Molecular targets and compounds, and methods to identify the same, useful in the treatment of fibrotic diseases

Abstract

The present invention relates to methods and assays for identifying agents useful in the treatment of fibrotic conditions. The invention provides polypeptide and nucleic acid TARGETs, siRNA sequences based on these TARGETs and antibodies against the TARGETs. The invention is further related to pharmaceutical composition comprising siRNA sequences based on the TARGETs and antibodies against the TARGETs for use in the treatment of fibrotic conditions. The invention further provides in vitro methods for reduction or inhibition of macrophage differentiation into alternatively-activated macrophages (M2).

1 . A method for identifying a compound useful for the treatment of fibrosis, said method comprising:
 a) contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-50, fragments and structurally functional derivatives thereof, or with a cell expressing said polypeptide;   b) determining a binding affinity of the test compound to said polypeptide, or measuring expression, amount or an activity of said polypeptide;   c) contacting the test compound with a population of macrophage cells;   d) measuring a property related to differentiation of macrophages into alternatively activated macrophages; and   e) identifying a compound capable of reducing or inhibiting macrophage differentiation into alternatively-activated macrophages and demonstrating binding affinity to said polypeptide or reducing or inhibiting the expression, amount or an activity of said polypeptide.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . A method for identifying a compound useful for reducing or inhibiting differentiation of macrophages to M2 macrophages, said method comprising:
 a) contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-50, functional fragments and functional derivatives thereof or with a nucleic acid encoding an amino acid selected from the group consisting of SEQ ID NOs: 26-50 or a functional derivative thereof;   b) measuring the expression or an activity of said polypeptide;   c) contacting the test compound with a population of macrophage cells;   d) measuring a property related to differentiation of macrophages into alternatively-activated macrophages; and   e) identifying a compound capable of reduction or inhibition of differentiation of macrophages into M2 macrophages and inhibiting the expression or an activity of said polypeptide.   
     
     
         5 . (canceled) 
     
     
         6 . The method according to  claim 4 , wherein the nucleic acid is selected from the group consisting of SEQ ID NOs: 1-25. 
     
     
         7 . The method according to  claim 1  or  4 , which additionally comprises the step of comparing the compound to be tested to a control. 
     
     
         8 . The method of  claim 1  or  4 , wherein said polypeptide is coupled to a detectable label. 
     
     
         9 . The method according to  claim 1 , wherein said polypeptide sequence in steps (a) and (b) is present in an in vitro cell-free preparation. 
     
     
         10 . The method according to  claim 1  or  4 , wherein said polypeptide sequence in steps (a) and (b) is present in a cell. 
     
     
         11 . The method according to  claim 10 , wherein the cell naturally expresses said polypeptide. 
     
     
         12 . The method according to  claim 10 , wherein the cell has been engineered so as to express said polypeptide. 
     
     
         13 . The method of  claim 10 , wherein said cell is a mammalian cell. 
     
     
         14 . The method of  claim 13 , wherein said cell is a macrophage cell. 
     
     
         15 . The method of  claim 1  or  4 , wherein said property is the inhibition of release or expression of a marker of alternatively-activated macrophages. 
     
     
         16 . The method of  claim 15  wherein said property is an expression or release of a marker selected from the group consisting of CCL18, CCL13, TGF13, CCL22, CCL17, soluble fibronectin, folate receptor β, CD206, and CD163. 
     
     
         17 . (canceled) 
     
     
         18 . The method according to  claim 4  wherein said cells have been triggered by a factor which induces macrophage differentiation into M2 macrophages (M2 inducing factor). 
     
     
         19 . The method according to  claim 4 , wherein said cells have been triggered by one or more M2 inducing factors selected from the group consisting of IL4, IL10, IL13, immune complexes and lipopolysaccharides. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1  or  4  wherein the method additionally comprises: measuring a property related to the differentiation of macrophages into classically-activated (M1) macrophages and identifying a compound that does not inhibit said differentiation. 
     
     
         22 . The method of  claim 21  wherein said property is the level and/or expression of a marker of the M1 macrophage phenotype, and a compound is identified which does not increase the levels of said marker. 
     
     
         23 . The method of  claim 22  wherein said marker is TNFα. 
     
     
         24 . The method according to  claim 1 , wherein said test compound is selected from the group consisting of an antisense polynucleotide, a ribozyme, short-hairpin RNA (shRNA), small interfering RNA (siRNA), and microRNA (miRNA). 
     
     
         25 . The method according to  claim 24 , wherein said test compound comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-25. 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 24 , wherein said antisense polynucleotide, said siRNA or said shRNA comprises an antisense strand of 17-25 nucleotides complementary to a sense strand, wherein said sense strand is selected from 17-25 continuous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-25. 
     
     
         29 . The method according to  claim 1  or  4 , wherein said compound is an antibody or an antibody fragment. 
     
     
         30 . A method for treatment of a fibrotic condition in a mammal comprising administering to said mammal a pharmaceutical composition comprising an antibody or a fragment thereof which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-50, or comprising an agent selected from the group consisting of an antisense polynucleotide, a ribozyme, a small interfering RNA (siRNA), microRNA (miRNA) and a short-hairpin RNA (shRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-25. 
     
     
         31 . The method according to  claim 30  wherein said antibody is a monoclonal antibody. 
     
     
         32 . The method according to  claim 30  wherein said antibody is a single chain antibody. 
     
     
         33 . (canceled) 
     
     
         34 . The method according to  claim 30 , wherein said fibrotic condition is a fibrotic condition associated with differentiation of macrophages into alternatively-activated (M2) macrophages. 
     
     
         35 . The method according to  claim 34 , wherein said fibrotic condition is selected from idiopathic pulmonary fibrosis (IPF), cystic fibrosis, other diffuse parenchymal lung diseases of different etiologies including iatrogenic drug-induced fibrosis, occupational and/or environmental induced fibrosis, granulomatous diseases (sarcoidosis, hypersensitivity pneumonia), collagen vascular disease, alveolar proteinosis, langerhans cell granulomatosis, lymphangioleiomyomatosis, inherited diseases (Hermansky-Pudlak Syndrome, tuberous sclerosis, neurofibromatosis, metabolic storage disorders, familial interstitial lung disease), radiation induced fibrosis, chronic obstructive pulmonary disease (COPD), scleroderma, bleomycin induced pulmonary fibrosis, chronic asthma, silicosis, asbestos induced pulmonary fibrosis, acute respiratory distress syndrome (ARDS), kidney fibrosis, tubulointerstitium fibrosis, glomerular nephritis, focal segmental glomerular sclerosis, IgA nephropathy, hypertension, Alport syndrome, gut fibrosis, liver fibrosis, cirrhosis, alcohol induced liver fibrosis, toxic/drug induced liver fibrosis, hemochromatosis, nonalcoholic steatohepatitis (NASH), biliary duct injury, primary biliary cirrhosis, infection induced liver fibrosis, viral induced liver fibrosis, autoimmune hepatitis, corneal scarring, hypertrophic scarring, Dupuytren disease, keloids, cutaneous fibrosis, cutaneous scleroderma, systemic sclerosis, spinal cord injury/fibrosis, myelofibrosis, vascular restenosis, atherosclerosis, arteriosclerosis, Wegener's granulomatosis and Peyronie's disease. 
     
     
         36 . (canceled) 
     
     
         37 . An in vitro method of reducing or inhibiting the differentiation of macrophages into alternatively-activated (M2) macrophages, said method comprising contacting a population of mammalian cells comprising macrophage cells with an inhibitor of the activity or expression of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-50. 
     
     
         38 . The method of  claim 37  wherein said inhibitor is an antibody. 
     
     
         39 . The method of  claim 37  wherein said antibody is a monoclonal antibody. 
     
     
         40 . The method of  claim 37  wherein said inhibitor is selected from the group consisting of an antisense polynucleotide, a ribozyme, a small interfering RNA (siRNA), microRNA (miRNA) and a short-hairpin RNA (shRNA), wherein said inhibitor comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid encoding said polypeptide.