presence or absence of AT-1001. Monocytes were suspended in RPMI medium supplemented with 5% heat inactivated human AB serum. Monocytes were incubated for 24 hours at 37°C with PTG (l mg/ml) or LPS (l μg/ml) in the presence or absence of test compounds being examined for the ability to suppress cytokine production. Following treatment cells were stained for accumulation of TNFα at the cell surface and separated by fluorescence flow cytometry using methods known in the art. AT-1001 treatment did not reduce the amount of TNFa protein present at the cell surface in response to LPS or PTG exposure (Figure 1 1).
[0246] 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.

Claims

What is claimed is:
1. A method of inhibiting tumor necrosis factor comprising: administering to a subject in need thereof a composition comprising a compound that inhibits production of tumor necrosis factor alpha.
2. The method of claim 1, wherein the subject is a human.
3. The method of claim 1, wherein the tight junction antagonist is a peptide.
4. The method of claim 3, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-24.
5. The method of claim 4, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 15.
6. The method of claim 3, wherein the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-24.
7. The method of claim 6, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 15.
8. The method of claim 1, wherein the composition is administered in conjunction with an additional therapeutic agent.
9. The method of claim 8, wherein the composition and the additional therapeutic agent are administered simultaneously.
10. The method of claim 8, wherein the composition and the additional therapeutic agent are not administered simultaneously.
1 1. The method of claim 8, wherein the composition further comprises the additional therapeutic agent.
12. The method of claim 8, wherein the additional therapeutic agent is selected from the group consisting of aminosalicylates, corticosteroids, immunomodulators, antibiotics, cytokines, chemokines and biologic therapeutics.
13. A pharmaceutical composition for inhibiting cytokine production, comprising, a compound described herein having an activity of inhibiting cytoikine production.
14. The composition of claim 13, wherein the cytokine is TNFα.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458925B1 (en) * 1998-08-03 2002-10-01 University Of Maryland, Baltimore Peptide antagonists of zonulin and methods for use of the same
US20040167174A1 (en) * 1999-12-21 2004-08-26 Celgene Substituted acylhydroxamic acids and method of reducing TNFalpha levels
US20060121132A1 (en) * 2000-12-18 2006-06-08 Meiji Dairies Corporation TNF-alpha production inhibitor comprising kavalactone as an active ingredient

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458925B1 (en) * 1998-08-03 2002-10-01 University Of Maryland, Baltimore Peptide antagonists of zonulin and methods for use of the same
US20040167174A1 (en) * 1999-12-21 2004-08-26 Celgene Substituted acylhydroxamic acids and method of reducing TNFalpha levels
US20060121132A1 (en) * 2000-12-18 2006-06-08 Meiji Dairies Corporation TNF-alpha production inhibitor comprising kavalactone as an active ingredient

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PORITZ ET AL.: 'Tumor Necrosis Factor Alpha Disrupts Tight Junction Assembly' JOURNAL OF SURGICAL RESEARCH vol. 116, 11 December 2002, pages 14 - 18 *

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