WO2018099363A1 - 含胰岛素样生长因子-2的药物组合物及其应用 - Google Patents

含胰岛素样生长因子-2的药物组合物及其应用 Download PDF

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WO2018099363A1
WO2018099363A1 PCT/CN2017/113341 CN2017113341W WO2018099363A1 WO 2018099363 A1 WO2018099363 A1 WO 2018099363A1 CN 2017113341 W CN2017113341 W CN 2017113341W WO 2018099363 A1 WO2018099363 A1 WO 2018099363A1
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macrophages
insulin
igf
growth factor
macrophage
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PCT/CN2017/113341
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English (en)
French (fr)
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时玉舫
王莹
杜黎明
林良宇
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中国科学院上海生命科学研究院
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Priority to US16/464,902 priority Critical patent/US20190381147A1/en
Priority to EP17876571.5A priority patent/EP3549598A4/en
Priority to AU2017370226A priority patent/AU2017370226B2/en
Priority to CN201780048982.3A priority patent/CN109562145B/zh
Publication of WO2018099363A1 publication Critical patent/WO2018099363A1/zh
Priority to US18/300,286 priority patent/US20230364201A1/en

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    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
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    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
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    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • the present invention relates to the field of biomedicine, and more particularly to pharmaceutical compositions containing insulin-like growth factor-2 and uses thereof.
  • Macrophages are distributed throughout the body and are specialized immune cells that can phagocytize senescent cells, cells that cannot be recognized as "self", cell debris, and the like. As a full-time antigen-presenting cell, macrophages can initiate an immune response to unsensitized T cells and trigger an inflammatory response. In this process, macrophages play an important role in the pathogenesis of various autoimmune diseases by expressing various inflammatory factors such as TNF ⁇ and IL-1. Removal of pathogenic macrophages can significantly inhibit the progression of autoreactive encephalomyelitis and inflammatory bowel disease.
  • macrophages can be regulated by different environmental factors into immunosuppressive cells, such as macrophages that express IL-10, Arginase I, etc., and evolved from monocytes to macrophages.
  • immunosuppressive macrophages formed by different immune stimuli directly or indirectly regulate the body's immune response. It has been found that spermidine-treated macrophages exhibit a strong and powerful ability to treat autoimmune diseases, suggesting that immunosuppressive macrophages have a significant effect in the treatment of autoimmune diseases.
  • an insulin-like growth factor-2 (IGF-2) for the preparation of a composition (including a chemical or pharmaceutical composition) for use in (i Promoting the expression of macrophage PD-L1, and/or (ii) inhibiting the expression of macrophage IL-1 ⁇ .
  • IGF-2 insulin-like growth factor-2
  • the insulin-like growth factor-2 comprises a full length insulin-like growth factor-2 and an insulin-like growth factor-2 active fragment.
  • the insulin-like growth factor-2 active fragment is an active fragment comprising amino acids 25-91 of insulin-like growth factor-2.
  • the insulin-like growth factor-2 active fragment is amino acid sequence 25-91 of insulin-like growth factor-2.
  • the insulin-like growth factor-2 is derived from a human or non-human mammal.
  • amino acid sequence of insulin-like growth factor-2 is as shown in SEQ ID NO.: 1.
  • the pharmaceutical composition is further for one or more uses selected from the group consisting of:
  • the autoimmune disease is selected from the group consisting of multiple sclerosis, inflammatory bowel disease, autoreactive encephalomyelitis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, Insulin resistance, diabetes, cirrhosis, or a combination thereof.
  • the insulin resistance or diabetes is obesity-induced insulin resistance or obesity-induced diabetes.
  • the pharmaceutical composition comprises (a) insulin-like growth factor-2; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition contains 0.001 to 99% by weight, preferably 0.1 to 90% by weight, more preferably 1 to 80% by weight, of insulin-like growth factor-2, based on the total weight of the pharmaceutical composition. .
  • the pharmaceutical composition further comprises a macrophage scavenger, which is a preparation for removing macrophages or inhibiting macrophage migration.
  • the pharmaceutical composition further comprises a PD-L1 promoter.
  • the pharmaceutical composition is a liquid preparation or a lyophilized preparation.
  • the pharmaceutical composition is an injection.
  • a macrophage which is an insulin-like growth factor-2 (IGF-2)-treated macrophage for preparing a pharmaceutical composition
  • the pharmaceutical composition is for one or more uses selected from the group consisting of:
  • the macrophages are cultured in the presence of insulin-like growth factor-2 to obtain the insulin-like growth factor-2-treated macrophages.
  • a pharmaceutical composition comprising (a) insulin-like growth factor-2 or an active fragment thereof (eg, an active fragment comprising amino acids 25-91), b) an optional macrophage scavenger, (c) an optional PD-L1 promoter, and (d) a pharmaceutically acceptable carrier.
  • At least one of the components (b) and (c) is present.
  • the macrophage scavenger is a preparation for removing macrophages or inhibiting macrophage migration.
  • the pharmaceutical composition is in the form of an injection, a sustained release, or a topical pharmaceutical dosage form.
  • the pharmaceutical composition is for one or more uses selected from the group consisting of:
  • the pharmaceutical composition contains a macrophage scavenger, which is a preparation for removing macrophages or inhibiting macrophage migration.
  • the macrophage scavenger is selected from the group consisting of a CCR2 inhibitor (inhibiting macrophage migration), a disodium clodronate liposome (clearing macrophages), or a combination thereof.
  • the mass ratio of component (a): component (b): component (c) is (1-100): (1-100): (1) -100) when the pharmaceutical composition contains components (a), (b) and (c).
  • the mass ratio of component (a): component (b) is (1-100): (1-100), when the pharmaceutical composition contains a group Points (a) and (b).
  • the mass ratio of the component (a): the component (c) is (1 to 100): (1 to 100), when the pharmaceutical composition contains the group Points (a) and (c).
  • the ratio of the active fragment of the amino acid 25-91 to the macrophage scavenger (mg: mg) of the insulin-like growth factor-2 is 1:100 to 100:1, preferably The ground is 1:20 to 20:1.
  • the total content of the active fragment of the amino acid 25-91 and the macrophage scavenger of the insulin-like growth factor-2 is from 1 to 99% by weight, more preferably from 5 to 5% of the pharmaceutical composition. 90wt%.
  • a pharmaceutical composition comprising (a1) an insulin-like growth factor-2 inhibitor, (b1) an optional macrophage promoter, (c1) An optional PD-L1 inhibitor, and (d1) a pharmaceutically acceptable carrier.
  • At least one of the components (b1) and (c1) is present.
  • the PD-L1 inhibitor comprises an antibody against PD-L1.
  • the mass ratio of component (a1): component (b1): component (c1) is (1-100): (1-100): (1) -100) when the pharmaceutical composition contains components (a1), (b1) and (c1).
  • the mass ratio of the component (a1):the component (b1) is (1 to 100): (1 to 100), when the pharmaceutical composition contains the group When (a1) and (b1) are divided.
  • the mass ratio of the component (a1): the component (c1) is (1 to 100): (1 to 100), when the pharmaceutical composition contains the group When (a1) and (c1) are divided.
  • a macrophage pretreated with insulin-like growth factor-2 in a fifth aspect of the invention, there is provided a macrophage pretreated with insulin-like growth factor-2.
  • the expression of PD-L1 in the macrophage is upregulated, and/or (ii) the expression of IL-1 ⁇ is downregulated.
  • the up-regulation refers to a ratio of the expression level M1 of PD-L1 in the treated macrophage to the expression level M0 of PD-L1 in the untreated macrophage M1/M0 ⁇ 1.5, Preferably it is ⁇ 2.0, more preferably ⁇ 3.0.
  • the down-regulation refers to a ratio of the expression level of IL-1 ⁇ in the untreated macrophage to the expression level N1 of IL-1 ⁇ in the treated macrophage N0/N1 ⁇ 1.5, Preferably it is ⁇ 2.0, more preferably ⁇ 3.0.
  • the treatment comprises contacting the macrophage with insulin-like growth factor-2 or an active fragment thereof for a period of time (e.g., 0.1-24 hours).
  • a pharmaceutical composition for regulating T cell differentiation comprising insulin-like growth factor-2-treated macrophages and a pharmaceutically acceptable carrier.
  • the modulating T cell differentiation refers to (b) promoting differentiation of regulatory T cells; and/or (c) inhibiting differentiation of Th1 cells and/or Th17 cells.
  • the pharmaceutical composition is also used to treat an autoimmune disease.
  • a method of non-therapeutically comprising the steps of:
  • the insulin-like growth factor-2 is administered at a concentration of from 1 ng/ml to 100 ⁇ g/ml, preferably from 3 ng/ml to 1 ⁇ g/ml, more preferably from 5 ng/ml to 50 ng/ Ml.
  • a polypeptide is provided, the sequence of which is shown at positions 25-91 of SEQ ID NO.: 1.
  • a ninth aspect of the invention there is provided a method of (i) promoting expression of macrophage PD-L1, and/or (ii) inhibiting expression of macrophage IL-1 ⁇ , comprising the steps of:
  • the subject comprises a non-human mammal and a human.
  • the insulin-like growth factor-2 is administered at a dose of from 1 ng to 1 mg/kg, preferably from 100 ng to 100 ⁇ g/kg, more preferably from 1 to 10 ⁇ g/kg.
  • the method further includes the steps of:
  • a macrophage scavenger (b) administering a macrophage scavenger to a subject in need thereof, the macrophage scavenger being a preparation for removing macrophages or inhibiting macrophage migration.
  • the macrophage clearing agent is treated at a conventional administration dose and frequency of administration.
  • the administration comprises simultaneous administration or sequential administration.
  • a kit comprising:
  • a second container and an active ingredient (b) macrophage scavenger contained in the second container, or a drug containing the active ingredient (b), wherein the macrophage scavenger is for removing giant a phagocytic or a preparation that inhibits the migration of macrophages;
  • the drug in the first container and the second container is a one-side preparation containing the active ingredient (a) and a single preparation containing the active ingredient (b).
  • the pharmaceutical composition of the third aspect wherein the kit of the tenth aspect of the invention is used for the preparation of a medicament for treating an autoimmune disease.
  • FIG 1 shows that insulin growth factor-2 (IGF-2) is effective in the treatment of EAE.
  • IGF-2 insulin growth factor-2
  • FIG 1a shows that IGF-2 can effectively alleviate the progression and extent of EAE.
  • FIG. 1b shows that IGF-2 can effectively inhibit the infiltration of monocytes in the central nervous system in EAE mice.
  • FIG. 1c shows that IGF-2 potently inhibits the proliferation of MOG-specific T cells in EAE mice.
  • Fig. 1d, Fig. 1e, Fig. 1f and Fig. 1g show that IGF-2 can effectively inhibit the ratio of Th1 and Th17 cells in EAE mice and promote the ratio of Treg.
  • FIG. 1h shows that IGF-2 does not directly affect the differentiation of Th1/Th17/Treg.
  • Figure 1i and Figure 1j show that IGF-2 promotes the expression of PD-L1 on CD11b+F4/80+ macrophages in the spinal cord of EAE mice and inhibits the expression of IL-1 ⁇ .
  • FIG. 1 shows that insulin growth factor-2 (IGF-2) induces anti-inflammatory properties of macrophages.
  • IGF-2 insulin growth factor-2
  • FIG. 1a shows that IGF-2 does not affect the number of macrophages in the spinal cord of EAE mice.
  • Figure 2b shows that IGF-2 does not affect the number of macrophages in the spleen of EAE mice.
  • Figure 2c shows that IGF-2 inhibits the expression of IL-1 ⁇ in CD11b+F4/80+ macrophages in the spleen of EAE mice.
  • Figure 2d shows that IGF-2 promotes the expression level of PD-L1 on CD11b+F4/80+ macrophages in the spinal cord of EAE mice.
  • Figure 2e shows that IGF-2 does not affect the expression levels of MHC-I, MHC-II, CD80 and CD86 on peritoneal macrophages induced by thioglycollate medium.
  • Figure 2f shows that IGF-2 does not affect the secretion of TNF- ⁇ , IL-6 and TGF- ⁇ by peritoneal macrophages induced by lipopolysaccharide-LPS-stimulated thioglycollate medium.
  • Figure 2g shows that IGF-2 inhibits the mRNA levels of inducible nitric oxide synthase (iNOS) in peritoneal macrophages induced by lipopolysaccharide-LPS-stimulated thioglycollate medium, and the regulation of this enzyme Production of nitrite.
  • iNOS inducible nitric oxide synthase
  • FIG. 3 shows that insulin growth factor-2 (IGF-2) treatment of macrophages can effectively treat EAE.
  • IGF-2 insulin growth factor-2
  • Figure 3a shows that after IGF-2 pretreatment, in the case of 100 ng/ml lipopolysaccharide stimulation, thiosyl acetate medium-induced peritoneal macrophages express IL-1 ⁇ messenger RNA, and the ability of the precursor protein is significantly decreased. .
  • Figure 3b shows that after IGF-2 pretreatment, the protein level of IL-1 ⁇ secreted by peritoneal macrophages induced by thioglycollate medium was significantly decreased in the case of 100 ng/ml lipopolysaccharide stimulation.
  • Figure 3c shows that after IGF-2 pretreatment, the thioglycollate medium-induced peritoneal macrophage-expressed PD-L1 protein levels were significantly increased in response to 100 ng/ml lipopolysaccharide stimulation.
  • Figure 3d and Figure 3e show that macrophage-dependent PD-L1 promotes Treg production after IGF-2 treatment.
  • FIG. 3f shows that IGF-2 treated macrophages can effectively inhibit EAE.
  • Figure 3g shows that IGF-2 treated macrophages can effectively promote the proportion of Treg cells in EAE mice.
  • FIG. 4 shows that insulin growth factor-2 (IGF-2) treatment confers the ability of bone marrow-derived macrophages to promote Treg cell differentiation.
  • IGF-2 insulin growth factor-2
  • Figure 4a shows that the expression level of PD-L1 was significantly increased in the bone marrow-derived macrophages treated with IGF-2 under the stimulation of lipopolysaccharide LPS.
  • Figure 4b and Figure 4c show that bone marrow-derived macrophages treated with IGF-2 promote Treg by PD-L1 Cell.
  • Figure 4d shows that the expression levels of MHC-I, MHC-II, CD80 and CD86 on bone marrow-derived macrophages after IGF-2 treatment were unchanged.
  • FIG. 5 shows that insulin-like growth factor-2 (IGF-2)-treated macrophages have the effect of alleviating experimental autoreactive encephalomyelitis and inflammatory bowel disease.
  • IGF-2 insulin-like growth factor-2
  • Figure 5a shows the effective treatment of EAE by bone marrow-derived macrophages after IGF-2 treatment.
  • Figures 5b and 5c show the proportion of bone marrow-derived macrophages treated with IGF-2 effective to promote Treg in EAE mice.
  • Figure 5d shows that bone marrow-derived macrophages treated with IGF-2 alleviated body weight loss in experimental enteritis mice. It was shown that IGF-2 treated macrophages have the ability to effectively treat IBD.
  • Figure 5e shows that IGF-2 treated macrophages can effectively prolong the survival of inflammatory bowel disease mice.
  • Figures 5f and 5g show that insulin-like growth factor-2 treated bone marrow macrophages treat inflammatory bowelitis mice and promote a significant increase in the proportion of spleen Treg cells.
  • FIG. 6 shows that insulin growth factor-2 (IGF-2) affects the anti-inflammatory properties of macrophages by regulating macrophage glucose metabolism.
  • IGF-2 insulin growth factor-2
  • Figure 6a shows a significant reduction in glucose consumption by macrophages after IGF-2 treatment.
  • Figure 6b shows that the amount of lactic acid accumulation in macrophages was significantly reduced after IGF-2 treatment.
  • Figure 6c shows a significant decrease in the ratio of NAD+/NADH in macrophages after IGF-2 treatment.
  • Figure 6d shows that the oxygen consumption and oxidative phosphorylation capacity of macrophages increased significantly after IGF-2 treatment.
  • Figure 6e shows that the extracellular acidification rate of macrophages was significantly reduced after IGF-2 treatment.
  • Figure 6f shows that macrophages tend to use oxidative phosphorylation metabolic pathways following IGF-2 treatment.
  • Figure 6g shows that IGF-2-mediated thiosyl acetate medium-induced increase in PD-L1 expression in peritoneal macrophages can be inhibited by oligomycin.
  • Figure 6h shows inhibition of oxidative phosphorylation with oligomycin, confirming that IGF-2 treated macrophages have aerobic respiration-dependent high expression of PD-L1 and promote Treg production.
  • FIG. 7 shows that insulin growth factor-2 (IGF-2) and IGF-2 treatment of macrophages are effective in the treatment of experimental enteritis.
  • IGF-2 insulin growth factor-2
  • IGF-2 treatment of macrophages are effective in the treatment of experimental enteritis.
  • Figure 7a shows that IGF-2 inhibits weight loss in mice with inflammatory bowel disease.
  • Figure 7b shows a significant increase in colon length in inflammatory bowelitis mice following IGF-2 treatment.
  • Figure 7c shows a significant decrease in the number of infiltration of monocytes in the colon following IGF-2 treatment.
  • Figure 7d and Figure 7e show a significant increase in the proportion of Treg cells in the colon lamina limbal growth factor-2 treatment.
  • Figure 7f shows that PD-L1 expression is significantly increased in CD11b+F4/80+ macrophages in the colon lamina basement of inflammatory bowelitis mice following IGF-2 treatment.
  • Figure 7g shows that IL-1 ⁇ expression was significantly decreased in CD11b+F4/80+ macrophages in the colonic lamina intestinal of inflammatory bowelitis mice following IGF-2 treatment.
  • Figure 7h shows that IGF-2 treated macrophages alleviated body weight loss in experimental enteritis mice.
  • Figure 7i shows that macrophages treated with IGF-2 can effectively prolong the survival of inflammatory bowel disease mice.
  • Figure 7j shows that the proportion of spleen Treg cells in inflammatory bowelitis mice was significantly increased after injection of IGF-2 treated macrophages.
  • FIG. 8 shows that insulin growth factor-2 (IGF-2) regulates the immune response in the spleen and abdominal lymph nodes of mice with inflammatory bowel disease.
  • IGF-2 insulin growth factor-2
  • Figures 8a and 8b show that IGF-2 significantly up-regulated the proportion of Treg cells in the peritoneal lymph nodes in experimental enteritis mice.
  • Figure 8c shows that IGF-2 has no effect on the proportion of spleen macrophages in experimental enteritis mice.
  • Figure 8d shows that IGF-2 up-regulates the proportion of CD11b+F4/80+ macrophages in the intestinal lamina limba of experimental enteritis mice.
  • Figure 8e shows that IGF-2 significantly down-regulated the expression of IL-1 ⁇ in spleen macrophages in mice with experimental enteritis.
  • Figure 8f shows that IGF-2 significantly up-regulated the expression of PD-L1 in spleen macrophages in mice with experimental enteritis.
  • Figure 9 shows that thiosyl acetate-induced peritoneal macrophages treated with insulin-like growth factor-2 (IGF-2) can up-regulate the peritoneal lymph nodes and colonic lamina intestinal of mice with inflammatory bowel disease. Treg cells.
  • IGF-2 insulin-like growth factor-2
  • Figure 9a shows that IGF-2 treated macrophages can significantly upregulate the proportion of Treg cells in the peritoneal lymph nodes of mice with inflammatory bowel disease.
  • Figure 9b shows that IGF-2 pretreated macrophages can significantly upregulate the proportion of Treg cells in the lamina intestinal of mice with inflammatory bowel disease.
  • FIG 10 shows that insulin-like growth factor-2 (IGF-2) and clodronate liposomes can significantly inhibit the progression of EAE and provide effective treatment compared with control mice. More importantly, the therapeutic effect of combined injection of IGF-2 and clodronate liposomes on EAE was more pronounced than in the other groups.
  • IGF-2 insulin-like growth factor-2
  • clodronate liposomes can significantly inhibit the progression of EAE and provide effective treatment compared with control mice. More importantly, the therapeutic effect of combined injection of IGF-2 and clodronate liposomes on EAE was more pronounced than in the other groups.
  • FIG 11 shows the tertiary structure of insulin growth factor-2 (IGF-2).
  • FIG 12 shows the key sites for binding of insulin growth factor-2 (IGF-2) to the receptor.
  • IGF-2 insulin growth factor-2
  • Figure 13A shows the results of the glucose tolerance test.
  • Figure 13B shows the results of an insulin tolerance test.
  • the present inventors have extensively and intensively studied, and for the first time, unexpectedly discovered a pharmaceutical composition containing insulin-like growth factor-2 and its use.
  • the present invention provides the use of insulin-like growth factor-2 and an active fragment thereof for the preparation of a pharmaceutical composition for (i) promoting the expression of macrophage PD-L1, and/or (ii) ) inhibits the expression of IL-1 ⁇ in macrophages.
  • the present invention also provides a pharmaceutical composition comprising insulin-like growth factor-2 as an active ingredient.
  • insulin-like growth factor -2 insulin growth factor-2, IGF-2) -2 and a second active fragments of 25 to 91 amino acid sequence of insulin-like growth factor (IGF 25-91)
  • IGF 25-91 insulin-like growth factor-2
  • IGF 25-91 insulin growth factor-2, IGF-2, IGF-2 and a second active fragments of 25 to 91 amino acid sequence of insulin-like growth factor (IGF 25-91)
  • IGF 25-91 promotes reprogramming of macrophages to immunosuppressive macrophages mainly by affecting the aerobic glycolysis and oxidative phosphorylation pathways of macrophages, and this is via IGF.
  • 25-91 treated macrophages have a very significant effect on the treatment of autoimmune diseases. So far, the present invention is based on the therapeutic effect of IGF 25-91 on autoimmune diseases, and clarifies the regulatory mechanism of IGF 25-91 on macrophages, and found that IGF 25-91 induces immunosuppressive function of macrophages in autoimmune diseases.
  • the application potential in therapy forms a new technology for macrophage immunotherapy.
  • IGF-2 Insulin-like growth factor-2
  • IGF-2 is a growth factor secreted mainly by the liver and abundantly present in the blood. It has anti-apoptosis, growth regulation, insulin-like and mitogenic functions.
  • IGF-2 from the mRNA precursor protein translated from a total of 180 amino acids, sequence shown in SEQ ID NO.:1 (mgipmgksmlvlltflafascciaayrpsetlcggelvdtlqfvcgdrgfyfsrpasrvsrrsrgiveeccfrscdlalletycatpakserdvstpptvlpdnfprypvgkffqydtwkqstqrlrrglpallrarrghvlakeleafreakrhrplialptqdpahggappemasnrk); an active form through post-translational modifications produced by the 67 amino acid sequence SEQ ID NO.
  • IGF-2 The tertiary structure formed by IGF-2 is shown in Figure 11, consisting of three ⁇ -helices and two ⁇ -sheets. Where 11G-21C is a spiral, 25G-27Y is the first fold, the second spiral is 42I-49R, and the third spiral 53L-58T, the second fold is 59Y-61A. It is predicted that T16 on the first helix, F19 and L63 on the third helix are sites where IGF-2 binds to the IGF-2 receptor, as shown in FIG.
  • insulin-like growth factor-2 plays an important role in embryonic development and can promote embryonic development and organ formation. It has also been reported to be related to memory and reproduction. Loss of signaling by insulin-like growth factor-2 causes impaired brain development.
  • the present inventors have found that the use of insulin-like growth factor-2 alone or an active fragment having amino acid sequence 25-91 of insulin growth factor-2 (IGF-2) (IGF 25-91 ) can be effective.
  • IGF-2 insulin growth factor-2
  • IGF 25-91 insulin growth factor-2
  • the proportion of regulatory T cells in the IGF 25-91 treatment group was significantly increased, and the proportion of TH1 and TH17 cells was significantly decreased.
  • IGF 25-91 could not directly affect the efficiency of T cell differentiation into Th1, Th17 and Treg.
  • IGF 25-91 is an indirect effect on the proportion of T cell subsets.
  • Significant changes in the macrophage phenotype were also observed when insulin-like growth factor-2 was used to treat autoimmune diseases.
  • the study found that the expression of IL-1 ⁇ in the macrophage of the injured part of mice treated with IGF 25-91 was significantly decreased, while the expression of PD-L1 was significantly increased, suggesting that IGF 25-91 may promote the anti-inflammatory macrophage.
  • the proportion of cells plays a role in inhibiting autoimmune diseases.
  • IGF 25-91 did not induce the expression of inflammatory genes directly macrophages, but the time to respond showed LPS-stimulated macrophages in the mature processed by the IGF 25-91 bone marrow-derived IL or abdominal
  • the expression level of -1 ⁇ was significantly decreased, while the expression level of PD-L1 was significantly increased.
  • Macrophages play an important role in the occurrence and development of autoimmune diseases. A series of studies have shown that macrophages can exert strong pro-inflammatory ability under the stimulation of inflammatory factors such as interferon and lipopolysaccharide, which exacerbates themselves. The course of immune disease.
  • IGF 25-91 insulin-like growth factor-2 alters the response of macrophages to inflammatory factors, and macrophages overexpress the anti-inflammatory molecule PD-L1 under proinflammatory conditions. Therefore, the treatment of autoimmune diseases by IGF 25-91 is likely to be achieved by reprogramming macrophages.
  • IGF 25-91- treated macrophages were co-cultured with T cells after lipopolysaccharide pre-stimulation, and it was found that after IGF 25-91 treatment Macrophages can significantly promote the differentiation of Treg, and this promotion depends on the high expression of PD-L1. Further studies have found that IGF 25-91- treated macrophages can also be directly used to treat autoreactive encephalomyelitis and inflammatory bowel disease. During the treatment, these macrophage infusions also promote Treg in mice. The rise in proportion.
  • IGF 25-91- treated macrophages and control cells were compared in detail, and studies have found that IGF 25-91 treated macrophages can significantly change.
  • the state of the glucose metabolism pathway of macrophages makes it more biased towards the metabolic pathway of oxidative phosphorylation.
  • the high expression of PD-L1 and the ability to promote Treg differentiation in macrophages induced by insulin-like growth factor-2 also disappeared.
  • IGF 25-91 up-regulates regulatory T cells by inducing immunosuppressive macrophages and exerts therapeutic effects on multiple sclerosis and inflammatory bowel disease.
  • Macrophages treated with IGF 25-91 can exert therapeutic effects on autoimmune diseases.
  • targeted regulation of aerobic glycolysis and oxidative phosphorylation in macrophages can induce the production of immunosuppressive macrophages and play a role in the treatment of various autoimmune diseases.
  • the present invention provides a pharmaceutical composition for (i) promoting the expression of macrophage PD-L1, and/or (ii) inhibiting the expression of macrophage IL-1 ⁇ , which contains a safe and effective amount of insulin-like growth.
  • Factor-2 or an active fragment thereof
  • macrophage containing insulin-like growth factor-2 treatment is provided.
  • the pharmaceutical composition of the present invention can be used to (i) promote the expression of macrophage PD-L1, and/or (ii) inhibit the expression of macrophage IL-1 ⁇ .
  • other therapeutic agents for treating autoimmune diseases can also be used at the same time.
  • the pharmaceutical composition of the present invention may further comprise a macrophage scavenger which is a preparation for removing macrophages or inhibiting migration of macrophages.
  • a macrophage scavenger which is a preparation for removing macrophages or inhibiting migration of macrophages.
  • the invention also provides a kit comprising:
  • a second container and an active ingredient (b) macrophage scavenger contained in the second container, or a drug containing the active ingredient (b), wherein the macrophage scavenger is for removing giant a phagocytic or a preparation that inhibits the migration of macrophages;
  • the pharmaceutical composition of the present invention contains a safe and effective amount of insulin-like growth factor-2 and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the pharmaceutical combination of the invention may also be formulated as a powder for nebulization.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 1 microgram per kilogram body weight to about 50 milligrams per kilogram body weight per day, from about 5 micrograms per kilogram body weight to about 10 milligrams per kilogram body weight, and about 10 micrograms per kilogram body weight to about 5 weight percent. Mg/kg body weight.
  • the present invention combines The substance can also be used with other therapeutic agents.
  • composition of the present invention can be administered to a subject (e.g., human and non-human mammal) by a conventional means.
  • a subject e.g., human and non-human mammal
  • Representative modes of administration include, but are not limited to, oral, injection, nebulization, and the like.
  • a safe and effective amount of the medicament is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably The dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • Insulin-like growth factor-2 and its active fragment can (i) promote macrophage PD-L1 expression, and/or (ii) inhibit macrophage IL-1 ⁇ expression
  • Insulin-like growth factor-2 and its active fragment-treated macrophages can promote the differentiation of regulatory T cells and inhibit the differentiation of Th1 cells and/or Th17 cells, thereby treating autoimmune diseases.
  • CFA complete Freund's adjuvant
  • Emulsification of antigen two glass needles were connected through a three-way tube, an equal volume of antigen solution (300 ⁇ g MOG in 100 ⁇ L PBS) and 100 ⁇ L of complete Freund's adjuvant were added to the needle tube, the bubbles in the needle tube were removed and pushed back and forth to obtain an emulsion. . It takes about 500 pushes, and the resistance is gradually increased. At this time, the ingredients can be fully mixed into an emulsified state.
  • Pertussis toxin (PT) solution preparation Pertussis toxin was dissolved in PBS to a working concentration of 1 ng/ ⁇ L, 200 ⁇ L per mouse, and injected into the tail vein.
  • insulin-like growth factor 2 was used for daily injection at a dose of 5 ng/dip, administered intraperitoneally.
  • the saline treated group served as a positive control.
  • the spinal cord was removed from EAE mice and washed with PBS.
  • the spinal cord was mechanically ground on a 70 ⁇ m pore size cell screen to obtain a single cell suspension.
  • the obtained cell suspension was centrifuged, and the cells were resuspended in 30% percoll, and an equal volume of 70% percoll solution was gently added to the bottom. Centrifuge at zero speed for 20 minutes at room temperature at 2000 rpm. After centrifugation, cells were aspirated from the high-low density liquid junction and washed twice with PBS to obtain lymphocytes.
  • mice After the EAE mice were sacrificed, the spleens of the mice were removed and a single cell suspension was obtained.
  • the spleen cells were plated in a U-bottom 96-well plate at a number of 3-5*105 per well per well, and MOG was added to 20 ug/ml. .
  • H3 labeled thymidine was added. After 6 hours, the well plate was repeatedly freeze-thawed and then vacuum-adsorbed onto a special filter, and the scintillation solution was added and detected on the machine.
  • DSS Sodium dextran sulfate
  • insulin-like growth factor-2 was used for daily injection at a dose of 50 ng/dip, administered intraperitoneally.
  • the saline treated group served as a positive control.
  • the colon removed from the IBD mice was excreted and washed with PBS, then the colon was cut into 2 cm sections and rinsed twice in HBSS (1 mM DTT and 5 mM EDTA) under the conditions of 250 rpm * 20 min * 37 degree.
  • the washed colon tissue was cut as much as possible with scissors and added to HBSS containing DNAase I, dispase and type VIII collegenase under the conditions of 250 rpm*30 min*37 degrees.
  • the obtained cell suspension was centrifuged, and the cells were resuspended in 40% percoll, and an equal volume of 80% percoll solution was gently added to the bottom. Centrifuge at zero speed for 20 minutes at room temperature at 2000 rpm. After centrifugation, cells were aspirated from the high-low density liquid junction and washed twice with PBS to obtain lymphocytes.
  • Test medium was formulated by adding 10 mM glucose, 2 mM glutamine and 2 mM pyruvate to the XF culture. The cells were washed twice with test medium before the machine was placed, and each cell culture well was fixed to 525 ⁇ L. Metabolic inhibitors were prepared using test medium: 1 [mu]M oligomycin, 0.75 [mu]M FCCP, 100 [mu]M rotenone + 1 [mu]M antimycin. The rate of oxygen consumption and the concentration of extracellular hydrogen ions in the basal state and drug stimulation conditions were measured. The instrument uses an extracellular flux analyzer.
  • the preparation process of bone marrow-derived macrophages is as follows: the mouse bone marrow is blown out with a 1 ml syringe, and after fully blowing, the cells are centrifuged according to the condition of 400 g*5 min, the supernatant is discarded, and the cells are blown into a single cell suspension. , DMEM/F12 complete medium (containing 10% FBS, 20% L929 supernatant, 100 U/ml Penicillin, 100 U/ml Streptomycin, 2 mM L-Glutamine) was plated in a non-coated dish and cultured to the fourth 5 ml of the culture solution was added in the daytime, and the cells were cultured for 6 days to obtain mature macrophages. IGF-2 stimulation was added on days 1, 3, and 5, respectively.
  • the abdominal cavity-derived macrophages were prepared by injecting 2 ml of 4% thioglycollate solution into the peritoneal cavity of 8-12 weeks old C57BL/6 mice, intraperitoneally injecting factor or PBS into the peritoneal cavity, and euthanizing the mice on the third day.
  • the 10 ml syringe was used to blow the macrophages in the peritoneal cavity with a volume of 10 ml of PBS. After centrifugation, the cells were blown into single cell suspensions, plated in uncoated cell culture dishes or 6-well plates for subsequent experiments.
  • C57/BL6 mice purchased from the Slack Shanghai Laboratory Animal Center of the Chinese Academy of Sciences) were fed high-fat diets for 5 weeks from 5 weeks.
  • mice were starved overnight, intraperitoneally injected with 1 g of glucose/kg body weight, and a drop of tail vein was inhaled into the blood glucose test strip at different time points, and blood glucose was measured by a blood glucose meter.
  • mice were starved overnight, starvation time was about 16 hours, and intraperitoneal injection of 0.75 U insulin/kg body weight.
  • a drop of tail vein blood of each mouse was sequentially inhaled into the blood glucose test paper, and blood glucose was measured by a blood glucose meter. Make accurate timings.
  • IGF 25-91 treatment of experimental autoimmune encephalomyelitis depends on its expression of macrophage genes
  • IGF 25-91 When IGF 25-91 was used to treat mice that induced EAE, IGF 25-91 was able to effectively alleviate the progression and severity of EAE, as evidenced by a decrease in clinical scores and a decrease in the number of monocytes infiltrated in the spinal cord. And a decrease in the response of MOG-specific T cells in EAE mice (Fig. 1a-c). At the same time, flow cytometry analysis showed that the use of IGF 25-91 can effectively reduce the ratio of Th1 and Th17 in the spinal cord of EAE mice, and increase the proportion of Treg (Fig. 1d-g).
  • IGF 25-91 When using flow cytometry to analyze immune cells in EAE mice, it was also found that the use of IGF 25-91 did not affect the proportion of macrophages in the spinal cord and spleen of EAE-affected mice (Fig. 2a-b). However, it can significantly affect the gene expression of macrophages, which shows that the expression of IL-1 ⁇ in macrophages is significantly decreased, while the expression of PD-L1 is significantly increased (Fig. 1i-j; Fig. 2c-d). In summary, IGF 25-91 can be used for the treatment of EAE and can affect the proportion of different T cell subsets and gene expression of macrophages in diseased mice.
  • IGF 25-91 treated macrophages promote Treg production and treat EAE
  • IGF 25-91- treated macrophages including bone marrow-derived macrophages and peritoneal-derived macrophages
  • PD-L1 in this process plays a crucial role, since neutralizing antibodies to PD-L1 used once, the ability to IGF 25-91-treated macrophages was also produced pro Treg inhibit ( Figure 3d-e; Figures 4a-c).
  • IGF 25-91- treated macrophages including bone marrow-derived macrophages and peritoneal-derived macrophages
  • IGF 25-91 pretreatment of macrophages can effectively alleviate the clinical scores of EAE, while promoting diseased EAE ratio of Treg in the spleen of mice (FIG. 3f-g; FIGS. 5a-c).
  • IGF 25-91 affects gene expression by increasing oxidative phosphorylation of macrophages
  • IGF 25-91 In order to study the mechanism of regulation of IGF 25-91 macrophages, and macrophages comparison traits relative to control-treated macrophages IGF 25-91 found that macrophages glucose consumption decreased after treatment IGF 25-91, while Lactic acid accumulation was reduced (Fig. 6a-b). At the same time, NAD+/NADH in macrophages also decreased significantly after IGF 25-91 treatment (Fig. 6c). Therefore, it is speculated that IGF 25-91 can regulate the metabolic pathway of glycolytic and oxidative phosphorylation in macrophages. When tracking the oxygen consumption and acid production of macrophages, it was found that IGF 25-91 can effectively increase the efficiency of macrophage consumption of oxygen while reducing the acid production of macrophages in the background state (Fig. 6d- f). This suggests that IGF 25-91 can modulate the metabolism of macrophages toward the pathway of oxidative phosphorylation.
  • IGF 25-91 and IGF 25-91 treated macrophages can be used to treat inflammatory bowel disease (IBD)
  • IGF 25-91 was used to treat the autoimmune disease, the inflammatory bowel disease, which is the leading immune system.
  • the weight loss of IBD-affected mice was alleviated, and colon damage and infiltration of monocytes in the colon were significantly reduced (Fig. 7a-c).
  • Analysis of T cell subsets and macrophage gene expression by flow cytometry revealed that IGF 25-91 was effective in increasing the proportion of Treg in colon and peritoneal lymph nodes (Fig. 7d-e; Fig. 8a-b).
  • IGF 25-91- treated macrophages including bone marrow-derived macrophages and peritoneal-derived macrophages
  • Rat survival rate Fig. 5d-e; Fig. 7h-i
  • IGF 25-91 macrophages treated diseased mice also increased the spleen, and the ratio of PAN junction of Treg retroperitoneal lymph nodes (FIG. 5f-g; FIG. 7h-j; FIG. 9).
  • IGF 25-91 is more effective in treating inflammatory diseases with macrophage clearance
  • IGF 25-91 can significantly inhibit inflammatory diseases such as the above-mentioned multiple sclerosis and inflammatory bowel disease, and this effect and IGF 25-91 regulate macrophages reprogramming into macrophages with immunosuppressive function, thereby inhibiting Excessive inflammatory response is closely related.
  • the system and inflammation are often accompanied by a large number of pro-inflammatory macrophage infiltration, and macrophage infiltration at the site of inflammation often depends on the recruitment of peripheral monocytes and the evolution to pro-inflammatory macrophages. Therefore, it has been studied whether the removal of intrinsic pro-inflammatory macrophages and the full play of the reprogramming effect of IGF 25-91 on macrophages can optimize the treatment of inflammatory diseases.
  • mice from different experimental groups received IGF 25-91 , Clodronate liposome (for clearing macrophages) or IGF 25 on days 9, 11 and 13 of EAE induction.
  • the combined injection of -91 and Clodronate liposome was used to observe the progression of EAE in mice.
  • a single injection of IGF 25-91 and Clodronate liposome were significantly inhibit the progression of EAE, to play an effective role in the treatment, more importantly, in conjunction with the injection of IGF 25-91 and Clodronate liposome for The therapeutic effect of EAE was more pronounced than in the other groups ( Figure 10). Therefore, the combination therapy of IGF 25-91 and macrophage clearance plays a more optimal therapeutic role for inflammatory diseases by reprogramming macrophages with immunosuppressive function.
  • IGF 25-91- treated macrophages can be used to treat obesity-induced insulin resistance
  • mice were fed a high fat diet administered 20 weeks PBS injection, macrophages (5 ⁇ 10 6) or macrophages (5 ⁇ 10 6) via a process IGF 26-91, 2 weeks later glucose tolerance test and insulin Tolerance experiments were performed to detect changes in glucose in each group of mice.
  • the study found that IGF 26-91- treated macrophages significantly increased glucose tolerance in insulin-resistant mice ( Figures 13A and 13B), indicating that IGF 26-91- treated macrophages are effective in treating insulin resistance and diabetes.

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Abstract

提供了胰岛素样生长因子-2在用于制备药物组合物中的用途，该药物组合物用于(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。还提供了含胰岛素样生长因子-2作为有效成分的药物组合物。

Description

含胰岛素样生长因子-2的药物组合物及其应用 技术领域
本发明涉及生物医药领域，更具体地涉及含胰岛素样生长因子-2的药物组合物及其应用。
背景技术
巨噬细胞分布于全身的各个部分，是一种特化的免疫细胞，可以以吞噬的方式清除衰老细胞、不能被识别为“自己”的细胞、细胞碎片等。作为专职抗原提呈细胞，巨噬细胞可以始动未致敏T细胞的免疫应答，触动炎症反应。在此过程中，巨噬细胞通过表达多种炎症因子，如TNFα、IL-1等在多种自身免疫性疾病致病过程中发挥重要作用。去除致病性巨噬细胞可以显著抑制自身反应性脑脊髓炎、炎症性肠病的进展。除了促炎作用，巨噬细胞可以被不同的环境因素调控为具有免疫抑制作用的细胞，例如以表达IL-10、ArginaseI等为标志的巨噬细胞，以及在单核细胞向巨噬细胞演变过程中由不同免疫刺激形成的具有免疫抑制功能的巨噬细胞，直接或间接调节机体免疫反应。已有研究发现，经过亚精胺处理的巨噬细胞表现为强而有力的治疗自身免疫性疾病的能力，提示具有免疫抑制功能的巨噬细胞在自身免疫性疾病治疗中的显著效果。
目前，本领域关于巨噬细胞在自身免疫性疾病治疗中的研究还有很多不足，本领域迫切需要开发应用巨噬细胞治疗自身免疫性疾病的新方法。
发明内容
本发明的目的在于提供含胰岛素样生长因子-2的药物组合物及其应用。
在本发明的第一方面，提供了一种胰岛素样生长因子-2(IGF-2)的用途，用于制备一组合物(包括化学制剂或药物组合物)，所述组合物用于(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。
在另一优选例中，所述的胰岛素样生长因子-2包括全长胰岛素样生长因子-2和胰岛素样生长因子-2活性片段。
在另一优选例中，所述的胰岛素样生长因子-2活性片段为包含胰岛素样生长因子-2的第25-91位氨基酸的活性片段。
在另一优选例中，所述的胰岛素样生长因子-2活性片段为胰岛素样生长因子-2的第25-91位氨基酸序列。
在另一优选例中，所述的胰岛素样生长因子-2来源于人或非人哺乳动物。
在另一优选例中，所述的胰岛素样生长因子-2的氨基酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述的药物组合物还用于选自下组的一种或多种用途：
(a)促进巨噬细胞的氧化磷酸化代谢途径；
(b)促进调节性T细胞的分化；
(c)抑制Th1细胞和/或Th17细胞的分化；
(d)治疗自身免疫性疾病。
在另一优选例中，所述的自身免疫性疾病选自下组：多发性硬化、炎症性肠炎、自身反应性脑脊髓炎、自身免疫性肝炎、系统性红斑狼疮、类风湿性关节炎、胰岛素抵抗、糖尿病、肝硬化、或其组合。
在另一优选例中，所述的胰岛素抵抗或糖尿病是肥胖诱导的胰岛素抵抗或肥胖诱导的糖尿病。
在另一优选例中，所述药物组合物包含(a)胰岛素样生长因子-2；和(b)药学上可接受的载体。
在另一优选例中，所述药物组合物中含有0.001-99wt％，较佳地0.1-90wt％，更佳地1-80wt％的胰岛素样生长因子-2，按药物组合物的总重量计。
在另一优选例中，所述的药物组合物还含有巨噬细胞清除剂，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂。
在另一优选例中，所述的药物组合物还含有PD-L1促进剂。
在另一优选例中，所述的药物组合物为液态制剂或冻干制剂。
在另一优选例中，所述的药物组合物为注射剂。
在本发明的第二方面，提供了一种巨噬细胞的用途，所述的巨噬细胞为胰岛素样生长因子-2(IGF-2)处理后的巨噬细胞，用于制备一药物组合物，所述药物组合物用于选自下组的一种或多种用途：
(b)促进调节性T细胞的分化；
(c)抑制Th1细胞和/或Th17细胞的分化；
(d)治疗自身免疫性疾病。
在另一优选例中，在胰岛素样生长因子-2存在的条件下培养巨噬细胞，从而得到所述的胰岛素样生长因子-2处理后的巨噬细胞。
在本发明的第三方面，提供了一种药物组合物，所述的组合物包含(a)胰岛素样生长因子-2或其活性片段(如含第25-91位氨基酸的活性片段)、(b)任选的巨噬细胞清除剂、(c)任选的PD-L1促进剂、和(d)药学上可接受的载体。
在另一优选例中，所述的组分(b)和(c)中至少存在一种。
在另一优选例中，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂。
在另一优选例中，所述的药物组合物的剂型为注射剂、缓释剂、或外用药物剂型。
在另一优选例中，所述的药物组合物用于选自下组的一种或多种用途：
(a)促进巨噬细胞PD-L1的表达；
(b)抑制巨噬细胞IL-1β的表达；
(c)促进调节性T细胞的分化；
(d)抑制Th1细胞和/或Th17细胞的分化；
(e)治疗自身免疫性疾病。
在另一优选例中，所述的药物组合物含有巨噬细胞清除剂，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂。
在另一优选例中，所述的巨噬细胞清除剂选自下组：CCR2抑制剂(抑制巨噬细胞迁移)、氯膦酸二钠脂质体(清除巨噬细胞)、或其组合。
在另一优选例中，在所述药物组合物中，组分(a)：组分(b)：组分(c)的质量比为(1-100)：(1-100)：(1-100)，当所述药物组合物含有组分(a)、(b)和(c)时。
在另一优选例中，在所述药物组合物中，组分(a)：组分(b)的质量比为(1-100)：(1-100)，当所述药物组合物含有组分(a)和(b)时。
在另一优选例中，在所述药物组合物中，组分(a)：组分(c)的质量比为(1-100)：(1-100)，当所述药物组合物含有组分(a)和(c)时。
在另一优选例中，所述的胰岛素样生长因子-2的第25-91位氨基酸的活性片段与巨噬细胞清除剂的比例(mg:mg)为1:100至100:1，较佳地为1:20至20:1。
在另一优选例中，所述的胰岛素样生长因子-2第25-91位氨基酸的活性片段与巨噬细胞清除剂的总含量为药物组合物的1～99wt％，更佳地为5～90wt％。
在本发明的第四方面，提供了一种药物组合物，所述的组合物包含(a1)胰岛素样生长因子-2抑制剂、(b1)任选的巨噬细胞促进剂、(c1)任选的PD-L1抑制剂、和(d1)药学上可接受的载体。
在另一优选例中，所述的组分(b1)和(c1)中至少存在一种。
在另一优选例中，所述的PD-L1抑制剂包括抗PD-L1的抗体。
在另一优选例中，在所述药物组合物中，组分(a1)：组分(b1)：组分(c1)的质量比为(1-100)：(1-100)：(1-100)，当所述药物组合物含有组分(a1)、(b1)和(c1)时。
在另一优选例中，在所述药物组合物中，组分(a1)：组分(b1)的质量比为(1-100)：(1-100)，当所述药物组合物含有组分(a1)和(b1)时。
在另一优选例中，在所述药物组合物中，组分(a1)：组分(c1)的质量比为(1-100)：(1-100)，当所述药物组合物含有组分(a1)和(c1)时。
在本发明的第五方面，提供了一种巨噬细胞，所述的巨噬细胞是经胰岛素样生长因子-2预处理的。
在另一优选例中，所述的巨噬细胞中PD-L1的表达是上调的，和/或(ii)IL-1β的表达是下调的。
在另一优选例中，所述的上调指：经处理的巨噬细胞中PD-L1的表达量M1与未处理的巨噬细胞中PD-L1的表达量M0的比值M1/M0≥1.5,较佳地≥2.0，更佳地≥3.0。在另一优选例中，所述的下调指：未处理的巨噬细胞中IL-1β的表达量N0与经处理的巨噬细胞中IL-1β的表达量N1的比值N0/N1≥1.5,较佳地≥2.0，更佳地≥3.0。
在另一优选例中，所述的处理包括：将巨噬细胞与胰岛素样生长因子-2或其活性片段接触一段时间(如0.1-24小时)。
在本发明的第六方面，提供了一种用于调节T细胞分化的药物组合物，所述的组合物包含胰岛素样生长因子-2处理后的巨噬细胞和药学上可接受的载体。
在另一优选例中，所述的调节T细胞分化是指(b)促进调节性T细胞的分化；和/或(c)抑制Th1细胞和/或Th17细胞的分化。
在另一优选例中，所述的的药物组合物还用于治疗自身免疫性疾病。
在本发明的第七方面，提供了一种非治疗性地(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达的方法，包括步骤：
(a)在胰岛素样生长因子-2存在的条件下，培养巨噬细胞，从而(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。
在另一优选例中，所述的胰岛素样生长因子-2的施用浓度为1ng/ml-100μg/ml，较佳地为3ng/ml-1μg/ml，更佳地为5ng/ml—50ng/ml。
在本发明的第八方面，提供了一种多肽，所述多肽的序列如SEQ ID NO.:1的第25-91位所示。
在本发明的第九方面，提供了一种(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达的方法，包括步骤：
(a)给需要的对象施用胰岛素样生长因子-2。
在另一优选例中，所述的对象包括非人哺乳动物和人。
在另一优选例中，所述的胰岛素样生长因子-2施用剂量为1ng-1mg/kg，较佳地100ng-100μg/kg，更佳地1-10μg/kg。
在另一优选例中，所述的方法还包括步骤：
(b)给需要的对象施用巨噬细胞清除剂，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂。
在另一优选例中，所述的巨噬细胞清除剂按常规的的施用剂量和施用频率进行治疗。
在另一优选例中，所述的施用包括同时施用或先后施用。
在本发明的第十方面，提供了一种药盒，所述药盒含有：
(i)第一容器，以及装于该第一容器中的活性成分(a)胰岛素样生长因子-2，或含有活性成分(a)的药物；
(ii)第二容器，以及装于该第二容器中的活性成分(b)巨噬细胞清除剂，或含有活性成分(b)的药物，其中，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂；以及
(iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而治疗自身免疫性疾病的说明。
在另一优选例中，所述的第一容器和第二容器中的药物是含活性成分(a)的单方制剂以及含活性成分(b)的单方制剂。
在本发明的第十一方面，提供了一种第三方面所述药物组合物，本发明第十方面所述的药盒的用途，用于制备治疗自身免疫性疾病的药物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示胰岛素样生长因子-2(insulin growth factor-2，IGF-2)有效治疗EAE。
图1a显示了IGF-2可以有效缓解EAE的发病进程和程度。
图1b显示IGF-2可以有效抑制EAE小鼠中单核细胞在中枢神经系统的浸润。
图1c显示了IGF-2有效抑制EAE小鼠MOG特异性T细胞的增殖。
图1d、图1e、图1f和图1g显示了IGF-2可以有效抑制EAE小鼠Th1和Th17的细胞比例，促进Treg的比例。
图1h显示了IGF-2并不直接影响Th1/Th17/Treg的分化。
图1i和图1j显示了IGF-2促进EAE小鼠脊髓中CD11b+F4/80+的巨噬细胞上PD-L1的表达，抑制IL-1β的表达。
图2显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)诱导巨噬细胞具有抗炎的特性。
图2a显示了IGF-2并不影响EAE小鼠脊髓中巨噬细胞的数目。
图2b显示了IGF-2并不影响EAE小鼠脾脏中巨噬细胞的数目。
图2c显示了IGF-2抑制EAE小鼠脾脏中CD11b+F4/80+的巨噬细胞中IL-1β的表达水平。
图2d显示了IGF-2促进EAE小鼠脊髓中CD11b+F4/80+的巨噬细胞上PD-L1的表达水平。
图2e显示了IGF-2并不影响巯基醋酸盐培养基诱导的腹腔巨噬细胞上MHC-I，MHC-II,CD80和CD86的表达水平。
图2f显示了IGF-2并不影响脂多糖-LPS刺激巯基醋酸盐培养基诱导的腹腔巨噬细胞分泌TNF-α、IL-6和TGF-β。
图2g显示了IGF-2抑制脂多糖-LPS刺激巯基醋酸盐培养基诱导的腹腔巨噬细胞中诱导性一氧化氮合成酶(inducible nitric oxide synthase,iNOS)的mRNA水平，以及该酶调控的亚硝酸盐的产生。
图3显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)处理巨噬细胞可以有效治疗EAE。
图3a显示了IGF-2预处理后，在100ng/ml的脂多糖刺激的情况下，巯基醋酸盐培养基诱导的腹腔巨噬细胞表达IL-1β的信使RNA，前体蛋白的能力显著下降。
图3b显示了IGF-2预处理后，在100ng/ml的脂多糖刺激的情况下，巯基醋酸盐培养基诱导的腹腔巨噬细胞分泌的IL-1β的蛋白水平显著下降。
图3c显示了IGF-2预处理后，在100ng/ml的脂多糖刺激的情况下，巯基醋酸盐培养基诱导的腹腔巨噬细胞表达PD-L1蛋白水平显著上升。
图3d和图3e显示了IGF-2处理后的巨噬细胞依赖PD-L1促进Treg的产生。
图3f显示了IGF-2处理的巨噬细胞可以有效抑制EAE。
图3g显示了IGF-2处理后的巨噬细胞可以有效促进EAE小鼠中Treg细胞的比例。
图4显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)处理赋予骨髓来源的巨噬细胞促进Treg细胞分化的能力。
图4a显示了IGF-2处理后的骨髓来源巨噬细胞在脂多糖LPS刺激下，PD-L1的表达水平显著上升。
图4b和图4c显示IGF-2处理后的骨髓来源巨噬细胞通过PD-L1促进Treg细 胞。
图4d显示了IGF-2处理后的骨髓来源巨噬细胞上MHC-I，MHC-II,CD80和CD86的表达水平未发生变化。
图5显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)处理的巨噬细胞具有缓解实验性自身反应性脑脊髓炎以及炎症性肠炎的作用。
图5a显示了IGF-2处理后的骨髓来源巨噬细胞有效治疗EAE。
图5b和图5c显示了IGF-2处理后的骨髓来源巨噬细胞有效促进EAE小鼠Treg的比例。
图5d显示了IGF-2处理后的骨髓来源巨噬细胞缓解了实验性肠炎小鼠的体重降低。显示IGF-2处理后的巨噬细胞具有有效治疗IBD的能力。
图5e显示了IGF-2处理的巨噬细胞可以有效延长炎症性肠炎患病小鼠的存活时间。
图5f和图5g显示了胰岛素样生长因子-2处理的骨髓巨噬细胞处理炎症性肠炎小鼠，促进脾脏Treg细胞的比例显著上升。
图6显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)通过调控巨噬细胞糖代谢影响巨噬细胞的抗炎特性。
图6a显示IGF-2处理后，巨噬细胞的葡萄糖消耗量显著降低。
图6b显示IGF-2处理后，巨噬细胞的乳酸积累量显著降低。
图6c显示IGF-2处理后，巨噬细胞中NAD+/NADH比例显著降低。
图6d显示IGF-2处理后，巨噬细胞的氧气消耗以及氧化磷酸化能力显著上升。
图6e显示IGF-2处理后，巨噬细胞胞外酸化速率显著降低。
图6f显示IGF-2处理后，巨噬细胞倾向于使用氧化磷酸化代谢途径。
图6g显示IGF-2介导的巯基醋酸盐培养基诱导的腹腔巨噬细胞的PD-L1表达上升可以被寡霉素所抑制。
图6h显示了以寡霉素抑制氧化磷酸化，明确IGF-2处理后的巨噬细胞具有有氧呼吸依赖的高表达PD-L1并促进Treg产生的能力。
图7显示胰岛素样生长因子-2(insulin growth factor-2，IGF-2)以及IGF-2处理巨噬细胞有效治疗实验性肠炎的治疗。
图7a显示了IGF-2抑制炎症性肠炎小鼠的体重下降。
图7b显示了IGF-2治疗后，炎症性肠炎小鼠的结肠长度显著增加。
图7c显示了IGF-2治疗后，结肠中单核细胞的浸润数目显著下降。
图7d和图7e显示了IGF-2治疗后，炎症性肠炎小鼠的结肠固有层中Treg细胞的比例显著上升。
图7f显示了IGF-2治疗后，炎症性肠炎小鼠的结肠固有层中，CD11b+F4/80+的巨噬细胞中，PD-L1表达显著上升。
图7g显示了IGF-2治疗后，炎症性肠炎小鼠的结肠固有层中，CD11b+F4/80+的巨噬细胞中，IL-1β的表达量显著下降。
图7h显示了IGF-2处理后的巨噬细胞缓解了实验性肠炎患病小鼠的体重降低。
图7i显示了IGF-2处理后的巨噬细胞可以有效延长炎症性肠炎患病小鼠的存活时间。
图7j显示了炎症性肠炎小鼠在注射IGF-2处理的巨噬细胞后，其脾脏Treg细胞的比例显著上升。
图8显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)可以调控炎症性肠炎患病小鼠脾脏以及腹腔淋巴结中的免疫反应。
图8a和图8b显示了IGF-2显著上调了实验性肠炎小鼠患病小鼠中腹腔淋巴结中Treg细胞的比例。
图8c显示了IGF-2对实验性肠炎小鼠患病小鼠中脾脏巨噬细胞的比例没有影响。
图8d显示了IGF-2上调了实验性肠炎小鼠患病小鼠肠道固有层中CD11b+F4/80+巨噬细胞的比例。
图8e显示了IGF-2显著下调了实验性肠炎小鼠患病小鼠中脾脏巨噬细胞IL-1β的表达量。
图8f显示了IGF-2显著上调了实验性肠炎小鼠患病小鼠中脾脏巨噬细胞PD-L1的表达量。
图9显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)处理后的巯基醋酸盐培养基诱导的腹腔巨噬细胞可以上调炎症性肠炎小鼠腹腔淋巴结以及结肠固有层的Treg细胞。
图9a显示了IGF-2处理后的巨噬细胞可以显著上调炎症性肠炎患病小鼠腹腔淋巴结中Treg细胞的比例。
图9b显示了IGF-2预处理后的巨噬细胞可以显著上调炎症性肠炎患病小鼠结肠固有层中Treg细胞的比例。
图10显示与对照组小鼠相比较，胰岛素样生长因子-2(insulin growth factor-2，IGF-2)和氯膦酸盐脂质体的单独注射均可以显著抑制EAE的进展，发挥有效治疗作用，更重要的是，IGF-2和氯膦酸盐脂质体的结合注射对于EAE的治疗作用与其他各组相比较，更为显著。
图11显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)的三级结构。
图12显示了胰岛素样生长因子-2(insulin growth factor-2，IGF-2)与受体结合的关键位点。
图13A显示了葡萄糖耐量实验的结果。
图13B显示了胰岛素耐量实验的结果。
在图1-图13中，附图标记及附图说明中的IGF-2表示具有胰岛素样生长因子-2的第25～91位氨基酸的活性片段，即IGF25-91。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现含胰岛素样生长因子-2的药物组合物及其用途。本发明提供了胰岛素样生长因子-2及其活性片段在用于制备一药物组合物中的用途，该药物组合物用于(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。本发明还提供了含胰岛素样生长因子-2作为有效成分的药物组合物。
具体地，在本发明中，发现胰岛素样生长因子-2(insulin growth factor-2,IGF-2)以及具有胰岛素样生长因子-2第25～91位氨基酸序列的活性片段(IGF25-91)在多发性硬化和结肠炎的动物模型(实验性自身反应性脑脊髓炎、DSS诱导的炎症性肠病)治疗中均表现显著的治疗效果，明显抑制疾病的进展和相应部位的组织损伤及炎症浸润。更重要的是，IGF25-91通过诱导低表达IL-1和高表达PD-L1的巨噬细胞，间接地上调调节性T细胞(Treg)，从而促进自身免疫性疾病的恢复。进一步的研究发现，IGF25-91主要通过影响巨噬细胞的有氧糖酵解和氧化磷酸化途径，促进巨噬细胞向具有免疫抑制功能的巨噬细胞方向的重编程，而这种经由IGF25-91处理的巨噬细胞对于自身免疫性疾病的治疗具有非常显著的作用。至此，本技术发明基于IGF25-91对自身免疫性疾病的治疗作用，明确IGF25-91对巨噬细胞的调节机制，发现IGF25-91诱导具有免疫抑制功能巨噬细胞在自身免疫性疾病治疗中的应用潜能，形成巨噬细胞免疫治疗的新技术。
胰岛素样生长因子-2
胰岛素样生长因子-2(IGF-2)是一种主要由肝脏分泌并在血液中大量存在的生长因子，它具有抗凋亡、生长调节、类胰岛素和促有丝分裂等功能。IGF-2由mRNA翻译而来的前体蛋白共180个氨基酸序列，序列SEQ ID NO.:1所示(mgipmgksmlvlltflafascciaayrpsetlcggelvdtlqfvcgdrgfyfsrpasrvsrrsrgiveeccfrscdlalletycatpakserdvstpptvlpdnfprypvgkffqydtwkqstqrlrrglpallrarrghvlakeleafreakrhrplialptqdpahggappemasnrk)；经过翻译后修饰产生的活性形式由67个氨基酸组成，序列SEQ ID NO.:2所示(ayrpsetlcggelvdtlqfvcgdrgfyfsrpasrvsrrsrgiveeccfrscdlalletycatpakse)。IGF-2形成的三级结构如图11所示，由三个α-螺旋及两个β-折叠组成。其中11G-21C是一个螺旋，25G-27Y是第一个折叠，第二个螺旋42I-49R，第三个螺旋 53L-58T,第二个折叠是59Y-61A。预测位于第一个螺旋上的T16，F19以及第三个螺旋上的L63为IGF-2与IGF-2受体结合的位点，如图12所示。
目前的研究成果普遍认为，胰岛素样生长因子-2在胚胎发育中具有重要的作用，可以促进胚胎发育和器官形成；也有报道称其与记忆和生殖有关，通过对基因缺陷小鼠的研究发现，胰岛素样生长因子-2的信号缺失会造成脑发育不健全。
本发明发现，单独使用胰岛素样生长因子-2或者具有胰岛素样生长因子-2(insulin growth factor-2,IGF-2)第25～91位氨基酸序列的活性片段(IGF25-91)可以有效的治疗实验性自体免疫性脑脊髓炎或者炎症性肠炎这两种自体免疫性疾病。在病灶部位，如患病小鼠的脊髓中或结肠中，IGF25-91治疗组中调节性T细胞的比例显著提高，同时，TH1和TH17细胞的比例显著下降。
然而，当利用体外T细胞分化体系去验证IGF25-91对T细胞分化的影响时，发现IGF25-91并不能直接影响T细胞向Th1、Th17、Treg方向分化的效率。这些研究提示IGF25-91对T细胞亚群比例的改变是一个间接的作用。在使用胰岛素样生长因子-2治疗自身免疫性疾病时，还观察到了巨噬细胞表型的明显变化。研究发现，经IGF25-91治疗的小鼠损伤部位的巨噬细胞中IL-1β表达量显著下降，而PD-L1表达量显著上升，提示IGF25-91可能通过促进具有抗炎作用巨噬细胞的比例发挥抑制自身免疫性疾病的作用。
研究发现，IGF25-91并不能直接诱导巨噬细胞中抗炎基因的表达，但在由IGF25-91处理的骨髓或腹腔来源的成熟巨噬细胞在应对脂多糖刺激的时候表现出了IL-1β表达量显著下降，而PD-L1表达量显著上升的特性。巨噬细胞在自身免疫性疾病的发生和发展中起到了重要的作用，一系列研究表现巨噬细胞在炎症因子例如干扰素 ，脂多糖的刺激下能发挥极强的促炎能力，加剧了自身免疫性疾病的进程。研究表明胰岛素样生长因子-2改变了巨噬细胞对炎症因子的应答，在原本促炎的刺激条件下巨噬细胞高表达了抑炎分子PD-L1。因此IGF25-91对自身免疫性疾病的治疗很可能是通过对巨噬细胞的重编程实现的。
为了明确IGF25-91处理后的巨噬细胞在调控Treg中的作用，将IGF25-91诱导的巨噬细胞在脂多糖预刺激后与T细胞共培养，结果发现，IGF25-91处理后的巨噬细胞可以显著促进Treg的分化，而这种促进作用依赖于PD-L1的高表达。进一步的研究发现，IGF25-91处理过的巨噬细胞还可以直接用于治疗自身反应性脑脊髓炎和炎症性肠炎，在治疗过程中，这些巨噬细胞的输注同样促进小鼠体内Treg比例的上升。
为了进一步阐释IGF25-91是如何影响巨噬细胞PD-L1的表达，详细地比较了IGF25-91处理的巨噬细胞与对照细胞，研究发现IGF25-91处理巨噬细胞可以显著地改变巨噬细胞的糖代谢途径状态，使其更偏向于氧化磷酸化的代谢途径。当阻断巨噬细胞线粒体氧化磷酸化时，胰岛素样生长因子-2所诱导的巨噬细胞的PD-L1的高表达以及促Treg分化的能力也都消失了，这些研究说明IGF25-91是通过改变巨噬细胞 的代谢倾向，从而赋予巨噬细胞促进Treg产生、治疗自身免疫性疾病的能力。
综上所述，IGF25-91通过诱导具有免疫抑制作用的巨噬细胞，上调调节性T细胞，发挥对多发性硬化和炎症性肠炎的治疗效果。经由IGF25-91处理的巨噬细胞便可以发挥对自体免疫性疾病的治疗作用。此外，靶向性调节巨噬细胞中有氧糖酵解和氧化磷酸化可以诱导免疫抑制性巨噬细胞的产生，发挥对多种自身免疫性疾病治疗的作用。
药物组合物和施用方法
本发明提供了一种用于(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达的药物组合物，它含有安全有效量的胰岛素样生长因子-2(或其活性片段)或含有胰岛素样生长因子-2处理后的巨噬细胞。
本发明的所述的药物组合物可用于(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。在使用本发明药物制剂时，还可同时使用其他治疗自身免疫性疾病的治疗剂。
本发明的药物组合物还可以含有巨噬细胞清除剂，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂。实验表明，IGF25-91和巨噬细胞清除的结合治疗通过重编程具有免疫抑制功能的巨噬细胞，发挥更为优化的炎症性疾病治疗作用。
本发明还提供了一种药盒，所述药盒含有：
(i)第一容器，以及装于该第一容器中的活性成分(a)胰岛素样生长因子-2，或含有活性成分(a)的药物；
(ii)第二容器，以及装于该第二容器中的活性成分(b)巨噬细胞清除剂，或含有活性成分(b)的药物，其中，所述的巨噬细胞清除剂为去除巨噬细胞或抑制巨噬细胞迁移的制剂；以及
(iii)说明书，所述说明书中记载了联合给予活性成分(a)和活性成分(b)从而治疗自身免疫性疾病的说明。
本发明所述药物组合物，含有安全有效量的胰岛素样生长因子-2以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。
本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。本发明的药物组合也可以被制成粉剂用于雾化吸入。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约50毫克/千克体重，约5微克/千克体重-约10毫克/千克体重，约10微克/千克体重-约5毫克/千克体重。此外，本发明化合 物还可与其他治疗剂一起使用。
对于本发明的药物组合物，可通过常规的方式施用于所需的对象(如人和非人哺乳动物)。代表性的施用方式包括(但并不限于)：口服、注射、雾化吸入等。
使用药物组合物时，是将安全有效量的药物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
(a)胰岛素样生长因子-2及其活性片段(第25～91位)能够(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达
(b)胰岛素样生长因子-2及其活性片段处理后的巨噬细胞能够促进调节性T细胞的分化，抑制Th1细胞和/或Th17细胞的分化，从而治疗自身免疫性疾病。
(c)胰岛素样生长因子-2及其活性片段与巨噬细胞清除的结合治疗能够用于自身免疫性疾病的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用材料和方法
1.实验性自身免疫性脑脊髓炎EAE模型的建立及治疗
(1)实验前准备过程
制备完全弗氏佐剂(CFA)：将热灭活的结核分支杆菌(购自sigma)加入不完全弗氏佐剂中直至终浓度达到5mg/ml，使用前充分颠倒混匀。
抗原的乳化：将两支玻璃针管通过三通管连接，将等体积的抗原溶液(300μg MOG于100μL PBS中)和100μL完全弗氏佐剂加入针管中，排除针管内气泡并来回推动获得乳化液。大约需要推动约500次，推动阻力会逐渐增加，此时各成分可充分混匀成乳化状态。
百日咳毒素(PT)溶液配制：将百日咳毒素)溶解于PBS至工作浓度1ng/μL，每只小鼠200μL，尾静脉注射。
(2)EAE模型的建立和治疗
第0天，将200μL乳化好的抗原皮下注射于C57BL/6小鼠(购自中国科学院斯莱克上海实验动物中心)背部进行抗原免疫，胸部对应的背部两侧每侧100μL； 同时通过尾静脉注射PT，200μL/只。
第2天，再次通过尾静脉注射PT，200μL/只。
从第九天开始，使用胰岛素样生长因子2治疗，每天注射，剂量为5ng/只，腹腔给药。生理盐水治疗组作为阳性对照。
使用巨噬细胞治疗时，分别在第9、11、13天腹腔注射的方式注射5*106巨噬细胞，每天记录临床评分。
(3)EAE临床评分标准
0分：无异常表现；0.5分：尾尖下垂；1分：整条尾巴瘫痪；2分：后肢无力；3分：一后肢瘫痪；4分：双后肢瘫痪；5分：死亡。
2.脊髓中淋巴细胞的提取
从EAE小鼠中取出脊髓，并用PBS进行清洗干净，将脊髓放在70μm孔径细胞筛网上进行机械研磨，得到单细胞悬液。将获得的细胞悬液离心，并重悬细胞于30％percoll中，轻轻于底部加入等体积的70％percoll溶液。在室温下以2000转每分钟的速度在零加速度的条件下离心20分钟。离心后，从高低密度液面交界层吸取细胞，并用PBS冲洗两次，得到淋巴细胞。
3.脾细胞体外增殖实验
处死EAE小鼠后，取出小鼠的脾并获取单细胞悬液，按照每孔3-5*105每孔的数量将脾细胞铺在U型底的96孔板中，加MOG至20ug/ml。
37摄氏度温箱中培养72小时后加入H3标记的胸腺嘧啶，6小时之后将孔板经反复冻融后上机通过真空吸附到专用滤膜上，加入闪烁液后上机检测。
4.炎症性肠炎IBD模型的建立和治疗
葡聚糖硫酸钠(DSS)按照质量体积比2.5:100配成溶液，使用22μm的筛网过滤以保证无菌，用封口膜封口备用。
选用8-10周的C57/BL6雌鼠(购自中国科学院斯莱克上海实验动物中心)，用配好的葡聚糖硫酸钠溶液替换饮用水诱导模型，每天称取小鼠体重并观察粪便情况，葡聚糖硫酸钠溶液每隔一天换液或补加。
从模型诱导当天开始，使用胰岛素样生长因子-2治疗，每天注射，剂量为50ng/只，腹腔给药。生理盐水治疗组作为阳性对照。
使用巨噬细胞治疗时，分别在第1、3、5天腹腔注射的方式注射1*107巨噬细胞，每天记录体重变化情况。
5.结肠组织淋巴细胞的提取
将从IBD小鼠中取出的结肠出去粪便，并用PBS进行清洗干净，然后将结肠剪成2cm每段，并置于HBSS(1mM DTT和5mM EDTA)中冲洗两次，条件为250rpm*20min*37度。将清洗好的结肠组织用剪刀尽量剪碎，并加入含有DNAase I,dispase和type VIII collegenase的HBSS中消化，条件为250rpm*30min*37度。将获得的细胞悬液离心，并重悬细胞于40％percoll中，轻轻于底部加入等体积的80％percoll溶液。在室温下以2000转每分钟的速度在零加速度的条件下离心20分钟。离心后，从高低密度液面交界层吸取细胞，并用PBS冲洗两次，得到淋巴细胞。
6.海马实验
在海马测试板上以1*105每孔的密度种植细胞贴壁。XF培养中加入10mM glucose,2mM glutamine和2mM pyruvate配成测试培养基。上机前用测试培养基将细胞润洗两遍，每个细胞培养孔定容至525μL。使用测试培养基配制代谢抑制剂：1μM oligomycin,0.75μM FCCP,100μM rotenone+1μMantimycin。测量基础状态和药物刺激条件下氧气的消耗速率以及胞外氢离子的浓度。仪器使用的是Extracellular flux analyzer。
7.巨噬细胞的制备和处理
骨髓来源的巨噬细胞制备过程为：将小鼠骨髓用1ml注射器吹出，充分吹散后，按照400g*5min的条件离心所的细胞，弃去上清，将所的细胞吹打为单细胞悬液，使用DMEM/F12完全培养基(含有10％FBS，20％L929上清，100U/ml Penicillin，100U/ml Streptomycin，2mM L-Glutamine)铺板于无包被的培养皿中，在培养到第四天时补充5ml培液，培养细胞6天，即可得到成熟巨噬细胞。IGF-2刺激分别在第1、3、5天加入。
腹腔来源的巨噬细胞的制备方法为：向8-12周龄的C57BL/6小鼠腹腔中注射2ml 4％thioglycollate溶液，每天向腹腔注射因子或者PBS，在第三天安乐死处死小鼠，用10ml注射器以10ml体积的PBS将腹腔中的巨噬细胞吹出，离心后将细胞吹打为单细胞悬液，铺板于无包被的细胞培养皿或者6孔板中，以用于后续实验。
8.巨噬细胞与
Figure PCTCN2017113341-appb-000001
T细胞共培养体系
将成熟的骨髓或腹腔来源的巨噬细胞用LPS刺激24小时之后，吸去刺激溶液，使用PBS清洗三遍以充分去除残留的LPS。然后将巨噬细胞(2.5*105)与所分选好的CD4+CD62L+T细胞(1*106)铺板于96孔板中。共培养72小时后，吹出细胞用于流式细胞染色和分析。
9.高脂饲料诱导肥胖建模
从5周起给C57/BL6小鼠(购自中国科学院斯莱克上海实验动物中心)饲喂高脂饲料，持续20周。
10.葡糖糖耐量试验
小鼠饥饿过夜，腹腔注射1g葡糖糖/kg体重，在不同时间点将一滴尾静脉学虹吸入血糖试纸中，用血糖仪测血糖。
11.胰岛素耐量试验
小鼠饥饿过夜，饥饿时间约16小时，腹腔注射0.75U胰岛素/kg体重，在不同时间点，依次将各小鼠的一滴尾静脉血虹吸入血糖试纸中，用血糖仪测血糖，用计时器进行准确计时。
实施例1
IGF25-91治疗实验性自体免疫性脑脊髓炎(EAE)依赖于其对巨噬细胞基因的表达
使用IGF25-91治疗诱导了EAE的小鼠时可以看到，IGF25-91能够有效地缓解EAE的发病进程和严重程度，表现为临床评分的降低、脊髓中浸润的单核细胞数的减少以及EAE小鼠中MOG特异性T细胞的反应的降低(图1a-c)。与此同时利用流式细胞仪分析发现，IGF25-91的使用可以有效地降低EAE小鼠中脊髓部位Th1、Th17的比例，同时升高Treg的比例(图1d-g)。然而，当发明人将IGF25-91直接加入到T细胞体外分化体系时，发现T细胞向Th1、Th17和Treg的分化效率并没有受到影响，说明IGF25-91对T细胞的影响是一个间接的作用(图1h)。
当利用流式细胞仪分析EAE小鼠中的免疫细胞时，还发现，IGF25-91的使用虽然不会影响EAE患病小鼠脊髓和脾脏中巨噬细胞的比例(图2a-b)，但是可以显著的影响巨噬细胞的基因表达，表现为巨噬细胞的IL-1β表达量显著下降，而PD-L1的表达量显著上升(图1i-j；图2c-d)。总结来说，IGF25-91可以用于EAE的治疗，并且可以影响患病小鼠体内不同T细胞亚群的比例和巨噬细胞的基因表达。
实施例2
IGF25-91处理后的巨噬细胞能够促进Treg的产生并治疗EAE
为了进一步研究IGF25-91对巨噬细胞的影响，使用了两种经典方法制备巨噬细胞用于研究，一种是利用巯基乙醇酸盐诱导的腹腔巨噬细胞，另一种是利用骨髓细胞在L929细胞培养上清的刺激下分化出巨噬细胞。发现巨噬细胞分化过程中接受IGF25-91处理后，腹腔巨噬细胞或者骨髓来源巨噬细胞在共刺激分子(CD80、CD86、MHC I、MHC II)以及TNF-α、IL-6、TGF-β表达上并未出现显著的变化(图2e-f；图 4d)。但是在脂多糖刺激后，与对照巨噬细胞相比胰岛素样生长因子-2处理的巨噬细胞的IL-1β表达量显著下降，而PD-L1的表达量显著上升，这与体内观察到的现象十分吻合(图3a-c)。为了研究IGF25-91处理后巨噬细胞对Treg分化的影响，将巨噬细胞与T细胞共培养，同时在培养体系中加入Treg分化所需的anti-CD3、anti-CD28和TGF-β。结果显示，相比于正常的巨噬细胞，IGF25-91处理后的巨噬细胞(包括骨髓来源的巨噬细胞和腹腔来源的巨噬细胞)在脂多糖预刺激后可以更有效地促进Treg的产生。同时，PD-L1在这一过程中发挥着至关重要的作用，因为一旦使用PD-L1的中和抗体，IGF25-91处理过的巨噬细胞促Treg产生的能力也就被抑制了(图3d-e；图4a-c)。
为了进一步验证IGF25-91处理后的巨噬细胞的免疫调控能力，将处理好的巨噬细胞(包括骨髓来源的巨噬细胞和腹腔来源的巨噬细胞)用于EAE的治疗中，结果显示，IGF25-91的预处理使巨噬细胞能够有效的缓解EAE的临床评分，同时促进EAE患病小鼠脾脏中Treg的比例(图3f-g；图5a-c)。
实施例3
IGF25-91通过提高巨噬细胞氧化磷酸化水平影响其基因表达
为了研究IGF25-91对巨噬细胞的调控机制，比较了IGF25-91处理的巨噬细胞和对照巨噬细胞的性状发现IGF25-91处理后的巨噬细胞会葡萄糖消耗量降低，同时乳酸积累减少(图6a-b)。与此同时，巨噬细胞中的NAD+/NADH在IGF25-91处理后也明显下降(图6c)。因此推测IGF25-91可以调控巨噬细胞的中糖酵解与氧化磷酸化的代谢途径。当对巨噬细胞的耗氧量和产酸量进行追踪时发现，IGF25-91可以有效地提高巨噬细胞消耗氧气的效率，同时降低本底状态巨噬细胞的产酸量(图6d-f)。这说明IGF25-91可以使巨噬细胞的代谢倾向于氧化磷酸化的途径。
为了确认氧化磷酸化的代谢状态是巨噬细胞PD-L1高表达和促进Treg分化的基础，使用了氧化磷酸化过程的抑制剂寡霉素。结果发现，一旦氧化磷酸化被阻断，IGF25-91引起的巨噬细胞的PD-L1的高表达以及促进Treg分化的能力的都会随之消失(图6g-h)。这说明，IGF25-91所引起的巨噬细胞高表达PD-L1和促Treg产生能力是依赖于氧化磷酸化的代谢方式的。
实施例4
IGF25-91以及IGF25-91处理的巨噬细胞能够用于治疗炎症性肠炎(IBD)
为了进一步验证IGF25-91对自体免疫性疾病的治疗效果和对巨噬细胞的调控作用，使用IGF25-91去治疗固有免疫系统主导的自体免疫性疾病—炎症性肠炎。使用IGF25-91治疗后，IBD患病小鼠的体重下降得到缓解，结肠的损伤以及结肠中单核细胞的浸润明显降低(图7a-c)。利用流式细胞仪分析T细胞分群和巨噬细胞基因表 达时发现，IGF25-91可以有效提高结肠和腹膜淋巴结中Treg的比例(图7d-e；图8a-b)。同时，在结肠和脾脏中，巨噬细胞的IL-1β表达量下降，PD-L1表达量上升(图7f-g；图8c-f)。更进一步的，发现IGF25-91处理的巨噬细胞(包括骨髓来源的巨噬细胞和腹腔来源的巨噬细胞)可以直接用于IBD的治疗，缓解IBD小鼠的体重降低，提高患病小鼠的存活率(图5d-e；图7h-i)。同时，IGF25-91处理的巨噬细胞也提高了患病小鼠脾脏、潘氏结和腹膜淋巴结中Treg的比例(图5f-g；图7h-j；图9)。
实施例5
IGF25-91与巨噬细胞清除更有效治疗炎症性疾病
由于IGF25-91可以显著抑制炎症性疾病，如上述的多发性硬化和炎症性肠病，并且该作用与IGF25-91调控巨噬细胞重编程为具有免疫抑制功能的巨噬细胞，从而抑制过强炎症反应密切相关。然而，在炎症性疾病发生时，系统及炎症局部往往伴随大量促炎的巨噬细胞浸润，并且炎症部位的巨噬细胞浸润往往依赖于外周单核细胞的补充和向促炎巨噬细胞的演变，因此，研究了清除内在促炎巨噬细胞和充分发挥IGF25-91对巨噬细胞的重编程作用是否更能优化炎症性疾病的治疗。为此，在EAE诱导第9天、11天、13天，不同实验组小鼠分别接受IGF25-91、氯膦酸二钠脂质体(Clodronate liposome，用于清除巨噬细胞)或IGF25-91和Clodronate liposome的结合注射，观察小鼠EAE进展情况。研究发现，与对照组小鼠相比较，IGF25-91和Clodronate liposome的单独注射均可以显著抑制EAE的进展，发挥有效治疗作用，更重要的是，IGF25-91和Clodronate liposome的结合注射对于EAE的治疗作用与其他各组相比较，更为显著(图10)。因此，IGF25-91和巨噬细胞清除的结合治疗通过重编程具有免疫抑制功能的巨噬细胞，发挥更为优化的炎症性疾病治疗作用。
实施例6
IGF25-91处理的巨噬细胞能够用于治疗肥胖诱导的胰岛素抵抗
给予小鼠饲喂高脂饮食20周，注射PBS、巨噬细胞(5×106)或经由IGF26-91处理的巨噬细胞(5×106)，2周后进行葡萄糖耐量试验和胰岛素耐量实验，检测各组小鼠葡萄糖变化情况。研究发现，IGF26-91处理的巨噬细胞可以显著提高胰岛素抵抗小鼠的葡萄糖耐受能力(图13A和13B)，说明IGF26-91处理的巨噬细胞可以有效治疗胰岛素抵抗和糖尿病。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种胰岛素样生长因子-2(IGF-2)的用途，其特征在于，用于制备一药物组合物，所述药物组合物用于(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。
  2. 如权利要求1所述的用途，其特征在于，所述的胰岛素样生长因子-2包括全长胰岛素样生长因子-2和胰岛素样生长因子-2活性片段。
  3. 如权利要求2所述的用途，其特征在于，所述的胰岛素样生长因子-2活性片段为包含胰岛素样生长因子-2的第25-91位氨基酸的活性片段。
  4. 如权利要求1所述的用途，其特征在于，所述的药物组合物还用于选自下组的一种或多种用途：
    (a)促进巨噬细胞的氧化磷酸化代谢途径；
    (b)促进调节性T细胞的分化；
    (c)抑制Th1细胞和/或Th17细胞的分化；
    (d)治疗自身免疫性疾病。
  5. 如权利要求4所述的用途，其特征在于，所述的自身免疫性疾病选自下组：多发性硬化、炎症性肠炎、自身反应性脑脊髓炎、自身免疫性肝炎、系统性红斑狼疮、类风湿性关节炎、胰岛素抵抗、糖尿病、肝硬化、或其组合。
  6. 一种巨噬细胞的用途，其特征在于，所述的巨噬细胞为胰岛素样生长因子-2(IGF-2)处理后的巨噬细胞，用于制备一药物组合物，所述药物组合物用于选自下组的一种或多种用途：
    (b)促进调节性T细胞的分化；
    (c)抑制Th1细胞和/或Th17细胞的分化；
    (d)治疗自身免疫性疾病。
  7. 一种药物组合物，其特征在于，所述的组合物包含(a)胰岛素样生长因子-2或其活性片段(如含第25-91位氨基酸的活性片段)、(b)任选的巨噬细胞清除剂、(c)任选的PD-L1促进剂、和(d)药学上可接受的载体。
  8. 一种药物组合物，其特征在于，所述的组合物包含(a1)胰岛素样生长因子-2抑制剂、(b1)任选的巨噬细胞促进剂、(c1)任选的PD-L1抑制剂、和(d1)药学上可接受的载体。
  9. 一种用于调节T细胞分化的药物组合物，其特征在于，所述的组合物包含 胰岛素样生长因子-2处理后的巨噬细胞和药学上可接受的载体。
  10. 一种非治疗性地(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达的方法，其特征在于，包括步骤：
    (a)在胰岛素样生长因子-2存在的条件下，培养巨噬细胞，从而(i)促进巨噬细胞PD-L1的表达，和/或(ii)抑制巨噬细胞IL-1β的表达。
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