WO2018054354A1 - 含有src同源区2的蛋白酪氨酸磷酸酶-1激动剂用于改善纤维化的用途 - Google Patents

含有src同源区2的蛋白酪氨酸磷酸酶-1激动剂用于改善纤维化的用途 Download PDF

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WO2018054354A1
WO2018054354A1 PCT/CN2017/102945 CN2017102945W WO2018054354A1 WO 2018054354 A1 WO2018054354 A1 WO 2018054354A1 CN 2017102945 W CN2017102945 W CN 2017102945W WO 2018054354 A1 WO2018054354 A1 WO 2018054354A1
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shp
fibrosis
cells
treatment
stat3
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PCT/CN2017/102945
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French (fr)
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陈昆锋
萧崇玮
苏东弘
王诚一
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陈昆锋
萧崇玮
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Priority to CN201780058335.0A priority Critical patent/CN109789116B/zh
Priority to US16/335,504 priority patent/US10993923B2/en
Priority to EP17852422.9A priority patent/EP3517108B1/en
Publication of WO2018054354A1 publication Critical patent/WO2018054354A1/zh

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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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • 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
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the use of a protein tyrosine phosphatase-1 (SHP-1) agonist comprising SRC homology 2, in particular to a protein tyrosine phosphatase-1 comprising SRC homology 2
  • SHP-1 protein tyrosine phosphatase-1
  • Fibrosis is a pathological change characterized by fibroblast activation and proliferation, increased fibrous connective tissue in tissues and organs, and decreased parenchymal cells. Continued progression can lead to tissue and organ structural damage and loss of function. Fibrosis of vital organs seriously affects the quality of life of patients and even life-threatening. Fibrotic diseases include diseases involving multiple systems, such as systemic sclerosis, multifocal fibrosis, scleroderma, renal multisystemic fibrosis, and organ-specific diseases such as skin, heart, lungs, Liver, kidney fibrosis, etc. The causes of different fibrotic diseases are different, such as tissue organ damage, infection, immune response, chronic inflammation, etc., but their common feature is excessive deposition of extracellular matrix (ECM) in tissues and organ tissue remodeling.
  • ECM extracellular matrix
  • Hepatic fibrosis refers to the pathological process of abnormal proliferation of connective tissue in the liver caused by various pathogenic factors and excessive precipitation of diffuse extracellular matrix in the liver.
  • liver fibrosis such as viral infections, inflammatory reactions and alcohol abuse.
  • the pathological features of liver fibrosis are massive fibrous tissue hyperplasia and deposition in the hepatic portal vein and hepatic lobule, but no interlobular septa have been formed.
  • pseudolobules are formed, and the central venous region and the hepatic portal region are separated, and the normal structure of the liver is destroyed.
  • the further development of liver fibrosis is cirrhosis.
  • Pulmonary fibrosis diseases include idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, pneumoconiosis, fibrosis caused by drugs and radiation, and fibrotic alveolitis associated with collagen vascular disease, and a wide range of causes Disease spectrum.
  • the main pathological features of pulmonary fibrosis include increased lung mesenchymal cells Colonization, extracellular matrix hyperplasia and remodeling of lung parenchyma.
  • anti-inflammatory, anti-oxidation, anti-fibroblast proliferation and collagen deposition and lung transplantation are mainly used to treat pulmonary fibrosis.
  • Idiopathic pulmonary fibrosis is the most common type of interstitial lung disease, mainly a fatal disease of unknown cause in middle-aged and elderly adults. Although the clinical manifestations of idiopathic pulmonary fibrosis are known to contain varying degrees of interstitial fibrosis and inflammation, other related diagnoses remain difficult to treat. Idiopathic pulmonary fibrosis is characterized by loss of respiratory function and significant deformation of the lung structure. The histopathological features of patients with idiopathic pulmonary fibrosis are called fibroblastic lesions, which are composed of aggregates of activated fibroblasts that produce excess extracellular matrix in the alveolar space at the site of loss of epithelial cells. .
  • lung fibroblasts play an important role in extracellular matrix deposition in pulmonary fibrosis, the origin of the amplified population of these cells in the lungs is of great interest.
  • a typical theory is that tissue damage causes activation of fibroblasts to proliferate and express components of the extracellular matrix.
  • Contemporary theory suggests that tissue damage in the presence of transforming growth factor-beta (TGF ⁇ ) induces the transformation of epithelial cells into mesenchymal phenotypes, ie fibroblasts/myofibroblasts, the latter contributing to fiber Hyperplasia and fibrosis. Therefore, fibroblast inactivation and inhibition of epithelial-mesenchymal transition (EMT) play an important role in improving pulmonary fibrosis.
  • TGF ⁇ transforming growth factor-beta
  • SHP-1 protein tyrosine phosphatase-1
  • Another object of the present invention is to provide a use of the following compounds for the preparation of a medicament for treating a disease characterized by inactivation of protein tyrosine phosphatase-1 (SHP-1) containing SRC homology 2
  • SHP-1 protein tyrosine phosphatase-1
  • the protein tyrosine phosphatase-1 agonist comprising SRC homology 2 increases the activity of protein tyrosine phosphatase-1 (SHP-1) containing SRC homology 2 .
  • the protein tyrosine phosphatase-1 agonist comprising SRC homology region 2 is the following compound
  • the disease characterized by the inactivation of SRC homologous region 2 protein tyrosine phosphatase-1 is fibrosis.
  • the fibrosis is skin hardening or fibrosis of the heart, lung, liver, pancreas, or kidney.
  • Embodiments of the present invention evaluate the anti-fibrotic efficacy produced by administering SHP-1 phosphatase agonists, such as SC-43 compounds, to cells and animals via the SHP-1/STAT3 signaling pathway in liver and pulmonary fibrosis.
  • SHP-1 phosphatase agonist SC-43 compound directly interacts with the catalytic protein tyrosine phosphatase region by reducing the SHP-1 inhibitory N-SH 2 region, thereby activating the SHP-1 and promoting the fiber.
  • the apoptosis of the mother cells reveals anti-fibrotic activity, indicating that the SHP-1 phosphatase agonist is a potential target for anti-fibrotic drugs, which may be a new strategy for the development of anti-fibrotic drugs.
  • FIG. 1 is a schematic diagram of an experimental acute lung injury model of bleomycin-induced pulmonary fibrosis in mice.
  • Figure 2 is a graph showing hematoxylin-eosin (H&E) tissue staining of lungs treated with SC-43 to improve bleomycin-induced pulmonary fibrosis in mice.
  • CTL indicates control mice;
  • BLM indicates bleomycin-induced mouse group;
  • BLM+SC43 indicates treatment of bleomycin-induced mice with SC-43.
  • Figure 3 is a view of the present invention treated with SC-43 to improve bleomycin-induced pulmonary fibrosis in mice Masson's trichrome tissue staining map.
  • CTL indicates control mice;
  • BLM indicates bleomycin-induced mouse group;
  • BLM+SC43 indicates treatment of bleomycin-induced mice with SC-43.
  • Figure 4 is a bar graph of the treatment of SC-43 to improve the hydroxyproline content of bleomycin-induced pulmonary fibrosis in mice.
  • CTL indicates control mice;
  • BLM indicates bleomycin-induced mouse group;
  • BLM+SC43 indicates treatment of bleomycin-induced mice with SC-43.
  • Columns represent the mean; bands indicate standard deviation; **P ⁇ 0.05.
  • Figure 5 is a bar graph of the treatment of SC-43 to improve the collagen content of bleomycin-induced pulmonary fibrosis in mice.
  • CTL indicates control mice;
  • BLM indicates bleomycin-induced mouse group;
  • BLM+SC43 indicates treatment of bleomycin-induced mice with SC-43.
  • the bars represent the mean; the bands represent the standard deviation.
  • Figure 6 is a colloidal electropherogram of epithelial-mesenchymal transition (EMT) treated with SC-43 to improve bleomycin-induced pulmonary fibrosis in mice.
  • EMT epithelial-mesenchymal transition
  • Figure 7A shows that treatment with SC-43 of the present invention reduces the survival rate of mouse fibroblasts (NIH3T3 cell line) via the SHP-1/STAT3 signaling pathway.
  • Figure 7B shows that treatment of SC-43 in the present invention reduces expression of p-STAT3 via the SHP-1/STAT3 signaling pathway.
  • Figure 7C shows that treatment with SC-43 of the invention induces apoptosis via the SHP-1/STAT3 signaling pathway.
  • Figure 7D shows that treatment with SC-43 in the present invention counteracts the apoptotic effect caused by SHP-1 knockout via the SHP-1/STAT3 signaling pathway.
  • Figure 8A shows the production of human lung epithelial cells (A549 cell line) exposed to TGF- ⁇ 1 to induce epithelial-mesenchymal transition.
  • the expression of E-cadherin is decreased; the expression of fibronectin and N-cadherin is up-regulated; STAT3 phosphorylation is also increased after exposure to TGF- ⁇ 1 (left panel); invasive cells and migration of EMT after TGF- ⁇ 1 stimulation Increased cells (right).
  • Figure 8B shows that the treatment of SC-43 in the present invention inhibits TGF- ⁇ 1-induced epithelial-mesenchymal transition in human lung epithelial cells (A549 cell line).
  • the expression of E-cadherin is increased; the expression of fibronectin and N-cadherin is decreased; STAT3 phosphorylation is also decreased after exposure to TGF- ⁇ 1.
  • Figure 8C shows that treatment with SC-43 of the present invention inhibits TGF- ⁇ 1-induced epithelial-mesenchymal transition in human lung epithelial cells (A549 cell line). EMT produced by TGF- ⁇ 1 stimulation after SC-43 treatment Invasive cells and migrated cells are reduced.
  • Fig. 8D shows invading cells and migrating cells which inhibit TGF- ⁇ 1-induced epithelial-mesenchymal transition in human lung epithelial cells (A549 cell line) treated with SC-43 in the present invention.
  • FIG. 9A shows that transfection of SHP-1 in A549 cells of the present invention reduces EMT marker expression of migrating cells and invading cells.
  • Figure 9B shows the effect of the present invention on the expression of EMT marker of migrating cells and invading cells by siRNA knockout of SHP-1 in A549 cells.
  • Figure 9C shows the effect of the expression of STAT3 in siRNA knockdown of EMT markers, migrating cells and invasive cells in A549 cells of the present invention.
  • 10A shows the expression of fibrosis induced by CCl 4 in the region of a mouse model of liver fibrosis in SHP-1 phosphatase; scale bar: 200 ⁇ m.
  • Figure 10B shows the expression of SHP-1 phosphatase in patients with chronic hepatitis B (CHB) advanced fibrosis; scale bar: 200 ⁇ m.
  • CHB chronic hepatitis B
  • Figure 10C shows that the expression of SHP-1 phosphatase is positively correlated with serum alanine aminotransferase concentration.
  • FIG. 10D shows that SHP-1 phosphatase is expressed in activated hepatic stellate cells (HSCs).
  • HSCs activated hepatic stellate cells
  • qCPA collagen - positive area
  • FIG 11D shows the prevention of CCl 4 induced liver fibrosis model mouse SC-43 treatment will improve the image of FIG ⁇ -SMA staining of hepatic fibrosis.
  • qCPA collagen - positive area
  • BDL bile duct ligation
  • Figure 14A is a bar graph showing the survival rate of hepatic stellate cells administered to SC-43 in a time- and dose-dependent manner; administration of SC-43 or sorafenib in HSC-T6 and LX2 cells for 24 hours; primary mouse HSCs cells Give SC-43 24 or 48 hours respectively.
  • PARP poly(ADP-ribose) polymerase
  • TGF transforming growth factor
  • PDGFR platelet-derived growth factor receptor
  • Figure 15A shows an electropherogram of SC-43 treatment in a dose-dependent down-regulation of p-STAT3 and cyclin D1 in LX2 and HSC-T6 cells.
  • Figure 15B shows an electropherogram of SC-43 that down-regulates interleukin (IL)-6-STAT3.
  • IL interleukin
  • Figure 15C shows that SC-43-induced apoptosis is significantly abolished in STAT3 overexpressing HSC; the bars represent the mean, the bands represent the standard deviation; ***P ⁇ 0.001 compared to the vehicle.
  • Figure 15D shows that SC-43 down-regulates p-Akt after administration of the specific PDGFR inhibitor AG1295 And p-STAT3.
  • Figure 16A is a schematic representation of SHP-1 and its mutant dN1 (containing the N-SH2 region deletion) and D61A (single mutation for D61).
  • Figure 17F is a schematic representation of the anti-fibrotic mechanism of SC-43 through the SHP-1-STAT3 pathway.
  • Fibrosis refers to the continuous damage of tissues and organs such as lung, liver, kidney, blood vessels, peritoneum, pancreas, skin, etc. due to various causes such as inflammation, infection, immune reaction, ischemia, chemical substances, radiation, etc. , fibroblasts activate proliferation, increased fibrous connective tissue in tissues and organs, decreased parenchymal cells, tissue and organ structure destruction and loss of function.
  • the term fibrotic lesions covers various stages of cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, vascular fibrosis, skin fibrosis (hardening) and other tissue and organ fibrotic lesions, including various diseases. And the development of the process of cardiac fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, vascular fibrosis, skin fibrosis (hardening) and other tissue fibrosis lesions.
  • liver fibrosis refers to intrahepatic conditions caused by or associated with various causes such as inflammation, infection (eg, viral infection), immune response, ischemia, chemicals, radiation, oxidative stress, and alcohol abuse. Abnormal hyperplasia of connective tissue, excessive precipitation of diffuse extracellular matrix in the liver, and pathological changes (lesions) in which the normal structure of the liver is destroyed. Further development of liver fibrosis is cirrhosis, and cirrhosis is also encompassed within the scope of the term “liver fibrosis" of the present invention.
  • pulmonary fibrosis refers to pulmonary tissue mesenchymal cell proliferation, extracellular matrix proliferation and deposition caused by or associated with various causes (eg, inflammation, infection, immune response, ischemia, chemicals, radiation). Pathological processes caused by the remodeling of lung parenchyma.
  • treatment includes the prevention of a particular disorder or condition, or the alleviation of a symptom associated with a particular disorder or condition and/or the prevention or elimination of that condition.
  • Prevention as described herein is the treatment to reduce or minimize the risk of a disease state in a patient who has not yet presented a clinical disease state, while secondary prevention is defined as minimizing or reducing the recurrence or second occurrence of the same or similar clinical disease state. risks of.
  • An "individual” as used herein is an animal, such as a human, but may also be a pet (eg, a dog, a cat, and the like), an economic animal (eg, cow, sheep, pig, horse, and the like) or experimental. Animals (eg, rats, mice, guinea pigs, and the like) that require treatment as described herein.
  • effective amount refers to the amount of active agent required to achieve a therapeutic effect in an individual, either alone or in combination with one or more other active agents. Depending on the route of administration, the use of the excipients, and the use in conjunction with other active agents, the effective amounts will vary, as will be recognized by those skilled in the art.
  • Suitable routes of administration may include, for example, oral, rectal, mucosal, or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary, and intrathecal, direct intraventricular, intravenous, peritoneal Intranasal or intraocular injection, and may be a supplemental or sustained release dosage form.
  • compositions of the present invention can be made in a manner known in the art, for example, by conventional mixing, dissolving, emulsifying, embedding, encapsulating, or lyophilizing processes.
  • the pharmaceutical compositions provided in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable excipients, including excipients and/or adjuvants, to aid in the processing of the active compound.
  • physiologically acceptable formulations means that the excipient must be compatible with the active ingredient of the composition (and preferably, the active ingredient can be stabilized) and not deleterious to the individual being treated.
  • acceptable dosage form will depend on the route of administration chosen.
  • the compounds of the invention may be formulated, for example, in physiologically compatible buffers such as Hank buffer, Ringer's buffer or physiological saline buffer.
  • physiologically compatible buffers such as Hank buffer, Ringer's buffer or physiological saline buffer.
  • the compounds of the invention may be formulated by combining the active compound with pharmaceutically acceptable excipients known in the art, such as lactose, sucrose, mannitol, sorbitol, corn starch, wheat starch, Rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) to enable the compound of the present invention to be formulated They are tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like.
  • PVP polyvinylpyrrolidone
  • the compound of the present invention can be formulated as an aerosol spray sprayed from a pressurized container or sprayer, in combination with a suitable propellant, for example, dichlorodifluoromethane, trichloro Fluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant for example, dichlorodifluoromethane, trichloro Fluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the progress of the reaction was determined by thin layer chromatography (TLC) analysis on a silica gel 60 F254 plate (Merck). Purification by chromatography was carried out on silica gel column 60 (0.063-0.200 mm or 0.040-0.063 mm, Merck) in basic silica gel. Commercially available reagents and solvents were used without further purification.
  • TLC thin layer chromatography
  • the present invention relates to Src-homology region 2 containing tyrosine phosphatase-1 (SHP-1)-signal transducer and transcription activator 3 (signal transducer) in fibrosis And activator of transcription 3, STAT3) Path, assessing the anti-fibrotic efficacy produced by administering SC-43 (SHP-1 phosphatase agonist) in vitro and in vivo.
  • SC-43 SHP-1 phosphatase agonist
  • Sorafenib supplied by Bayer HealthCare AG (Berlin, Germany). --smooth muscle actin ( ⁇ -SMA), phosphorylated STAT3 (Tyr705), STAT3, cyclin D1, glyceraldehyde-3-phosphate dehydrogenase, P-Smad2 (Ser465/467), P-Smad3 (Ser423 /425) Smad2, Smad3, poly(ADP-ribose) polymerase (PARP), platelet-derived growth factor receptor (PDGFR)- ⁇ , P-PDGFR- ⁇ (Tyr857) and P-Akt (Ser473) were purchased from cell signals Cell Signaling (Massachusetts, USA).
  • Akt was purchased from Santa Cruz Biotechnology (California, USA). Sodium vanadate was purchased from Cayman Chemical (Michigan, USA). PTP inhibitor III was purchased from Calbiochem (California, USA). Polyoxyethylene castor oil (Cremophor) was obtained from Sigma (Missouri, USA).
  • mice Male C57BL/6J (6-8 weeks old) was obtained from the National Laboratory Animal Center (Taipei, Taiwan). All experimental procedures performed using these mice were performed according to protocols approved by the Animal Care and Use Committee of the Institutional Laboratory of the clergy Hospital. Animals were housed in individual ventilation cages (IVC) for 12 hours per day; room temperature was maintained at 22 to 25 ° C; humidity was maintained at 60 to 70%; aeration rate was maintained at 16 to 18 times per hour; food And drinking water (Altromin 1326 mouse feed, Germany) was given freely. All experimental procedures were performed in accordance with standard procedures for animal laboratory animal methods. Animal management and feeding are in line with actual use (NRC 1996) Basis for implementation of the rule method
  • BLM
  • the paraffin-embedded tissue array block was cut into 4 ⁇ m thick stained sections of hematoxylin-eosin staining (H&E Stain). For each case, two pathologists re-examine cancer type, cell differentiation, growth pattern, tumor cell nuclear morphology, tissue transformation, calcification, necrosis, mitotic number, invasion status, and other specific differences. Immunohistochemistry (IHC) staining was performed using a Ventana BenchMark XT automatic staining machine (Arizona, USA). Briefly, 4 [mu]m thick sections were continuously cut from formalin-fixed paraffin-embedded tissue. The sections were then placed on a silanized slide and allowed to dry overnight at 37 °C. After deparaffinization and rehydration, the slides were reacted with 3% hydrogen peroxide solution for 5 minutes. After washing with the supplied buffer, the tissue sections were repaired with ethylenediamine tetraacetic acid (EDTA) for 40 minutes.
  • EDTA ethylened
  • Mouse fibroblasts (NIH3T3 cell line) and human lung epithelial cells (A549 cell line) were purchased from the Center for Conservation and Research of Biological Resources (Hsinchu, Taiwan).
  • DMEM Dulbecco's modified eagle medium
  • FBS fetal bovine serum
  • penicillin 100 ⁇ g/mL
  • streptomycin 100 ⁇ g/mL
  • the cells were cultured in a box and subcultured every 4 to 5 days in a ratio of 1:4.
  • Rat immortal hepatic stellate cell line HSC-T6 and LX2 human hepatic stellate cell line were provided by Professor Scott Friedman of Mount Sinai Hospital in New York.
  • Primary mouse HSCs were intubated from mouse hepatic portal vein and used Leffert's and B.
  • EGTA Glycol bis-aminoethyl ether tetraacetic acid
  • HSC-T6 cells were cultured in Waymouth medium; LX2 cells and primary mouse HSCs cells were treated with 10% fetal bovine serum (FBS), 100 ⁇ g/mL penicillin, 100 ⁇ g/mL streptomycin, and 2 mM L-glutamine.
  • FBS fetal bovine serum
  • the modified Eagle medium (DMEM) was cultured in a humidified incubator containing 5% carbon dioxide at 37 °C. After adding the FBS, the sleeping HSC is started.
  • various concentrations of SC-43 and sorafenib were dissolved in dimethyl sulfoxide and subsequently added to cells in 5% FBS for a predetermined period of time.
  • Hydroxyproline analysis was performed using a hydroxyproline assay kit according to the manufacturer's protocol (Cell BioLabs, Inc., USA).
  • the lung tissue in 12N HCl 120 °C hydrolyzed for 3 hours and then washed and resuspended in H 2 O in.
  • the tissue suspension was then mixed with chloramine-T and allowed to react at room temperature for 30 minutes.
  • Ehrlich's reagent was then added and reacted at 60 ° C for 90 minutes. Thereafter, the absorbance at 540 nm was measured.
  • Mouse lung collagen was measured in lung homogenate using the Sircol assay (Biocolor, UK) according to the manufacturer's protocol. Briefly, lung tissue homogenates were mixed with Sircol dye and centrifuged. The precipitate was resuspended in NaOH and the absorbance at 550 nm was measured. The absorbance obtained is directly proportional to the concentration of the recently synthesized collagen.
  • Antibodies for immunoblotting techniques such as E-cadherin, fibronectin (Fibronectin), N-cadherin (Abcam, UK), other antibodies purchased from Cell Signaling Corporation (Massachusetts, USA) such as anti-SHP-1, phosphorylated STAT3 (Tyr705), STAT3 ⁇ -smooth muscle actin ( ⁇ -SMA) and caspase-9 (Caspase-9).
  • Mouse NIH3T3 fibroblasts were seeded into 96-well plates (1 x 10 3 cells/well). To measure cell viability and proliferation, 10% WST-1 (cell proliferation reagent WST-1, Roche) was added to each well and cultured for 3 hours. The reaction was catalyzed by mitochondrial reductase in active cells, and the absorbance at 450 nm was measured by a Bio-Rad ELISA analyzer to calculate the optical density (OD).
  • WST-1 cell proliferation reagent WST-1, Roche
  • HSC-T6, LX2 or primary mouse HSCs cells were seeded in 96-well flat pans (5,000 cells/well) for 24 hours, and the cells were each at various concentrations of SC-43 or Sorafil. Treatment for 24 and 48 hours.
  • CellTiter 96AQueous one solution cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, an inner, MTS) The company, USA) evaluated the effect of each reagent on cell viability and performed three replicates according to the manufacturer's operating manual.
  • BD FACS Verse flow cytometry (BD, USA) was used to detect double staining with annexin V and propidium iodide.
  • the proportion of apoptotic cells was measured after mixing of early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells.
  • Apoptotic cells were metered using flow cytometry (sub-G1) and cell death was detected by enzyme-bound immunosorbent assay (ELISA) for cytoplasmic histone-associated DNA fragmentation. Measurement of 570 nm by using an ELISA analyzer The optical density (OD) was calculated from the absorbance of the (formazan) dyeing dose.
  • ELISA enzyme-bound immunosorbent assay
  • STAT3 cDNA (KIAA1524) was purchased from Addgene Platinum Corporation.
  • Mouse fibroblasts (NIH3T3 cell line) and human lung epithelial cells (A549 cell line) transiently transfected with STAT3, SHP-1, SHP-1 (dN1) mutants and SHP-1 (61A) mutants (Transient transfection) And then reacted separately with the SC-43 compound and subjected to Western blotting. Transfection is used X-tremeGENE HP Transfection Reagent (Roche, Germany), according to the manufacturer's operating manual.
  • mouse fibroblasts (NIH3T3 cell line), human lung epithelial cells (A549 cell line) or LX2 cell protein extract were reacted with anti-SHP-1 antibody overnight in immunoprecipitation buffer.
  • Protein A/G Sepharose Fast Flow Beads (GE) were added to each sample, followed by a rotation reaction at 4 ° C for 3 hours.
  • the tyrosine phosphatase assay kit (R-22067) was used for SHP-1 activity assay (Invitrogen, USA).
  • siRNA including control (sc-37007) siRNA, siRNA anti-SHP-1, and STAT3, were purchased from Santa Cruz Biotechnology (California, USA).
  • Hepatic fibrosis was induced using carbon tetrachloride in male C57BL/6J mice. Mice were given peritoneal injection of carbon tetrachloride for 4 or 8 weeks, and a polyoxyethylene castophor carrier, SC-43 (5, 10, or 20 mg/kg, respectively) was administered via feeding over a specified period of time. Kg) or sorafenib was sacrificed every Friday; sorafenibine has been shown to have anti-fibrotic activity and is therefore used as a positive control.
  • SC-43 polyoxyethylene castophor carrier
  • bile duct ligation BDL mice
  • Vehicle or SC-43 was administered daily by feeding on Day 1 or Day 8 until sacrifice on Day 14.
  • Mouse liver samples were stored in 10% formaldehyde, dehydrated with gradient alcohol, embedded in paraffin blocks, sectioned to a thickness of 3 ⁇ m, placed on glass slides, and stained with Sirius Red Stain (Picysirius Red Stain) kit (ScyTek) , United States), according to the manufacturer's operating manual. The severity of liver fibrosis was graded according to the Ishak fibrosis scoring system. Collagen-positive area (qCPA) was quantified via densitometry via using ImageJ software. Hepatic hydroxyproline concentrations were measured using a hydroxyproline kit assay (Biovision, USA USA) according to the manufacturer's instructions. Use Leica Immunohistochemical staining was performed on a BOND-MAX automatic chromatograph (Leica Biosystems, Germany) according to the manufacturer's operating manual.
  • TGF- ⁇ induction HSC serum was treated for 4 hours, followed by treatment with 10 ⁇ M SC-43 for 4 hours, and treated with 10 ng/mL recombinant human TGF- ⁇ 1 (R&D Systems, Inc., USA) for 20 minutes.
  • PDGF-BB induction LX2 or HSC-T6 cells were sera for 4 hours, followed by 10 ⁇ M SC-43 for 4 hours, followed by 100 ng/mL recombinant human or rat PDGF-BB (R&D) Systems, Inc., USA, processed for 10 minutes.
  • R&D platelet-derived growth factor
  • IL-6 stimulation HSC serum was treated for 4 hours, then treated with 10 ⁇ M SC-43 for 4 hours, and treated with 100 ng/mL IL-6 (R&D Systems, Inc., USA) for 30 minutes.
  • Rat STAT3 (Open Biiosystem, USA) was constructed on pLVX-AcGFP-N1 expression vector (Clontech, USA), followed by the use of lipids in addition to lentiviral wrap and expression vectors (P8.91 and VSV-G) Lipofectamine 2000 transfection reagent (Invitrogen, USA) was co-transfected into 293FT cells. After 48 hours of transfection, the lentiviral supernatant was taken for infection of 5 ⁇ 10 5 HSC-T6 cells, which were seeded on a 6-cm culture dish; cells stably expressing STAT3 in rats were obtained.
  • a plastid containing human wild-type SHP-1 and SHP-1 mutants is a truncated N-SH 2 region (dN1) or an aspartic acid at position 61
  • the plastid which is an alanine residue (D61A)
  • D61A was cloned into the pCMV6-entry vector with the myc tag.
  • Smart-pool siRNA including the control group (D-001810-10) and SHP-1 (PTPN6, L-009778-00-0005) were purchased from Dharmacon (Chicago, USA).
  • SHP-1 plastids or siRNA final concentration 100 nM were transfected into LX2 cells for 24 hours with Lipofectamine 2000 transfection reagent according to the manufacturer's protocol.
  • LX2 cells were plated on 10-cm culture dishes (1500 to 5000 cells per dish) and cultured in DMEM medium for 2 weeks. The cells were then fixed with 4% formaldehyde and stained with 0.1% crystal violet.
  • Example 3 SC-43 is used to treat lung fibrosis in mice
  • Protein tyrosine phosphatase-1 which contains SRC homology 2
  • SRC homology 2 is a regulator of different intracellular signaling molecules, such as signal transduction and activator of transcription 3, KIT, CD22, CD5, CD72, SHPS- 1.
  • TIMP metaloproteinase
  • CDK2 p27
  • SRC p27
  • SRC p27
  • SRC ZAP70
  • IL-10 interleukin 10
  • NF- ⁇ B NF- ⁇ B
  • Lck interleukin 10
  • 3BP2 3BP2, Lyn
  • cyclin D1 cyclin D1.
  • STAT3 is a transcription factor that regulates cell growth and survival by regulating the expression of a target gene.
  • SHP-1 is a key regulator of STAT3 activity.
  • the present invention demonstrates the anti-pulmonary fibrosis activity of SC-43, showing that SC-43 compounds initiate SHP-1 activity, down-regulate STAT3, and then reduce fibroblast survival and epithelial-mesenchymal transition in epithelial cells.
  • the in vivo experimental data of the present invention also confirmed that SC-43 improves bleomycin-induced pulmonary fibrosis in mice, indicating that SHP-1 agonists are useful for the treatment of idiopathic pulmonary fibrosis (IPF) and other fibrotic diseases. .
  • the present invention establishes an experimental acute lung injury model of bleomycin-induced pulmonary fibrosis (as shown in Figure 1). Using this animal model, treatment with a 7.5 mg/kg dose of SC-43 via daily gavage was well tolerated because no drug-related adverse events were observed. According to the hematoxylin-eosin staining (H&E Stain) of lung sections, intranasal injection of BLM resulted in destruction of normal lung structure, significant proliferation of fibroblasts, infiltration of inflammatory cells, and massive deposition of fibrillar collagen (Figure 2 ). More importantly, these pathological changes will be significantly improved after SC-43 administration (Figure 2 right); Fibrous collagen deposition was significantly reduced after administration of SC-43, as shown by the Masson's trichrome staining positive area ( Figure 3).
  • H&E Stain hematoxylin-eosin stain staining
  • the present invention detects the pulmonary hydroxyproline (Hyp) content of each group of mice to quantify the degree of pulmonary fibrosis.
  • the Hyp content decreased by about 22% after SC-43 treatment compared to the BLM group, indicating that SC-43 has a protective effect on extracellular matrix (ECM) accumulation.
  • the present invention detects the collagen content of mice (Fig. 5) and finds that SC-43 improves the accumulation of collagen in the pulmonary fibrosis model.
  • the present invention further detects whether SC-43 reduces epithelial-mesenchymal transition, which is a critical step in fiber development; as shown in Figure 6, bleomycin induction is small compared to the control group (lane 1 and lane 2). E-cadherin (epithelial phenotype protein) was reduced in murine pulmonary fibrosis samples (lane 3 and lane 4). However, bleomycin-induced pulmonary fibrosis in mice was treated with SC-43 (the expression of E-cadherin was increased after bleomycin-induced pulmonary fibrosis in mice in lanes 5 and 6.
  • Fibronectin is an interstitial phenotype marker that increases the expression of bleomycin-induced pulmonary fibrosis in mice (lanes 3 and 4), but after SC-43 treatment (p. Reduction of lane 5 and lane 6.
  • P-STAT3 is increased in bleomycin-induced pulmonary fibrosis, but is also reduced after SC-43 treatment.
  • the main effector cells responsible for excessive epithelial-mesenchymal transition are activated fibroblasts, which are the proliferation of alveolar extracellular matrix of alveolar epithelial cells and resident fibroblasts. cause.
  • the present invention utilizes A549 cells to evaluate the effect of SC-43 on EMT, an alveolar type II that has been widely used in an ideal in vitro model for studying EMT.
  • Epithelial cell line Exposure of A549 cells to TGF- ⁇ for 18 hours induced EMT.
  • the present invention transfects SHP-1 in A549 cells, and overexpression of SHP-1 reduces EMT markers, migrating cells, and invading cells (Fig. 9A).
  • the present invention then knocks out SHP-1 expression by siRNA, counteracting the effects of SC-43, including expression of p-STAT3, EMT marker expression, migrating cells, and invading cells (Fig. 9B).
  • SC-43 inhibits EMT via the activity of SHP-1, down-regulating the expression of p-STAT3, thereby reducing epithelial-mesenchymal transition in epithelial cells.
  • the present invention knocks out the expression of STAT3, which is a downstream molecule in the SHP-1/STAT3 signaling pathway, confirming that knocking out STAT3 counteracts the effects of SC-43, including EMT marker expression (Fig. 9C, left panel), migrating cells And invading cells (Fig. 9C, right panel).
  • the present invention demonstrates that SC-43 treatment is effective in improving pulmonary fibrosis in a model of bleomycin-induced pulmonary fibrosis in mice.
  • SC-43 can inhibit epithelial-mesenchymal transition of epithelial cells, reduce proliferation and collagen synthesis in fibroblasts, and promote fibroblast apoptosis.
  • SC-43 increases SHP-1 activity and inhibits STAT3 phosphorylation.
  • Enhanced SHP-1 activity significantly inhibits EMT of epithelial cells and promotes fibroblast apoptosis, whereas inhibition of SHP-1 expression counteracts EMT inhibition and fibroblast apoptosis of epithelial cells induced by SC-43 treatment.
  • the present inventors have also found that SC-43 interacts with the N-SH 2 region of SHP-1 to enhance SHP-1 activity and inhibit STAT3 signaling, which is the mechanism of action of SC-43 against fibrosis.
  • Example 4 SC-43 is used to treat liver fibrosis in mice
  • SHP-1 is overexpressed in the fibrotic region of human and mouse liver.
  • CHSC hepatic stellate cells
  • SC-43 increases SHP-1 activity and inhibits STAT3 phosphorylation, which is independent of the platelet-derived growth factor receptor pathway.
  • Enhanced SHP-1 activity significantly inhibited HSC proliferation, whereas SHP-1 inhibition counteracted SC-43-induced HSC apoptosis.
  • SC-43 interacts with the N-SH 2 region of SHP-1 to increase the anti-fibrotic effect of SHP-1.
  • the present invention investigates the expression of SHP-1 of a P-STAT3 inhibitor in fibrotic liver.
  • SHP-1 was overexpressed in regions with significant fibrosis (Fig. 10A).
  • CHB chronic hepatitis B
  • SHP-1 expression is significantly increased in patients with advanced fibrosis (Fig. 10B).
  • SHP-1 expression was positively correlated with serum alanine aminotransferase concentration (Fig. 10C).
  • SHP-1 was colocalized with the activated HSC marker ⁇ -SMA (Fig. 10D).
  • SC-43 enhances the expression of SHP-1 activity over the known anti-fibrosis drug sorafenib (Fig. 2A). Therefore.
  • the present invention assumes that SC-43 has more anti-fibrotic ability than sorafenib in a mouse model of fibrosis prevention, after simultaneously administering a mouse model of SC-43 or sorafenib and CCl 4 to induce liver fibrosis.
  • SC-43 or sorafenib treatment a reduction in fibrosis in liver tissue was clearly observed, using Piriusrius Red Stain (Figure 11A), to quantify collagen-positive area via densitometry ( qCPA) (Fig. 11B), ⁇ -SMA expression (Fig. 11D) and hydroxyproline concentration (Fig. 11E) were examined according to the Ishak fibrosis scoring system fraction (Fig. 11C).
  • liver fibrosis treatment In a mouse model of liver fibrosis treatment, mild hepatic fibrosis (Ishak classification, 2-3) was observed 2 weeks after hepatic fibrosis was induced by mouse CCl 4 . In the next 6 weeks, a mouse model of SC-43 (5, 10 or 20 mg/kg) or sorafenib (10 mg/kg) and CCl 4 induced liver fibrosis was administered simultaneously, as observed by Sirius red staining. At that time, treatment with SC-43 and sorafenib significantly improved liver fibrosis (Fig. 12A), qCPA via densitometry (Fig. 12B), grading according to Ishak fibrosis scoring system (Fig.
  • the present invention further detects the anti-fibrotic effect of SC-43 in a mouse model of bile duct ligation.
  • SC-43 10 mg/kg
  • the present invention confirms the anti-fibrotic mechanism of SC-43 in vitro.
  • the dose-dependent treatment of SC-43 has the effect of reducing the survival rate of HSC-T6 and LX2 cells, and the effect of sorafenib is more significant; SC-43 also reduces the survival rate of primary mouse HSCs in time and dose-dependent manner.
  • the effect ( Figure 14A).
  • SC-43 significantly increased HSC cell apoptosis at dose-dependent compared to sorafenib (Fig. 14B), and Western blot showed SC-43 dose-dependent increase in poly(ADP-ribose) polymerase (PARP) Fragmentation of the fragment (Fig. 14C).
  • PARP poly(ADP-ribose) polymerase
  • TGF- ⁇ and PDGFR are major pathways involved in fibrogenesis and HSC activation and proliferation, and the present inventors investigated the effect of SC-43 on TGF- ⁇ and PDGFR pathways.
  • SC-43 down-regulates p-Smad2 and p-Smad3 in the TGF- ⁇ pathway in LX2 cells (Fig. 14D); it also down-regulates p-PDGFR and p-Akt in the PDGFR pathway in HSC-T6 and LX2 cells. (Fig. 14E).
  • the present invention further investigates the effect of SC-43 on the STAT3 pathway, which is a key regulator of fibrogenesis.
  • SC-43 treatment in LX2 and HSC-T6 cells showed a more significant dose-dependent down-regulation effect of sorafenib on p-STAT3 and cyclin D1 (Fig. 15A).
  • SC-43 also inhibited IL-6-induced up-regulation of p-STAT3 (Fig. 15B).
  • SC-43-induced apoptosis was significantly abolished in STAT3 overexpressing HSCs (Fig. 15C).
  • SHP-1 comprises two N-terminal SH 2 regions (N-SH 2 and C-SH 2 ) followed by a catalytic protein tyrosine phosphatase (PTPase) domain and a C-terminus.
  • PTPase catalytic protein tyrosine phosphatase
  • SHP-1 activity was increased in the dN1 and D61A mutants (Fig. 16A).
  • the present invention further detects wild-type SHP-1, dN1 and D61A using cell colony formation analysis Correlation between ectopic expression of mutants and HSC proliferation.
  • ectopic expression of the SHP-1, dN1 and D61A mutants significantly inhibited cell population formation compared to the vehicle control group.
  • using the solution cell proliferation assay of MTS, dN1 and D61A mutant expression significantly reduced cell viability (Fig. 17B).
  • the present invention detects the effect of SC-43-induced SHP-1 activity via ectopic expression of different SHP-1 mutants.
  • SC-43 treatment significantly increased the SHP-1 activity of the vector and wild-type SHP-1, but did not increase the SHP-1 activity of the dN1 and D61A mutants (Fig. 17C). Since SC-43 treatment increases SHP-1 activity, the present invention further investigates phenotypic changes (apoptosis) and p-STAT3 expression of ectopic expression of SHP-1 mutants after SC-43 treatment.
  • SC-43 treatment significantly increased apoptotic LX2 cells and down-regulated p-STAT3 (Fig. 17D and Fig.
  • SC-43 showed anti-fibrotic activity in both hepatotoxic and cholestatic fibrosis mouse models.
  • the present invention further demonstrates that SC-43 induces HSC apoptosis via a critical anti-fibrotic mechanism.
  • the SHP-1/STAT3 signaling pathway has proven to be extremely important in fibrogenesis and is involved in the survival of HSCs.
  • SC-43 Through interaction with the inhibitory N-SH 2 region of SHP-1, SC-43 increased SHP-1 activity and down-regulated p-STAT3, independent of PDGFR signaling.
  • the present invention demonstrates the anti-fibrotic activity of SC-43 in different animal models.
  • SC-43 treatment improved the survival rate of fibrotic mice compared to the control group.
  • the present invention observed recovery of liver cirrhosis in mice after stopping the causative factor CCl 4 , showing that fibrosis can be reversed after withdrawal of the virulence factor toxic agent, and similar to recent clinical findings, antiviral therapy
  • Continued viral suppression reverses cirrhosis in patients with chronic hepatitis B.
  • Administration of SC-43 significantly reduced fibrosis even during fibrosis recovery, indicating that HSC apoptosis is critical in remission of fibrosis.
  • the present invention demonstrates a correlation between the SHP-1/STAT3 signaling pathway and fibrogenesis, and SC-43 initiates SHP via direct interaction with the inhibitory N-SH 2 region of SHP-1. 1 activity and anti-fibrosis by promoting apoptosis of fibroblasts.
  • SC-43 showed anti-fibrotic activity in a mouse model of hepatotoxicity and cholestatic fibrosis caused by bile duct ligation, indicating that the SHP-1 phosphatase agonist is a potential target for anti-fibrotic drugs.
  • the SHP-1/STAT3 signaling pathway is a key signaling pathway in fiber generation. SC-43 promotes upregulation of SHP-1 activity and inhibits STAT3 phosphorylation to significantly improve fibrosis.
  • SHP-1 targeted anti-fibrotic therapy may be a strategy for the development of anti-fibrotic drugs.

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Abstract

提供一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂式(I)在制备治疗、降低纤维化的药物中的用途，优选所述激动剂为SC-43，所述纤维化为皮肤硬化或心脏、肺、肝、胰或肾脏的纤维化。

Description

含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂用于改善纤维化的用途
相关申请的交叉引用
本申请主张在2016年9月22日在美国提交的美国专利申请号No.62/398,274的优先权，其全部内容通过引用包含于此。
技术领域
本发明涉及一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)激动剂的用途，特别是涉及一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂用于改善纤维化的用途。
背景技术
纤维化是一种病理变化，表现为纤维母细胞激活增殖、组织器官内纤维结缔组织增多，实质细胞减少，持续进展可致组织、器官结构破坏和功能丧失。重要脏器的纤维化严重影响患者生存质量，甚至危及生命。纤维化疾病包括累及多系统的疾病，如系统性硬化症、多灶性纤维化、硬皮病、肾源性的多系统纤维化，也包括器官组织特异性疾病，如皮肤、心、肺、肝、肾纤维化等。不同纤维化疾病的病因各不相同，例如组织器官的损伤、感染、免疫反应、慢性炎症等，但其共同的特征是细胞外基质(ECM)在组织中的过度沉积和器官组织重构。
肝纤维化是指由各种致病因素所致的肝内结缔组织异常增生，肝内弥漫性细胞外基质过度沉淀的病理过程。多种因素均可引起肝纤维化，如病毒感染、炎症反应和酗酒等。肝纤维化的病理特点为肝门脉区和肝小叶内有大量纤维组织增生和沉积，但尚未形成小叶内间隔。肝硬化则有假小叶形成，中心静脉区和肝门脉区出现间隔，肝的正常结构遭到破坏。肝纤维化进一步发展即为肝硬化。
肺纤维化疾病包括特发性肺纤维化、结节病、过敏性肺炎、尘肺、药物和放射线导致的纤维化，以及与胶原血管病有关的致纤维化肺泡炎等病因各异、范围广泛的疾病谱。肺纤维化的主要病理特点包括肺组织间充质细胞增 殖、细胞外基质增生沉积及肺实质的重构等。目前主要采用抗炎、抗氧化、抗纤维母细胞增殖和胶原沉积及肺移植等措施治疗肺纤维化。特发性肺纤维化(idiopathic pulmonary fibrosis，IPF)是间质性肺疾病最常见的类型，主要是发生在中年和老年成人中未知病因的致命性疾病。虽然特发性肺纤维化的临床表现已知包含不同程度的间质性纤维化和炎症，但其他相关的诊断仍然很难治疗。特发性肺纤维化的特点是呼吸功能丧失，肺结构明显变形。特发性肺纤维化患者的组织病理学特征称为纤维母细胞病灶，其由活化的纤维母细胞的聚集体组成，该纤维细胞是在上皮细胞损失部位的肺泡空间内产生过量的细胞外基质。
由于肺纤维母细胞在肺纤维化中的细胞外基质沉积中起重要作用，所以肺中这些细胞的扩增群的起源是非常有意义的。对纤维母细胞/肌纤维母细胞的起源有一个典型及当代的理论。典型的理论是组织损伤会引起纤维母细胞的活化，以增殖和表现细胞外基质的成分。当代的理论认为，存在转化生长因子β(transforming growth factor-beta；TGFβ)的组织损伤会诱导上皮细胞转变为间充质的表现型，即纤维母细胞/肌纤维母细胞，后者有助于纤维增生和纤维化。因此，纤维母细胞的失活以及上皮-间质转化(epithelial mesenchymal transition，EMT)的抑制对改善肺纤维化有重要作用。
发明内容
有鉴于此，本发明的一个目的在于提供一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)激动剂在制备治疗纤维化的药物中的用途。
本发明的另一个目的在于提供一种以下化合物在制备治疗以含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)不活化为特征的疾病的药物中的用途
Figure PCTCN2017102945-appb-000001
本发明的再一个目的在于提供一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)激动剂在制备治疗以含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)不活化为特征的疾病的药物中的用途。
在本发明的一个实施例中，该含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂提高含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)的活性。
在本发明的一个实施例中，该含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂为以下化合物
Figure PCTCN2017102945-appb-000002
在本发明的一个实施例中，该以含有SRC同源区2蛋白酪氨酸磷酸酶-1(SHP-1)不活化为特征的疾病为纤维化。
在本发明的一个实施例中，该纤维化为皮肤硬化或心脏、肺、肝、胰、或肾脏的纤维化。
本发明的实施例经由肝、肺纤维化中的SHP-1/STAT3信号传导路径，评估给予细胞和动物SHP-1磷酸酶激动剂，例如SC-43化合物，而产生的抗纤维化效力。SHP-1磷酸酶激动剂SC-43化合物经由降低SHP-1具有抑制性的N-SH2区域与有催化性的蛋白酪氨酸磷酸酶区域直接相互作用，而活化SHP-1，并促进纤维母细胞凋亡而展现抗纤维化活性，表明SHP-1磷酸酶激动剂是抗纤维化药物的潜在靶标，其可为一种发展抗纤维化药物的新策略。
以下将结合附图进一步说明本发明的实施方式，下述所列举的实施例用以阐明本发明，并非用以限定本发明的范围，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可做些许更动与润饰，因此本发明的保护范围当视后附的权利要求所限定的范围为准。
附图说明
图1为本发明的博来霉素(bleomycin)诱导小鼠肺纤维化的实验性急性肺损伤模型的示意图。
图2为本发明以SC-43治疗以改善博来霉素诱导的小鼠肺纤维化的肺的苏木精-伊红(H&E)组织染色图。CTL表示对照组小鼠；BLM表示博来霉素诱导小鼠组；BLM+SC43表示以SC-43治疗博来霉素诱导小鼠组。
图3为本发明以SC-43治疗以改善博来霉素诱导的小鼠肺纤维化的肺的 梅生三色(Masson′s trichrome)组织染色图。CTL表示对照组小鼠；BLM表示博来霉素诱导小鼠组；BLM+SC43表示以SC-43治疗博来霉素诱导小鼠组。
图4为本发明以SC-43治疗以改善博来霉素诱导的小鼠肺纤维化的羟脯氨酸含量的柱状图。CTL表示对照组小鼠；BLM表示博来霉素诱导小鼠组；BLM+SC43表示以SC-43治疗博来霉素诱导小鼠组。柱表示平均值；条带表示标准偏差；**P＜0.05。
图5为本发明以SC-43治疗以改善博来霉素诱导的小鼠肺纤维化的胶原蛋白含量的柱状图。CTL表示对照组小鼠；BLM表示博来霉素诱导小鼠组；BLM+SC43表示以SC-43治疗博来霉素诱导小鼠组。柱表示平均值；条带表示标准偏差。
图6为本发明以SC-43治疗以改善博来霉素诱导的小鼠肺纤维化的上皮-间质转化(epithelial mesenchymal transition，EMT)的胶体电泳图。
图7A表示本发明以SC-43处理经由SHP-1/STAT3信号传导路径会降低小鼠纤维母细胞(NIH3T3细胞株)存活率。
图7B表示本发明以SC-43处理经由SHP-1/STAT3信号传导路径会降低p-STAT3的表达。
图7C表示本发明以SC-43处理经由SHP-1/STAT3信号传导路径会诱导细胞凋亡。
图7D表示本发明以SC-43处理经由SHP-1/STAT3信号传导路径会因SHP-1剔除而导致细胞凋亡作用抵消。
图8A表示本发明将人肺上皮细胞(A549细胞株)暴露于TGF-β1诱导上皮-间质转化的产生。E-钙黏蛋白的表达降低；纤连蛋白和N-钙黏蛋白的表达向上调节；暴露于TGF-β1后STAT3磷酸化也增加(左图)；TGF-β1刺激后EMT的侵袭细胞和迁移细胞增加(右图)。
图8B表示本发明以SC-43处理在人肺上皮细胞(A549细胞株)中会抑制TGF-β1-诱导上皮-间质转化的产生。E-钙黏蛋白的表达增加；纤连蛋白和N-钙黏蛋白的表达降低；暴露于TGF-β1后STAT3磷酸化也降低。
图8C表示本发明以SC-43处理在人肺上皮细胞(A549细胞株)中会抑制TGF-β1-诱导上皮-间质转化的产生。以SC-43处理后TGF-β1刺激产生的EMT 侵袭细胞和迁移细胞减少。
图8D表示本发明以SC-43处理在人肺上皮细胞(A549细胞株)中会抑制TGF-β1-诱导上皮-间质转化的侵袭细胞和迁移细胞。
图9A表示本发明在A549细胞中转染SHP-1会减少迁移细胞及侵袭细胞的EMT标记表达。
图9B表示本发明在A549细胞中以siRNA剔除SHP-1的对迁移细胞及侵袭细胞的EMT标记表达的影响。
图9C表示本发明在A549细胞中以siRNA剔除STAT3的表达对EMT标记、迁移细胞及侵袭细胞的影响。
图10A表示在CCl4诱导肝纤维化小鼠模型中的纤维化区域中SHP-1磷酸酶的表达；比例尺：200μm。
图10B表示在慢性乙型肝炎(CHB)晚期纤维化患者中SHP-1磷酸酶的表达；比例尺：200μm。
图10C表示SHP-1磷酸酶的表达与血清丙氨酸氨基转移酶浓度有正相关性。
图10D表示SHP-1磷酸酶表达于活化的肝星状细胞(hepatic stellate cells，HSC)。SHP-1磷酸酶与活化的肝星状细胞标志物(marker)α-SMA共定位(colocalized)，证实SHP-1磷酸酶表达于活化的肝星状细胞。
图11A表示在CCl4诱导肝纤维化小鼠预防模型中SC-43治疗会改善肝纤维化的天狼星红染色(Picrosirius Red Stain)影像图；每个组n＝7-9。
图11B表示在CCl4诱导肝纤维化小鼠预防模型中SC-43治疗会改善肝纤维化的天狼星红染色经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝7-9。
图11C表示在CCl4诱导肝纤维化小鼠预防模型中SC-43治疗会改善肝纤维化的Ishak纤维化评分系统分级柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝7-9。
图11D表示在CCl4诱导肝纤维化小鼠预防模型中SC-43治疗会改善肝纤维化的α-SMA染色影像图。α-SMA为活化的肝星状细胞标志物(marker)；比例 尺：200μm，每个群组n＝7-9。
图11E表示在CCl4诱导肝纤维化小鼠预防模型中SC-43治疗会改善肝纤维化的羟脯氨酸浓度柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝7-9。
图12A表示在CCl4诱导肝纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的天狼星红染色影像图；比例尺：200μm；每个组n＝6-8。
图12B表示在CCl4诱导肝纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的天狼星红染色经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05以及**P＜0.01，每个组n＝6-8。
图12C表示在CCl4诱导肝纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的Ishak纤维化评分系统分级的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05，每个组n＝6-8。
图12D表示在CCl4诱导肝纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的羟脯氨酸浓度的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05，每个组n＝6-8。
图12E表示在CCl4诱导肝纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的小鼠存活率(对数秩(log-rank)P＝0.0291)；每个组n＝6-10。
图12F表示在CCl4诱导肝纤维化小鼠复原模型中SC-43治疗会改善肝纤维化的天狼星红染色影像图；比例尺：200μm；每个组n＝6-8。
图12G表示在CCl4诱导肝纤维化小鼠复原模型中SC-43治疗会改善肝纤维化的天狼星红染色经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05，每个组n＝6-8。
图12H表示在CCl4诱导肝纤维化小鼠复原模型中SC-43治疗会改善肝纤维化的Ishak纤维化评分系统分级的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05，每个组n＝6-8。
图13A表示在胆管结扎(bile duct ligation，BDL)小鼠预防模型中SC-43治疗会改善肝纤维化的天狼星红染色影像图；比例尺：200μm；每个组n＝7-8。
图13B表示在BDL诱导胆汁淤积纤维化小鼠预防模型中SC-43治疗会改善肝纤维化的天狼星红染色经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05，每个组n＝7-8。
图13C表示在BDL诱导胆汁淤积纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的天狼星红染色影像图；比例尺：200μm；每个组n＝7-8。
图13D表示在BDL诱导胆汁淤积纤维化小鼠治疗模型中SC-43治疗会改善肝纤维化的天狼星红染色经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)的柱状图；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)*P＜0.05，每个组n＝7-8。
图14A表示在时间及剂量依赖下给予SC-43的肝星状细胞存活率的柱状图；在HSC-T6及LX2细胞中给予SC-43或索拉非尼24小时；原代小鼠HSCs细胞中分别给予SC-43 24或48小时。柱代表平均值、条带代表标准偏差，每个组n＝3-4。
图14B表示在时间及剂量依赖下给予SC-43的肝星状细胞凋亡的柱状图；在HSC-T6及LX2细胞中给予SC-43或索拉非尼24小时。柱代表平均值、条带代表标准偏差，每个组n＝3-4。
图14C表示SC-43经由增加切割的聚(ADP-核糖)聚合酶(PARP)片段诱导肝星状细胞凋亡的电泳图；每个组n＝3-4。
图14D表示在LX2细胞中SC-43会诱导转化生长因子(TGF)-β路径p-Smad2以及p-Smad3向下调节的电泳图；每个组n＝3-4。
图14E表示在LX2以及HSC-T6细胞中SC-43会诱导血小板衍生生长因子受体(PDGFR)路径p-PDGFR以及p-Akt向下调节的电泳图；每个组n＝3-4。
图15A表示在LX2和HSC-T6细胞中SC-43治疗会剂量依赖的向下调节p-STAT3和细胞周期蛋白D1的电泳图。
图15B表示SC-43会向下调节介白素(IL)-6-STAT3的电泳图。
图15C表示SC-43诱导的细胞凋亡在STAT3过度表达的HSC中会被显著消除；柱代表平均值、条带代表标准偏差；相较于载体(vehicle)***P＜0.001。
图15D表示给予特异性PDGFR抑制剂AG1295后SC-43会向下调节p-Akt 和p-STAT3。
图16A为SHP-1及其突变体dN1(包含N-SH2区域缺失)以及D61A(D61的单一突变)的示意图。
图16B表示SHP-1过度表达会显著减少LX2细胞存活率；柱代表平均值、条带代表标准偏差；*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝3-5。
图16C表示在LX2及HSC-T6细胞中分别给予索拉非尼(5μM)或SC-43(2.5或5μM)后会增加SHP-1活性使细胞凋亡率提高；柱代表平均值、条带代表标准偏差；*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝3-5。
图16D表示以非专一性磷酸酶抑制剂钒酸盐治疗会向上调节p-STAT3及减少细胞凋亡；柱代表平均值、条带代表标准偏差；*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝3-5。
图16E表示SHP-1的专一性抑制剂(PTP抑制剂III)会向上调节p-STAT3以及减少细胞凋亡；柱代表平均值、条带代表标准偏差；*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝3-5。
图16F表示利用siRNA沉默SHP-1基因而逆转SC-43对p-STAT以及细胞凋亡的效果；柱代表平均值、条带代表标准偏差；*P＜0.05、**P＜0.01以及***P＜0.001，每个组n＝3-5。
图17A表示SHP-1、dN1以及D61A突变体的异位表达显著抑制细胞群落形成的柱状图；柱代表平均值、条带代表标准偏差；***P＜0.001，每个组n＝3-5。
图17B表示dN1以及D61A突变体表达显著使细胞存活率降低；柱代表平均值、条带代表标准偏差；***P＜0.001，每个组n＝3-5。
图17C表示SC-43处理会显著增加载体和野生型SHP-1的SHP-1活性但不会增加dN1和D61A突变体的SHP-1活性；柱代表平均值、条带代表标准偏差；*P＜0.05，每个组n＝3-5。
图17D表示载体对照组以及野生型SHP-1的异位表达在SC-43处理(5μM，24小时)后会显著增加凋亡的LX2细胞并向下调节p-STAT3；柱代表平均值、条带代表标准偏差，每个组n＝3-5。
图17E表示在过度表达野生型SHP-1的细胞中，SC-43处理会显著增加凋亡的LX2细胞并向下调节p-STAT3，但dN1和D61A突变体对SC-43处理并不敏 感；柱代表平均值、条带代表标准偏差；***P＜0.001，每个组n＝3-5。
图17F为SC-43通过SHP-1-STAT3路径的抗纤维化机制的示意图。
具体实施方式
除非另有定义，本文所用的所有技术及科学术语与本发明所属技术领域的技术人员通常理解的含义相同。本文所提及的所有出版物皆以引用方式并入本文，以揭露及描述与该被引用的出版物有关的方法和/或材料。
如本文所使用，单数形式“一个/一种”(a、an)和“该”(the)包括复数对象，除非本文中清楚地另有规定。因此，例如，参照“一种样品”包括复数个此类样品以及本领域的技术人员已知的等同物。
本文所述的“纤维化”是肺、肝、肾、血管、腹膜、胰脏、皮肤等组织和器官由于炎症、感染、免疫反应、缺血、化学物质、辐射等各种原因导致持续损伤后，纤维母细胞激活增殖、组织器官内纤维结缔组织增多，实质细胞减少，组织、器官结构破坏和功能丧失的病变。该纤维化病变术语涵盖各种原因导致的心脏纤维化、肺纤维化、肝纤维化、肾纤维化、血管纤维化、皮肤纤维化(硬化)等组织器官纤维化病变，还包括各种疾病发生、发展过程中伴随产生的心脏纤维化、肺纤维化、肝纤维化、肾纤维化、血管纤维化、皮肤纤维化(硬化)等组织器官纤维化病变。
本文所述的“肝纤维化”是指由各种原因(例如炎症、感染(例如病毒感染)、免疫反应、缺血、化学物质、辐射、氧化应激和酗酒)导致的或伴随的肝内结缔组织异常增生，肝内弥漫性细胞外基质过度沉淀、肝的正常结构遭到破坏的病理变化(病变)。肝纤维化进一步发展即为肝硬化，肝硬化也涵盖在本发明“肝纤维化”术语的范围内。
本文所述的“肺纤维化”是指各种原因(例如炎症、感染、免疫反应、缺血、化学物质、辐射)导致的或伴随的肺组织间充质细胞增殖、细胞外基质增生沉积及肺实质的重构而造成的病理过程。
本文所述的“治疗”包括特定失调或病症的预防，或与特定失调或病症有关的症状的减轻和/或该症状的预防或消除。
本文所述的“预防”为治疗以降低或最小化尚未呈现临床疾病状态的患者的疾病状态的风险，而次级预防定义为最小化或降低相同或类似临床疾病状态的复发或第二次发生的风险。
本文所述的“个体”为动物，如人类，但也可以是宠物(例如，狗、猫及其类似物)、经济动物(例如，牛、羊、猪、马及其类似物)或实验用动物(例如，大鼠、小鼠、天竺鼠及其类似物)，该动物需要如本文所述的治疗。
本文所述的“有效量”是指在个体上达到治疗效果所需要的活性剂的量，不论是单独或与一种或多种其他活性剂组合使用。根据给药途径、赋形剂的使用，以及与其他活性剂共同使用等不同情况，在由本领域技术人员确认下，有效量各不相同。
适合的给药途径可能包括如口服、直肠给药、黏膜给药、或肠道给药；非经口递送，包括肌内、皮下、髓内注射，以及鞘内、直接心室内、静脉、腹膜、鼻内或眼内注射，并可为补充或缓释剂型。
本发明的医药组合物可以本领域已知的方式制造，例如，通过惯用的混合、溶解、乳化、包埋、包封、或冻干过程制造。因此，根据本发明所提供使用的医药组合物可以常规的方式配制，使用一个或多个生理上可接受的辅料，包含赋形剂和/或助剂，以有助于活性化合物的加工而形成医药上可使用的制剂。如本文所用，“可接受”是指该辅料必须与该组合物的活性成分相容(且较佳地，能够稳定该活性成分)，且不会对受治疗的个体有害。适当的剂型取决于所选择的给药途径。
具体而言，对于注射给药，本发明的化合物可以被配制于例如生理上相容的缓冲液内，如汉克缓冲液、林格氏缓冲液或生理盐水缓冲液。针对口服给药，本发明的化合物的配制可经由将该活性化合物与本领域已知的医药上可接受的辅料结合，如乳糖、蔗糖、甘露糖醇、山梨糖醇、玉米淀粉、小麦淀粉、米淀粉、马铃薯淀粉、明胶、黄蓍胶、甲基纤维素、羟丙基甲基纤维素、羧甲基纤维素钠，和/或聚乙烯吡咯烷酮(PVP)，以使本发明的化合物可以配制为片剂、丸剂、糖衣丸、胶囊、液体、凝胶、糖浆、浆液、悬浮液及其类似物。对于吸入法给药，本发明的化合物可被配制为自加压容器或喷雾器喷出的气雾喷雾，并搭配使用适当的推进剂，例如，二氯二氟甲烷、三氯 氟甲烷、二氯四氟乙烷、二氧化碳或其它合适的气体。
实施例1 本发明的SC-43化合物的制备方法
将4-氯-3-(三氟甲基)苯氨(0.21g，1.1mmol)和2当量的三乙胺加入50mL含有三光气(0.30g，1.0mmol)的THF溶液中。将混合物加热至50℃后加热30分钟。待温度降至室温后，将溶于10mL THF溶液的4-(4-氨基苯氧基)芐腈加入该混合物中，并再次加热至50℃后加热30分钟。将该混合物蒸发，用水稀释，并用乙酸乙酯(EtOAc)萃取。以食盐水洗涤萃取液，以无水硫酸镁干燥，并进行减压浓缩，得到1-(3-(4-氰基苯氧基)苯基)-3-(4-氯-3-(三氟甲基)苯)脲(0.34g，80％)，本发明命名为SC-43。
Figure PCTCN2017102945-appb-000003
质子核磁共振(1H-NMR)光谱记录于核磁共振光谱仪(Bruker DPX300(400MHz))。化学位移被报告为δ值(ppm)低磁场，该低磁场来自指示用有机溶液内部的氘代氯仿。峰的多重性表示如下：s，单峰；d，双峰；t，三重峰；q，四重峰；dd，双重双峰；ddd，双重峰组成的双重双峰；dt，双重三重峰；brs，宽单峰；m，多重峰。偶合常数(J值)以赫兹(Hz，hertz)表示。反应进程以在硅胶60F254板(Merck)上进行薄层色谱分析法(TLC，thin layer chromatography)分析而确定。色谱分析的纯化是于硅胶管柱60(0.063-0.200mm或0.040-0.063mm，Merck)，碱性硅胶中进行。使用市售的试剂和溶剂而无需再纯化。化学式缩写如下所示：CDCl3，氘代氯仿；DMSO-d6，二甲亚砜-d6；EtOAc，乙酸乙酯；DMF，N，N-二甲基甲酰胺；MeOH，甲醇；THF，四氢呋喃；EtOH，乙醇；DMSO，二甲亚砜；NMP，N-甲基吡咯烷酮。高解析质谱纪录于FINNIGAN MAT 95S质谱仪上。
H NMR(400MHz，DMSO)：δ9.17(s，1H)，9.03(s，1H)，8.04(d，J＝2.4Hz，1H)，7.83(d，J＝8.8Hz，2H)，7.64-7.55(m，2H)，7.41-7.32(m，2H)，7.23(d，J＝7.2Hz，1H)，7.11(d，J＝8.0Hz，2H)，6.75(dd，J＝8.0Hz，2.4Hz，1H)；C21H12N3O2F3Cl[M-H]-的计算值：430.0570。实测值：430.0576。
实施例2 生物活性检测的方法
本发明研究纤维化中的含有SRC同源区2的蛋白酪氨酸磷酸酶 -1(Src-homology region 2 containing tyrosine phosphatase-1，SHP-1)-信号转导及转录激活因子3(signal transducer and activator of transcription 3，STAT3)路径，评估给予活体外(in vitro)和活体内(in vivo)SC-43(SHP-1磷酸酶激动剂)而产生的抗纤维化效力。本发明在纤维化的肺、肝中提高SHP-1活性。通过鼻内滴注博来霉素在雄性C57BL/6J小鼠中建立实验性肺纤维化小鼠模型；以及经由在雄性C57BL/6小鼠中注射四氯化碳(CCl4)以及经由Balb/C小鼠胆管结扎(BDL)建立实验性肝纤维化小鼠模型，并给予SC-43进行试验。人肺上皮细胞(A549细胞株)、小鼠纤维母细胞(NIH3T3细胞株)及大鼠、人类和小鼠肝星状细胞(HSC)则用于体外细胞研究，特别是着眼于SHP-1/STAT3信号传导路径。
2.1材料与方法
索拉非尼(蕾莎瓦膜衣锭
Figure PCTCN2017102945-appb-000004
)由拜耳制药公司(Bayer HealthCare AG)(柏林，德国)提供。α-平滑肌肌动蛋白(α-SMA)、磷酸化STAT3(Tyr705)、STAT3、细胞周期蛋白D1、甘油醛-3-磷酸脱氢酶、P-Smad2(Ser465/467)、P-Smad3(Ser423/425)Smad2、Smad3、聚(ADP-核糖)聚合酶(PARP)、血小板衍生生长因子受体(PDGFR)-β，P-PDGFR-β(Tyr857)和P-Akt(Ser473)购自细胞信号转导公司(Cell Signaling)(麻州，美国)。Akt购自圣克鲁斯生物技术公司(Santa Cruz Biotechnology)(加州，美国)。钒酸钠购自昶安生物科技有限(Cayman Chemical)(密西根，美国)。PTP抑制剂III购自Calbiochem公司(加州，美国)。聚氧乙烯蓖麻油(Cremophor)从Sigma公司(密苏里州，美国)获得。
2.2实验方法
2.2.1动物实验
雄性C57BL/6J(6-8周龄)自国家实验动物中心(National Laboratory Animal Center)(台北，台湾)而来。所有使用这些小鼠进行的实验程序，根据由天主教耕莘医院机构实验室动物护理和使用委员会批准的方案进行。将动物饲养在个别通气笼(individual ventilation cage，IVC)中，每天光照射12小时；室温维持在22至25℃；湿度保持在60至70％；通气率保持在16至18次/小时；食物和饮用水(Altromin 1326小鼠饲料，德国)自由给予。所有实验程序均按照动物实验动物法标准程序执行。动物的管理和喂养符合实际使用 (NRC 1996)规则方法实施的依据
2.2.2博来霉素诱导肺纤维化
在雄性C57BL/6J小鼠中，使用博来霉素硫酸盐诱导肺纤维化(图1)。在阿佛丁(Avertin)(240mg/Kg)麻醉下小鼠腹膜内注射(IP)。在第0天(n＝20)，经由鼻内滴注，以3.5mg/kg接受单剂量的博来霉素(BLM)(1mg＝1000IU，临床级，Bleomycine Bellon，Sanofi-Aventis，法国)。对照组(n＝10)中的小鼠只给予食盐水。在鼻内BLM滴注后的第14天，将BLM处理的小鼠随机分成接受载体(vehicle)或SC-43的两组，直到实验结束。从第14天至第28天，每天腹膜内给予SC-43 7.5mg/kg/天，施用体积为10mL/kg体重。研究时间为28天，SC-43化合物在14至28天内给药。
2.2.3免疫组织分析
将石蜡包埋的组织数组块切成4μm厚的苏木精-伊红染色(H&E Stain)的染色切片。对于每种病例，由两名病理学家重新检查癌症类型、细胞分化、生长模式、肿瘤细胞核形态、组织转化、钙化、坏死、有丝分裂数、侵袭状态和其他特异性差异。使用Ventana BenchMark XT自动染色机(亚利桑那，美国)进行免疫组织化学(IHC)染色。简言之，从福尔马林固定的石蜡包埋的组织中连续切割4μm厚的切片。然后将这些切片放置在硅烷化载玻片上，并使其在37℃下干燥过夜。脱石蜡和再水化后，将载玻片与3％过氧化氢溶液反应5分钟。用供应的缓冲液洗涤后，用乙二胺四乙酸(ethylenediamine tetraacetic acid，EDTA)将组织切片修复40分钟。
2.2.4细胞培养
小鼠纤维母细胞(NIH3T3细胞株)和人肺上皮细胞(A549细胞株)购自生物资源保存及研究中心(新竹，台湾)。使用添加10％胎牛血清(FBS)、100μg/mL青霉素、100μg/mL链霉素的杜氏改良Eagle培养基(dulbecco′s modified eagle medium，DMEM)，于37℃、含有5％二氧化碳的加湿培养箱中培养，每4至5天以1∶4比例继代培养。
大鼠永生肝星状细胞株HSC-T6以及LX2人类肝星状细胞株由纽约西奈山医院的Scott Friedman教授亲自提供，原代小鼠HSCs细胞由小鼠肝门静脉插管后，利用Leffert’s及乙二醇双氨乙基醚四乙酸(EGTA)缓冲液原位循环灌 流，再灌入胶原酶(collagenase)将肝组织进行消化，取得细胞后利用Histodenz梯度分离的方式获得。HSC-T6细胞培养在Waymouth培养基；LX2细胞及原代小鼠HSCs细胞使用添加10％胎牛血清(FBS)、100μg/mL青霉素、100μg/mL链霉素以及2mM L-谷氨酰胺的杜氏改良Eagle培养基(DMEM)，于37℃、含有5％2氧化碳的加湿培养箱中培养。在添加FBS后，休眠的HSC被启动。对于活体外研究中，将各种浓度的SC-43和索拉非尼溶解在二甲基亚砜中，随后加入至在5％FBS中的细胞中处理预定时间。
2.2.5羟脯氨酸测定
依据制造商的操作手册进行(Cell BioLabs，Inc公司，美国)，使用羟脯氨酸检测试剂盒进行羟脯氨酸分析。将肺组织在120℃的12N HCl中水解3小时，然后洗涤并悬浮于H2O中。然后将组织悬浮液与氯胺-T(chloramines-T)混合，并在室温下反应30分钟。然后加入Ehrlich’s试剂并在60℃下反应90分钟。之后，测量540nm下的吸光值。
2.2.6胶原蛋白含量
依据制造商的操作手册进行，使用Sircol测定(Biocolor公司，英国)在肺均质物(homogenate)中测量小鼠肺胶原蛋白。简言的，将肺组织均质物与Sircol染料混合并离心。将沉淀物重新悬浮在NaOH中，并测量550nm下的吸光值。获得的吸光值与最近合成的胶原蛋白的浓度成正比。
2.2.7蛋白质印记法(Western blotting)
经由RIPA缓冲液(Millipore公司，美国)获得全细胞萃取物，且经由BCA蛋白质分析试剂盒(Thermo Fisher scientific公司，美国)定量蛋白质浓度。将20或25μg的蛋白质加入至各种百分比SDS-丙烯酰胺凝胶上，并印迹至聚偏二氟乙烯(polyvinylidene fluoride，PVDF)膜上。然后，将膜与第一抗体进行反应；大量清洗后，将膜与含有结合第二抗体的辣根过氧化物酶(horseradish peroxidase，HRP)阻拦缓冲液反应。蛋白质由以Immobilon Western Chemiluminescent HRP底物(Millipore公司，美国)或ECL检测系统(UVP公司，美国)检测。
2.2.8抗体
用于免疫印迹技术的抗体如E-钙黏蛋白(E-cadherin)、纤维连接蛋白 (Fibronectin)、N-钙黏蛋白(N-cadherin)(Abcam公司，英国)，购自细胞信号转导公司(麻州，美国)的其它抗体如抗SHP-1、磷酸化STAT3(Tyr705)、STAT3α-平滑肌肌动蛋白(α-SMA)和半胱天冬酶-9(Caspase-9)。
2.2.9细胞存活率分析
将小鼠NIH3T3纤维母细胞接种至96-孔盘中(1×103个细胞/孔)中。为测量细胞存活率及增殖，加入10％WST-1(细胞增殖试剂WST-1，罗氏)至每个孔并培养3小时。该反应在活性细胞中以线粒体还原酶进行催化，并经由Bio-Rad ELISA分析仪测量450nm下的吸光值，计算光密度(OD)。
将HSC-T6、LX2或原代小鼠HSCs细胞接种至96-孔平底盘(5，000个细胞/孔)中培养24小时，将该细胞分别以各种浓度的SC-43或索拉非尼处理24及48小时。使用含有3-(4，5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺基苯基)-2H-四氮唑内盐(3-(4，5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium，an inner，MTS)的CellTiter 96AQueous one溶液细胞增殖分析(Promega公司，美国)评估各试剂对细胞存活率的影响，依据制造商的操作手册进行，进行三个重复。在SC-43或索拉非尼治疗24小时后，以BD FACS Verse流式细胞仪(BD公司，美国)，利用膜联蛋白(Annexin)V和碘化丙锭(propidium iodide)双重染色检测，在早期(Annexin V+/PI-)和晚期(Annexin V+/PI+)凋亡细胞混合后检测凋亡细胞的比例。
2.2.10细胞凋亡分析
使用流式细胞仪(sub-G1)对凋亡的细胞进行计量，且以细胞质组蛋白相关的DNA片段化的酵素结合免疫吸附分析法(ELISA)检测细胞死亡。经由使用ELISA分析仪以测量570nm下甲
Figure PCTCN2017102945-appb-000005
(formazan)染剂量的吸光值计算光密度(OD)。
2.2.11 STAT3的异位表达(ectopic expression)
由Addgene质体工厂公司购买STAT3cDNA(KIAA1524)。小鼠纤维母细胞(NIH3T3细胞株)和人肺上皮细胞(A549细胞株)与STAT3、SHP-1、SHP-1(dN1)突变体以及SHP-1(61A)突变体瞬时转染(Transient transfection)，然后分别与SC-43化合物一起反应，并进行蛋白质印记法。转染是使用 X-tremeGENE HP转染试剂(罗氏公司，德国)，依据制造商的操作手册进行。
2.2.12 SHP-1磷酸酶活性
药物治疗后，将小鼠纤维母细胞(NIH3T3细胞株)、人肺上皮细胞(A549细胞株)或LX2细胞蛋白质萃取物在免疫沉淀缓冲液中与抗-SHP-1抗体一起反应过夜。蛋白质A/G琼脂糖快速流珠(GE)加入每个样本中，随后在4℃下旋转反应3小时。将
Figure PCTCN2017102945-appb-000006
酪氨酸磷酸酶分析试剂盒(R-22067)用于SHP-1活性检测(Invitrogen公司，美国)。
2.2.13使用siRNA进行基因剔除
Smart-pool siRNA，包括对照组(sc-37007)siRNA、siRNA抗SHP-1以及STAT3，均购自圣克鲁斯生物技术公司(Santa Cruz Biotechnology)(加州，美国)。
2.2.14在肝纤维化患者中SHP-1的表达
本发明收集25个具有不同程度肝纤维化的慢性乙型肝炎患者(n＝5，分别为F0、F1、F2、F3和F4)。他们的活组织检查肝组织进行SHP-1或α-SMA染色。该研究符合1975年“赫尔辛基宣言”的道德准则，并获得台湾大学附设医院伦理委员会的批准。所有患者在入院时获得书面知情同意书。
2.2.15肝纤维化小鼠模型
在雄性C57BL/6J小鼠中，使用四氯化碳诱导肝纤维化。每两周腹膜注射给予小鼠四氯化碳持续4或8周，在指定期间内，经由灌食给予聚氧乙烯蓖麻油(cremophor)载体、SC-43(分别为5、10、或20mg/kg)或索拉非尼每周五天至牺牲；索拉非尼因已证实具有抗纤维化活性，故作为阳性对照组。
在胆管结扎(BDL)小鼠模型中，在Balb/C小鼠中将胆总管双重结扎然后切除。第1天或第8天每天经由灌食给予载体或SC-43，直到在第14天牺牲。
2.2.16肝纤维的组织分析
将小鼠肝脏样本保存在10％甲醛中，以梯度酒精脱水，包埋至石蜡块中，切片至3μm厚度，放置在载玻片上，并使用天狼星红染色(Picrosirius Red Stain)试剂盒(ScyTek公司，美国)，依据制造商操作手册进行。肝纤维化严重程度根据Ishak纤维化评分系统分级。经由使用ImageJ软件，经由光密度测定以定量胶原蛋白-阳性面积(qCPA)。使用羟脯氨酸试剂盒测定(Biovision公司，USA美国)测量肝羟脯氨酸浓度，依据制造商操作手册进行。使用Leica  BOND-MAX自动色谱仪(Leica Biosystems公司，德国)进行免疫组织化学染色，依据制造商操作手册进行。
2.2.17转化生长因子-β诱导
关于转化生长因子(TGF)-β诱导，将HSC血清处里4小时，随后用10μM SC-43处理4小时，再用10ng/mL重组人类TGF-β1(R&D Systems公司，美国)处理20分钟。
2.2.18血小板衍生生长因子-BB诱导
关于血小板衍生生长因子(PDGF)-BB诱导，将LX2或HSC-T6细胞血清处里4小时，随后用10μM SC-43处理4小时，再用100ng/mL重组人类或大鼠PDGF-BB(R&D Systems公司，美国)处理10分钟。
2.2.19介白素-6刺激
关于介白素(IL)-6刺激，将HSC血清处里4小时，随后用10μM SC-43处理4小时，再用100ng/mL IL-6(R&D Systems公司，美国)处理30分钟。
2.2.20质体、siRNA以及转染
大鼠STAT3(Open Biiosystem，美国)建构在pLVX-AcGFP-N1表达载体(Clontech公司，美国)上，其后除了慢病毒包裹以及表达载体(P8.91以及VSV-G)外，经由使用脂质体(Lipofectamine)2000转染试剂(Invitrogen公司，美国)共转染至293FT细胞。转染48小时后取慢病毒上清液，用于感染5×105个HSC-T6细胞，将其接种在6-cm培养皿上；获得大鼠STAT3稳定表达的细胞。
含有人类野生型SHP-1以及SHP-1突变体的质体，含SHP-1突变体的质体是将N-SH2区域截短(dN1)或位点61处的一个天冬氨酸改变为丙氨酸残基(D61A)的质体被选殖至具有myc标签的pCMV6-entry载体。经由DNA测序确认该些突变体的序列。Smart-pool siRNA，包括对照组(D-001810-10)以及SHP-1(PTPN6，L-009778-00-0005)均购自Dharmacon公司(芝加哥，美国)。关于瞬时基因表达，依据制造商的操作手册进行，以脂质体(Lipofectamine)2000转染试剂将SHP-1质体或siRNA(最终浓度为100nM)转染至LX2细胞24小时。
2.2.21细胞群落形成分析
将LX2细胞置于10-厘米的培养皿上(每个培养皿1500至5000个细胞)，以DMEM培养基培养2周。该细胞之后以4％甲醛固定并以0.1％结晶紫染色。
2.2.22统计分析
连续变量以平均值(标准误差)表示，分类数据以适当的数量(百分比)表示。使用学生t检验(Student′s t test)确定亚组(subgroup)之间的差异。使用斯皮尔曼相关性(Spearman′s coefficient)用于确认SHP-1和血清丙氨酸氨基转移酶(ALT)之间的关联性。使用Kaplan-Meier法评估小鼠存活率。对数秩(Log-rank)检验用于确定不同实验组的存活率的统计学差异。使用STATA(第13版，Stata Corp公司，美国)进行统计学分析。所有检验均为双侧，P＜0.05认为显著差异有统计学意义。
实施例3 SC-43用于治疗减少小鼠肺纤维化
含有SRC同源区2的蛋白酪氨酸磷酸酶-1，是一种对不同的细胞内信号分子的调节剂，例如信号转导及转录激活因子3、KIT、CD22、CD5、CD72、SHPS-1、TIMP(金属蛋白酶)、CDK2、p27、SRC、ZAP70、介白素10(IL-10)、NF-κB、Lck、3BP2、Lyn，以及细胞周期蛋白D1。STAT3是一种通过调控目标基因的表达来调节细胞生长和存活的转录因子。SHP-1是STAT3活性的一个关键调节因子。
本发明证明SC-43的抗肺纤维化活性，显示SC-43化合物启动SHP-1活性，向下调节STAT3，然后降低上皮细胞的纤维母细胞存活率和上皮-间质转化。本发明于活体内实验数据也证实SC-43改善博来霉素诱导小鼠的肺纤维化，这表示SHP-1激动剂有可用于治疗特发性肺纤维化(IPF)和其他纤维化疾病。
3.1 SC-43会改善在博来霉素诱导小鼠肺纤维化的肺纤维化
本发明建立博来霉素诱导肺纤维化的实验性急性肺损伤模型(如图1所示)。使用这种动物模型，利用7.5mg/kg剂量的SC-43经由日常管饲法进行治疗有良好耐受性，因为没有观察到药物相关的不良事件。根据肺切片的苏木精-伊红染色(H&E Stain)检测，BLM鼻内注射导致正常肺结构的破坏、纤维母细胞的显著增殖、发炎细胞的浸润和纤维胶原的大量沉积)(图2中间)。更重要的是，给予SC-43后这些病理变化会显著改善(图2右图)；同样，在 给予SC-43后纤维胶原沉积会大幅降低，如梅生三色(Masson′s trichrome)染色阳性区域所示(图3)。
接着，由于肺羟脯氨酸(Hyp)是胶原蛋白的主要成分，本发明检测每组小鼠的肺羟脯氨酸(Hyp)含量，以定量肺纤维化的程度。如图4所示，与BLM组相比，SC-43治疗后Hyp含量降低约22％，表示SC-43在对细胞外基质(ECM)积累中具有保护作用。本发明检测小鼠的胶原蛋白含量(图5)，发现SC-43会改善肺纤维化模型中胶原蛋白的积累。本发明进一步检测SC-43是否减少上皮-间质转化，这是纤维发生的关键步骤；如图6所示，与对照组(第1泳道和第2泳道)相比，博来霉素诱导小鼠肺纤维化样本(第3泳道和第4泳道)中E-钙黏蛋白(E-cadherin)(上皮表型蛋白)减少。但是，博来霉素诱导小鼠肺纤维化样本经过SC-43处理后(第5泳道和第6泳道博来霉素诱导小鼠肺纤维化样本后，E-钙黏蛋白的表达量增加。纤维连接蛋白(Fibronectin)是间质表现型标记物，在博来霉素诱导小鼠肺纤维化样本的表达量会增加(第3泳道和第4泳道)，但在SC-43处理后(第5泳道和第6泳道)减少。博来霉素诱导的肺纤维化中P-STAT3增加，但在SC-43治疗后也会降低。
3.2在小鼠肺纤维化中SC-43经由SHP-1诱导STAT3抑制进而抑制细胞增殖以及诱导细胞凋亡
上述实验结果促使本发明进一步研究SC-43抗纤维化活性的机制。使用SC-43治疗会以剂量依赖方式降低小鼠纤维母细胞(NIH3T3细胞株)存活率(图7A)；且SC-43的治疗亦会降低p-STAT3的表达(图7B)；STAT3的过度表达会因SC-43诱导细胞凋亡而拯救NIH3T3(图7C)；除此之外，SC-43的凋亡作用会因SHP-1剔除而抵消(图7D)。这些结果表示SC-43的治疗会下调节p-STAT3并诱导纤维化凋亡，而SHP-1的抑制会抵消SC-43的影响。
3.3在人肺上皮细胞(A549细胞株)中SC-43会抑制TGF-β1-诱导上皮-间质转化的产生
在肺纤维化的疾病发生过程，负责过量的上皮-间质转化产生的主要效应细胞为活化的纤维母细胞，其是由肺泡上皮细胞的肺泡细胞外基质以及驻留的纤维母细胞的增殖所引起。本发明利用A549细胞评估SC-43在EMT的效应，A549细胞是一种已被广泛应用于研究EMT的理想体外模型的肺泡II型 上皮细胞株。将A549细胞暴露于TGF-β18小时诱导EMT。黏着连接(adherens junction)蛋白E-钙黏蛋白的表达降低，中间丝蛋白(intermediate filament)纤连蛋白和N-钙黏蛋白的表达向上调节；此外，暴露于TGF-β1后STAT3磷酸化也增加(图8A，左图)。还观察到TGF-β1刺激后EMT的侵袭和迁移细胞增加(图8A，右图)。另一方面，使用SC-43处理A549细胞以剂量依赖性方式会逆转TGF-β1诱导的EMT现象，如EMT标记的表达图谱(图8B)所示，迁移细胞和侵袭细胞会减少(图8C和图8D)。
3.4 SC-43经由SHP-1/STAT3信号传导路径抑制上皮-间质转化
为确认经由SHP-1/STAT3信号传导路径SC-43的影响，本发明在A549细胞中转染SHP-1，SHP-1过量表达会减少EMT标记、迁移细胞及侵袭细胞(图9A)。本发明接着以siRNA剔除SHP-1的表达，而抵消SC-43的作用，包含p-STAT3的表达、EMT标记表达、迁移细胞及侵袭细胞(图9B)。该些结果显示SC-43会经由SHP-1的活性抑制EMT，向下调节p-STAT3的表达，进而减少在上皮细胞中的上皮-间质转化。最后，本发明剔除STAT3的表达，其为在SHP-1/STAT3信号传导路径中的下游分子，确认剔除STAT3会抵消SC-43的作用，包含EMT标记表达(图9C，左图)、迁移细胞及侵袭细胞(图9C，右图)。
因此，本发明证实在博来霉素诱导小鼠肺纤维化模型中，SC-43治疗能有效地改善肺纤维化。同时，在活体外研究显示，SC-43能抑制上皮细胞的上皮-间质转化，并减少纤维母细胞中的增殖和胶原蛋白的合成，促进纤维母细胞凋亡。SC-43会增加SHP-1活性并抑制STAT3磷酸化。增强的SHP-1活性显著抑制上皮细胞的EMT和促进纤维母细胞凋亡，而SHP-1表达的抑制会抵消SC-43治疗导致的上皮细胞的EMT抑制和纤维母细胞凋亡。本发明另发现SC-43与SHP-1的N-SH2区域会相互作用而增强SHP-1的活性并抑制STAT3信号传递，其为SC-43抗纤维化的作用机制。
实施例4 SC-43用于治疗减少小鼠肝纤维化
SHP-1在人和小鼠肝脏的纤维化区域中过度表达，在注射四氯化碳(CCl4)以及胆管结扎小鼠模型中，以SC-43治疗能有效预防和减少肝纤维化，进而改善小鼠的存活率。体外研究显示SC-43促进肝星状细胞(HSC)凋亡；SC-43 增加SHP-1活性，并且抑制STAT3磷酸化，其与血小板衍生生长因子受体途径无关。增强的SHP-1活性显著抑制HSC增殖，而SHP-1抑制会抵消SC-43诱导的HSC凋亡。此外，SC-43与SHP-1的N-SH2区域会相互作用而提高SHP-1的抗纤维化作用。
4.1 SHP-1磷酸酶与肝纤维化的关系
因此，本发明在纤维化的肝脏中研究P-STAT3抑制剂的SHP-1的表达。在CCl4诱导肝纤维化4周的小鼠模型中，SHP-1在具有显著纤维化区域中会过度表达(图10A)。本发明进一步研究具有不同程度纤维化的慢性B型肝炎(CHB)患者的SHP-1表达，晚期纤维化患者的SHP-1表达会显著增加(图10B)。SHP-1表达与血清丙氨酸氨基转移酶浓度正相关(图10C)。SHP-1与活化的HSC标志物α-SMA共定位(colocalized)表达(图10D)。这些数据显示SHP-1可能参与肝纤维化的发生。
4.2 SC-43可改善CCl4诱导肝纤维化小鼠模型中的纤维化
本发明已证实SC-43可提高SHP-1活性的表达优于已知抗肝纤维化药物索拉非尼(图2A)。因此。本发明假设SC-43比索拉非尼更具有抗纤维化的能力，在纤维化预防小鼠模型中，同时给予SC-43或索拉非尼与CCl4诱导肝纤维化的小鼠模型后。接着给予SC-43或索拉非尼治疗，明显观察到在肝组织中纤维化的减少，使用天狼星红染色(Picrosirius Red Stain)(图11A)，经由光密度测定以定量的胶原-阳性面积(qCPA)(图11B)，依据Ishak纤维化评分系统分级(图11C)，检测α-SMA的表达(图11D)以及羟脯氨酸浓度(图11E)。
在肝纤维化治疗小鼠模型中，在小鼠CCl4诱导肝纤维化2周后为轻度肝纤维化(Ishak分级，2-3)。在接下来的6周内，同时给予SC-43(5、10或20mg/kg)或索拉非尼(10mg/kg)与CCl4诱导肝纤维化的小鼠模型，如经由天狼星红染色观察到的，SC-43以及索拉非尼的治疗能显著改善肝纤维化(图12A)，经由光密度测定qCPA(图12B)、依据Ishak纤维化评分系统分级(图12C)以及羟脯氨酸浓度(图12D)观察到SC-43的抗纤维化活性增加。此外，与对照组相比，SC-43(10mg/kg)治疗后显著提高存活率(对数秩P＝0.0291)，并且具有比索拉非尼治疗更高的存活率(对数秩P＝0.0671；图12E)。
在纤维化复原小鼠模型中，在小鼠CCl4诱导肝纤维化8周后为晚期纤维 化和肝硬化(Ishak分级，4-6)。接下来的四周在没有CCl4诱导的情况下，给予SC-43(10mg/kg)或索拉非尼(10mg/kg)。在小鼠牺牲时，观察到肝脏纤维化甚至在对照组(图12F，与图12A相比)显示停止CCl4诱导8周后自发性纤维化减少。然而，观察到SC-43治疗组的qCPA和Ishak纤维化评分显著降低(图12G和图12H)。
4.3 SC-43可改善胆汁淤积性纤维化小鼠模型中的纤维化
本发明进一步检测SC-43在胆管结扎小鼠模型中的抗纤维化效果。在纤维化预防小鼠模型中，第一天直到第十四天后小鼠牺牲，给予BDL小鼠模型载体或SC-43(10mg/kg)。使用天狼星红染色，经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)，显示SC-43可显著减少定量的胶原蛋白-阳性面积(qCPA)(图13A及图13B)。
在胆汁淤积性纤维化治疗小鼠模型中，BDL之后第八天直到第十四天小鼠牺牲，给予BDL小鼠模型载体或SC-43(10mg/kg)。使用天狼星红染色，经由光密度测定以定量的胶原蛋白-阳性面积(qCPA)，显示SC-43可显著减少定量的胶原蛋白-阳性面积(qCPA)(图13C及D)。该些数据表示在预防、治疗及复原小鼠模型中，SC-43具有抗纤维化活性。
4.4 SC-43经由血小板衍生生长因子受体(PDGFR)依赖性信号转导及转录激活因子3的抑制诱导肝星状细胞凋亡
本发明在活体外(in vitro)确认SC-43的抗纤维化机制。SC-43的剂量依赖治疗具有减少HSC-T6及LX2细胞存活率的效果，且比索拉非尼的效果更显著；在时间及剂量依赖下SC-43亦具有减少原代小鼠HSCs细胞存活率的效果(图14A)。此外，相较于索拉非尼，在剂量依赖下SC-43显著增加HSC细胞凋亡(图14B)，蛋白质印记法显示SC-43剂量依赖的增加聚(ADP-核糖)聚合酶(PARP)片段的切割(图14C)。
转化生长因子(TGF)-β和PDGFR是参与纤维化发生以及HSC活化与增殖的主要路径，本发明研究SC-43对TGF-β以及PDGFR路径的影响。SC-43会向下调节LX2细胞中TGF-β路径的p-Smad2以及p-Smad3(图14D)；亦会向下调节HSC-T6和LX2细胞中PDGFR路径中的p-PDGFR以及p-Akt(图14E)。
本发明进一步研究SC-43对STAT3路径的影响，其是纤维发生的关键调节因素。在LX2和HSC-T6细胞中SC-43治疗显示比索拉非尼对p-STAT3和细胞周期蛋白D1的有更显著的剂量依赖下向下调节的效果(图15A)。SC-43也抑制IL-6诱导p-STAT3向上调节(图15B)。此外，SC-43诱导的细胞凋亡在STAT3过度表达的HSC中被显著消除(图15C)。这些结果显示，SC-43经由抑制STAT3路径能比索拉非尼更显著地诱导HSC凋亡。
另外，在给予特异性PDGFR抑制剂AG1295后，本发明观察到SC-43仍然会向下调节p-Akt和p-STAT3，与PDGFR讯号无关(图15D)。该结果显示SC-43与PDGFR各自独立下会向下调节p-Akt以及p-STAT3讯号传导，两者均与细胞增殖和存活有关。
4.5 SHP-1在SC-43诱导STAT3抑制中扮演重要的角色
SHP-1包含N-端两个SH2区域(N-SH2以及C-SH2)，接着其后是催化蛋白酪氨酸磷酸酶(PTPase)结构域和C末端。在非活性形式下，N-SH2结构域的D61位点与PTPase结构域上的WPD位点相互作用，并阻碍PTPase的活性。dN1和D61A突变体中SHP-1活性增加(图16A)。
为研究SHP-1在HSC凋亡中的作用，本发明观察到SHP-1过度表达自转染第2天起显著降低细胞存活率(图16B)。相较于索拉非尼，在LX2以及HSC-T6细胞中，SC-43在低浓度下治疗能显著提高SHP-1活性(图16C)。此外，经由非特异性磷酸酶抑制剂钒酸盐(vanadate)对SHP-1的抑制，由向上调节p-STAT3抵消SC-43诱导的LX2细胞凋亡(图16D)。同样地，SHP-1特异性抑制剂PTP抑制剂III亦会向上调节p-STAT3并抵消SC-43诱导的LX2细胞凋亡(图16E)。经由使用siRNA(图16F)，SHP-1基因剔除显著抵抗SC-43的抗增殖活性，显示SC-43主要靶向于SHP-1，且HSC增殖受SHP-1表达和活性显著影响。
这些结果显示SHP-1活性启动会降低HSC增殖，以SC-43处理会增加SHP-1活性，向下调节p-STAT3以促进HSC凋亡，而SHP-1抑制则抵消SC-43的作用。
4.6 SHP-1突变体的表达及HSCs的增殖
本发明使用细胞群落形成分析进一步检测野生型SHP-1、dN1以及D61A 突变体的异位表达与HSC增殖的相关性。如图17A所示，与载体对照组比较，SHP-1、dN1以及D61A突变体的异位表达显著抑制细胞群落形成。此外，使用MTS的溶液细胞增殖分析，dN1以及D61A突变体表达显著使细胞存活率降低(图17B)。这些结果显示，增加的SHP-1活性与降低的HSC增殖有相关性。
4.7 SC-43经由与SHP-1具有抑制性的N-SH2区域交互作用而活化SHP-1
接着，本发明经由不同的SHP-1突变体的异位表达检测SC-43诱导SHP-1活性的影响。SC-43处理会显著增加载体和野生型SHP-1的SHP-1活性，但不会增加dN1和D61A突变体的SHP-1活性(图17C)。因为SC-43处理会增加SHP-1活性，本发明进一步研究SC-43处理后SHP-1突变体异位表达的表现型变化(细胞凋亡)和p-STAT3表达。由载体对照组以及野生型SHP-1的异位表达，SC-43处理会显著增加凋亡的LX2细胞并向下调节p-STAT3(图17D及图17E)。然而，经由dN1和D61A突变体的细胞凋亡以及向下调节p-STAT3的结果，dN1和D61A突变体对SC-43处理并不敏感(图17E)，显示SC-43因不能与dN1和D61A突变体相互作用而不能达到启动SHP-1活性的效果。
上述结果显示出SHP-1和dN1和D61A突变体的异位表达能显著抑制细胞增殖。SHP-1具有抑制性的N-SH2区域的D61位点对SC-43诱导SHP-1向上调节极具有关键性。dN1和D61A突变体的过度表达会抵消SC-43诱导的SHP-1活性提高、细胞凋亡以及向下调节p-STAT3。图17F中总结SHP-1/STAT3信号传导路径中SC-43的抗纤维化机制。
因此，人类和小鼠肝脏的纤维化区域中表现出SHP-1过度表达。SC-43在肝毒性以及胆汁淤积性纤维化小鼠模型中均表现出抗纤维化活性。本发明进一步说明SC-43经由关键性的抗纤维化机制诱导HSC凋亡。SHP-1/STAT3信号传导路径证明在纤维发生中极为重要，并参与HSC的存活。经由与SHP-1的具有抑制性的N-SH2区域交互作用，SC-43增加SHP-1活性并向下调节p-STAT3，与PDGFR信号无关。这些结果显示SHP-1磷酸酶激动剂是抗纤维化治疗药物的潜在靶标。
本发明经由在不同动物模型中证实SC-43的抗纤维化活性。此外，与对照组比较，SC-43治疗改善纤维化小鼠的存活率。更重要的是，本发明在停 止致病因子CCl4后观察到小鼠的肝硬化的恢复，显示纤维化在撤回致病因子毒性剂后可逆转，并且类似于最近的临床发现，抗病毒治疗持续的病毒抑制可逆转慢性乙型肝炎患者的肝硬化。即使在纤维化恢复的过程中，给予SC-43治疗显著降低纤维化，表示HSC凋亡在纤维化缓解是极为关键的。
综上所述，本发明证实SHP-1/STAT3信号传导路径与纤维发生中的相关性，SC-43经由与SHP-1的具有抑制性的N-SH2区域的直接相互作用而启动SHP-1活性，并经由促进纤维母细胞凋亡而抗纤维化。此外，SC-43在肝毒性以及胆管结扎造成的胆汁淤积性纤维化小鼠模型中显示出抗纤维化活性，表示SHP-1磷酸酶激动剂是抗纤维化药物的潜在靶标。SHP-1/STAT3信号传导路径是纤维发生中的关键信号通路。SC-43促进SHP-1活性上调并抑制STAT3磷酸化而显著改善纤维化。SHP-1靶向抗纤维化治疗可能是一种发展抗纤维化药物的策略。

Claims (11)

  1. 一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)激动剂在制备治疗纤维化的药物中的用途。
  2. 如权利要求1所述的用途，其中所述含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂提高含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)的活性。
  3. 如权利要求1所述的用途，其中所述含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂为以下化合物
    Figure PCTCN2017102945-appb-100001
  4. 如权利要求1所述的用途，其中所述纤维化为皮肤硬化或心脏、肺、肝、胰、或肾脏的纤维化。
  5. 一种以下化合物在制备治疗以含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)不活化为特征的疾病的药物中的用途
    Figure PCTCN2017102945-appb-100002
  6. 如权利要求5所述的用途，其中所述以含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)不活化为特征的疾病为纤维化。
  7. 如权利要求6所述的用途，其中所述纤维化为皮肤硬化或心脏、肺、肝、胰、或肾脏的纤维化。
  8. 一种含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)激动剂在制备治疗以含有SRC同源区2的蛋白酪氨酸磷酸酶-1(SHP-1)不活化为特征的疾病的药物中的用途。
  9. 如权利要求8所述的用途，其中所述含有SRC同源区2的蛋白酪氨酸磷酸酶-1激动剂为以下化合物
    Figure PCTCN2017102945-appb-100003
  10. 如权利要求8所述的用途，其中所述以含有SRC同源区2的蛋白酪 氨酸磷酸酶-1(SHP-1)不活化为特征的疾病为纤维化。
  11. 如权利要求10所述的用途，其中所述纤维化为皮肤硬化或心脏、肺、肝、胰、或肾脏的纤维化。
PCT/CN2017/102945 2016-09-22 2017-09-22 含有src同源区2的蛋白酪氨酸磷酸酶-1激动剂用于改善纤维化的用途 WO2018054354A1 (zh)

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