JP2007515162A - Screening method using CTGF and TRKA receptors for the identification of compounds used in the treatment of fibrosis - Google Patents

Abstract

  The present invention provides a method for identifying and / or making compounds for use in reducing and / or preventing fibrosis and comprises the following steps: providing a CTGF receptor; providing a test sample; providing a CTGF receptor agonist; Exposure of the receptor to the test sample; subsequent or simultaneous exposure of the CTGF receptor to the CTGF receptor agonist; detection and / or measurement of the amount of CTGF receptor activation; CTGF receptor activation detected and / or measured in the presence of the test sample The amount of CTGF receptor activation detected and / or measured in the absence of the test sample; based on not increasing or decreasing CTGF receptor activation, the compound reduces fibrosis and / or Includes a decision to prevent or not. There are also compounds provided for the reduction and / or prevention of fibrosis, or the use of such compounds.

Description

  The present invention relates to methods of identifying and / or making compounds for use in reducing and / or preventing fibrosis. The invention also relates to compounds and uses of the compounds for reducing and / or preventing fibrosis.

  When a biological tissue is damaged, both the damage and the associated inflammatory response can cause cell death. As cell death occurs, new tissue is synthesized and replaced with dead or dying cells. New tissue synthesis falls into two categories: regeneration of differentiated cells and increase in connective tissue. In certain pathologies, an increase in connective tissue determines the healing process and causes the formation of fibrosis. In fibrosis, the new tissue repairs any structural defect in the tissue, but weakens its function by replacing differentiated cells with connective tissue and cells produced by connective tissue.

  Both the occurrence of fibrosis or the actual extent of fibrosis is affected by various factors including the nature, severity, and location of the injury being treated. Fibrosis is most commonly known as a scar on the surface of the skin that is relatively difficult, except for large areas of wound. However, fibrosis can also occur in tissues of internal organs such as the liver, lungs, and kidneys. In most cases, it is fibrosis in these areas that is most serious because the special activity of the organ is weakened. In the most extreme cases, organ weakness or death can occur due to the weakening.

  An example of the importance of fibrosis in the pathology is shown by the occurrence of renal fibrosis (diabetic nephropathy) in diabetes, which is currently prevalent throughout the world.

  The incidence of diabetes has increased worldwide in recent years. In particular, this is due to a dramatic increase in type 2 diabetes (late onset diabetes) (Silink M (2002), Horm. Res. 57 (Suppl 1) pp. 1-5). Diabetes is closely related to many secondary complications, particularly those related to the microcirculatory system. These complications, including fibrotic condition nephropathy, usually develop many years after the onset of diabetes.

  Diabetic nephropathy is characterized by excessive deposition of extracellular matrix proteins in the glomerular and basement membranes of the glomeruli and the renal tubular stroma.

  Although genetic background has been considered important in determining susceptibility to diabetic nephropathy (DN) (Quinn M et al., (1996) Diabetologica 39 pp 940-945), the determinant is It is understood that the tissue is exposed to chronic hyperglycemia (UKPDS Group, (1998) Lancet 352 pp.837-853). The nephropathy epidemic varies by region, type of diabetes, and length of time since diagnosis. Without countering influencing factors, the prevalence of diabetic nephropathy is predicted to increase in the coming decades (Bagust A et al. (2002) Diabetes Med 19 (Suppl 4): ppl-5). Diabetic nephropathy is a major cause of end-stage renal disease, and new therapies are needed to limit its development.

  The pathology of diabetic nephropathy is similar in type 1 and type 2 diabetes. Both types of diabetes are associated with similar ultrastructural changes that occur in the glomeruli (Osterby R, (1992) Diabetologica 35 pp 803-812). The basement membrane of the renal glomerulus increases in thickness and the extracellular matrix of the glomerular stroma increases.

  Increased glomerular stroma is believed to be a major cause of decreased kidney function in diabetic nephropathy (Steffes M et al., (1989) Diabetes 38 pp1077-1081). As the glomerular interstitial matrix increases, it acts on the glomerular capillaries, reducing the surface available for filtration and lumen constriction or occlusion. Tubular stromal fibrosis also occurs in diabetic nephropathy in addition to glomerulosclerosis. Progressive loss of renal function is associated with progressive onset of interstitial fibrosis in other kidney diseases (Risdon R et al. (1968) Lancet 2 7564 pp363-366).

  Fibrosis is generally associated with growth factor and hormone imbalances that subsequently affect the production of protein expression. Subsequent abnormal protein expression causes the formation of fibrosis. For example, fibrosis is generally affected by an increase in transforming growth factor β present in fibrotic tissue.

  Fibrosis is partly due to the influence of various factors on the mechanisms underlying these diseases, and the complete cellular mechanism has not been elucidated. One of a large group. As such, there is a lack of understanding of molecular targets, or their nature, that may provide peripheral targets that may be the basis of therapeutic methods for anti-fibrotic diseases.

  In the case of diabetic nephropathy, studies have shown that glucose can induce matrix synthesis, at least in part, by the action of transforming growth factor β (TGF-β) (Ziyadeh F et al. 2000) Proc Natl Acad Sci USA 97 pp. 8015-8020).

  However, TGF-β has many physiological roles including involvement in immunity and epithelial proliferation (McCartney-Francis N et al. (1998) Int. Rev. Immunol. 16 pp. 553-580). These different physiological effects mean that TGF-β should not be a clinically advantageous target. Interfering with the function of TGF-β can have a number of effects on the organism, causing unwanted and potentially serious side effects.

  Transforming growth factor β causes fibrosis by direct induction of collagen and matrix synthesis. In addition, TGF-β can also induce the expression of other molecules involved in and / or affecting the pathways that cause fibrosis. One such protein is connective tissue growth factor (CTGF) that induces proliferation in mesenchymal cells, collagen synthesis, and chemotaxis (Moussad E et al. (2000) Molec Genet Metab. 71 pp. 276-292). CTGF (CCN2) is a 38 kDa secreted protein encoded by the immediate early gene and having many domains that are members of the CCN protein family (Bork et al. (1993) Febs Lett. 327 pp 125-130; Perbal et al. al. (2001) Mol. Pathol. 54 pp 57-79). However, the molecular mechanism by which it functions has not been fully elucidated. The presence of many domains in CTGF indicates that it interacts with a number of other factors. CTGF has been shown to bind directly to BMP4 and TGF-β via the Von Willebrand type C domain, causing inhibition of BMP and promotion of TGF-β signaling (Abreu et al. (2002) Nat. Cell. Biol. 4 pp. 599-604).

  CTGF has also been shown to bind integrins (Babic et al. (1999) Mol. Cell Biol. 19 pp. 3811-3815), and this interaction mediates several cellular phenomena induced by CTGF. May be important.

  CTGF is overexpressed in various fibrosis including diabetic nephropathy (Wahab N et al. (2001) Biochem J. 359 pp. 77-87). Indeed, increased expression levels of CTGF have been shown to be associated with increased severity and rate of progression of diabetic nephropathy (Ito Y et al. (1998) Kidney Int. 53 pp.853-886). .

  As such, CTGF may be a potentially useful molecular indicator of fibrotic response. CTGF has not yet been shown to induce renal fibrosis directly in vivo, but it induces persistent cutaneous fibrosis in rats when injected subcutaneously with TGF-β (Mori T et al. (1999) J. Cell. Physiol. 181 pp 153-159).

  In the course of developing the present invention, the inventors have shown that CTGF interacts with the cellular receptor TrkA receptor to induce an intracellular signaling cascade associated with fibrosis formation.

There are three Trk receptor tyrosine kinase genes (TrkA, TrkB, and TrkC). In binding to its ligand, the Trk receptor dimerizes, autophosphorylates, activates several low molecular weight G proteins, including the Ras, Rap-1, and Cdc42-Rac-Rho families, and Causes activation of pathways regulated by MAP kinase, PI3 kinase, and phospholipase C-γ (PLCγ) (Segal, 2003). Activated Trk receptors interact directly or indirectly with various cytoplasmic adapter proteins, cell proliferation and survival, axon and dendrite growth and remodeling, cytoskeleton construction and remodeling, It produces many biological responses including membrane trafficking and fusion, and synapse formation, function, and plasticity (Huang E and Reichardt L, (2003) Annu. Rev. Biochem. 72 pp. 609-642).
Silink M (2002), Horm. Res. 57 (Suppl 1) pp. 1-5 Quinn M et al., (1996) Diabetologica 39 pp 940-945 UKPDS Group, (1998) Lancet 352 pp.837-853 Bagust A et al. (2002) Diabetes Med 19 (Suppl 4): ppl-5 Osterby R, (1992) Diabetologica 35 pp 803- 812 Steffes M et al., (1989) Diabetes 38 pp1077-1081 Risdon R et al. (1968) Lancet 2 7564 pp363-366 Ziyadeh F et al. (2000) Proc Natl Acad Sci USA 97 pp. 8015-8020 McCartney-Francis N et al. (1998) Int. Rev. Immunol. 16 pp. 553-580 Moussad E et al. (2000) Molec Genet Metab. 71 pp. 276-292 Bork et al. (1993) Febs Lett. 327 pp 125-130 Perbal et al. (2001) Mol. Pathol. 54 pp 57-79 Abreu et al. (2002) Nat. Cell. Biol. 4 pp. 599-604 Babic et al. (1999) Mol. Cell Biol. 19 pp.3811-3815 Wahab N et al. (2001) Biochem J. 359 pp. 77-87 Ito Y et al. (1998) Kidney Int. 53 pp.853-886 Mori T et al. (1999) J. Cell. Physiol. 181 pp 153-159

  Experiments by the inventors described in the examples show that Trk tyrosine kinase activity is required for CTGF-dependent induction of intracellular signaling molecules associated with fibrosis. This experiment has led to the establishment of methods for identifying and making compounds that can interact with CTGF receptors and / or CTGF receptor agonists to reduce or prevent fibrosis.

Thus, in a first aspect of the invention, there is provided a method for identifying and / or making a compound for use in reducing and / or preventing fibrosis,
(a) Provision of CTGF receptor
(b) Providing test samples
(c) Provision of CTGF receptor agonists
(d) exposure of the CTGF receptor to the test sample
(e) subsequent or simultaneous exposure of the CTGF receptor to the CTGF receptor agonist
(f) Detection and / or measurement of the amount of CTGF receptor activation
(g) Comparison of the amount of CTGF receptor activation in the presence of the test sample and the amount of CTGF receptor activation detected and / or measured in the absence of the test sample
(h) Determination of whether a compound reduces and / or prevents fibrosis based on not increasing or decreasing CTGF receptor activation.
These steps are included.

  By “CTGF receptor agonist” is used to mean a compound that acts on a CTGF receptor that produces substantially the same effect as that produced by CTGF interacting with the receptor. Also included are derivatives, analogs, and fragments of CTGF receptor agonists that have the ability to produce effects substantially similar to CTGF that interact with the CTGF receptor. CTGF receptor agonists other than CTGF itself can be readily identified by measuring and / or detecting CTGF receptor autophosphorylation, receptor-induced protein phosphorylation, and TIEG expression using the method described in Example 1.

  “Derivative” is used to mean a CTGF receptor agonist compound further having at least one chemical modification of one or more amino acid side chains, alpha carbon atoms, terminal amino groups, or terminal carboxyl groups. Chemical modification includes the addition of groups with chemical functions, the creation of new bonds, and the removal of groups with chemical functions. Amino acid side chain modifications include acylation of the ε-amino group of lysine, N-alkylation of arginine, histidine, or lysine, alkylation of the carboxylic acid group of glutamic acid or aspartic acid, and deamidation of glutamine or asparagine. Terminal amino group modifications include deamination, N low alkyl, N di low alkyl, and N acyl modifications. Terminal carboxyl group modifications include amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Low alkyl is C1-C4 alkyl. Furthermore, one or more side chains, or terminal groups, may be protected by protecting groups known to protein chemists with ordinary skill. The alpha carbon of the amino acid may be mono- or dimethylated.

  With respect to “analogs”, the ability to produce an effect that is substantially similar to that produced by CTGF that has one or more amino acid substitutions, deletions, inversions, or additions that interact with the receptor Is used to mean a CTGF receptor agonist.

  By “fragment” is used to mean a portion of a CTGF receptor agonist that has the ability to produce an effect substantially similar to the effect produced by CTGF interacting with the receptor.

  Optionally, the method further comprises the step of isolating compounds having the ability to reduce and / or prevent fibrosis. The isolated compound may then optionally be formulated into a composition further comprising a pharmaceutically acceptable carrier, excipient, and / or diluent.

  Preferably, CTGF receptor activation is detected and / or measured by detecting and / or measuring at least one of the following activities: CTGF receptor autophosphorylation, CTGF receptor-induced protein phosphorylation, or CTGF Induced TIEG expression. Exemplary methods for measuring these activities are provided in Examples 1 and 2.

  Preferably, the CTGF receptor agonist is CTGF.

  Preferably, the CTGF receptor is a TrkA receptor.

  Conveniently, the compound acts directly on the interaction between the CTGF receptor and its agonist. In other words, the compound interacts directly with the CTGF receptor or an agonist thereof to reduce CTGF receptor activation.

  Alternatively, the compound acts indirectly on the interaction between the CTGF receptor and its agonist. In other words, the compound interacts indirectly with the CTGF receptor or an agonist thereof via at least one additional compound to reduce CTGF receptor activation.

  Conveniently, the compound identified and / or produced by the above method is an antagonist of tyrosine kinase.

  By “antagonist” is used to mean a compound that acts on the CTGF receptor that inhibits and / or prevents the effects produced by CTGF or a CTGF receptor agonist that interacts with the receptor. Also included are derivatives, analogs, and fragments of CTGF receptor antagonists that have the ability to inhibit and / or prevent the effects of CTGF and / or CTGF receptor agonists that interact with the CTGF receptor. CTGF receptor antagonists can be readily identified by measuring and / or detecting CTGF receptor autophosphorylation, receptor-induced protein phosphorylation, and TIEG expression using the method described in Example 1.

  In the second aspect of the present invention, CTGF receptor activation is inhibited and / or prevented, more preferably the following activities (CTGF receptor autophosphorylation, CTGF receptor induced protein phosphorylation, and / or TIEG: Provided are compounds for use in reducing and / or preventing fibrosis characterized in inhibiting and / or preventing at least one of (induction).

  Preferably, said compound is identified and / or made by the method of the first aspect of the invention.

  Conveniently, the compound is at least one selected from a polypeptide, an antibody molecule, and an antisense nucleotide. Preferably, the compound is an antibody molecule.

  The term “antibody molecule” should be construed with reference to any one of an antibody, antibody fragment, or antibody derivative. Without limitation, single-chain modified antibody molecules produced by phage display of immunoglobulin light and / or heavy chain variable and / or constant regions, or antigens in immunoassay formats known to those skilled in the art. It is intended to include wild-type antibodies, synthetic antibodies, recombinant antibodies, or hybrid antibodies, such as other immunointeractive molecules that have the ability to bind.

The term “antibody derivative” refers to a fragment of an antibody (eg, a Fab or Fv fragment), or one or more amino acids to facilitate association of the antibody with other peptides or polypeptides, large carrier proteins, or solid supports. Or known to those skilled in the art, such as antibody molecules modified by the addition of amino acids such as tyrosine, lysine, glutamic acid, aspartic acid, cysteine, and derivatives thereof, especially NH ₂ acetyl group or COOH terminal amide group Reference is made to any modified antibody molecule capable of binding to an antigen in an immunoassay format.

  With respect to “antisense oligonucleotide”, it is used to mean a single-stranded nucleic acid capable of specifically binding to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are usually referred to as “antisense” because they are complementary to the sense or encoding strand of a gene. Recently, the possibility of triple helix formation has been demonstrated when oligonucleotides bind to DNA duplexes. It has been shown that the oligonucleotide will recognize sequences in the major groove of the DNA duplex. A triple helix was thereby formed. This indicates that it is possible to synthesize a sequence-specific molecule that specifically binds to double-stranded DNA through recognition of the major groove-type hydrogen bonding site.

  By binding to the target nucleic acid, the above-described oligonucleotide can inhibit the function of the target nucleic acid. This is the result of promotion of cellular inhibitory mechanisms such as, for example, transcription, processing, poly (A) addition, replication, interference with translation, or promotion of RNA degradation.

  Antisense oligonucleotides are prepared in the laboratory and then introduced into cells by, for example, microinjection or cell culture medium uptake into cells. Alternatively, antisense oligonucleotides are expressed in cells after transfection using plasmids, retroviruses, or other vectors that have antisense genes.

  Alternatively, the compound is an inhibitor of a tyrosine kinase receptor. Preferably, the tyrosine kinase inhibitor is BSF-466895, AP-23451, AP23464, AP-23485, AZD-0530, AP-22408, RG-13022, RG-13291, RG-14620, RP 53801, CEP-075, CEP-2563 dihydrochloride, CHIR-200131, CHIR-285, c-jun kinase, KST-638, KF-250706, MNAC-13, anti-EphA2 Mab, MLN-608, AG-957, lavenderine A analog, NSC -330507, NSC-680410, phenylalanine derivative, SH2 inhibitor, AG-1295, EGF-genistein, arbustatin, genistein, neuT Mab, PP1, TT-232, CGP-52411, CGP-53716, CGP-57148, imatinib, NVP -AAK980-NX, NV-50, Phenoxdiol, FAK inhibitor, IGF-1, Met receptor inhibitor, TIE-2 inhibitor, CP-564959, PN-355, CP-127374, FCE-26806, FGFR-3 inhibitor, Met RTK antagonist, PD-171026, PD-173956, PD-180970, Src non-RTK antagonist, Kahalalide F, CCX2, celastrol, TAK- 165, TG-100-13, TG-100-96, desmal, U3-1566, and SKI-606.

  Preferably, the compound is a CTGF receptor antagonist.

  In a third aspect of the invention, there is provided a compound of the second aspect of the invention for use in the treatment and / or prevention and / or diagnosis of fibrosis.

  Preferably, the compound of the second aspect of the invention is used in the manufacture of a medicament for the treatment and / or prevention and / or diagnosis of fibrosis.

  Conveniently, said fibrosis is diabetic nephropathy, non-diabetic kidney fibrosis, lung fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, myocardial fibrosis, atheroma Atherosclerosis, systemic sclerosis, scleroderma, retinal fibrosis, radiation-induced fibrosis, keloid scar formation, and tumor-related fibrosis.

  Preferably, the fibrosis is diabetic nephropathy.

  In a fourth aspect of the invention, the treatment or prophylactically effective administration of a single or multiple doses of a compound identified and / or made according to the method of the first aspect of the invention comprises A method of treating and / or preventing fibrosis is provided.

  Conveniently, said fibrosis is diabetic nephropathy, non-diabetic kidney fibrosis, lung fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, myocardial fibrosis, atheroma Atherosclerosis, systemic sclerosis, scleroderma, retinal fibrosis, radiation-induced fibrosis, keloid scar formation, and tumor-related fibrosis.

  Preferably, the fibrosis is diabetic nephropathy.

  In a fifth aspect of the invention, there is provided the use of an agent having the ability to bind to a CTGF receptor agonist in the treatment and / or prevention and / or diagnosis of fibrosis.

  In a sixth aspect of the invention there is provided the use of an agent having the ability to bind to a CTGF receptor agonist in the manufacture of a medicament for the treatment and / or prevention and / or diagnosis of fibrosis.

  Preferably, said fibrosis is one or more diabetic nephropathy, non-diabetic renal fibrosis, pulmonary fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, myocardial fibrosis, atheroma Selected from atherosclerosis, systemic sclerosis, scleroderma, retinal fibrosis, fibrosis induced by radiation, keloid scar formation, and fibrosis associated with tumors.

  In a seventh aspect of the invention, there is provided the use of an agent having the ability to bind to a CTGF receptor agonist in a method that reduces and / or interferes with the binding of a CTGF receptor agonist to a CTGF receptor in vivo or in vitro.

  With respect to “agent having the ability to bind to a CTGF receptor agonist”, a compound, nucleic acid, polypeptide, or antibody having the ability to physically bind to a CTGF receptor agonist is used. Binding between such agents and CTGF receptor agonists can result in ionic, electrostatic, and / or covalent bonds. Preferably, the binding of the agent to the CTGF receptor agonist will reduce and / or prevent the action of the CTGF receptor agonist to bind and / or associate and / or activate the CTGF receptor.

  Preferably, in the seventh aspect of the invention, the agent capable of binding to a CTGF receptor agonist is a CTGF receptor. Alternatively, the agent having the ability to bind to a CTGF receptor agonist is a CTGF receptor joined to the Fc region of an immunoglobulin.

  For the “Fc region”, the “fragment that is crystallizable” region of the antibody is used. With respect to “immunoglobulin”, polypeptides including one or more immunoglobulin complementarity determining regions (CDRs) such as antibodies, B cell receptors, T cell receptors, or fragments thereof are used. Preferably, the immunoglobulin is an antibody. More preferably, the immunoglobulin is IgG.

  Preferably, the seventh aspect of the present invention provides the use wherein the CTGF receptor is a TrkA receptor. Alternatively, the CTGF receptor is a soluble form of the TrkA receptor.

It will be understood that the term “soluble form” includes forms of the polypeptide that are not bound or inserted into the cell membrane and that can exist in a soluble state without aggregation.

  Preferably, in the fifth, sixth and seventh aspects of the present invention, the CTGF agonist is CTGF.

  In an eighth aspect of the invention, there is provided a nucleic acid encoding a TrkA receptor joined to the Fc region of an immunoglobulin.

  In a ninth aspect of the invention, there is provided a vector comprising a nucleic acid according to the eighth aspect of the invention.

  In a tenth aspect of the invention, there is provided a polypeptide comprising a TrkA receptor joined to an immunoglobulin Fc region.

  In an eleventh aspect of the invention there is provided a cell comprising a nucleic acid according to the eighth aspect of the invention and / or a vector according to the ninth aspect of the invention and / or a polypeptide according to the tenth aspect of the invention. .

  In a twelfth aspect of the invention, a nucleic acid according to the eighth aspect of the invention and / or a vector according to the ninth aspect of the invention and / or a polypeptide according to the tenth aspect of the invention and / or a Comprising a cell according to 11 embodiments, and a pharmaceutically acceptable carrier or excipient, wherein the nucleic acid and / or the vector and / or the polypeptide and / or the cell are used to treat and / or prevent fibrosis and A pharmaceutical composition is provided that is present in an amount effective for diagnosis.

  With respect to “effective amount”, fibrosis is used in an amount sufficient to treat and / or prevent and / or diagnose. An effective amount may be determined by use of methods known to those skilled in the art.

Example 1
Identification of CTGF TrkA-interacting materials and methods Cell cultures, antibodies, and reagents. Maintained in culture as described in (Wahab N et al. (1996) Biochem J. 316pp. 985-992).

  Cultures after confluent logarithmic growth phase of HMC (passage 6-8) were treated with 10% (v / v) fetal calf serum and 4 mM (normoglycemic), 11, 15, or 30 mM for a period of up to 4 weeks. (Hyperglycemic) d Maintained in culture medium containing glucose. At the end of each week, the cultures were extensively washed with PBS and used either for RNA extraction or for 24 hours of culture in glucose-supplemented medium lacking serum.

  Phosphorylated Akt antibody (P-Ser472 / 473/474) (Pharmingen, San Diego, CA, USA), phosphorylated Akt (P-Thr 308) (Sigma, Gillingham, Dorset, UK) and ERK5 antibody (Sigma, Gillingham, Dorset, UK) was used.

  Phosphorylated ERK1 / 2 pathway sampler, phosphorylated JNK pathway sampler, phosphorylated P38MAPK pathway sampler, phosphorylated PKCδ, phosphorylated PKCα, phosphorylated TrkA (Tyr674 / 675), phosphorylated TrkA (Tyr490) antibodies from New England BioLab Obtained (Hitchen, herts., UK). Phosphorylated CaMKII (P-Thr286) antibody was obtained from Promega (Southampton, Hants., UK), and anti-phosphotyrosine antibody was obtained from Santa Cruz (Autogen Bioclear, Calne, Wilts., UK). Anti-TrkA antibody was obtained from Upstate Biotechnology (Milton Keynes, UK). Anti-TIEG-1 antibody was a gift from Dr. Steven Johnson (Mayo Foundation, Minnesota, USA). K-252a was purchased from Calbiochem (Nottingham, UK). Recombinant CTGF (CTGF / V5 fusion protein) was expressed in transformed HMC and purified from media using Talon metal affinity resin (Wahab N et al. (2001) Biochem J. 359 pp.77- 87). Alternatively, r-CTGF (non-fusion protein) was expressed in the baculovirus system and was donated by FibroGen Inc. (South San Francisco, CA, USA). Rabbit anti-CTGF (pAb2) and chicken anti-CTGF (pIgY3) are also provided by FibroGen Inc.

Crosslinking and membrane preparation Cell layers were washed twice using cold binding buffer (PBS and 0.5% glucose) and incubated with CTGF in binding buffer for 2 hours at 4 ° C. After incubation, the cell layer is washed 5 times using cold binding buffer and 1 mM 3,3′-dithiobis (sulfosuccinimidylpropionate) (DTSSP) or disc in PBS at room temperature for 30 minutes. Incubated with cinimidyl suberate (DSS) (Pierce Biotechnology, Tattenhall, Cheshire, UK). The reaction was stopped at room temperature for 15 minutes by the addition of 50 mM Tris buffer pH 7.5.

  The cell layer was washed using a washing buffer (10 mM Tris buffer (pH 7.5), 5 mM MgCl, 150 mM NaCl), and homogenized buffer (10 mM Tris buffer (pH 7.5), 250 mM sucrose, 1 mM EDTA, 5 mM MgCl. , 150 mM NaCl, and 1 × protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany)), passed through a 25 gauge needle, and homogenized on ice for 30-40 revolutions in a Dounce homogenizer.

  The product obtained by homogenization was centrifuged at 2500 × g for 10 minutes at 4 ° C. The obtained supernatant was centrifuged at 4 ° C. and 45000 × g for 90 minutes. Membrane-rich pellet can be placed in solubilization buffer (10 mM Tris buffer (pH 7.5), 5 mM MgCl, 150 mM NaCl, 1% Triton-X100, 1 × protease inhibitor cocktail (Roche, as above)) for 1 hour Solubilized. Soluble membrane proteins were recovered after further centrifugation at 45000 × g for 1 hour at 4 ° C.

  For the use of rCTGF / V5 fusion protein, CTGF cross-linked protein was immunoprecipitated using rabbit anti-CTGF antibody or captured on a pull-down poly His column (Pierce Biotechnology, Tattenhall, Cheshire, UK).

  In the use of rCTGF, CTGF-crosslinked protein was captured on a goat anti-CTGF-C-terminal domain sepharose immunoaffinity column. An IgG sepharose column (FibroGen Inc.) was used as a control. After extensive washing of the column using solubilization buffer, the bound proteins were solubilized in reducing SDS-PAGE loading buffer, boiled for 5 minutes and analyzed on a 4-12% gradient gel by SDS-PAGE. Coomassie blue was used to stain the gel or used for Western blots.

RNA extraction or RT-PCR analysis Total RNA was extracted from 6 × 10 ⁶ glomerular stromal cells using the RNAzol B method (AMS biotechnology (UK) Ltd., Oxfordshire, UK). An equal amount (2 μg) of total RNA from each sample was reverse transcribed into cDNA using SuperScriptII RNase H + reverse transcriptase (Gibco BRL, Paisley, Scotland, UK) and random primers.

Specific to each of 10 μl 10 × PCR buffer, 16 μl dNTP (1.25 mM each), 2 mM MgCl2, 5 M betaine (Sigma), 0.5 μM using an equal volume (0.5 μl) of reverse transcription reaction (20 μl) PCR amplification in a 100 μl volume containing typical primers and 1.25 U Amplitaq DNA polymerase (Gibco BRL). Amplification began at 94 ° C at 5 minutes of denaturation, followed by 30 PCR cycles for all genes. Each cycle consisted of 94 ° C for 60 seconds, 55 ° C for 60 seconds, and 72 ° C for 60 seconds. Final extension was at 72 ° C. for 10 minutes. The sequences of primers for amplifying TrkA, TrkB, and TrkC p75 ^NTR , NGF, BDGF are described by Anderson et al. (2002) J. Clin. Endocrinol. Metab. 87 pp. 890-897. 1 (applied from Table 1 in Anderson et al. (2002)).

Western blotting Cells were lysed in reducing SDS-PAGE loading buffer and plates were scraped immediately. The cell lysate was sonicated for 10 seconds to cleave the DNA. Samples were then boiled for 5 minutes and analyzed on a 4-12% gradient gel by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membrane filters (Immobilin-P, Millipore, Bedford, UK) using a BioRad transfer device. Blotted membranes were incubated for 1 hour in blocking buffer containing 1 × TBS, 0.1% Tween-20, and 5% (w / v) nonfat dry milk.

  Immunodetection was performed by incubating the membranes blotted in primary antibody appropriately diluted in antibody dilution buffer (1 × TBS, 0.1% Tween-20, and 5% BSA) at 4 ° C. overnight. The blotted membrane was then washed 3 times with wash buffer (1 × TBS, 0.1% Tween-20) and incubated with secondary horseradish peroxidase (HRP) -conjugated antibody for 1 hour at room temperature. .

  The bound antibody was visualized using the chemiluminescence enhancing reagent Luminol (Autogen Bioclear UK Ltd, Wiltshire, UK). A pre-stained molecular weight standard was used to measure protein migration.

Immunofluorescence staining
Cells were fixed using 3.7% paraformaldehyde and permeabilized using PBS containing 0.5% Triton X-100 for 10 minutes at room temperature. The coverslips were then incubated overnight at 4 ° C. using serum from the same species from which the secondary antibody was obtained (5% in PBS). After the first incubation, the coverslips were incubated for 1 hour at 37 ° C. using a primary antibody (optimally diluted in PBS containing 3% BSA).

  The coverslips were then washed and incubated for 1 hour in the dark using a fluorescein-conjugated secondary antibody (Sigma Aldrich, Dorset, UK). After staining, the cover glass was placed on a slide glass with anti-fading packing medium (Vector Labs, Peterborough, U.K.) and examined using a fluorescence microscope.

result
CTGF activates several intracellular signaling pathways
To identify intracellular signaling pathways activated in response to growth factors in HMC, purified rCTGF-V5 fusion protein was used. CTGF has been shown to immediately cause activation of the classical MAPK (ERK1 / 2) and JNK pathways (FIGS. 1A and B) but not p38MAPK. FIG. 1 shows the maximum activation of these kinases 15 minutes after CTGF stimulation.

  CTGF stimulation also caused activation of AKT, also known as protein kinase B (PKB), in both known phosphorylation sites (Thr-308 and Ser-473) (FIG. 1C). The activation of Thr-308 appears to be fast and persistent compared to that of ser-473.

  Activation of Thr-308 is affected by phosphoinositide-dependent kinase 1, which has an activity strictly dependent on inositol triphosphate lipid, or PDK1 (Dawnward J (1998) Curr. Opin. Cell Biol. 10 pp.262 -267). Ser-473 phosphorylation is carried out by integrin-linked kinase (ILK) (Attwell et al. (2000) Oncogene 19 pp. 3811-3815), transiently maximal in 15 minutes after CTGF exposure, It seems to return to near the standard level within 30 minutes.

  CTGF stimulation also causes transient activation of CaMKII (FIG. 1C). Other kinases that are activated in response to CTGF are PKCδ and PKCα (FIG. 1D).

CTGF interacts with receptors FIG. 1 shows that CTGF provides signals to downstream signaling proteins via receptors that activate the kinases described above. These kinases are normally activated by receptor tyrosine kinases (RTK).

  The possibility of CTGF acting through receptor tyrosine kinase (RTK) was tested by exposing HMC to CTGF for different times. Cell lysates were prepared from HMC cells and Western blot analysis was performed using anti-phosphotyrosine antibodies.

  The results showed that CTGF fusion protein (40 ng / ml) stimulated tyrosine phosphorylation of at least two major proteins with apparent molecular weights of about 75-80 and 140-150 kDa in HMC within 10 minutes. (FIG. 2).

  Other phosphorylated tyrosine proteins (molecular weight 45 kDa) were detected in control cell lysates but decreased in response to CTGF treatment.

CTGF interacts with human glomerular stromal cell (HMC) surface proteins
The interaction between CTGF and HMC surface protein was studied by binding CTGF to the cell surface. Subsequently, a cross-linking method was performed and a membrane rich fraction was isolated from the cells. After solubilization, this fraction was immunoprecipitated using a rabbit anti-CTGF antibody. The covalently bound CTGF complex was then analyzed by Western blot using PAGE and chicken anti-CTGF antibody.

  In FIG. 3, CTGF appears to be cross-linked with membrane proteins to form complexes with apparent molecular weights of 85 kDa, 180 kDa, and> 220 kDa (the latter being a highly diffuse band (lane 2)). . These complexes were not immunoprecipitated from the membrane-rich fraction when the cross-linking step was excluded (lane 1).

  To confirm whether CTGF activates RTK, serum starved HMCs were incubated for 15 minutes in the presence and absence of CTGF. Cells were lysed and phosphorylated tyrosine protein was immunoprecipitated. Immunoprecipitated proteins were analyzed by Western blot using antibodies against a number of known tyrosine kinase receptors.

  FIG. 4A shows a cross-reacted anti-TrkA antibody (a band of about 140 kDa). The intensity of this band became stronger when incubated with CTGF (lane 2), indicating activation by CTGF.

  The interaction between CTGF and the TrkA receptor involves binding either His-tagged CTGF / V5 fusion protein or rCTGF expressed by the baculovirus system to its ligand using the reversible cross-linker DTSSP. It was confirmed by different experiments to crosslink. The latter was then cleaved with a reducing agent.

  Subsequently, membrane fractions were prepared and any cross-linked CTGF complex was captured on affinity metal beads or anti-C-terminal CTGF antibody affinity beads. Captured complexes were SDS-PAGEd in the reduced state and analyzed by Western blot.

  Figures 4B, 4C and 4D show that CTGF interacts with the TrkA receptor.

  In the past, the Trk receptor has been shown to interact with p75NTR, the all neurotrophin receptor. Therefore, blotted membranes were stripped and tested again using anti-p75NTR antibody. FIG. 4E shows that the antibody cross-reacts with the correct molecular weight protein of p75NTR.

  Thus, the results indicate that CTGF interacts with TrkA and p75NTR receptors.

HMC expresses the Trk receptor.
The expression of Trk receptor by HMC was studied by extraction of total RNA from HMC for RT-PCR analysis to be performed. FIG. 5 shows that HMC expresses all three members of the Trk receptor family (TrkA, TrkB and TrkC) as well as all receptors p75NTR.

CTGF activates TrkA in HMC
TrkA autophosphorylates several tyrosine residues upon binding by its ligand, causing the binding and activation of numerous effector molecules. Phosphorylation of Tyr490 is required for Shc binding and activation of the Ras-MAP kinase cascade. Phosphorylation of Tyr674 / 675 exists in the catalytic domain and affects Trk kinase activity. Therefore, it was tested whether cells stimulated with CTGF caused phosphorylation of these residues of TrkA. The results in FIG. 6 clearly show that CTGF induces phosphorylation of these residues of the receptor.

Inhibition of CTGF-induced signaling by K252a
K252a is an alkaloid-like kinase inhibitor that is known to selectively inhibit tyrosine kinases. K252a inhibited protein phosphorylation of ERK1 / 2, JNK, and ERK5 stimulated using CTGF in HMC cells. This indicated that phosphorylation of these kinases was induced by the tyrosine kinase receptor TrkA and that tyrosine kinase inhibitors had the ability to inhibit CTGF-mediated signaling.

CTGF induces expression of TIEG FIG. 7 shows that exposure to CTGF causes a rapid induction of the expression level of TIEG.

  The action of TIEG directly mediating CTGF-dependent down-regulation of Smad7 levels was studied by cells treated with TIEG antisense and control oligonucleotides. FIG. 8 shows that the constitutive levels of TIEG and Smad7 proteins in HMC are low (lane 1).

  Incubation of cells for 24 hours with CTGF significantly increased TIEG levels and decreased Smad7 levels to almost undetectable levels (lane 2). This effect disappeared completely in the presence of TIEG antisense oligonucleotide (lane 5), but not with the control oligonucleotide (lane 6).

  Incubation of cells at the same time using only TGF-b caused a mild increase in both TIEG and Smad7 (lane 3). However, incubation of cells using TGF-b in the presence of CTGF antisense oligonucleotide completely abolished the mild induction of TIEG and increased the induction of Smad7 (lane 7). This is not confirmed in the presence of the control antisense oligonucleotide (lane 8) and is consistent with the TGF-b inducible CTGF being involved in the moderate increase observed in TIEG expression levels.

  Similar results were obtained by treatment of cells using TIEG antisense and control antisense oligonucleotides (lanes 9 and 10). Incubation of cells using both TGF-b and CTGF significantly increased the expression level of TIEG and decreased the expression level of Smad7. These results clearly show that TIEG mediates CTGF-dependent down-regulation of Smad7 expression.

(Example 2)
Screening methods for the identification of compounds that inhibit CTGF-induced fibrosis
Screening for compounds with fibrosis-inhibiting properties that depend on CTGF-CTGF receptor interaction is performed, for example, by testing the action of each compound to inhibit the induction of TIEG in HMC treated with CTGF .

  The screening method is performed using human glomerular stromal cells (HMC) that have been pre-incubated for 30 minutes with or without potential inhibitors. These cells are then stimulated using CTGF-V5 fusion protein (40 ng / ml) in the presence or absence of potential inhibitors. After washing the cell layer with cold PBS, lyse the cells in RIPA buffer and use the lysate for testing for TIEG by ELISA.

  For ELISA studies (Voller A et al. (1976) in Manual of Clinical Immunology (Rose, N and Fishman H, eds.) Pp 506-512, American Society of Microbiology, Washington, DC.), NUNC microtiter plate Are coated overnight at 4 ° C. using a lysate or a reference dilution of r-TIEG that provides a reference curve.

  After removing the coating solution and washing the wells briefly with PBS, non-specific by incubating the wells for 1 hour using PBS containing 1% (w / v) bovine serum albumin at 37 degrees Interfere with proteins. The wells are then incubated for 60 minutes using anti-TIEG antibody at optimal dilution followed by 60 minutes at 37 degrees with peroxidase-conjugated secondary antibody. After washing the wells three times using PBS, bound antibody is detected using the substrate 2,2′-azinobis-3-ethylbenzthiazoline 6 sulfonic acid and an absorbance reading at 405 nm.

  Recombinant TIEG protein is generated from full-length TIEG cCNA by cloning into PcDNA3.1 / V5-His Topo vector (InVitrogen). This vector can be transfected into a mammalian cell line to express the TIEG fusion protein.

  The TIEG fusion protein is purified from cell lysate using Probond nickel chelate resin. Anti-TIEG antibodies are obtained from Dr. Steven Johnson (Mayo Foundation, Minnesota, USA) or raised against TIEG fusion proteins in rabbits by use of conventional methods.

(Example 3)
Reduced development of diabetic nephropathy in diabetic mice by interfering with access to cell surface receptors for CTGF agonists
It should be possible to prevent and / or reduce the development of diabetic nephropathy in diabetic mice by interfering with access to cell surface receptors for CTGF agonists. This would be done by using an agent that has the ability to bind to the CTGF receptor agonist, thereby preventing the agonist from binding to the cell surface receptor. For example, a soluble mouse TrkA receptor (sTrkA) can be used.

  A cDNA sequence representing the extracellular region of the CTGF receptor can be produced by RT-PCR using total RNA extracted from mouse kidney and specifically designed oligonucleotide primers. The PCR product can be cloned in frame into the pSecTag2 mammalian expression vector (Invitrogen) designed for efficient secretion of the expressed protein. Fusion of the Fc part of immunoglobulin G (IgG) has been shown to delay elimination of other soluble receptors in vivo (Zhou A, Ueno H, Shimomura M, Tanaka R, Shirakawa T, Nakamura H, Matsuo M , Iijima K, Kidney Int. 2003, 64: 92-101), the cDNA of the Fc portion of mouse IgG can be amplified by RT-PCR and cloned so that the frame matches the 3 ′ end of CTGF.

  The mouse Fc antisense primer should contain a stop codon to prevent fusing with the myc epitope present in the pSecTag2 vector. These constructs can be used to produce stably transfected mouse fibroblast cell lines by using the zeocin resistance gene present in the pSecTag2 vector for selection according to standard techniques.

  Cell lines that efficiently express and secrete the receptor can usually be grown on a large scale (T150 flask). FPLC Mono S (Pharmacia) can be used to purify the expressed recombinant protein from the medium, and the purified protein is checked by Coomassie blue stained SDS-PAGE and phosphate buffered saline (PBS ) Can be dialyzed. The dialyzed protein can be passed through a Detox-igel column (Pierce), endotoxin checked by the Limulus amebocyte test (Sigma), and passed through a sterile filter (0.2 μm).

  To test this in vivo, four groups of mice ((a), (b), and (c) db / db mice (n = 10 each), and (d) db / m mice (n = 10)) can be prepared. For these experiments, diabetic db / db mice and non-diabetic db / m mice (Jackson Laboratory) can be used. The db / db mouse is a transgenic mouse model that represents type 2 diabetes and expresses hyperglycemia associated with obesity and insulin resistance in the first few weeks of life. This model will be selected to show glomerular glomerular stroma expansion by 16 weeks of age (Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J , Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Cell. 1996, 84: 491-5; Like AA, Lavine RL, Poffenbarger PL, Chick WL. Am J Pathol. 1972, 66: 193-224. ).

  Immediately after the onset of diabetes, group (a) will receive daily subcutaneous injections of 50 μg sTrkA. This dose will be selected based on similar successful experiments designed by other researchers (Park L, Raman KG, Lee KJ, Lu Y, Ferran LJ Jr, Chow WS, Stern D, Schmidt AM. Nat Med. 1998, 4: 1025-31; Wendt TM, Tanji N, Guo J, Kislinger TR, Qu W, Lu Y, Bucciarelli LG, Rong LL, Moser B, Markowitz GS, Stein G, Bierhaus A, Liliensiek B, Arnold B, Nawroth PP, Stern DM, D'Agati VD, Schmidt AM. Am J Pathol. 2003,162: 1123-37; Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, Ioffe E, Huang T, Radziejewski C, Bailey K, Fandl JP, Daly T, Wiegand SJ, Yancopoulos GD, Rudge JS. Proc Natl Acad Sci US A. 2002,99: 11393 -8.).

  The subcutaneously injected s-receptor-Fc attractant for VEGF has a serum half-life (t1 / 2) of about 2 days. Group (b) should receive an equal amount of vehicle. Groups (c) and (d) should be untreated. Mice are killed at 20 weeks of age and tested for the development of diabetic nephropathy.

  The development of diabetic nephropathy can be examined by urinary albumin, serum and urine creatinine, changes in glomerular tissue structure, and accumulation of ECM in the glomeruli. Mice should be housed in separate metabolic cages for 24-hour urine collection. The urinary albumin concentration can be determined by ELISA using a mouse microalbuminuria kit (Albuwell M; Exocell, Philadelphia). Renal function can be assessed by calculation of creatinine removal (ml / min / 100 g body weight). Serum and urine creatinine levels can be measured by enzymatic methods using the picric acid colorimetric method (Sigma).

Example 4
Pharmaceutical and Administration The compounds of the present invention may be administered in a pharmaceutically acceptable dosage form, in the form of a pharmaceutical formulation containing the active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base, addition salt. Usually, it will be administered by oral or any parenteral route. Depending on the disease and the patient being treated, as well as the route of administration, the composition may be administered at different dosages.

  In human therapy, the compounds of the invention may be administered alone, but generally will be suitable pharmaceutical excipients selected with regard to the intended route of administration and standard pharmaceutical practice. As a mixture with a diluent or carrier.

  For example, the compounds of the present invention can be administered orally, buccally, or sublingually in the form of tablets, capsules, eggs, elixirs, solutions, or suspensions. It may also contain flavoring or coloring agents and is administered for short release, delayed release or controlled release applications. The compounds of the present invention may be administered via intravenous injection.

  Tablets include excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine, starch (preferably corn, potato, or tapioca starch), sodium starch glycolate, cloth Disintegrants such as carmellose sodium and certain silicate complexes, and granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia May be included. In addition, smoothing agents such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.

  Similar types of solid compositions may be used as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the present invention may contain various sweetening or flavoring agents, coloring or staining agents, emulsifying agents and / or suspending agents, water, ethanol, propylene glycol, And diluents such as glycerin, and combinations thereof.

  The compounds of the invention may be administered parenterally, for example intravenously, intraarterially, intraperitoneally, intracapsularly, intraventricularly, intrasternally, intracranial, intramuscularly or subcutaneously, or transplantation techniques May be administered by. They are best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately neutralized (preferably pH 3 to 9). The preparation of suitable parenteral formulations in a sterile state is readily accomplished by standard pharmaceutical techniques known to those skilled in the art.

  Formulations suitable for parenteral administration are aqueous and non-aqueous sterilizations that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient. Injectable solutions, as well as sterile aqueous and non-aqueous suspensions that may contain suspending agents and thickening agents. The formulation may be present in single or multiple dose containers (eg, sealed ampoules and vials) and only requires the addition of a sterile liquid carrier (eg, water for injection) just prior to use. May be stored in a freeze-dried state. Injection solutions and suspensions formulated as needed may be prepared from sterile powders, granules and tablets of the kind previously described.

  For oral and parenteral administration to human patients, the daily dosage level of the compounds of the invention will usually be from 1 mg / kg to 30 mg / kg. Thus, for example, a tablet or capsule of the compound of the invention may suitably contain a dosage of the compound having activity for one or more administrations. In any event, the physician will determine the actual dosage that will be most appropriate for any individual patient. They will vary in relation to the age, weight and response of a particular individual patient. The above doses are typical of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

  The compounds of the present invention may also be administered intranasally or by inhalation, and suitable high pressure gases such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane Pressurized vessels, pumps with the use of (HFA134A3) or 1,1,1,2,3,3,3-hepatafluoropropane (HFA227EA3), hydrofluoroalkanes, carbon dioxide, or other suitable gas) It is conveniently administered in the form of a dry powder inhaler such as a spray or nebulizer or an aerosol spray. In the case of a pressurized aerosol, the single dose may be determined by providing a valve for metered dose. Pressurized containers, pumps, sprays, or nebulizers may contain solutions or suspensions of the active compound (eg, use of a mixture of ethanol and high pressure gas as a solvent) and lubricants (eg, sorbitan trioles). E)). Capsules and capsules (eg, made from gelatin) for use in an inhaler or device for inhalation contain a powder mixture of a compound of the invention and a suitable powder base such as lactose or starch. It may be formulated with.

  Aerosol or dry powder formulations are preferably prepared so that each metered dose or “one breath” doses an appropriate amount of a compound of the invention for administration to a patient. It will be appreciated that the overall daily dosage using an aerosol will vary from patient to patient, and that it may be administered in a single dose, or more generally divided throughout the day.

  Alternatively, the compounds of the present invention may be administered in the form of a suppository or pessary, or may be applied topically in the form of a lotion, solution, cream, ointment, or spray. The compounds of the present invention may be administered transdermally (eg, by use of a dermal patch). The compounds of the invention may be administered by the eye route, particularly for the treatment of eye diseases.

  For use in the eye, the compounds of the present invention are micronized suspensions in sterile physiological saline that is isotonic and pH-adjusted or, preferably, sterile physiological that are isotonic and pH-adjusted. As a solution in saline, it can be formulated optionally in combination with a preservative such as benzylalkonium chloride. Alternatively, the compounds of the present invention may be formulated in ointments such as petrolatum.

  For topical application to the skin, the compounds of the present invention may be one or more of, for example, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, and water. And may be formulated as a suitable ointment containing the active compound. Alternatively, the compound of the present invention is, for example, one of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Suspended or dissolved in a mixture with one or more and formulated as a suitable lotion or cream.

  Formulations suitable for topical administration in the mouth include flavored bases, usually troches containing ingredients active in sucrose and acacia or tragacanth, gelatin and glycerin, or inert groups such as sucrose and acacia Including pastilles containing ingredients active in the agent as well as mouthwashes containing the active ingredient in a suitable liquid carrier.

  In general, in humans, oral or topical administration of the compounds of the present invention is the preferred route and most convenient. In situations where the recipient suffers from dysphagia or impaired drug absorption after oral administration, the drug may be administered parenterally (eg, sublingual or buccal).

  For veterinary use, the compounds of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice, and animal surgeons use the most appropriate usage and route of administration for a particular animal. Will decide.

CTGF incubated serum starved human glomerular stromal cells (HMCs) that activate intracellular signaling pathways in the presence of CTGF / V5 fusion proteins for the times indicated. Equal amounts of cell lysate proteins were analyzed by SDS-PAGE and analyzed by Western blot using phosphorylation specific antibodies against (A) MAPK pathway, (B) JNK, and (C) PKB and CamKII constituent proteins. . β-actin is shown as a marker for loading of equal amounts of protein. D) Cells are grown on coverslips and pre-serum starved for 48 hours prior to absence ((a) and (c)) or presence ((b) and 40 ng / ml CTGF fusion protein. (d)) was incubated in the medium for 30 minutes. Cells were fixed, permeabilized, tested using anti-phosphorylated PKCδ ((a) and (b)) and anti-phosphorylated PKCα ((c) and (d)) primary antibodies, then fluorescein-conjugated secondary Tested using antibodies. The results are representative of three separate experiments. CTGF was incubated with serum-starved HMCs that induce tyrosine phosphorylation of different proteins in the presence of 40 ng / ml CTGF / V5 fusion protein for the times indicated. Equal amounts of cell lysate proteins were SDS-PAGE and analyzed by Western blot using anti-phosphorylated antibodies. The results are representative of three separate experiments. CTGF interacts with HMC surface protein CTGF / V5 fusion protein is bound to the cell surface and then chemically cross-linked with its ligand using BS ³ , followed by a cell membrane rich fraction Prepared from cells. Cross-linked CTGF complexes were immunoprecipitated using rabbit anti-CTGF antibody, SDS-PAGE and analyzed by Western blot using chicken anti-CTGF antibody (lane 2). The cross-linking step was omitted for some cultures (lane 1). The results are representative of three separate experiments. CTGF interacts with TrkA and p75NTR in HMC (A) Serum-starved HMC for 15 minutes in the absence (lane 1) or presence (lane 2) of CTGF / V5 fusion protein (40 ng / ml) After incubation, cell lysates were prepared in RIPA buffer. Antiphosphorylated tyrosine beads were used to immunoprecipitate equal amounts of lysate protein. Bound protein was SDS-PAGE and analyzed by Western blot using an antibody against TrkA. (B) HMC was incubated at 4 ° C. for 2 hours in the absence (lane 1) or presence (lane 2) of a His-tagged CTGF / V5 fusion protein (200 ng / ml) to obtain a cell surface receptor. After binding, the protein was chemically cross-linked using DTSSP. A membrane rich fraction was prepared and solubilized. An equal amount of solubilized protein was incubated with metal affinity beads. Bound proteins were SDS-PAGE in the reduced state and Western blotted using an antibody against TrkA. (C) HMC was incubated with 200 ng / ml rCTGF (FibroGen Inc.) for 2 hours at 4 ° C. Bound CTGF was cross-linked as described above, and a membrane-rich fraction was prepared and solubilized. The cross-linked CTGF complex of the solubilized fraction was captured on anti-C-terminal CTGF antibody affinity beads (lane 1) or the fraction was incubated with control IgG affinity beads (lane 2). Bound protein was analyzed by Western blot using anti-TrkA antibody. (D) The sample in lane 1 of FIG. 4C was boiled for a long time and then Western blotted using anti-TrkA antibody. (E) Western blots from (D) were stripped and retested using anti-p75NTR antibody. The results are representative of 4 separate experiments. For HMC, total RNA expressing Trk receptor was extracted from HMC and used for RT-PCR. After amplification, 10 μl of each PCR reaction product was electrophoresed using a 1.2% (w / v) agarose gel containing ethidium bromide (0.5 μg / ml). The results are representative of three separate experiments. CTGF was incubated with serum starved HMC that activates TrkA in HMC for 15 minutes in the absence (lane 1) or presence (lane 2) of CTGF / V5 (40 ng / ml). Equal amounts of cell lysate proteins were SDS-PAGE and analyzed by Western blot. Blot A was tested using anti-TrkA antibody. Blot B was tested using anti-phosphorylated TrkA (Tyr490) antibody. Blot C was tested using anti-phosphorylated TrkA (Tyr674 / 675). The results are representative of three separate experiments. Stimulation of TIEG levels by CTGF in HMC After exposing serum-starved HMC to rCTGF / V5 fusion protein for different times, cell lysates were prepared and analyzed for TIEG and b-actin levels by Western blot. Shown are representative blots of 3 independent experiments performed (3 homotypic cultures per experiment, per condition). TIEG exposed serum starved HMCs that mediate CTGF-dependent down-regulation of Smad7 expression levels to the conditions shown in the figure. After 24 hours, cell lysates were prepared and TIEG, Smad7, and b-actin levels were analyzed by Western blot. Shown are representative blots of three independent experiments performed (3 homotypic cultures per experiment, per condition).

Claims (37)

A method of identifying and / or making a compound for use in reducing and / or preventing fibrosis comprising:
(a) Provision of CTGF receptor
(b) Providing test samples
(c) Provision of CTGF receptor agonists
(d) exposure of the CTGF receptor to the test sample
(e) subsequent or simultaneous exposure of the CTGF receptor to the CTGF receptor agonist
(f) Detection and / or measurement of the amount of CTGF receptor activation
(g) a comparison of the amount of CTGF receptor activation in the presence of the test sample and the amount of CTGF receptor activation detected and / or measured in the absence of the test sample; and
(h) a method comprising the step of determining whether the compound reduces and / or prevents fibrosis based on not increasing or decreasing CTGF receptor activation. 2. The method of claim 1, further comprising the step of (i) isolating a compound having the ability to reduce and / or prevent fibrosis. 3. The method of claim 2, further comprising the step of formulating (j) the isolated compound into a composition further comprising a pharmaceutically acceptable carrier, excipient, and / or diluent.   CTGF receptor activation is measured and / or measured by detecting and / or measuring the activity of at least one of CTGF receptor autophosphorylation, receptor-induced protein phosphorylation, and / or CTGF receptor-induced TIEG expression. 4. The method according to any one of claims 1 to 3, wherein the method is detected.   The method according to any one of claims 1 to 4, wherein the CTGF receptor agonist is CTGF.   The method according to any one of claims 1 to 5, wherein the CTGF receptor is a TrkA receptor.   7. A method according to any one of claims 1 to 6, wherein the compound acts directly on the interaction between the CTGF receptor and its agonist.   7. The method according to any one of claims 1 to 6, wherein the compound acts indirectly on the interaction between the CTGF receptor and its agonist.   9. The method according to any one of claims 1 to 8, wherein the compound is a CTGF receptor antagonist.   A compound for use in the reduction and / or prevention and / or diagnosis of fibrosis, characterized by inhibiting or preventing CTGF receptor activation.   A compound identified and / or produced by a method according to any one of claims 1 to 9 for use in the reduction and / or prevention and / or diagnosis of fibrosis.   The compound according to claim 10 or 11, which is at least one selected from a polypeptide, an antibody molecule, and an antisense nucleotide.   13. A compound according to claim 12, which is an antibody molecule.   13. A compound according to claim 12, which is a CTGF receptor antagonist.   Use of a compound identified and / or produced by the method according to any one of claims 1 to 9 in the treatment and / or prevention and / or diagnosis of fibrosis.   Use of a compound identified and / or produced by the method according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment and / or prevention and / or diagnosis of fibrosis.   Use of a compound according to claim 10 or 11 in the treatment and / or prevention and / or diagnosis of fibrosis.   Use of a compound according to claim 10 or 11 in the manufacture of a medicament for the treatment and / or prevention and / or diagnosis of fibrosis.   Said fibrosis is one or more diabetic nephropathy, non-diabetic renal fibrosis, pulmonary fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, myocardial fibrosis, atherosclerosis 19. Any one of claims 15-18, selected from sclerosis, systemic sclerosis, scleroderma, retinal fibrosis, radiation-induced fibrosis, keloid scar formation, and tumor-related fibrosis. Use as described in.   20. Use according to claim 19, wherein the fibrosis is diabetic nephropathy.   10. A fibrosis comprising administration of a therapeutically or prophylactically effective single dose or multiple doses of a compound identified and / or produced by the method of any one of claims 1-9. Methods of treatment and / or prevention.   15. A method for the treatment and / or prevention of fibrosis comprising the administration of a single or multiple doses of a compound as claimed in any one of claims 10 to 14 in a therapeutically or prophylactically effective amount.   Said fibrosis is one or more diabetic nephropathy, non-diabetic renal fibrosis, pulmonary fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, myocardial fibrosis, atherosclerosis 23. A method according to claim 21 or 22, wherein the method is selected from: sclerosis, systemic sclerosis, scleroderma, retinal fibrosis, radiation-induced fibrosis, keloid scar formation, and tumor-related fibrosis.   24. The method of claim 23, wherein the fibrosis is diabetic nephropathy.   Use of an agent capable of binding to a CTGF receptor agonist in the treatment and / or prevention and / or diagnosis of fibrosis.   Use of an agent capable of binding to a CTGF receptor agonist in the manufacture of a medicament for the treatment and / or prevention and / or diagnosis of fibrosis.   Said fibrosis is one or more diabetic nephropathy, non-diabetic renal fibrosis, pulmonary fibrosis, liver fibrosis (cirrhosis), skeletal muscle fibrosis, myocardial fibrosis, atherosclerosis 27. Use according to claim 25 or 26, selected from symptom, systemic sclerosis, scleroderma, retinal fibrosis, radiation-induced fibrosis, keloid scar formation and tumor-related fibrosis.   Use of an agent capable of binding to a CTGF receptor agonist in a method for reducing and / or preventing binding of a CTGF receptor agonist to a CTGF receptor in vivo or in vitro.   29. Use according to any one of claims 26 to 28, wherein the agent capable of binding to a CTGF receptor agonist is a CTGF receptor.   29. Use according to any one of claims 26 to 28, wherein the agent having the ability to bind to a CTGF receptor agonist is a CTGF receptor conjugated to an immunoglobulin Fc region.   Use according to claim 29 or 30, wherein the CTGF receptor is a TrkA receptor.   Use according to claim 29 or 30, wherein the CTGF receptor is a soluble form of the TrkA receptor.   A nucleic acid encoding the TrkA receptor joined to the Fc region of an immunoglobulin.   34. A vector comprising the nucleic acid of claim 33.   A polypeptide comprising a TrkA receptor joined to the Fc region of an immunoglobulin.   A cell comprising the nucleic acid according to claim 33 and / or the vector according to claim 34 and / or the polypeptide according to claim 35.   A nucleic acid according to claim 33 and / or a vector according to claim 34 and / or a polypeptide according to claim 35 and / or a cell according to claim 36, and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising an agent, wherein the nucleic acid and / or the vector and / or the polypeptide and / or the cell are present in an amount effective for the treatment and / or prevention and / or diagnosis of fibrosis.

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