WO2006110577A2 - Methodes et compositions pour reduire la production de cytokines pro-inflammatoires - Google Patents

Methodes et compositions pour reduire la production de cytokines pro-inflammatoires Download PDF

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WO2006110577A2
WO2006110577A2 PCT/US2006/013149 US2006013149W WO2006110577A2 WO 2006110577 A2 WO2006110577 A2 WO 2006110577A2 US 2006013149 W US2006013149 W US 2006013149W WO 2006110577 A2 WO2006110577 A2 WO 2006110577A2
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fril
patient
protein
fril protein
pro
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PCT/US2006/013149
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WO2006110577A3 (fr
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Charles A. Dinarello
Jeffrey G. Moore
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Morningside Venture Investments Limited
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Priority to CA002604007A priority Critical patent/CA2604007A1/fr
Priority to EP06740758A priority patent/EP1901766A2/fr
Priority to AU2006235254A priority patent/AU2006235254A1/en
Publication of WO2006110577A2 publication Critical patent/WO2006110577A2/fr
Publication of WO2006110577A3 publication Critical patent/WO2006110577A3/fr

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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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention relates to methods and compositions for reducing the production of pro-inflammatory cytokines. More specifically, the invention relates to methods of treatment of disorders characterized by pathologic or deleterious production of proinflammatory cytokines using proteins which are members of the FLT3-receptor interacting ligand (FRIL) family of progenitor cell preservation factors.
  • FRIL FLT3-receptor interacting ligand
  • Inflammation is a physiological response by the body to restore and maintain homeostasis following insults such as infections and tissue injury (Kuby, J., Immunology , Second Edition, (W.H. Freeman and Company, New York)). Most of the immune cells responsible for the body's defense are located in the blood, and inflammation is the . means by which these cells leave the blood and enter the tissues around the infected or injured site. The inflammatory response provides early protection by restricting the tissue damage to the site of the infection or tissue injury. Secreted cytokine proteins provide signals between immune cells to coordinate the inflammatory response.
  • Cytokines are a group of low molecular weight regulatory proteins secreted in response to inducing stimuli by cells such as lymphocytes, monocytes/macrophages, mast cells, eosinophils, and endothelial cells lining blood vessels.
  • cytokines There are four major categories of cytokines: interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), colony stimulating factors (e.g., G-CSF, M-CSF, GM-CSF), tumor necrosis factors (e.g., TNF- ⁇ , TNF- ⁇ ), and interleukins (e.g., IL-4, IL-5, IL-10).
  • Cytokines can exert their effect through the bloodstream on distant target cells (endocrine), on target cells adjacent to those that produce them (paracrine), or on the same cell that produces the cytokine (autocrine). Physiologically, cytokines appear to exert their most important effects in a paracrine/autocrine manner (Kuby, J. supra). Defects in the complex regulatory networks governing the expression ot cytol ⁇ nes have been implicated in several diseases.
  • pro-inflammatory cytokines such as IL-l ⁇ , IL-l ⁇ , IL-2, IL-4, IL-6, IL-8, IL-IO, IL-13, TNF- ⁇ , and IFN- ⁇
  • diseases such as psoriasis, septic shock, periodontal disease, rheumatoid arthritis, inflammatory bowel disease, toxic shock syndrome, inflammatory sequelae of viral infections, eczema, autoimmune disorders, and neurodegenerative diseases.
  • diseases such as psoriasis, septic shock, periodontal disease, rheumatoid arthritis, inflammatory bowel disease, toxic shock syndrome, inflammatory sequelae of viral infections, eczema, autoimmune disorders, and neurodegenerative diseases.
  • the FRIL family of proteins was previously identified and described as mannose- binding plant lectins having the ability to preserve progenitor cells by inhibiting proliferation and/or differentiation (e.g., Moore et al. (1997), Blood 90, Suppl. 1, 308 (abstract); Mo et al. (1999), Glycobiology 9:173-179; Colucci etal. (1999), Proc. Natl. Acad. ScL USA 96:646-650; Moore et al. (2000), Biochim. Biophys. Acta 25027: 1-9).
  • the FRIL proteins were not known to have any activity or utility with respect to treating or alleviating conditions characterized by pathologic or deleterious production of pro-inflammatory cytokines.
  • the present invention depends, in part, upon the discovery that certain lectins are useful in reducing undesired or pathological levels of one or more pro-inflammatory cytokines.
  • the invention relates to the discovery that FRIL proteins reduce the production of pro-inflammatory cytokines by cells that have been exposed to inducers of pro-inflammatory cytokines.
  • the FRIL proteins of the invention can selectively reduce the levels of pro-inflammatory cytokines and, therefore, they can be used in treatments for patients diagnosed with disorders characterized by pathologic or deleterious production of a pro-inflammatory cytokine.
  • the invention provides a method for reducing the levels and/or production of a pro-inflammatory cytokine in a mammal in need thereof by administering an effective amount of a FRIL protein to the mammal.
  • the pro-inflammatory cytokine is selected from me group consisting or IFN- ⁇ , IL-6, TNF- ⁇ , and combinations thereof.
  • the mammal is a human patient.
  • the patient has been diagnosed with a condition selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease.
  • a condition selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease.
  • the invention provides a method for treating a patient diagnosed with a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine by administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the undesired or pathological levels of the pro-inflammatory cytokine.
  • the patient has been diagnosed with a condition selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease.
  • the invention provides a method for treating a pro-inflammatory cytokine-mediated disorder in a cell by administering a therapeutically effective amount of a FRIL protein to the patient.
  • the invention provides a method for treating inflammation by administering a therapeutically effective amount of a FRIL protein to the patient.
  • the inflammation is chronic inflammation, or acute inflammation.
  • the inflammation is associated with a condition selected from the group consisting of psoriasis, eczema, dermatitis, and arthritis.
  • the invention provides a method for treating a patient diagnosed with an autoimmune disease by administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with the autoimmune disease.
  • the invention provides a method for treating a patient diagnosed with psoriasis by administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein re ⁇ uces me levels or a proinflammatory cytokine in the patient, thereby treating the patient with psoriasis.
  • the invention provides a method for treating a patient diagnosed with multiple sclerosis by administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with multiple sclerosis.
  • the invention provides a method for treating a patient diagnosed with rheumatoid arthritis by administering a therapeutically effective amount of a FRIL protein to the patient, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby treating the patient with rheumatoid arthritis.
  • the invention provides a method for suppressing rejection to a graft in a patient by administering a therapeutically effective amount of a FRIL molecule to the patient prior to, at the same time as, or subsequent to the graft, wherein the administration of the FRIL protein reduces the levels of a pro-inflammatory cytokine in the patient, thereby suppressing rejection by the patient of the graft.
  • the graft is an allograft or a xenograft.
  • the invention provides a method for treating mucositis by administering a therapeutically effective amount of a FRIL protein to the patient.
  • the mucositis is selected from the group consisting of radiation-induced mucositis, chemotherapy-induced mucositis, and infection-induced mucositis.
  • the invention provides a composition comprising a FRIL protein and a second agent.
  • the second agent is selected from the group consisting of an IL-I receptor antagonist, a soluble cytokine receptor that binds IL-2, a soluble cytokine receptor that binds IL-4, a soluble cytokine receptor that binds IL-6, a soluble cytokine receptor that binds IL-7, a soluble cytokine receptor that binds IFN- ⁇ , a soluble cytokine receptor that binds TNF- ⁇ , a soluble cytokine receptor that binds TNF- ⁇ , a soluble cytokine receptor that binds LIF, an endogenous anti-endotoxin antibody, and any combinations thereof.
  • the FRIL protein can be selected from the group consisting of a native FRIL protein and a recombinant FRIL protein.
  • the native FRIL protein is selected from the group consisting of a native Dl-FRIL protein, a native Pv-FRIL protein, and a native Yam-FRIL protein.
  • the FRIL protein is a mature FRIL protein lacking an N-terminal leader sequence.
  • the FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, or SEQ ID NO.: 8.
  • the FRIL protein is expressed from a nucleic acid including SEQ ID NO.: 1, SEQ ID NO.:5, or SEQ ID NO.:7.
  • the invention provides for the use of a FRIL protein in the manufacture of a medicament for reducing the levels and/or production of a pro- inflammatory cytokine.
  • the FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, or SEQ ID NO.: 8.
  • the FRIL protein is expressed from a nucleic acid including SEQ ID NO.:1, SEQ ID NO.:5, or SEQ ID NO.:7.
  • the FRIL protein is a native FRIL protein or a recombinant FRIL protein.
  • the pro-inflammatory cytokine is selected from the group consisting of IFN- ⁇ , IL-6, TNF- ⁇ , and combinations thereof.
  • the invention provides for the use of a FRIL protein in the manufacture of a medicament for the treatment of a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine.
  • the condition characterized by undesired or pathological levels of a pro-inflammatory cytokine is selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, lupus, Crohn's disease, amyotrophic lateral sclerosis, atherosclerosis, septic shock, inflammatory bowel disease, endotoxemia, and graft-versus-host disease.
  • the FRIL protein corresponds to an amino acid sequence included in at least one of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, or SEQ ID NO.: 8.
  • the FRIL protein is expressed from a nucleic acid including SEQ ID NO.:1, SEQ ID NO.:5, or SEQ ID NO.:7.
  • the FRIL protein is a native FRIL protein or a recombinant FRIL protein.
  • the pro- inflammatory cytokine is selected from the group consisting of IFN- ⁇ , IL-6, TNF- ⁇ , and combinations thereof.
  • Figure 1 is a schematic representation of a whole blood assay to detect the effect of various stimuli on cytokine levels (e.g., effect of addition of LPS with or without FRIL on IL-6 levels).
  • Figure 2 is a schematic representation of a bar graph showing the reduction of IL-
  • Figure 3 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatants of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl- FRIL.
  • Figure 4 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatants of human blood cultures from donor 2, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl- FRIL.
  • Figure 5 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatants of human blood cultures from donor 3, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl- FRIL.
  • Figure 6 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatants of human blood cultures from donor 4, 24 hours after stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of Dl-FRIL.
  • Figure 7 is a schematic representation of a bar graph showing the reduction of
  • IFN- ⁇ (pg/mL) in the lysate of human blood cultures from donor 4, 48 hours after stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of Dl-FRIL.
  • Figure 8 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatant of human blood cultures from donor 5, 24 hours after stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of Dl-FRIL.
  • Figure 9 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatant of human blood cultures from donor 7, 24 hours after stimulation with LPS in the presence or absence of Dl-FRIL.
  • Figure 10 is a schematic representation of a bar graph showing the reduction of
  • IL- l ⁇ (pg/mL) in the lysate of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl- FRIL.
  • Figure 11 is a schematic representation of a bar graph showing the reduction of TNF- ⁇ (pg/mL) in the supernatant of human blood cultures from donor 1, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl- FRIL.
  • Figure 12 is a schematic representation of a bar graph showing the reduction of TNF- ⁇ (pg/mL) in the supernatant of human blood cultures from donor 3, 24 hours after stimulation with heat-killed Staphylococcus epidermidis in the presence or absence of Dl- FRIL.
  • Figure 13 is a schematic representation of a bar graph showing the reduction of TNF- ⁇ (pg/mL) in the supernatant of human blood cultures from donor 4, 24 hours after stimulation with heat-killed Staphylococcus epidermidis or LPS in the presence or absence of Dl-FRIL.
  • Figure 14 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatant of human blood cultures from donors 8 and 9, 24 hours after stimulation with IL- 12 and IL- 18 in the presence or absence of Dl-FRIL.
  • Figure 15 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the supernatant of human blood cultures from donors 8 and 9, 48 hours after stimulation with IL- 12 and IL- 18 in the presence or absence of Dl-FRIL.
  • Figure 16 is a schematic representation of a bar graph showing the reduction of IFN- ⁇ (pg/mL) in the lysate of human blood cultures from donors 8 and 9, 48 hours after stimulation with IL- 12 and IL- 18 in the presence or absence of Dl-FRIL.
  • FRIL-protein means a FRIL protein isolated from a legume in which the protein is naturally expressed.
  • recombinant FRIL-protein means a FRIL protein isolated from an organism in which the protein is expressed by a recombinant gene including, without limitation, bacteria, yeast, plant, or animal cells which have been transfected with a recombinant construct encoding the FRIL protein.
  • a recombinant FRIL protein can have an amino acid sequence identical to a native FRIL protein, or can have an amino acid sequence including one or more amino acid insertions, deletions, and/or substitutions including, without limitation, N-terminal additions or deletions, C- terminal additions or deletions, and chimeric proteins.
  • FRIL-protein without further modification, means any native FRIL protein or recombinant FRIL protein.
  • the term "inflammation,” or “inflammatory response” means a non-specific immune response that occurs in reaction to a bodily injury.
  • the term "pro-inflammatory cytokine” means a cytokine that is capable of promoting, inducing, or increasing inflammation in a responsive tissue.
  • Non- limiting examples of such cytokines include IL-l ⁇ , IL-l ⁇ , IL-2, IL-4, IL-6, IL-8, IL-IO, IL-13, IL-15, IL-16, IL-17, IL-18, TNF- ⁇ , TGF- ⁇ , IFN- ⁇ , and IFN- ⁇ .
  • sequence identity means a measure of the degree of similarity of two sequences based upon an alignment of the sequences which maximizes identity and which is a function of the number of identical nucleotides or residues, the number of total nucleotides or residues, and the presence and length of gaps in the sequence alignment.
  • a variety of algorithms and computer programs are available for determining sequence identity using standard parameters, for example, Gapped BLAST or PSI-BLAST (Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402), BLAST (Altschul et al. (1990), J. MoI. Biol.
  • the term "substantially pure” means a preparation which contains at least 60% (by dry weight) of the protein of interest, exclusive of the weight of other intentionally included compounds. In some embodiments, the preparation is at least 75%, at least 90%, or at least 99%, by dry weight the protein of interest, exclusive of the weight of other intentionally included compounds. Purity can be measured by any appropriate method, e.g., column chromatography, gel electrophoresis, or HPLC analysis.
  • a "substantially pure" preparation means a preparation in which the total dry weight of the proteins of the invention is at least 60% of the total dry weight, exclusive of the weight of other intentionally included compounds.
  • the total weight of the proteins of the invention can be at least 75%, at least 90%, or at least 99%, of the total dry weight of the preparation, exclusive of the weight of other intentionally included compounds.
  • proteins of the invention are mixed with one or more other proteins (e.g., serum albumin) or compounds (e.g., diluents, detergents, excipients, salts, polysaccharides, sugars, lipids) for purposes of administration, stability, storage, and the like, the weight of such other proteins or compounds is ignored in the calculation of the purity of the preparation.
  • other proteins e.g., serum albumin
  • compounds e.g., diluents, detergents, excipients, salts, polysaccharides, sugars, lipids
  • the term "therapeutically effective amount” means the total amount of each active component of a pharmaceutical composition or method that is sufficient to show a meaningful patient benefit (e.g., a statistically significant decrease in the production of a pro-inflammatory cytokine).
  • a meaningful patient benefit e.g., a statistically significant decrease in the production of a pro-inflammatory cytokine.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.
  • the term “reduce” means to cause a decrease of at least 5% in a specified characteristic, such as a level or concentration of a molecule in the circulation or in a specified tissue, or a rate of cell proliferation (i.e., cell reproduction) or survival, relative to a baseline level or a level which would have been expected in the absence of a specified treatment.
  • the term "inhibiting/reducing inflammation,” means to reduce the level of one or more pro-inflammatory cytokines in a mammal suffering from an inflammatory response, thereby reducing the damage to the cells, tissues, and/or organs affected by the inflammation.
  • suppressing rejection means inhibiting a graft recipient's immune response which would otherwise occur, e.g., in response to the introduction of a non-self MHC antigen into the recipient.
  • suppressing rejection of a graft refers to an inhibition of the immune system of the recipient to a graft of a donor compared to a situation where a method of the invention was not used.
  • the term "statistically significant" means having a probability of less than 10% under the relevant null hypothesis (i.e., p ⁇ 0.1).
  • the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range.
  • the variable can be equal to any integer value within the numerical range, including the end-points of the range.
  • the variable can be equal to any real value within the numerical range, including the end-points of the range.
  • a variable which is described as having values between 0 and 2 can take the values 0, 1, or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values > 0 and ⁇ 2 if the variable is inherently continuous.
  • the present invention depends, in part, upon the discovery that certain lectins are useful in reducing undesired or pathological levels of one or more pro-inflammatory cytokines.
  • the invention relates to the discovery that FRIL proteins can reduce the production of pro-inflammatory cytokines by cells that have been exposed to inducers of pro-inflammatory cytokines.
  • the FRIL proteins of the invention can selectively reduce the levels of pro-inflammatory cytokines and, therefore, they can be used in treatments for patients diagnosed with disorders characterized by pathologic or deleterious production of a pro-inflammatory cytokine.
  • FRIL proteins are mannose/glucose-specific legume lectins which were initially identified as having the ability to preserve progenitor cells, in the sense of inhibiting differentiation, with or without inducing proliferation, and were referred to as "pylartin” ⁇ see, e.g., U.S. Pat. No. 6,084,060).
  • the proteins were also shown in a biological assay to stimulate the proliferation of NIH 3T3 cells transfected with the flk2/Flt3 receptor but not untransfected cells and, therefore, were designated as Flt3 Receptor Interacting Lectins (FRIL) (See, e.g., Moore et al. (1997), Blood 90, Suppl.
  • FRIL protein was identified in the hyacinth bean (Dolichos lab lab), but FRIL proteins have now been identified in other legumes (tribe Phaseoleae) including, without limitation, Phaseolus vulgaris, Sphenostylis stenocarpa, Cicer arietinum, Phaseolus acutifolius, Phaseolus lunatus, Vigna sinensis, and Voandzeia subterranea.
  • Native FRIL proteins useful in the invention include, but are not limited to, the FRIL proteins of Dolichos lab lab (“Dl-FRIL”), Phaseolus vulgaris (“Pv-FRIL”), Phaseolus acutifolius (“Pa-FRIL”), and Sphenostylis stenocarpa (“Yam-FRIL”).
  • Dl-FRIL Dolichos lab lab
  • Pv-FRIL Phaseolus vulgaris
  • Pa-FRIL Phaseolus acutifolius
  • Yam-FRIL Sphenostylis stenocarpa
  • the native FRIL proteins are expressed as heterodimers of ⁇ and ⁇ chains and have calculated molecular weights of approximately 15-20 kD for the ⁇ chain and approximately 12-20 kD for the ⁇ chain.
  • the ⁇ and ⁇ chains are initially expressed as a single polypeptide but are subsequently cleaved.
  • the proteins also appear to possess N- linked glycosylation sites.
  • the amino acid sequence of one Dl-FRIL protein is provided in SEQ ID NO.: 2.
  • the sequence begins with a 22 amino acid leader sequence which is cleaved from the mature protein. Residues 23-145 constitute the ⁇ chain, and residues 146-286 constitute the ⁇ chain. In the mature native protein, the C-terminus is often truncated to varying degrees, including deletions of the last 14 residues.
  • the protein of SEQ ID NO.: 2 is based on the sequence of Colucci et al. (1999), Proc. Natl. Acad. ScL USA 96:646-650, but with several changes based on subsequent data. Kotlarczyk et al.
  • SEQ ID NO.: 3 begins with an 8 amino acid leader sequence derived from an immunoglobulin kappa chain and is not part of the native protein. Residues 9 to approximately 129-135 constitute the ⁇ chain, and the residues from approximately 130-136 to 276 constitute the ⁇ chain. As before, the C-terminus can be truncated to varying degrees, including deletions of the last approximately 14 residues.
  • the amino acid sequence of one Pv-FRIL protein is provided in SEQ ID NO.: 6.
  • the sequence begins with a 22 amino acid leader sequence which is cleaved from the mature protein. Residues 23-145 constitute the ⁇ chain, and residues 146-301 constitute the ⁇ chain. In the mature native protein, the C-terminus is often truncated to varying degrees.
  • the amino acid sequence of one Yam-FRIL protein is provided in SEQ ID NO.: 8.
  • various N-terminal or C-terminal deletions or additions, as well as internal insertions, deletions, and substitutions can be made without affecting biological activity.
  • Such variants can be produced by routine methods known to those of skill in the art, and can be tested for therapeutic utility without undue experimentation.
  • the variants can be tested using the whole blood cell assay described in Example 1.
  • Those variants or newly identified FRIL family proteins that reduce pro-inflammatory cytokine production in this assay are considered useful for the methods and compositions of the present invention.
  • a recombinant FRIL protein can have an amino acid sequence identical to a native FRIL protein, or can have an amino acid sequence including one or more amino acid insertions, deletions, and/or substitutions.
  • the N-terminal leader sequence of a native FRIL protein can be deleted or can be replaced with an alternative leader sequence.
  • the C-terminal sequences can also be truncated or replaced.
  • Fusion proteins can also be produced, adding purification tags or epitopes ⁇ e.g., poly-His tag, Flag tag, c-myc epitope, HA tag, MBP epitope, GST epitope), or targeting sequences (e.g., ligands for cell surface receptors or immunoglobulin domains). Internal substitutions, deletions, and insertions are also possible.
  • the recombinant proteins are chimeric sequences produced by intermingling the sequences of two or more native FRIL proteins.
  • the recombinant proteins can also differ from native FRIL proteins due to differences in post-translational processing, such as cleavage of the ⁇ and ⁇ chains, removal of N-terminal leader sequences, and/or C-terminal truncation or degradation.
  • recombinant FRIL proteins including chimeric proteins, can be produced which have at least 45% amino acid sequence identity, at least 55% amino acid sequence identity, at least 65% amino acid sequence identity, at least 75% amino acid sequence identity, at least 85% amino acid sequence identity, at least 90% amino acid sequence identity or at least 95% amino acid sequence identity with a native FRIL protein.
  • a recombinant FRIL protein can have at least 85%-95% amino acid sequence identity with a native FRIL protein (e.g., SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, SEQ ID NO.: 8). In certain other embodiments, a recombinant FRIL protein can have at least 85%-95% amino acid sequence identity with a native FRIL protein (e.g., SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 6, SEQ ID NO.: 8), wherein the recombinant FRIL protein reduces the level of a pro-inflammatory cytokine (e.g., by the assay described in Examplel).
  • a pro-inflammatory cytokine e.g., by the assay described in Examplel.
  • Amino acid sequence identity and nucleic acid sequence identity between two proteins or two nucleic acid molecules can be measured according to standard methods (see, e.g., Pearson and Lipman (1988), Proc. Natl. Acad. ScL USA 85:2444-2448; George et al., in
  • FRIL protein whether native or recombinant, which is capable of reducing the levels of a pro-inflammatory cytokine produced by cells that have been exposed to an inducer of pro-inflammatory cytokines is considered useful in the invention.
  • FRIL proteins can be tested for their ability to reduce the levels of pro-inflammatory cytokines using simple in vitro assays such as that described below in the examples. Purification of FRIL proteins
  • FRIL proteins are readily purified using standard techniques. Methods for purifying proteins are known in the art and include, without limitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation, immunosorption, high performance liquid chromatography (HPLC), size-exclusion chromatography (SEC), immunoaffinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, or a combination of any of these methods.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • HPLC high performance liquid chromatography
  • SEC size-exclusion chromatography
  • immunoaffinity chromatography ion-exchange chromatography
  • hydrophobic interaction chromatography hydrophobic interaction chromatography
  • a FRIL family member molecule can also be purified by binding to mannose, which can be coupled to a solid support ⁇ e.g., a sepharose bead).
  • FRIL proteins can be purified from extracts of ground legumes by mannose-affinity chromatography, or by ovalbumin affinity chromatography. FRIL proteins are relatively abundant in legumes. For example, Dl-FRIL can account for approximately 0.02% of the mass of hyacinth beans.
  • FRIL proteins can also be made by recombinant methods.
  • a FRIL protein can be produced by introducing a nucleic acid sequence encoding the FRIL protein into any appropriate host cell type including bacterial ⁇ e.g., E. coli), yeast ⁇ e.g., S. cerevisiae), plant ⁇ e.g., Arabidopsis, Lemna, tobacco, or corn), insect ⁇ e.g., Drosophila), or mammalian cells ⁇ e.g., CHO cells), by recombinant techniques well-known to those with skill in the art.
  • a FRIL protein-encoding nucleic acid sequence can be inserted into a baculovirus vector which can be used to generate recombinant baculovirus particles.
  • Insect cells ⁇ e.g., Sf 9 cells
  • the FRIL protein can be purified.
  • recombinant FRIL proteins can be produced in dicotyledonous plants, such as Nicotiana tabacus ox Arabidopsis thaliana.
  • Arabidopsis plants can be transformed using a strain of Agrobacterium tumefaciens carrying a nucleic acid molecule encoding a FRIL protein.
  • Methods for making vectors for producing Agrobacterium with a desired nucleic acid molecule are known in the art (see, e.g., McBride and Summerfelt (1990), Plant MoI Biol. 14(2):269-276; U.S. Pat. No. 4,940,838, and U.S. Pat. No. 5,464,763).
  • the FRIL protein can be purified from the transformed plant by standard methods (see, e.g., Ausubel et al, supra).
  • Nucleic acid sequences encoding a FRIL protein include, without limitation, any sequence encoding the proteins of SEQ ID NOs.: 2, 3, 6, or 8, including the nucleic acid sequences of SEQ ID NOs.: 1, 5, or 7.
  • nucleic acid sequences can be designed and produced encoding any of the recombinant FRIL variants described herein.
  • FRIL proteins reduce the levels of several pro-inflammatory cytokines.
  • the FRIL proteins of the present invention are useful in treating or alleviating disorders characterized by pathologic or deleterious production of a pro-inflammatory cytokine.
  • Such treatment could be beneficial for treating acute and chronic conditions in which inflammation is key to the pathogenesis of the diseases including, but not limited to, psoriasis, eczema, dermatitis, and/or arthritis.
  • the present invention provides a method for preventing or reducing inflammation, or treating diseases characterized by inflammation, comprising the administration of a therapeutically effective amount of a FRIL protein.
  • FRIL proteins of the present invention have uses in the prevention and/or treatment of inflammatory conditions.
  • FRIL proteins can reduce production of cytokines by cells involved in an inflammatory response, these proteins can be used to prevent and/or treat chronic and acute inflammatory conditions.
  • Such inflammatory conditions include, but are not limited to, inflammation associated with infection (e.g., septic shock, toxic shock syndrome, sepsis, systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine- or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, over-production of cytokines (e.g., IFN- ⁇ , IL-6, TNF- ⁇ ), respiratory disorders (e.g., asthma, allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric cancer, ovarian cancer, lung cancer, bladder cancer, liver cancer, breast cancer, cervical cancer, lymphoid cancers, cardiac myxoma, myeloma, plasmacytoma); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke; traumatic brain injury; neurodegenerative
  • tissue-specific inflammatory disorders including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis
  • tissue-specific inflammatory disorders including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendici
  • Inflammation can also be a life-threatening complication of severe physical trauma (e.g., traumatic head injury), burns, cardiopulmonary bypass surgery, renal ischemia-reperfusion, and organ transplant surgery.
  • FRIL proteins can be administered to subjects in these situations who are in need of a treatment that reduces inflammation.
  • chronic inflammation increases the risk of cancer (Wiseman and
  • FRIL can be administered to patients that are at a risk of cancer due to chronic inflammation.
  • the invention features a method for treating or reducing inflammation in a mammal by administering a pharmaceutical composition including a therapeutically effective amount of a FRIL protein.
  • the inflammation is characterized by deregulated levels of a pro-inflammatory cytokine.
  • the pro-inflammatory cytokine is selected from the group consisting of IFN- ⁇ , IL-6 and TNF- ⁇ .
  • the invention features a method for treating a mammal diagnosed with a disorder characterized by undesired or pathological levels of a pro- inflammatory cytokine by administering to the mammal a pharmaceutical composition including a therapeutically effective amount of a FRIL protein to reduce the levels of the pro-inflammatory cytokine.
  • the pro-inflammatory cytokine is selected from the group consisting of IFN- ⁇ , IL-6 and TNF- ⁇ .
  • the disorder is selected from the group consisting of psoriasis, multiple sclerosis, mucositis, endotoxemia, inflammatory bowel disease, and rheumatoid arthritis.
  • the mucositis is selected from the group consisting of radiation- induced mucositis, chemotherapy-induced mucositis, and infection-induced mucositis.
  • the invention provides a method for treating a mammal diagnosed with an autoimmune disease or a neurodegenerative disease by administering to the mammal a pharmaceutical composition including a therapeutically effective amount of a FRIL protein to reduce the levels of the pro-inflammatory cytokine.
  • the method is directed to treatment or a mammal diagnosed with an autoimmune disease selected from the group consisting of multiple sclerosis, rheumatoid arthritis, juvenile arthritis, myocarditis, colitis, nephritis, scleroderma, tendonitis, lupus, chronic inflammatory demyelinating polyneuropathy, psoriasis, and autoimmune hepatitis.
  • the autoimmune disease is psoriasis. In another specific embodiment, the autoimmune disease is multiple sclerosis. In yet another specific embodiment, the autoimmune disease is rheumatoid arthritis. In another embodiment of this aspect, the method is directed to treatment of a mammal diagnosed with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, and Parkinson's disease.
  • the invention features a method for treating a mammal diagnosed with a cancer by administering to the mammal a pharmaceutical composition including a therapeutically effective amount of a FRIL protein to reduce the progression of the cancer.
  • Abnormalities in the production of cytokines or their receptors have been associated with several types of cancer.
  • the cancer is selected from the group consisting of a cardiac myxoma, a myeloma, a plasmacytoma, a cervical cancer, a bladder cancer, and a lymphoid cancer.
  • the cancer is a cardiac myxoma.
  • the cancer is a myeloma.
  • the cancer is a lymphoid cancer.
  • the invention provides a method for suppressing rejection of a graft, or increasing tolerance to the graft, in a mammal undergoing transplantation.
  • administration of the FRIL proteins of the present invention to the mammal prior to, at the same time as, or subsequent to transplantation reduces the levels of pro- inflammatory cytokines thereby permitting the graft ⁇ i.e., autograft, graft from an MHC- matched donor, allograft, xenograft) to be accepted by the mammal.
  • the mammal can be a human patient or, in some embodiments, the mammal can be a non-human primate, laboratory animal ⁇ e.g., mouse, rat, rabbit, hamster), a livestock or breeding animal ⁇ e.g., horse, sheep, cow, pig, goat), or a pet ⁇ e.g., cat, dog).
  • the invention provides a method for determining whether a subject ⁇ e.g., a human patient) diagnosed with a condition characterized by undesired or pathological levels of a pro-inflammatory cytokine will benefit from treatment with a pharmaceutical composition including a FRIL protein.
  • This method includes contacting a cell from diseased tissue of the subject with a FRIL protein and determining whether the FRIL protein reduces the production of pro-inflammatory cytokines (e.g., by comparison to an extrinsic standard or to an untreated control sample of cells from the diseased tissue of the same subject).
  • a FRIL protein is chosen which is known to be effective against the category of cancer from which the patient is suffering. If it is determined that the cancer is FRIL-sensitive, the patient can undergo treatment with the FRIL protein.
  • Any route of administration can be employed which is suitable to the particular formulation chosen for the FRIL protein pharmaceutical composition including, without limitation, parenteral routes such as intravenous, intra-arterial, intra-muscular, subcutaneous, intraperitoneal, intranasal, intrapulmonary, intrarectal, and intravaginal. Oral administration can also be employed for certain formulations.
  • the pharmaceutical preparations can be administered locally to an affected area or can be administered systemically.
  • a therapeutically effective amount when administered systemically, can be in the range of 500 ng/kg (i.e., 500 ng of the FRIL protein per kg total body weight of the subject) to 100 mg/kg per day. In some embodiments, a therapeutically effective amount is in the range of 1 ⁇ g/kg to 50 mg/kg per day, or 5 ⁇ g/kg to 25 mg/kg per day.
  • a FRIL protein can be administered prophylactically to a subject that is considered to be at a risk of developing a condition characterized by undesired or pathological levels of a pro- inflammatory cytokine.
  • a human patient newly diagnosed with a cytokine- related disorder e.g., by virtue of high levels of a pro-inflammatory cytokine
  • a FRIL protein can be treated with a FRIL protein as a first line of therapy.
  • the FRIL protein can be administered as an adjuvant therapy in combination with other, Standard treatments for the disease/disorder being treated.
  • FRIL may be given as an adjuvant therapy for the treatment of a patient diagnosed with rheumatoid arthritis, multiple sclerosis, or psoriasis.
  • the method comprises administering a therapeutic amount of a FRIL protein and a second agent.
  • the "second agent” is any substance that is capable of alleviating the condition being treated.
  • the second agent is an inhibitor of the production or activity of one or more cytokines.
  • Non-limiting examples of such a second agent include natural cytokine antagonists such as IL-I receptor antagonist, soluble cytokine receptors for IL-2, IL-4, IL-6, IL-7, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , and LIF, and endogenous anti-endotoxin antibody (EndoCAb); and antibodies that block the ability of pro-inflammatory cytokines to bind their cognate receptors.
  • natural cytokine antagonists such as IL-I receptor antagonist, soluble cytokine receptors for IL-2, IL-4, IL-6, IL-7, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , and LIF, and endogenous anti-endotoxin antibody (EndoCAb); and antibodies that block the ability of pro-inflammatory cytokines to bind their cognate receptors.
  • the second agent is a drug that is used to treat inflammatory diseases selected from the group consisting of non-steroidal anti-inflammatory drugs, anti-malarial drugs (e.g., hydroxychloroquine), corticosteroids, methotrexate, sulfasalazine, penicillamine, cyclophosphamide, and cyclosporine.
  • the second agent is any drug that is known to be useful to treat the specific disease in the patient.
  • the patient can be treated with a FRIL protein and a known treatment for psoriasis including, but not limited to, AMEVIVE ® , ENB REL ® , HUMIRA ® , RAPTIVA ® , and REMICADE ® .
  • the patient can be treated with a FRIL protein and a known treatment for multiple sclerosis including, but not limited to, COPAXONE ® , BETASERON ® , AVONEX ® , NOVANTRONE ® , and REBIF ® .
  • the patient can be treated with a FRIL protein and a treatment for rheumatoid arthritis including, but not limited to, RITUXAN ® , ZEVALIN ® , AND BEXXAR ® .
  • a treatment for rheumatoid arthritis including, but not limited to, RITUXAN ® , ZEVALIN ® , AND BEXXAR ® .
  • the FRIL protein can be administered to the patient prior to, at the same time as, or subsequent to the second agent.
  • a substantially pure FRIL protein can be formulated in combinations with other pharmaceuticals or therapeutics useful in the treatment of inflammatory disorders.
  • the FRIL protein can be combined with natural cytokine antagonists such as IL- 1 receptor antagonist, soluble cytokine receptors for IL-2, IL-4, IL-6, IL-7, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , and LIF, and endogenous anti-endotoxin antibody (EndoCAb).
  • natural cytokine antagonists such as IL- 1 receptor antagonist, soluble cytokine receptors for IL-2, IL-4, IL-6, IL-7, IFN- ⁇ , TNF- ⁇ , TNF- ⁇ , and LIF, and endogenous anti-endotoxin antibody (EndoCAb).
  • the invention provides pharmaceutical preparations including a substantially pure FRIL protein for use in the treatment of a condition characterized by undesirable levels of a pro-inflammatory cytokine or the manufacture of a medicament for use in such treatments.
  • the pharmaceutical preparations can include a FRIL protein in dry form (e.g., lyophilized alone or with a stabilizer) or in liquid solutions or suspensions (e.g., in a pharmaceutically acceptable carrier or diluent).
  • Pharmaceutically acceptable carriers for parenteral administration of liquids include, without limitation, water, buffered saline, polyols (e.g., glycerol), polyalkylene glycols (e.g., propylene glycol, liquid polyethylene glycol), vegetable oils, hydrogenated napthalenes, or suitable mixtures thereof.
  • the FRIL proteins can also be formulated with buffers or excipients.
  • the FRIL proteins are formulated in sustained-release particles or implantable devices.
  • such particles or devices can be formed from biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, polyoxyethylene-poloxypropylene copolymers, ethylene-vinyl acetate copolymers, and the like, to control the release of the FRIL protein.
  • Other potentially useful parenteral delivery systems include osmotic pumps, implantable infusion systems, and liposomes.
  • the FRIL proteins are delivered to a patient using DUROS ® Implant or ALZAMER ® Depot technology (Alza Corporation).
  • the invention provides a kit comprising, in a suitable container, a therapeutically effective amount of one or more substantially pure FRIL proteins and a second agent.
  • the FRIL protein and the second agent may be in separate containers or formulated together in one container.
  • the components of the kit may be provided in a liquid solution(s), and/or as a dried powder(s). When the components are provided in liquid solution, the liquid solution is a sterile solution. When the reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent ⁇ e.g., water or buffered saline).
  • the solvent may be provided as part of the kit.
  • cytokine assay kits Whole blood from nine healthy human volunteers was collected and stored in separate sterile heparinized tubes. A portion of the blood from each of these subjects was diluted 1:4 in a 4 mL polypropylene tube containing medium that either contained or lacked stimulants (e.g., E.coli lipopolysaccharide (LPS) or heat-killed Staphylococcus epidermidis), and which contained or lacked a FRIL protein (e.g., Dl-FRIL). The mixture was incubated at 37 0 C for 24 or 48 hrs. At the end of 24 and 48 hours, the supernatant was isolated and tested using commercially available cytokine assay kits.
  • stimulants e.g., E.coli lipopolysaccharide (LPS) or heat-killed Staphylococcus epidermidis
  • FRIL protein e.g., Dl-FRIL
  • Human whole blood cultures were stimulated with heat-killed S. epi in the presence or absence of Dl-FRIL (1 ⁇ g, 10 ⁇ g or 100 ⁇ g), and these mixtures were incubated at 37 0 C.
  • human whole blood cultures also were stimulated in separate experiments with LPS in the presence or absence of Dl-FRIL (1 ⁇ g or 10 ⁇ g).
  • Supernatants from these cultures were isolated 24 hours after the incubation and assayed for levels of secreted IFN- ⁇ .
  • lysates of the whole blood cells were prepared after 48 hours of incubation and assayed for levels of IFN- ⁇ .
  • Lysates collected at 48 hours showed the same pattern of IFN- ⁇ expression as seen with the supernatants at 24 hours ⁇ see, Fig. 7).
  • Example 5 Reduction of IL- l ⁇ in Human Whole Blood Cultures Stimulated by Staphylococcus epidermidis Human whole blood cultures were stimulated with heat-killed S. epi in the presence or absence of Dl-FRIL (1 ⁇ g, 10 ⁇ g or 100 ⁇ g), and these mixtures were incubated at 37 0 C. Lysates from these cultures were isolated 24 hours after the incubation and assayed for levels of IL- l ⁇ .
  • Lysates of cultures treated only with S. epi had high levels of IL-I ⁇ ⁇ see, Fig. 10), whereas supernatants of cultures treated only with FRIL (100 ng, 1 ⁇ g or 10 ⁇ g) had only background levels of IL- l ⁇ .
  • Supernatants of cultures treated with S. epi and Dl-FRIL (1 ⁇ g, 10 ⁇ g, or 100 ⁇ g) showed no significant reduction in the levels of IL-I ⁇ .
  • Human whole blood cultures were stimulated with IL-12 and IL- 18 in the presence or absence of Dl-FRIL (1 ⁇ g or 10 ⁇ g), and these mixtures were incubated at 37°C for 24 and 48 hours. Supernatants from these cultures were isolated 24 and 48 hours after the incubation and assayed for levels of secreted IFN- ⁇ . In addition, lysates of the whole blood cells were prepared after 48 hours of incubation and assayed for levels of IFN- ⁇ .
  • EAE allergic encephalomyelitis
  • MS multiple sclerosis
  • FRIL can reduce the levels of pro-inflammatory cytokines
  • FRIL is tested as a therapeutic agent for the amelioration of the autoimmune demyelinatory disease in EAE.
  • Female Lewis rats (Charles River Breeding Laboratories, Wilmington, Mass.,
  • EAE myelin basic protein
  • CFA complete Freund's adjuvant
  • One group of rats is injected with FRIL up to 4 times starting on the first day of immunization and continuing daily for the duration of the study.
  • one group of rats is induced for EAE but left untreated (positive control group), and another group of animals is not induced for EAE and used as the negative control group.
  • FRIL Treatment with FRIL will reduce the development of EAE disease in the Lewis female rats.
  • IFN- ⁇ and iNOS a known inducer of EAE
  • brain sections of all groups of rats is studied.
  • the FRIL treated rats will show low levels of IFN- ⁇ and iNOS in the central nervous system.
  • FRIL graft Rejection in a Patient by Administration of FRIL
  • a patient who is to undergo a transplantation of an organ, a tissue, or a group of cells from an allogeneic or xenogeneic donor is prepared for this procedure with immunosuppressive therapy.
  • the immunosuppressive therapy depletes the patient's immune cells facilitating the transplant of the graft.
  • the patient may be administered FRIL.
  • FRIL may be administered to the patient prior to (e.g., 3-5 days), at the same time as, or subsequent to the transplant procedure.
  • FRIL is administered up to four times daily at a dosage of 0.05 mg/kg to 5 mg/kg.
  • FRIL treatment is continued 5 to 10 days following transplantation. In some instances the FRIL treatment is extended until 30 to 60 days after transplantation of the graft.
  • Rats are administered FRIL (i.v. or i.p., up to 4 doses daily at a dosage of 0.05 mg/kg to 5 mg/kg) 1-7 days before indomethacin (7-9 mg/kg) is administered subcutaneously.
  • FRIL i.v. or i.p., up to 4 doses daily at a dosage of 0.05 mg/kg to 5 mg/kg
  • rats are either administered buffer (negative control) or repifermin (positive control) in place of FRIL.
  • intestinal ulceration in the rats is determined using standard methods.
  • Rats treated with FRIL will show significantly reduced ulceration compared to rats treated with buffer, and comparable protection to rats treated with repifermin.
  • Rats are administered FRIL (i.v. or i.p., up to 4 doses daily at a dosage of 0.05 mg/kg to 5 mg/kg) a day before being provided with cyclophosphamide (150, 200 or 250 mg/kg, intraperitoneally).
  • rats are either administered buffer (negative control) or repifermin (positive control) in place of FRIL.
  • FRIL FRIL
  • rats are either administered buffer or HSA (negative controls), or repifermin (positive control) in place of FRIL.
  • cytokines e.g., TNF- ⁇ , IFN- ⁇ , IL-6
  • FRIL-treated mice will show reduced levels of pro-inflammatory cytokines compared to the negative controls and comparable levels of pro-inflammatory cytokines as repifermin.
  • FRIL Platelet Activating Factor
  • FRIL proteins are used to test whether they attenuate the progression of contact dermatitis using a tetradecanoylphorbol acetate (TPA)-induced cutaneous inflammation model in mice.
  • TPA tetradecanoylphorbol acetate
  • the use of the female B ALB/c and male Swiss Webster mice in experimental cutaneous inflammation are well-characterized and reproducible models of contact dermatitis. These strains of mice have been shown to develop a long-lasting inflammatory response, following topical application of TPA, which is comprised of local hemodynamics, vascular permeability and local migration of leukocytes, and these pathological changes are similar to those of human dermatitis (Rao et al, Inflammation 17(6):723, 1993; Rao et al, J. Lipid Mediators Cell Signalling 10:213, 1994).
  • mice receive either vehicle or FRIL intraperitoneally, sub-cutaneously, or intravenously 60 min. after the topical application of TPA (4 ⁇ g/ear), which is applied as a solution in acetone (200 ⁇ g/ml), 10 ⁇ l each to the inner and outer surface of ear.
  • the control group receives 20 ⁇ l of acetone as a topical application.
  • TPA 4 ⁇ g/ear
  • acetone 200 ⁇ g/ml
  • mice receives 20 ⁇ l of acetone as a topical application.
  • Four hours following the application of TPA increase in ear thickness is measured and ears are excised for histology.
  • mice are intravenously injected through tail veins with Evans blue (300 mg/kg) at selected times after topical application of TPA and are sacrificed 15 min. thereafter. Ears are excised and removed then extracted into dimethylformamide and centrifuged. Absorbance readings are spectrophotometrically measured at 590 nm.
  • FRIL will be found to inhibit inflammation mediated by TPA.
  • Example 16 Dextran Sulfate Sodium-Induced Colitis: Mouse Model of Inflammatory Bowel Disease
  • DSS- induced colitis is characterized by histological events and an influx of neutrophils, macrophages, and mediators of inflammation similar to those observed with human inflammatory bowel diseases (IBD).
  • IBD inflammatory bowel diseases
  • drugs known to be useful for treating IBD such as corticosteroids and 5-ASA, are known to have activity in this model.
  • One hundred test animals female, 6 week old Swiss Webster mice, 18-30 g are divided into ten groups. Each animal is dosed daily (i.p.
  • FRIL drug-induced pulmonary disease induced by the administration ad libitum in drinking water of dextran sulfate sodium (DSS) as a 5% solution in tap water (10 mL/mouse/day for 5-6 days), with no other fluid source for animals in the DSS arm of the study. Filtered tap water is available ad libitum except for animals receiving 5% DSS as the sole source of fluid. After four days, signs of acute disease will occur with the loss of weight, diarrhea, and bloody stools.
  • DSS dextran sulfate sodium
  • Histological changes will include initial shortening of the crypts, then areas of separation of the crypts and the muscularis mucosae in the absence of destructive inflammatory filtrate. After five days, pathological changes will become confluent with the appearance of erosions and early hyperplastic epithelium. Inflammation scores will be high with neutrophils, lymphocytes, and plasma cells in the lamina intestinal, but sparing the epithelium. FRIL is evaluated for prophylactic activity, and FRIL given after the disease state is established is also evaluated for therapeutic activity.
  • Test animals are weighed daily from Day 0 to Day 8, or until completion of the study. The total duration of the study with DSS arm of the study varies depending on the time for progression of colitis. The condition of the test animals and consistency of stools is noted. At the conclusion of the study, test animals are euthanized (CO 2 ), a midline incision is made, and a stool sample is obtained. The sample is placed on a slide and tested for occult blood (Quic-CultTM, Laboratory Diagnostics Co., Morganville, NJ.). Occult blood is determined by placing two drops of the reagent onto the sample and observing any color change.
  • Occult blood presence is graded using a scoring protocol assigning a score of 0 for no color; 1 for a very light blue color (+/-) forming in >30 seconds; 2 for a blue color developing in 30 seconds or more (+); 3 for a change in color occurring in less than 30 seconds (++); and 4 for gross blood observable on the slide.
  • the colon is gently stretched and the length from the colon-cecal junction to the end of the distal rectum is measured to the nearest 0.1 cm. The data will show that FRIL provides significant protection from the inflammatory responses to DSS-induced colitis.
  • UVB light is a potent stimulus for pro-inflammatory cytokine release by keratinocytes. Reduction in the levels of such cytokines is important in treating psoriasis. Keratinocytes are UVB irradiated before and/or after FRIL treatment (1 ⁇ g to 100 ⁇ g). As a control, keratinocytes are treated with buffer alone.
  • the levels of IFN- ⁇ , IL-6, IL- l ⁇ , and TNF- ⁇ are determined by quantitative two- site enzyme immunoassay using commercially available ELISA kits for these cytokines. FRIL treatment will reduce the levels of one or more of these pro-inflammatory cytokines compared to the buffer control.

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Abstract

L'invention concerne l'utilisation de protéines de la famille FRIL (ligand en interaction avec le récepteur de FTL3), les protéines FRIL peuvent s'utiliser dans des méthodes de traitement de maladies inflammatoires, qui sont caractérisées par des taux indésirables de cytokines pro-inflammatoires, ainsi que dans la fabrication de médicaments utilisés dans le traitement de telles maladies. Les maladies inflammatoires pouvant être traitées par les protéines FRIL comprennent, sans limitation: les troubles auto-immuns, les maladies neurodégénératives et les cancers. Les protéines FRIL de l'invention peuvent en outre s'utiliser comme thérapie complémentaire pour augmenter la tolérance chez des patients faisant l'objet de transplantations.
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Cited By (13)

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Publication number Priority date Publication date Assignee Title
US20110092589A1 (en) * 2008-03-10 2011-04-21 The Charlotte-Mecklenburg Hospotal Authority Treatment of Inflammatory Conditions and Diseases with Metal-Thiols
US9339528B2 (en) 2009-10-26 2016-05-17 General Regeneratives, Ltd. Methods for treating epithelium trauma of the intestinal mucosa using interleukin-1 receptor antagonist
EP2494981A1 (fr) * 2009-10-26 2012-09-05 Shanghai Jiaotong University Utilisation d'un antagoniste du récepteur de l'interleukine-1 pour préparer un médicament pour prévenir ou traiter un traumatisme épithélial de la muqueuse intestinale
EP2494981A4 (fr) * 2009-10-26 2014-03-12 Univ Shanghai Jiaotong Utilisation d'un antagoniste du récepteur de l'interleukine-1 pour préparer un médicament pour prévenir ou traiter un traumatisme épithélial de la muqueuse intestinale
EP3173094A1 (fr) * 2009-10-26 2017-05-31 General Regeneratives (shanghai) limited Utilisation d'un antagoniste du récepteur de l'interleukine -1 pour la préparation d'un médicament de prévention ou de traitement des traumatismes de l'épithélium de la muqueuse intestinale
US20120094931A1 (en) * 2010-06-30 2012-04-19 Collins Colm Compositions and methods to modulate progression and onset of inflammatory bowel disease
US20140017713A1 (en) * 2011-02-14 2014-01-16 Atgen Co. Ltd. Method of diagnosing cancer and diagnosis kit using measurement of nk cell activity
JP2014510517A (ja) * 2011-02-14 2014-05-01 エイティージェン カンパニー リミテッド がんを診断する方法およびｎｋ細胞活性の測定を使用した診断キット
JP2019207242A (ja) * 2011-02-14 2019-12-05 エヌケーマックス カンパニー リミテッドＮＫＭＡＸ ＣＯ．， Ｌｔｄ． がんを診断する方法およびｎｋ細胞活性の測定を使用した診断キット
JP2022043119A (ja) * 2011-02-14 2022-03-15 エヌケーマックス カンパニー リミテッド がんを診断する方法およびｎｋ細胞活性の測定を使用した診断キット
US11442069B2 (en) * 2011-02-14 2022-09-13 Atgen Co. Ltd. Method of diagnosing cancer and diagnosis kit using measurement of NK cell activity
CN113631178A (zh) * 2018-09-06 2021-11-09 台湾地区“中央研究院” 抗病毒凝集素及其用途
EP3846841A4 (fr) * 2018-09-06 2022-05-18 Academia Sinica Lectine antivirale et ses utilisations

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WO2006110577A3 (fr) 2007-05-18

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