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WO2013090991A1 - Thérapie par le tgf-bêta - Google Patents

Thérapie par le tgf-bêta Download PDF

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Publication number
WO2013090991A1
WO2013090991A1 PCT/AU2012/001550 AU2012001550W WO2013090991A1 WO 2013090991 A1 WO2013090991 A1 WO 2013090991A1 AU 2012001550 W AU2012001550 W AU 2012001550W WO 2013090991 A1 WO2013090991 A1 WO 2013090991A1
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Prior art keywords
tgf
catenin
tissue
inflammation
cells
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PCT/AU2012/001550
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English (en)
Inventor
David Harris
Zheng GUOPING
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The University Of Sydney
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Priority claimed from AU2011905390A external-priority patent/AU2011905390A0/en
Application filed by The University Of Sydney filed Critical The University Of Sydney
Publication of WO2013090991A1 publication Critical patent/WO2013090991A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect

Definitions

  • the invention relates to minimising tissue inflammation and to treatment of fibrosis.
  • TGF- ⁇ There are two basic but opposing biological functions of TGF- ⁇ in inflammatory diseases: anti-inflammatory and profibrotic (Blobe et al., 2000). The former is beneficial, while the latter is detrimental causing organ fibrosis and loss of function. Also, in cancer, TGF- ⁇ inhibits proliferation and induces apoptosis of tumor cells, but promotes metastasis by inducing an invasive phenotype of tumor cells through induction of epithelial-mesenchymal transition (EMT) (Kudo-Saito et al., 2009; Sahai, 2005). The conflicting roles of TGF- ⁇ , especially TGF- ⁇ make it very difficult to target TGF- ⁇ therapeutically.
  • EMT epithelial-mesenchymal transition
  • WO 2006/101858 Al and WO2007056513 discuss alpha-helix mimetic structures and use of same for the treatment of fibrotic disease. These documents discuss that the Wnt ⁇ -catenin pathway is the appropriate pathway to target with the mimetic structures discussed in those documents. These documents discuss that a particular structure, namely ICG- 001 (FW 548) blocks >95% of bleomycin-induced TCF/ ⁇ -catenin transcription in a murine model of pulmonary fibrosis in transgenic Bat-Gal mice. This compound and other mimetic structures discussed in these documents, through inhibition of dependent transcription, offer a novel therapeutic approach to fibrotic disease. That is, the mimetic structures, such as ICG-001 are said in these documents to be the therapeutic agents for treatment of fibrotic disease.
  • the invention seeks to address one or more of the above mentioned limitations concerning the use of TGF- ⁇ for treatment of disease and/or to provide improvements in the treatment of disease and in one embodiment provides a method of minimising inflammation in one or more tissues of an individual including the following steps:
  • TGF- ⁇ applying TGF- ⁇ to said one or more tissues in an amount effective for inducing an anti-inflammatory response in said one or more tissues, thereby minimising inflammation in said one or more tissues of said individual.
  • the tissue may be fibrotic or susceptible to fibrosis.
  • TGF- ⁇ written instructions for use of TGF- ⁇ to minimise inflammation in a tissue that has been depleted of substantially all complexes.
  • TGF- ⁇ to minimise inflammation in a tissue of an individual, wherein said individual is one who has been treated to deplete substantially all p-catenin/Smad3 complexes from inflamed tissue.
  • a method of minimising inflammation in one or more tissues of an individual including the following steps:
  • TGF- ⁇ 1 provides TGF- ⁇ 1 to the one or more tissues of the individual; thereby minimising inflammation in one or more tissues of an individual.
  • the p-catenin/Smad3 complex inhibitor and TGF- ⁇ may be provided sequentially or simultaneously.
  • kits including:
  • TGF- ⁇ and a p-catenin/Smad3 complex inhibitor to minimise inflammation in a tissue of an individual.
  • TGF- ⁇ 1 provides TGF- ⁇ 1 to the one or more tissues of the individual; thereby minimising fibrosis in one or more tissues of an individual.
  • FIG. 1 ⁇ -catenin knockdown chimera F-TrCP-Ecad inhibits TGF- ⁇ -induced EMT in Cl.l cells.
  • B-D Immunofluorescence images of E-cadherin, (B) vimentin (C) and fibronectin (D) staining in Cl.l cells exposed to respective treatments.
  • FIG. 1 ⁇ -catenin knockdown chimera F-TrCP-Ecad abolishes TGF- ⁇ -induced E- cadherin promoter repression, Snail transcription and MMP-9 expression in Cl.l cells.
  • (A) Relative E-cadherin promoter (-201 to +131) activity in Cl.l cells of control, pcDNA3 or F- TrCP-Ecad transfection treated with TGF- ⁇ (3 ng/ml) for 24 hours, and F-TrCP-Ecad transfection alone. Values are means ⁇ s.d. **P ⁇ 0.01 vs TGF- ⁇ 1 with or without vector control (pcDNA3), n 6.
  • FIG. 3 F-TrCP-Ecad promotes soluble nuclear/cytosolic ⁇ -catenin degradation.
  • (B) Western blot analysis for total ⁇ -catenin in Cl.l cells untreated, treated with TGF- ⁇ (3 ng/ml) for 24 hours, or transfected with pcDNA3 or F-TrCP-Ecad. Values are means ⁇ s.d. n 3 (C) Western blot analysis of ⁇ -catenin levels in membrane protein fraction extracted from Cl.l cells untreated, treated with TGF- ⁇ (3ng/ml) for 24 hours, or transfected with vector control (pcDNA3) or F-TrCP-Ecad alone, al Sodium Potassium ATPase (ATPase) was used as membrane protein control. Values are means ⁇ s.d.
  • (B) Co- immunoprecipitation of ⁇ -catenin, LEF-1, P-Smad3 and P-Smad2 from Cl.l cell nuclear proteins with rabbit LEF-1 antibody. Purified rabbit IgG was used as a negative control. Immunoprecipitates were analyzed by Western blot with ⁇ -catenin, LEF-1, P-Smad3 and P- Smad2 antibodies. Values are means ⁇ s.d.
  • FIG. 5 ⁇ -catenin is dispensable for macrophage activation and F-TrCP-Ecad or ICG- 001 has no effect on anti-inflammatory effects of TGF- ⁇ in macrophage.
  • A Total ⁇ -catenin protein levels in J774 cells stimulated by LPS (500ng ml) in absence or presence of TGF- ⁇ for 24 hours, or treated by TGF- ⁇ alone. Quantitation is shown by relative densitometry intensity. Values are means ⁇ s.d.
  • B Total ⁇ -catenin protein levels in J774 cells stimulated by IFN- ⁇ (20ng/ml) in absence or presence of TGF- ⁇ (lOng/ml) for 24 hours, or treated by TGF- ⁇ alone.
  • ⁇ -catenin is not involved in smad3 -dependent TGF- ⁇ inhibition of macrophage activation in J774 cells.
  • A Co-immunoprecipitation of ⁇ -catenin, P-Smad3 and P-Smad2 from J774 cell nuclear proteins with rabbit P-Smad3 antibody. Purified rabbit IgG was used as a negative control. Immunoprecipitates were analyzed by Western blot with ⁇ -catenin, P-Smad3 and P-Smad2 antibodies. Quantitation of immunoblots (relative intensity) is shown by fold increase in densitometry intentsity (the intensity of control is arbitrarily defined as 1). Values are means ⁇ s.d.
  • ⁇ -catenin had been observed to be associated with profibrotic effects such as epithelial to mesenchymal transition (EMT) induced by exposure of epithelial cells to TGF- ⁇ .
  • EMT epithelial to mesenchymal transition
  • ⁇ -catenin was not known to be involved in TGF- ⁇ - signalling in immune cells, particularly in TGF- ⁇ anti-inflammatory actions.
  • the inventors have found that ⁇ -catenin is involved in TGF- ⁇ induced profibrotic function as a cofactor with Smad3. Specifically, as described herein, the inventors have discovered a nuclear p-catenin/Smad3 complex in epithelial cells that have been exposed to TGF- ⁇ . They have also shown that that this complex is associated with EMT. Further, the inventors have shown that EMT is not observed when nuclear ⁇ -catenin is degraded thereby disrupting the -catenin/Smad3 complex.
  • the inventors have also found that the p-catenin/Smad3 complex observed in the nucleus of epithelial cells exposed to TGF- ⁇ is not formed in immune cells when these cells are exposed to TGF- ⁇ . Put in other words, the inventors have found that the very cells that provide an anti-inflammatory function in response to TGF- ⁇ exposure do not contain a nuclear complex. In this sense, the anti-iriflammatory response of immune cells to TGF- ⁇ exposure, while dependent on Smad3, is independent of ⁇ -catenin.
  • TGF- ⁇ full anti-inflammatory potential of TGF- ⁇ can be realised by providing TGF- ⁇ in circumstances where, because of sequential or simultaneous administration of a ⁇ -catenin inhibitor, the TGF- ⁇ 1 is introduced into an environment that has no complexes that would otherwise lead to EMT, and therefore into one where the profibrotic effects of administered TGF- ⁇ can be avoided.
  • inflamed tissue generally refers to a tissue having one or more of the classical hallmarks of acute or chronic inflammation including redness, heat, pain, swelling and potentially loss of function.
  • acute inflammation generally refers to the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and polymorphonuclear cells from the blood into the injured tissues.
  • chronic inflammation generally refers to a prolonged period of inflammation whereby there is a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.
  • fibrosis generally refers to the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process.
  • Fibrosis or fibroproliferative disease may arise from chronic inflammation resulting in excess deposition of extracellular matrix and loss of structure and function of relevant tissue. Fibrosis may also arise as a normal part of wound healing whereby following granulation, fibrotic tissue or scar is formed.
  • the methods of the embodiments disclosed herein relate especially to the treatment of fibrotic disease, particularly to fibrosis that is associated with chronic inflammation, rather than scar formation.
  • ⁇ -catenin generally refers a protein that in humans is encoded by the CTNNB1 gene. In Drosophila, the homologous protein is called armadillo, ⁇ -catenin is a subunit of the cadherin protein complex and has been implicated as an integral component in the Wnt signaling pathway.
  • Smad3 generally refers to a protein known as "Mothers against decapentaplegic homolog 3" also known as "SMAD family member 3".
  • the human SMAD3 gene is located on Chromosome 15.
  • Smad 3 is one of several human homologues of a gene that was originally discovered in Drosophila.
  • p-catenin/SmadS complexes generally refers to heteromeric complexes including ⁇ -catenin and Smad3. These complexes may be observed in the nucleus of cells that have been exposed to TGF- ⁇ and that have undergone, or that are in the process of undergoing epithelial to mesenchymal cell transition (EMT). TGF- ⁇ induced EMT generally cannot proceed in the absence of these complexes.
  • EMT epithelial to mesenchymal cell transition
  • deplete substantially all ⁇ -catenin/Smadi _ complexes generally refers to the removal of an amount of complexes from cell nuclei, especially epithelial cell nuclei, or mesenchymal nuclei derived therefrom, to preclude TGF- ⁇ induced EMT.
  • the complexes may be depleted by preventing association of ⁇ -catenin with Smad3, or of Smad3 with ⁇ -catenin, for example by degradation of either or both of ⁇ -catenin and Smad3, or by provision of an inhibitor that binds to ⁇ -catenin and/or Smad3, thereby preventing association of these proteins to form the complex.
  • TGF- ⁇ Transforming growth factor beta
  • TGF- ⁇ is a protein that controls proliferation, cellular differentiation and other functions in most cells. It plays a role in immunity, cancer, heart disease, diabetes and other syndromes.
  • TGF- ⁇ is a secreted protein that exists in at least three isoforms called TGF- ⁇ , TGF- 2, and TGF- 3. It was also the original name for TGF- ⁇ , which was the founding member of this family.
  • the TGF- ⁇ family is part of a superfamily of proteins known as the transforming growth factor beta superfamily which includes inhibins, activin, bone morphogenetic protein and others.
  • an anti-inflammatory response generally refers to a cellular response, in particular a response mediated by immune cells such as macrophages and like antigen presenting cells on exposure to TGF- ⁇ .
  • the response includes an inhibition of the functions of activated immune cells and induction of suppressive immune cells.
  • An anti-inflammatory response is beneficial insofar as it suppresses inappropriate inflammation, especially chronic inflammation, thereby minimising fibrosis and fibroproliferative disease* culminating in minimisation of formation of fibrotic tissue.
  • a profibrotic response generally refers to a cellular response, in particular a response mediated by epithelial cells on exposure to TGF- ⁇ . Hallmarks of a profibrotic response include the change in morphology of epithelial cells to represent mesenchymal cells. In certain organs, functional cells such as stellate cells may convert to myofibroblasts, leading to production of further pathologic cytokine profiles and cellular activation.
  • TGF- ⁇ As discussed herein, the anti-inflammatory properties of TGF- ⁇ have been recognised for some time. However, the therapeutic potential of TGF- ⁇ has not been realised in the clinic because of the profibrotic function of TGF- ⁇ 1.
  • a method of minimising inflammation in one or more tissues of an individual including the following steps: - selecting and/or providing an individual having one or more inflamed tissues, and in whom the one or more inflamed tissues have been treated to deplete substantially all ⁇ -catenin/Smad3 complexes in the tissues;
  • TGF- ⁇ - applying TGF- ⁇ to said one or more tissues in an amount effective for inducing an anti-inflammatory response in said one or more tissues, thereby minimising inflammation in said one or more tissues of said individual.
  • TGF- ⁇ to minimise inflammation in a tissue of an individual, wherein said individual is one who has been treated to deplete substantially all -catenin/Smad3 complexes from inflamed tissue.
  • the individual may have acute or chronic inflammation in tissues.
  • the inflammation may arise from any form of tissue injury including accidental injury and intended injury (the latter including injury arising through clinical procedure).
  • the tissues may be fibrotic, susceptible to fibrosis, or otherwise have symptoms of fibroproliferative disease.
  • the tissue is organ tissue, for example kidney, liver, ovary, heart, lung, eye or neural tissue.
  • kidney or renal tissue it may be associated with scarring or fibroproliferative disorder.
  • diseases include polycystic kidney disease, renal fibrotic disease, glomerular nephritis and nephritis associated with systemic lupus.
  • the tissue is liver tissue, it may be associated with cirrhosis or other fibrosis.
  • the tissue is ovarian, it may be associated with polysystic ovarian syndrome.
  • tissue is myocardium, it may be associated with myocardial fibrosis.
  • tissue is lung tissue, it may be associated with pulmonary fibrosis or interstitial lung disease including idiopathic pulmonary fibrosis.
  • tissue is ocular tissue, it may be associated with Grave's ophthalmopathy, glaucoma, diabetic retinopathy and the like.
  • tissue is neural tissue, it may be associated with Alzheimer's disease or other neurodegenerative disorder.
  • the individual provided for, or selected for treatment is one who has been prior or pre-treated to deplete substantially all p-catenm/Smad3 complexes in the inflamed tissue.
  • this depletion is achieved by treating the individual with a ⁇ -catenin inhibitor in the form of a compound that prevents the formation of the P-catenin/Smad3 complex in the epithelial cells, especially the nucleus of epithelial cells.
  • the ⁇ -catenin inhibitor may not interfere with the function of membrane bound ⁇ -catenin, or ⁇ -catenin that is located in the cytoplasm.
  • the inhibitor may selectively inhibit activity of ⁇ -catenin in the cell nucleus while leaving other ⁇ -catenin activities elsewhere, including for example complexing with Smad3, unaffected.
  • ⁇ -catenin inhibitors useful in the invention is described further herein, although it will be understood that having demonstrated the principle of the invention in the form of the ⁇ depletion of P-catenin/Smad3 complexes in the nucleus of epithelial cells for minimising profibrotic TGF- ⁇ induced responses and a screening or discovery method for identifying other useful inhibitors, the invention is not limited to the use of a specific structural class of inhibitor and in fact extends to all compounds capable of inhibiting the formation of p-catenin/Smad3 complexes that are formed in epithelial cells in response to TGF- ⁇ exposure.
  • the individual selected for, or provided for treatment may be one having inflammatory and/or fibrotic disease and who has not been treated to deplete substantially all p-catenin/Smad3 complexes. Accordingly, in one embodiment there is provided a method of minimising inflammation in one or more tissues of an individual including the following steps:
  • TGF- ⁇ provides TGF- ⁇ to the one or more tissues of the individual; thereby minimising inflammation in one or more tissues of an individual.
  • the P-catenin/Smad3 complex inhibitor and TGF- ⁇ may be provided sequentially or simultaneously. Examples of compositions suitable for this purpose are discussed further herein.
  • the individual may not have fibrosis at the time of treatment, in particular at the time of administration of complex inhibitor to the individual.
  • the individual may be susceptible to fibrosis or have active fibrotic disease.
  • Individuals who are susceptible to fibrosis are generally those who have a prolonged or chronic form of inflammation. It will be understood therefore that the methods of the invention are generally applicable for prophylaxis or therapy of fibrotic disorders and fibroproliferative disease.
  • a method for minimising fibrosis in a tissue of an individual including the following steps: - providing a p-catenin/Smad3 complex inhibitor to the one or more tissues of the individual;
  • the p-catenin/Smad3 complex inhibitor may be administered by any means known to one of ordinary skill in the art including orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, ihtrastemal, intracranial, and intraosseous injection and infusion techniques. The exact administration protocol will vary depending upon various factors including the age, body weight, general health, gender and diet of the patient; the determination of specific administration procedures would be routine to an one of ordinary skill in the art.
  • the p-catenin/Smad3 complex inhibitor may be administered by a single dose, multiple discrete doses or continuous infusion.
  • Pump means particularly subcutaneous pump means, are useful for continuous infusion.
  • Dose levels on the order of about 0.001 mg/kg/d to about 100 mg kg/d of a P-catenin/Smad3 complex inhibitor are useful in the above described methods.
  • the dose level is about 0.1 mg/kg/d to about 100 mg/kg/d.
  • the dose level is about 1 mg/kg/d to about 10 mg/kg/d.
  • the specific dose level for any particular patient will vary depending upon various factors, including the activity and the possible toxicity of the specific compound employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the rate of excretion; the drug combination; the severity of the disease; and the form of administration.
  • in vitro dosage-effect results provide useful guidance on the proper doses for patient administration.
  • the considerations for determining the proper dose levels are well known in the art and within the skills of an ordinary physician. Any known administration regimen for regulating the timing and sequence of drug delivery may be used and repeated as necessary to effect treatment in the methods described above.
  • the -catenin/Smad3 - complex inhibitor may include pretreatment and/or co- administration with additional therapeutic agent(s).
  • the p-catenin/Smad3 complex inhibitor can be administered alone or in combination with one or more additional therapeutic agent(s) for simultaneous, separate, or sequential use.
  • additional therapeutic agent include, without limitation steroids (e.g., hydrocortisones such as methylprednisolone); anti-immune drugs, such as methotrexate, azathioprine, cyclophosphamide or cyclosporin A; interferon- ⁇ ; antibodies, such as anti-CD4 antibodies; chemotherapeutic agents; immunotherapeutic ' compositions; electromagnetic radiosensitizers; and morphine.
  • the - ⁇ 3 ⁇ /8 ⁇ 3 ⁇ 3 complex inhibitor may be co-administered with one or more additional therapeutic agent(s) either (i) together in a single formulation, or (ii) separately in individual formulations designed for optimal release rates of their respective active agent.
  • additional therapeutic agent(s) either (i) together in a single formulation, or (ii) separately in individual formulations designed for optimal release rates of their respective active agent.
  • the objective of the -catenin/Smad3 complex inhibitor is to facilitate the removal of an amount of p-catenin/Smad3 complexes from cell nuclei, especially epithelial cell nuclei, or mesenchymal nuclei derived therefrom, to preclude TGF- ⁇ induced EMT and related fibrotic responses.
  • epithelial cells are cultured in vitro with TGF- ⁇ in the presence or absence of inhibitor and the amount of complex formed in the nucleus of cells exposed to inhibitor is determined by one or more of co-immunoprecipitation, Western blotting and immunofluorescence.
  • the untreated cell culture is used as a positive control for determining the amount of reduction of complexes.
  • Another approach is simply to compare the morphology of treated and untreated cells exposed to TGF- ⁇ and to determine whether exposure to inhibitor prevents the transition to a mesenchymal phenotype.
  • WO2006/101858 discusses that ICG-001 (FW 548) blocks >95% of bleomycin-induced TCF ⁇ -catenin transcription in a murine model of pulmonary fibrosis in transgenic Bat-Gal mice.
  • This compound and other mimetic structures discussed in WO2006/101858 are considered in WO2006/101858, through inhibition-of Wnt/ -catenin/CBP dependent transcription, to offer a novel therapeutic approach to fibrotic disease. That is, the mimetic structures, such as ICG-001 are said in WO2006/101858 to be the therapeutic agents for treatment of fibrotic disease.
  • these compounds are not employed in the methods of the invention herein as active agents having antiinflammatory function, but rather as agents capable of blocking the profibrotic effects of TGF- ⁇ , thereby enabling TGF- ⁇ to be used as an active agent for treatment of inflammation.
  • the ⁇ -catenin inhibitor takes the form of an alpha-helix mimetic as generally disclosed in WO2006/101858.
  • One particularly useful ⁇ -catenin inhibitor is ICG-001.
  • B is -(CHRi 0 - or -N-R4-
  • Y is oxygen or sulfur
  • X and Z is independently nitrogen or CH
  • n 0 or 1
  • Rj, R 2 , R3, R , R 5 , R6, R , R 8 , R9 and R 10 are the same or different and independently selected from an amino acid side chain moiety or derivative thereof, the remainder of the molecule, a linker and a solid support, and
  • , R 2 , R 3 , R4, R 5 , R6, R 7 , R 8 , R 9 and Rio are independently selected from the group consisting of hydrogen, aminoC .5alkyl, guanidineC 2 -salkyl, Ci.
  • Ry, 2 , R 6 , and R 7 , R 8 and R9 of G are the same or different and represent the remainder of the compound, and R 3 , R4 or R5 are selected from an amino acid side chain moiety or derivative thereof.
  • the term "remainder of the compound” means any moiety, agent, compound, support, molecule, linker, amino acid, peptide or protein covalently attached to the.a-helix mimetic structure at Rj, R 2 , R 5 , ⁇ , R7, Rs and/or R9 positions. This term also includes amino acid side chain moieties and derivatives thereof.
  • amino acid side chain moiety represents any amino acid side chain moiety present in naturally occurring proteins including (but not limited to) the naturally occurring amino acid side chain moieties identified in Table 1.
  • Other naturally occurring amino acid side chain moieties of this invention include (but are not limited to) the side chain moieties of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine, ⁇ -carboxyglutamate, phosphotyrosine and phosphoserine.
  • glycosylated amino acid side chains may also be used in the practice of this invention, including (but not limited to) glycosylated threonine, serine and asparagine.
  • amino acid side chain moieties of the present invention also include various derivatives thereof.
  • a "derivative" of an amino acid side chain moiety includes modifications and/or variations to naturally occurring amino acid side chain moieties.
  • the amino acid side chain moieties of alanine, valine, leucine, isoleucine and phenylalanine may generally be classified as lower chain alkyl, aryl, or arylalkyl moieties.
  • amino acid side chain moieties include other straight chain or branched, cyclic or non-cyclic, substitutes or unsubstituted, saturated or unsaturated lower chain alkyl, aryl or arylalkyl moieties.
  • amino acid side chain derivative is selected from a C ⁇ n alkyl, a C 6- i2 aryl and a C 7 - 12 arylalkyl, and in a more preferred embodiment, from a C 1-7 alkyl, a C6- 10 aryl and a C 7- n arylalkyl.
  • Amino side chain derivatives of this invention further include substituted derivatives of lower chain alkyl, aryl, and arylalkyl moieties, wherein the substituents are selected from (but are not limited to) one or more of the following chemical moieties: -OH, -OR, -COOH, -COOR, -CONH2, -NH 2 , -NHR, -NRR, -SH, -SR, -S0 2 R, -S0 2 H, -SOR and halogen (including F, CI, Br and I), wherein each occurrence of R is independently selected from straight chain or branched, cyclic or non-cyclic, substituted or unsubstituted, saturated or unsaturated lower chain alkyl, aryl, and arylalkyl moieties.
  • substituents are selected from (but are not limited to) one or more of the following chemical moieties: -OH, -OR, -COOH, -COOR, -
  • cyclic lower chain alkyl, aryl and arylalkyl moieties of this invention include naphthalene, as well as heterocyclic compounds such as thiophene, pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole.
  • Amino acid side chain derivatives further include heteroalkyl derivatives of the alkyl portion of the lower chain alkyl and arylalkyl moieties, including (but not limited to) alkyl and arylalkyl phosphonates and silanes.
  • R 2 , R 5 , Rg, R 7 , R 8 and R 9 moieties specifically include (but are not limited to) -OH, -OR, -COR, -COOR, -CONH 2 , -CONR, -CONRR, -NH 2 , -NHR, -NRR, -S0 2 R and -COSR, wherein each occurrence of R is as defined above.
  • Ri, R 2 , R 5 , R6, R7, e or R 9 may be a linker facilitating the linkage of the compound to another moiety or compound.
  • the compounds of this invention may be linked to one or more known compounds, such as biotin, for use in diagnostic or screening assay.
  • Ri, R 2 , R 5 , R 6 , R7, 3 ⁇ 4 or R9 may be a linker joining the compound to a solid support (such as a support used in solid phase peptide synthesis) or alternatively, may be the support itself.
  • linkage to another moiety or compound, or to a solid support is preferable at the Ri, R 2 , R 7 or R 3 ⁇ 4 position, and more preferably at the Ri or R 2 position.
  • Ri , R 2 , R4, Re, 3 ⁇ 4 W and X are as defined above, Z is nitrogen or CH (when Z is CH, then X is nitrogen).
  • Ri, R 2 , R 3 ⁇ 4 , and R9 represent the remainder of the compound, and R4 is selected from an amino acid side chain moiety.
  • A is -0-CHR 3 -
  • B is -NR4-
  • E is -(ZR 6 )-
  • G is (XR 7 ) n -
  • the compounds of this invention have the following formula (III):
  • Ri, R 2 , R4, R$, R 7 , W, X and n are as defined above, and Z is nitrogen or CH (when Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen and n is not zero).
  • R 1; R 2 , and R 7 represent the remainder of the compound, and R4 is selected from an amino acid side chain moiety.
  • 3 ⁇ 4 or R 7 may be selected from an amino acid side chain moiety when Z and X are CH, respectively.
  • the compound of the present invention is the compound of formula (IV):
  • the compounds of the present invention can be synthesised by any methods known to the skilled person, including the methods described in WO2006/101858 and US 6013458.
  • ICG-001 described herein is a preferred compound for use as a p-catenin/Smad3 complex inhibitor.
  • Other potential inhibitors can be identified by a system exemplified by the inventors in the Examples described below.
  • epithelial cells are cultured in vitro with TGF- ⁇ in the presence or absence of inhibitor and the amount of complex formed in the nucleus of cells exposed to inhibitor is determined by one or more of co-immunoprecipitation, Western blotting and immunofluorescence.
  • the untreated cell culture is used as a positive control for determining the amount of reduction of complexes.
  • Another approach is simply to compare the morphology of treated and untreated cells exposed to TGF- ⁇ and to determine whether exposure to inhibitor prevents the transition to a mesenchymal phenotype.
  • the present invention provides for administration of TGF- ⁇ to one or more tissues to induce an anti-inflammatory response therein.
  • TGF- ⁇ compositions to be used in the therapy will be formulated and dosed in a fashion consistent with good medical practice, the condition of the individual patient, the site of delivery of the TGF- ⁇ , the method of administration and other factors known to practitioners.
  • Conventional routes of administration include oral administration, peritoneal injection, or repetitive peripheral injections
  • TGF- ⁇ can be formulated into a sterile sustained release composition suitable for injection or implantation or capsule.
  • the TGF- ⁇ is formulated into suppositories with pharmaceutically acceptable oleaginous substances as is generally known in the art.
  • Sustained release formulations will be selected from the classes of microcapsular particles and implantable articles such as a biodegradable matrix or microcapsule.
  • a suitable material for this purpose is a polylactide, although other polymers of poly (a-hydroxycarboxylic acids), such as poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), can be used.
  • Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters) or poly(orthocarbonates).
  • the initial consideration here must be that the carrier itself, or its degradation products, is nontoxic in the target tissue and will not further aggravate the disease. This can be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals.
  • the present invention contemplates methods of systemic treatment with
  • TGF- ⁇ which are minimally invasive.
  • systemic means nonlocal.
  • non-local can include a method whereby a protein or other bioactive agent is introduced to a subject at a single local site, such as but not limited to a peripheral percutaneous site or a central site, so as to effectuate treatment of the subject's whole body rather than just at the single local site.
  • minimally-invasive means non-invasive or non-open-field surgical methods.
  • minimally-invasive methods can include procedures involving an incision(s) or implantation of a medical device(s).
  • the TGF- ⁇ can be targeted to a specific cell-type by complexing of the TGF- ⁇ with an appropriate immunoglobulin (Ig).
  • the provided complex contains an immunoglobulin (Ig) portion and a TGF- ⁇ 1 portion.
  • the Ig portion can contain one Ig protein, or a plurality of Ig proteins, which can be full length Ig molecules or functional regions thereof, and include wild-type and variant Ig proteins as described herein.
  • the Ig protein can be any known Ig protein, including a full-length immunoglobulin (such as a human IgGl, IgG2, IgG3 or IgG4) or functional region thereof such as an Fc region or portion thereof, including the Ig proteins disclosed herein.
  • the Ig protein is an IgG or functional region thereof.
  • the TGF- ⁇ portion contains one or more TGF- ⁇ protein.
  • the TGF- ⁇ portion contains two TGF- ⁇ proteins, that are monomers associated to form a dimer via covalent bond, such as an activated, mature, TGF- ⁇ homodimer, for example, a 25 kDa TGF- ⁇ homodimer or variant thereof.
  • the present invention provides for use of suitable derivatives of monomeric TGF ⁇ ls that may be utilised to induce an anti-inflammatory response in one or more tissues.
  • therapeutically effective peptide derivatives of monomeric TGF ⁇ ls include: therapeutically effective peptide derivatives of monomeric TGF ⁇ ls (or their fragments); therapeutically effective fragments or derivatives comprising or based on the pharmacophore of monomeric TGF ⁇ ls (or their fragments); therapeutically effective peptoid derivatives of monomeric TGF ⁇ ls (or their fragments); therapeutically effective D- amino acid derivatives of monomeric TGF-pis (or their fragments); therapeutically effective peptidomimetics based on monomeric TGF ⁇ ls (or their fragments); therapeutically effective peptide analogues of monomeric TGF ⁇ ls (or their fragments); therapeutically effective pseudopeptides based on monomeric TGF ⁇ ls (or their fragments); therapeutically effective retro-inverso peptides based on monomeric TGF-pls (or their fragments); therapeutically effective depsipeptide derivatives based on monomeric TGF ⁇ ls (or their fragments); therapeutically effective ⁇
  • the present invention provides for use of modified or chimeric TGF- ⁇ polypeptides for inducing an anti-inflammatory response.
  • the chimeric TGF- ⁇ comprises a core domain from a first TGF-beta superfamily member and a variable domain from a second TGF-beta superfamily member.
  • the chimeric protein may be an agonist of the second TGF-beta superfamily member. Agonist forms will generally retain dimerization and receptor binding activities that are similar to those of the second TGF-beta superfamily member.
  • the chimeric protein may be an antagonist of the second TGF-beta superfamily member.
  • Antagonists may, for example, compete for binding to one or more receptors but fail to form a complex with the components or conformation necessary for triggering a signal transduction cascade.
  • the chimeric protein may be an agonist or an antagonist of a third TGF-beta superfamily member.
  • a chimeric TGF-beta superfamily protein comprises core domains from at least two different naturally-occurring TGF-beta superfamily members.
  • a chimeric TGF-beta superfamily protein comprises variable domains from at least two different naturally-occurring TGF-beta superfamily members.
  • variable and/or core domains are randomized or otherwise altered so as not to correspond precisely to a variable domain of any naturally occurring TGF-beta superfamily protein.
  • a core domain and/or a variable domain of a chimeric TGF-beta superfamily protein are randomized or otherwise altered so as not to correspond precisely to a variable domain of any naturally occurring TGF-beta superfamily protein.
  • Bioly active polypeptides herein are defined as those having the ability to cross- react with antisera raised against native TGF- ⁇ (where native TGF- ⁇ is that which is obtained from platelets or other natural sources). Immunological cross-reactivity is a measure of a single active epitope and does not necessarily encompass an active domain involved in immunosuppressive activity.
  • “Therapeutic biological activity” and “therapeutically biologically active” refer to activity that not only encompasses biological activity as defined above, but also the ability to inhibit the proliferation of a cultured established mink lung cell line CCL 64 and/or to inhibit the proliferation of human lymphocytes to an antigen such as tetanus toxoid and/or to mitogens such as phytohemagglutimn P (PHA-P) or Concanavalin A (ConA). Polypeptides with therapeutic biological activity- are preferred.
  • TGF-pi TGF-pi
  • a pharmaceutically acceptable diluent, excipient or carrier a pharmaceutically acceptable diluent, excipient or carrier.
  • composition may comprise one or more additional pharmaceutically acceptable ingredient(s), including without limitation one or more wetting agent(s), buffering agent(s), suspending agent(s), lubricating agent(s), emuisifier(s), disintegrant(s), absorbent(s), preservative(s), surfactant(s), colorant(s), flavorant(s), sweetener(s) and additional therapeutic agent(s).
  • additional pharmaceutically acceptable ingredient(s) including without limitation one or more wetting agent(s), buffering agent(s), suspending agent(s), lubricating agent(s), emuisifier(s), disintegrant(s), absorbent(s), preservative(s), surfactant(s), colorant(s), flavorant(s), sweetener(s) and additional therapeutic agent(s).
  • the inventive pharmaceutical composition may be formulated into solid or liquid form for the following: (1) oral administration as, for example, a drench (aqueous or non-aqueous solution or suspension), tablet (for example, targeted for buccal, sublingual or systemic absorption), bolus, powder, granule, paste for application to the tongue, hard gelatin capsule, soft gelatin capsule, mouth spray, emulsion and microemulsion; (2) parenteral administration by, for example, subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution, suspension or sustained-release formulation; (3) topical application as, for example, a cream, ointment, or controlled-release patch or spray applied to the skin; (4) intravaginal or intrarectal administration as, for example, a pessary, cream or foam; (5) sublingual administration; (6) ocular administration; (7) transdermal administration; or (8) nasal administration.
  • oral administration as, for example, a drench (aqueous or non-
  • composition can be advantageously combined with traditional wound dressings including primary and secondary dressings, wet-to-dry dressings, absorbent dressings, nonadherent dressings, semipermeable dressings, transparent dressings, hydrocolloid dressings, hydrogels, foam dressings, alginate dressings, surgical tapes and the like as is appropriate for the type of tissue being treated.
  • Additional active ingredients may include arginine, glutamine, zinc, copper, vitamin C, vitamin Bl, vitamin B2, vitamin B3, vitamin B6, vitamin B12, and folate or growth factors such as epidermal growth factor, platelet derived growth factor, insulin-like growth factor, keratinocyte growth factor, vascular endothelial growth factor, transforming growth factor alpha, nerve growth factor, connective tissue growth factor and granulocyte-monocyte colony stimulating factor.
  • Inflammation inhibitor such as interleukin-1 inhibitor, interleukin-6 inhibitor and tumor necrosis factor-alpha inhibitor may also be added to the composition.
  • pain relief agents, disinfectants, antibiotics and other active ingredients suitable for particular antiinflammatory applications may also be added thereto.
  • kits including: - TGF- ⁇
  • kits including: - TGF- ⁇
  • TGF- ⁇ is known to be both anti-inflammatory and profibrotic.
  • Cross-talk between TGF- ⁇ /Smad and Wnfc ⁇ -catenin pathways in epithelial-mesenchymal transition (EMT) suggests a specific role for ⁇ -catenin in profibrotic effects of TGF- ⁇ .
  • EMT epithelial-mesenchymal transition
  • F-TrCP-Ecad selectively degraded TGF- ⁇ - induced cytoplasmic ⁇ -catenin and blocked EMT of C 1.1 (renal tubular epithelial) cells.
  • Both F- TrCP-Ecad and ICG-001 blocked TGF- ⁇ -induced and Smad reporter activity in C 1.1 cells, suggesting that TGF- ⁇ -induced EMT depends on ⁇ -catenin binding to Smad3 but not LEF-1 downstream of Smad3 through canonical Wnt.
  • TGF- ⁇ -induced EMT in Cl.l cells is inhibited by cytosolic ⁇ -catenin knockdown chimera F-TrCP-Ecad.
  • Subconfluent tubular epithelial cells (Cl.l) cultured in the presence of 3 ng/ml TGF- ⁇ for 24 hours showed a change in morphology from cuboid clustered epithelial cells to spindle-shaped scattered fibroblast-like cells.
  • Cells treated with TGF- ⁇ 1 lost expression of the epithelial marker E-cadherin and acquired expression of the mesenchymal markers vimentin and fibronectin (Fig 1 A-F).
  • F-TrCP-Ecad chimera preferentially targets soluble nuclear/ cytosolic ⁇ -catenin for degradation.
  • Knockdown of cytosolic ⁇ -catenin inhibits TGF- ⁇ -induced EMT through inhibition of ⁇ - catenin/P-Smad3 but not ⁇ -catenin/LEF-l.
  • ICG-001 which selectively inhibits ⁇ -catenin nuclear activity by reducing its binding to the cAMP-response element-binding protein binding protein (CBP) (Teo et al., 2005), was also found to inhibit the -c&ttmnlV-Sma.d ' h complex formation in Smad3 co-IP (Fig 4A).
  • CBP cAMP-response element-binding protein binding protein binding protein binding protein
  • TrCP-Ecad transfection or ICG-001 treatment in the absence of TGF- ⁇ did not change the base line level of nuclear ⁇ -catenin in Smad3 co-IP.
  • TrCP-Ecad and ICG-001 jnay also block ⁇ -catenin interaction with LEF/TCF of the canonical Wnt pathway, thereby not excluding the dependence on ⁇ -catenin as a downstream effect of Smad.
  • co-IP of ⁇ -catenin with LEF-1 and TOP-Flash assay to determine canonical signaling in Cl . l cells.
  • TGF- ⁇ treatment did not increase the level of ⁇ -catenin co-precipitated by anti-LEF-1 antibody (Fig 4B), indicating that no ⁇ -catenin/LEF-l complex formation was stimulated by TGF- ⁇ .
  • the TOP-flash/FOP- flash luciferase activity was increased approximately 15-fold in Cl.l cells of LiCl-treated a positive control (Fig 4C), but only minimal TOP-flash/FOP-flash luciferase activity was detected and was almost unchanged (P>0.05) when Cl.l cells were treated with TGF- ⁇ with or without F-TrCP-Ecad transfection or ICG-001 treatment.
  • TGF- ⁇ -induced EMT To confirm ⁇ -catenin as a transcriptional co-factor of Smad in TGF- ⁇ -induced EMT, we performed a SMAD reporter assay as an indicator of TGF- ⁇ 1 -induced Smad signaling. As shown in Fig 4D, TGF- ⁇ induced a 7 fold increase in Smad reporter activity in Cl.l cells, However, disruption of complex formation by F-TrCP-Ecad transfection, or by direct inhibition of ⁇ -03 ⁇ 6 ⁇ /8 ⁇ 3 using ICG-001 (shown in Fig 4A) blocked TGF- ⁇ -induced Smad reporter activity to about 1/3, proving a dependence on ⁇ -catenin of Smad3 binding to Smad transcriptional responsive elements. The remaining TGF- ⁇ -induced Smad reporter activity after blockade with F-TrCP-Ecad or ICG-001 was presumably ⁇ - ⁇ -catenin dependent.
  • TGF- ⁇ -induced EMT in Cl.l cells involves ⁇ -catenin as a transcriptional co-factor for Smad3, but not as a downstream factor of Smad involving ⁇ - catenin/LEF-1 interaction.
  • Degradation of cytosolic ⁇ -catenin or direct inhibition of ⁇ -catenin binding to its co-factors diminished ⁇ -03 ⁇ / ⁇ -8 ⁇ 3 ⁇ 3 complex formation and blocked TGF- ⁇ - induced EMT in non-cancer Cl.l cells.
  • ⁇ -catenin is dispensible for TGF- ⁇ inhibition of macrophage activation.
  • TGF- ⁇ inhibited the expression of LPS-induced inflammatory mediator TNF-a mRNA, and of iNOS mRNA expression stimulated by IFN- ⁇ in J774 cells (Fig 5E, F).
  • TGF- ⁇ -induced down-regulation of macrophage activation was not affected in J774 cells transfected with F-TrCP-Ecad or WT ⁇ -cat, or treated by ICG-001 (Fig 5E, F).
  • F-TrCP-Ecad chimera only transfected and ICG-001 or TGF- ⁇ only treated J774 cells maintained a similar state of activation to controls (Fig.5E, F).
  • ⁇ -catenin The steady state level of ⁇ -catenin was low (Fig.5A, B) and there was no membrane staining of ⁇ -catenin in J774 cells (data not shown). IFN- ⁇ or LPS with or without TGF- ⁇ had no effect on ⁇ -catenin expression (Fig.5A, B). Similar to Cl.l cells, transfection of F-TrCP-Ecad chimera significantly reduced endogenous ⁇ -catenin levels (Fig 5C, D. P ⁇ 0.05) proving the effectiveness of the chimera in knocking down cytosolic ⁇ -catenin in macrophages. ⁇ -catenin is not involved in smad3 -dependent TGF- ⁇ inhibition of macrophage activation in J774 cells.
  • F-TrCP-Ecad chimera or ICG-001 did not change the levels of nuclear ⁇ -catenin of either total input or Smad3 co-IP, suggesting no ⁇ -catenin nuclear translocation or binding to Smad3, No Smad2 was present in P-Smad3 co-IP.
  • TOP-flash/FOP-flash luciferase activity was low and not changed in J774 cells treated with TGF- ⁇ with or without F-TrCP-Ecad transfection (Fig.6B), showing an absence of ⁇ -catenin/LEF/TCF dependent canonical Wnt signaling.
  • LiCl stimulated TOP-flash luciferase activity was blocked by F-TrCP-Ecad transfection or ICG-001 treatment (Fig 6B), proving their effectiveness as inhibitors of ⁇ -catenin-dependent canonical Wnt signaling in J774 macrophages.
  • TGF- ⁇ -induced Smad reporter activity in J774 cells was about the same level as in C 1.1 cells when ⁇ -catenin/Smad interaction was inhibited by F-TrCP-Ecad chimera or ICG-001, further corafirming an absence of ⁇ -catenin from the Smad complexes in macrophages.
  • ⁇ -catenin can be targeted to dissect the profibrotic effect of TGF- ⁇ in renal tubular epithelial cells from its anti -inflammatory effects through macrophages.
  • targeted degradation of cytosolic ⁇ -catenin or inhibition of ⁇ -catenin binding to Smad3 blocked TGF- ⁇ -induced EMT, and that this depends on P-catenin/Smad3 interaction as transcriptional co-activators but does not depend on canonical Wnt signaling in murine renal tubular epithelial Cl.l cells.
  • TGF- ⁇ -mediated inhibition of macrophage LPS-induced TNF-a and IFN-y-stimulated iNOS expression does not involve interaction or canonical ⁇ -catenin/LEF-l signaling. Thereby, TGF- ⁇ inhibition of macrophage activation was not compromised by the targeted inhibition of ⁇ - catenin/Smad3 interaction.
  • EMT Epithelial-mesenchymal transition
  • EMT extracellular matrix
  • Smads have been shown to cross-talk with components of other pathways such as RhoA, Ras, MAPK, Notch and Wnt/p-catenin.
  • TGF-p/Smad cross-talks with MAPK, ILK and Wnt (Guo and Wang, 2009) which are three major EMT pathways (Chun et al., 2008; Nieto, 2011) that converge at direct or indirect inactivation of GSK-3P (Hu et al., 2008; Thornton et al., 2008). Consequently, activating ⁇ -catenin whose target genes (e.g. Snail, Twist, LEF1, Jagged 1) are key transcription factors inducing EMT.
  • target genes e.g. Snail, Twist, LEF1, Jagged 1
  • Smad-dependent transcription is further dependent on other transcription co-factors which are both constitutive and cell type specific (Kim et al., 2009a).
  • the transcription co-factors especially those that are cell type specific, not only stabilize Smad association with DNA, but may also determine which specific gene(s) that the Smad complex targets, ⁇ -catenin of the Wnt pathway has been found to form ⁇ -catenin- Smad complexes in TGF ⁇ -induced EMT in lung alveolar epithelial cells (Kim et al., 2009a) and to facilitate ⁇ -catenin transcriptional activity in chondrocytes (Zhang et al., 2010).
  • the detailed molecular mechanisms remain poorly understood.
  • TGF- ⁇ is anti-inflammatory (Li et al, 2006). TGF- ⁇ down-regulates inflammatory processes by inhibiting activated immune cells and by inducing suppressive immune cells (Schmidt- Weber and Blaser, 2004). The concomitant profibrotic effect of TGF- ⁇ has limited its clinical use as an anti-inflammatory agent. We hypothesized that dependence on transcriptional co-factors to Smads in immune cells may differ from that of non- immune cells thereby generating different effects of TGF- ⁇ 1.
  • ⁇ -catenin is unique in that it has not, according to the published literature, been linked to anti-inflammatory processes. This suggests that by targeting ⁇ -catenin, it may be possible to separate the antiinflammatory and profibrotic effects of TGF- ⁇ . Stimulation of macrophages with IFN- ⁇ and /or microbial products such as LPS induces their activation (Bogdan et al., 1992; Takaki et al., 2006). Smad3 is reported to be a critical transcription factor responsible for TGF- ⁇ -mediated inhibition of macrophage activation (Werner et al., 2000).
  • F-TrCP-Ecad constructs encoding a chimeric protein with the ⁇ -catenin binding domain of E- cadherin fused to ⁇ ubiquitin-protein ligase, were kindly provided by Dr. Feng Cong. Wild- type (Buchert et al.) ⁇ -catenin construct: Flag-tagged WT ⁇ -cat fused into pcDNA3. E-cadherin promoter luciferase reporter constructs: pmoEcad (-201 to -131)/GL3, with mouse E-cadherin promoter -201 to -131 driving firefly luciferase reporter gene. pRL-RSV with RSV promoter driving Renilla luciferase as a transfection control. Plasmids were prepared using EndoFree plasmid kits (Qiagen).
  • Lipofectamine 2000 Invitrogen
  • PromoFectin-Macrophage Banksia Scientific
  • Subcellular Protein Extraction Cultured Cl.l and J774 cell lines were harvested, washed and pelleted in phosphate-buffered saline (PBS) at 850g for 2 minutes. Each cell pellet, containing 5x10 6 cells, was subjected to membrane protein extraction using ProteoJETTM Membrane Protein Extraction Kit (Thermo Scientific) and cytoplasmic and nuclear protein extraction using ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit (Thermo Scientific) as described by the manufacturer. The isolated membrane and cytoplasmic protein fractions were used directly in SDS-PAGE and Western blotting, and nuclear protein was used in co-immunoprecipitation.
  • PBS phosphate-buffered saline
  • Cl.l cells were split into 12-well plates and each well received 0.5 g E-cadherin promoter luciferase reporter construct, 0.02 ⁇ g Renilla luciferase construct pRL-RSV, with or without 1.0 ⁇ g of listed plasmids. After treatment for 24 h, cells were lysed and Firefly luciferase and Renilla luciferase activities were measured using Dual-Glo luciferase assay system (Promega) by a luminometer (Wallac 1420 Victor Plate Reader, Perkin Elmer Life Sciences). The Firefly luciferase activity was normalized against Renilla luciferase activity.
  • TOP -Flash assay The ⁇ -catenin reporter plasmid, TOP-flash, and its mutant control, FOP-flash, were purchased from Millipore Corporation. Cells were plated onto 12-well plates as described above. DNA quantities used in transfections were as follows: TOP-flash or FOP-flash at 1 ⁇ g/well; F-TrCP- Ecad at 0.5 ⁇ g/well; pRL-RSV renilla luciferase plasmid (0.1 ⁇ g/well) was used as an internal control for transfection efficiency. After 24 hours, cells were treated as described above.
  • Luciferase activities were measured 24 hours after treatment using a Dual-Glo Luciferase Assay System (Promega) in Wallac 1420 Victor Plate Reader. Experiments were performed in triplicate and repeated at least 3 times. Relative luciferase activities were expressed as the ratio of TOP-flash/FOP-flash luciferase activity, each normalized against Renilla luciferase activity. The mean values- of the normalized ratios were compared. SMAD report assay
  • the SMAD reporter assay kit (SABiosciences) was used for detecting TGF- ⁇ -induced Smad signaling.
  • the TGFP-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal CMV promoter and tandem repeats of the SMAD transcriptional response element (AGCCAGACA).
  • Cells were plated onto 24-well plates. DNA quantities used in transfections were as follows: SMAD reporter, negative control or positive control at 1 ⁇ g/well; F-TrCP-Ecad at 0.5 ⁇ g/well. Twenty-four hours after transfection, medium was changed and cells were.
  • RLA Relative luciferase activity
  • RRA SMAD reporter relative response activity
  • Co-IP Co-immunoprecipitation
  • Cell nuclear proteins were extracted and then quantified by Bradford assay (Bio-Rad).
  • Co-IP was carried out with 2 ⁇ g of affinity-purified antibody per 800 ⁇ g nuclear proteins supplemented with 30 ⁇ 1 of Protein-G agarose beads overnight at 4°C.
  • the agarose beads were collected by pulse centrifugation, the supernatant was discarded and beads were washed 5 times with 1ml IP buffer.
  • irnmunoprecipitated proteins were subjected to SDS- PAGE protein separation followed by western blot with specific primary antibodies. Western blot analyses of nuclear proteins before immunoprecipitation served as "input”.
  • the membrane was blocked by 5% Skim milk in PBS at 37°C for lh, and subsequently exposed to specific primary antibodies [anti-P-catenin, anti-E-cadherin (1 :500; BD Bioscience), anti-vimentin (1 :200; Abeam), anti-fibronectin (1 :100; Santa Cruz Biotechnology), anti-flag (1 :500; Sigma), anti-P- Smad2, anti-P-Smad3 and anti-LEF- 1(1 :200; Cell Signaling)] overnight at 4°C.
  • the membranes were washed and incubated with their respective horseradish peroxidase-conjugated secondary antibodies for 40 min at room temperature. Finally, enzyme activity was detected with the chemiluminescence detection kit. Protein expression was measured with image software (KODAK) by quantifying the relative expression of target protein versus-p-actinf
  • Subconfluent cells grown on glass cover slips, were treated with listed agents for 24 hours.
  • Cells were washed twice with PBS, fixed with 100% cold methanol for 20 min at -20°C, and nonspecific binding sites were blocked by cellular incubation for 1 h with 2% BSA (Sigma) and 0.2% Triton X-100 in PBS at room temperature.
  • Cells were then incubated in primary antibodies diluted in 2% BSA in PBS for 1 hour at room temperature, followed by four washes with PBS.
  • MMP-9 activity in the supernatants of CI .1 cells was determined with gelatin zymography as described previously (Tan et al., 2010). Briefly, supernatants were mixed with Tris-Glycine SDS native sample buffer (1 :1) (Invitrogen) and electrophoresed through 10% No vex ® zymogram gelatin gels (Invitrogen). After incubation with zymogram renaturing buffer (Invitrogen) for 30 min, the gel was washed and further incubated with developing buffer (Invitrogen) for 24 hours at 37°C.
  • a band was visualized by staining with 0.5% (wt vol) Coomassie Blue R-250 (Bio-Rad) in 50% (vol/vol) methanol, 10% (vol/vol) acetic acid for 30 min and destaining with 50% (vol/vol) methanol, 10% (vol/vol) acetic acid for 30 min at room temperature.
  • MMP-9 activity was quantified by densitometry using Adobe Photoshop 8 software.
  • Results from at least three independent experiments were expressed as mean ⁇ standard deviation (SD). Student's t-test was used to determine the significance of differences between two groups, whereas the one way analysis of variance (ANOVA) was used for comparison of multiple groups. *: PO.05 was used to indicate statistical significance.
  • beta-Catenin a pivot between cell adhesion and Wnt signalling. Curr- #io/ 15, R64-7.
  • Beta-catenin is dispensable for hematopoiesis and lymphopoiesis. / Exp Med 199, 221-9. Cong, F., Zhang, J., Pao, W., Zhou, P. and Varmus, H. (2003). A protein knockdown strategy to study the function of beta-catenin in tumorigenesis. BMC Mol Biol 4, 10.
  • Jun N-terminal kinase 1 interacts with and negatively regulates Wnt/beta-catenin signaling through GSKSbeta pathway. Carcinogenesis 29, 2317-24.
  • the cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover. J Biol Chem 276, 12301-9.
  • Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin. Am J Pathol 165, 1955-67.
  • TGF-betal suppresses IFN-gamma-induced NO production in macrophages by suppressing STATl activation and accelerating iNOS protein degradation.

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Abstract

L'invention concerne un procédé de minimisation de l'inflammation dans un ou plusieurs tissus d'un individu incluant le fait de sélectionner et/ou de faire appel à un individu présentant un ou plusieurs tissus enflammés, et chez lequel ledit un ou lesdits plusieurs tissus enflammés ont été traités pour réduire substantiellement la quantité de tous les complexes de bêta-caténine/Smad3 dans les tissus; l'administration de TGF-bêta 1 audit un ou auxdits plusieurs tissus en une quantité efficace pour induire une réponse anti-inflammatoire dans ledit un ou lesdits plusieurs tissus, minimisant ainsi l'inflammation dans ledit un ou lesdits plusieurs tissus dudit individu.
PCT/AU2012/001550 2011-12-22 2012-12-18 Thérapie par le tgf-bêta WO2013090991A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016205215A1 (fr) * 2015-06-15 2016-12-22 Vital Therapies, Inc. Composition et procédé permettant d'induire une réponse anti-inflammatoire
CN108026104A (zh) * 2015-09-18 2018-05-11 国立大学法人鸟取大学 利用低分子化合物的癌及纤维化的抑制和再生促进的效果

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1984001106A1 (fr) * 1982-09-24 1984-03-29 Us Health Reparation de tissus chez les animaux

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1984001106A1 (fr) * 1982-09-24 1984-03-29 Us Health Reparation de tissus chez les animaux

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BAARSMA H. ET AL.: "p-Catenin signaling is required for TGF-beta1-induced extracellular matrix production by airway smooth muscle cells", AMERICAN JOURNAL OF PHYSIOLOGY - LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, vol. 301, 9 September 2011 (2011-09-09), pages L956 - L965, XP055071975 *
CHEON S. ET AL.: "Beta-catenin regulates wound size and mediates the effect ofTGF-beta in cutaneous healing", THE FASEB JOURNAL, vol. 20, 2006, pages 692 - 701, XP055071976 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016205215A1 (fr) * 2015-06-15 2016-12-22 Vital Therapies, Inc. Composition et procédé permettant d'induire une réponse anti-inflammatoire
CN108026104A (zh) * 2015-09-18 2018-05-11 国立大学法人鸟取大学 利用低分子化合物的癌及纤维化的抑制和再生促进的效果
CN108026104B (zh) * 2015-09-18 2021-06-18 国立大学法人鸟取大学 利用低分子化合物的癌及纤维化的抑制和再生促进的效果

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