US20190381147A1 - Pharmaceutical composition containing insulin-like growth factor-2 and use thereof - Google Patents

Pharmaceutical composition containing insulin-like growth factor-2 and use thereof Download PDF

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US20190381147A1
US20190381147A1 US16/464,902 US201716464902A US2019381147A1 US 20190381147 A1 US20190381147 A1 US 20190381147A1 US 201716464902 A US201716464902 A US 201716464902A US 2019381147 A1 US2019381147 A1 US 2019381147A1
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igf
macrophages
macrophage
insulin
growth factor
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Yufang Shi
Ying Wang
Liming Du
Liangyu Lin
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Shanghai Institute of Nutrition and Health of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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    • 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
    • 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
    • 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
    • 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
    • A61K31/66—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • 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
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
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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
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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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
    • A—HUMAN NECESSITIES
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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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • This invention relates to the field of biological medicine, and in particular to a pharmaceutical composition containing insulin-like growth factor-2 and use thereof.
  • Macrophages are specialized immune cells distributed in all parts of the body. They remove the senescence cells, certain cells that cannot be recognized as their own, cell debris and so on through phagocytosis. As the professional antigen presenting cells, macrophages initiate the immune responses of unsensitized T cells and trigger the inflammatory response. However, inflammatory cytokines expressed by macrophages such as TNF ⁇ and IL-1 also play important roles in the pathogenesis of many autoimmune diseases. Removal of pathogenic macrophages has been shown to significantly inhibit autoimmune encephalomyelitis and inflammatory bowel diseases.
  • macrophages can be regulated by different circumstances factors into cells with immunosuppressive effect, such as macrophages marked by expressing IL-1 ⁇ , Arginase I, etc, as well as macrophages with immunosuppressive function which are formed by different immune stimuli in the evolution process of monocytes to macrophages, thereby directly or indirectly regulating the body's immune response. It has been reported that spermidine treated macrophages are able to powerfully treat autoimmune diseases, indicating that macrophages with immunosuppressive effect have significant therapeutic effects in autoimmune diseases.
  • the present invention provides a composition containing insulin-like growth factor-2 and the uses thereof.
  • the first aspect of the invention provides a use of insulin-like growth factor-2 (IGF-2) to prepare a composition (including a chemical preparation and a pharmaceutical composition) to (i) increase PD-L1 expression in macrophage and/or (ii) inhibit IL-1 ⁇ expression in macrophage.
  • IGF-2 insulin-like growth factor-2
  • the IGF-2 includes full length IGF-2 and an active fragment of IGF-2.
  • the active fragment of IGF-2 is an active fragment having amino acids from position 25 to position 91 of IGF-2.
  • the active fragment of IGF-2 is the amino acid sequence of position 25 to position 91 of IGF-2.
  • the IGF-2 is from human and non-human mammals.
  • amino acid sequence of the IGF-2 is shown in SEQ ID NO.:1.
  • the pharmaceutical composition is also used for one or more of the purposes selected from the group consisting of:
  • the autoimmune disease is selected from the group consisting of multiple sclerosis, inflammatory bowel disease, autoimmune encephalomyelitis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, insulin resistance, diabetes, liver cirrhosis or a combination thereof.
  • the insulin resistance or the diabetes is obesity-induced insulin resistance or obesity-induced diabetes.
  • the pharmaceutical composition comprises (a) IGF-2; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises 0.001-99 wt %, preferred 0.1-90 wt %, and more preferred 1-80% IGF-2, based on the total weight of the pharmaceutical composition.
  • the pharmaceutical composition comprises macrophage scavenger, and the scavenger is a formulation for removing macrophage or inhibiting macrophage migration.
  • the pharmaceutical composition comprises agonist of PD-L1.
  • the pharmaceutical composition is liquid or lyophilized formulation.
  • the pharmaceutical composition is an injection formulation.
  • the second aspect of the invention provides a use of macrophage treated with insulin-like growth factor-2 (IGF-2) for preparing a pharmaceutical composition for one or more of the purposes selected from the group consisting of:
  • IGF-2 insulin-like growth factor-2
  • macrophage is cultured in the presence of IGF-2 to obtain the macrophage treated with the IGF-2.
  • the third aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) IGF-2 or the active fragment thereof (such as active fragment having amino acids from position 25 to position 91), (b) optionally, macrophage scavenger, (c) optionally, PD-L1 promoter, and (d) a pharmaceutically acceptable carrier.
  • At least one of the components (b) and (c) is present.
  • the macrophage scavenger is an agent for removing macrophage or inhibiting macrophage migration.
  • the formulation of the pharmaceutical composition is an injection formulation, sustained release formulation or topical formulation.
  • the pharmaceutical composition is used for one or more of the purposes selected from the group consisting of:
  • the pharmaceutical composition comprises macrophage scavenger which depletes macrophage or suppresses macrophage migration.
  • the macrophage scavenger is selected from the group consisting of: CCR2 inhibitor (to inhibit macrophage migration), clodronate disodium liposome (to remove macrophage) and a combination thereof.
  • the pharmaceutical composition comprises component (a), component (b) and component (c)
  • the mass ratio of component (a): component (b): component (c) is (1-100): (1-100): (1-100).
  • the pharmaceutical composition comprises component (a) and component (b)
  • the mass ratio of component (a): component (b) is (1-100): (1-100).
  • the pharmaceutical composition comprises component (a) and component (c)
  • the mass ratio of component (a): component (c) is (1-100): (1-100).
  • the ratio (mg: mg) of IGF-2 active fragment having amino acids from position 25 to position 91 to macrophage scavenger ranges from 1:100 to 100:1, preferably, from 1:20 to 20:1.
  • the total content of IGF-2 active fragment having amino acids from position 25 to position 91 and macrophage scavenger is 1 to 99 wt % of the pharmaceutical composition, preferably, 5 to 90wt % of the total pharmaceutical composition.
  • the fourth aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a1) IGF-2 inhibitor, (b1) optionally, macrophage agonist, (c1) optionally, PD-L1 inhibitor, and (d1) a pharmaceutically acceptable carrier.
  • At least one of the components (b1) and (c1) is present.
  • the PD-L1 inhibitor includes PD-L1 antibodies.
  • the pharmaceutical composition comprises component (a1), component (b1) and component (c1)
  • the mass ratio of component (a1): component (b1): component (c1) is (1-100): (1-100): (1-100).
  • the pharmaceutical composition comprises component (a1) and component (b1)
  • the mass ratio of component (a1): component (b1) is (1-100): (1-100).
  • the pharmaceutical composition comprises component (a1) and component (c1)
  • the mass ratio of component (a1): component (c1) is (1-100): (1-100).
  • the fifth aspect of the invention provides a macrophage pre-treated with IGF-2.
  • the PD-L1 expression is up-regulated and/or (ii) the IL-1 ⁇ expression is down-regulated.
  • the up-regulation referred to that the ratio of PD-L1 expression quantity in treated macrophage (M1) to PD-L1 expression quantity in untreated macrophage (M0) M1/M0 is ⁇ 1.5, preferably, M1/M0 ⁇ 2.0, more preferably, M 1/M0 ⁇ 3.0.
  • the down-regulation referred to that the ratio of IL-1 ⁇ expression quantity in untreated macrophage (N0) to IL-1 ⁇ expression quantity in treated macrophage (N1) N0/N1 is ⁇ 1.5, preferably, N0/N1 ⁇ 2.0, more preferably, N0/N1 ⁇ 3.0.
  • the treatment includes exposing macrophage to IGF-2 or IGF-2 active fragment for a period of time (such as 0.1-24 hours).
  • the sixth aspect of the invention provides a pharmaceutical composition for regulating T cell differentiation, which comprises IGF-2 treated macrophage and a pharmaceutically acceptable carrier.
  • the regulating T cell differentiation referred to (b) promoting differentiation of regulatory T cell; and/or (c) inhibiting differentiation of Th1 and/or Th17 cell.
  • the pharmaceutical composition is used for treating autoimmune disease.
  • the seventh aspect of the invention provides a non-therapeutic method for (i) promoting PD-L1 expression and/or (ii) inhibiting IL-1 ⁇ expression in macrophage, which comprises the following steps:
  • the concentration of IGF-2 is 1 ng/ml-100 ⁇ g/ml, preferably, 3 ng/ml-1 ⁇ g/ml, and more preferably, 5 ng/ml-50 ng/ml.
  • the eighth aspect of the invention provides a polypeptide with a sequence of position 25 to position 91 of SEQ ID NO.: 1.
  • the ninth aspect of the invention provides a method for (i) enhancing PD-L1 expression and/or (ii) inhibiting IL-1 ⁇ expression, which comprises the following steps:
  • the subject includes human and nonhuman mammal.
  • the dose of IGF-2 administered is 1 ng-1 mg/kg, preferably, 100 ng-100 ⁇ g/kg, and more preferably, 1-10 ⁇ g/kg.
  • the method further comprises the steps of:
  • macrophage scavenger which is an agent for removing macrophage or inhibiting macrophage migration
  • the dose and frequency of macrophage scavenger are administered in a conventional manner.
  • the administration includes administrating simultaneously or successively.
  • the tenth aspect of the invention provides a medicine kit, which contains:
  • a first container which contains an active ingredient (a) IGF-2 or a medicine containing the active ingredient (a);
  • a second container which contains an active ingredients
  • macrophage scavenger or a medicine containing the active ingredient (b)
  • the macrophage scavenger is an agent for removing macrophage or inhibiting macrophage migration
  • the medicine in the first container or the second container is a formulation containing one single active ingredient (a) or a formulation containing one single active ingredient (b).
  • the eleventh aspect of the invention provides a use of the pharmaceutical composition in the third aspect and the medicine kit in the tenth aspect in preparation of a medicine for treating an autoimmune disease.
  • FIG. 1 shows that insulin-like growth factor 2 (IGF-2) is effective in treating EAE.
  • IGF-2 insulin-like growth factor 2
  • FIG. 1 a shows that IGF-2 can effectively alleviate the progress and degree of EAE.
  • FIG. 1 b shows that IGF-2 can effectively inhibit mononuclear cell infiltration in the central nervous system.
  • FIG. 1 c shows that IGF-2 can effectively inhibit MOG specific T cell proliferation in EAE mice.
  • FIG. 1 d , 1 e , 1 f , and 1 g show that IGF-2 can effectively inhibit the cell proportion of Th1 and Th17 in EAE mice and promote the proportion of Treg.
  • FIG. 1 h shows that IGF-2 does not directly effect the differentiation of Th1/Th17/Treg.
  • FIG. 1 i and 1 j show that IGF-2 promotes PD-L1 expression and inhibits IL-1 ⁇ expression in the CD11b+F4/80+ macrophage in the spinal cord of EAE mice.
  • FIG. 2 shows that insulin-like growth factor-2 (IGF-2) can induce anti-inflammatory macrophages.
  • IGF-2 insulin-like growth factor-2
  • FIG. 2 a shows that IGF-2 does not affect the number of macrophages in the spinal cord of EAE mice.
  • FIG. 2 b shows that IGF-2 does not affect the number of macrophages in the spleen of EAE mice.
  • FIG. 2 c shows that IGF-2 inhibits the expression level of IL-1 ⁇ in CD11b+F4/80+ macrophages in the spleen of EAE mice.
  • FIG. 2 d shows that IGF-2 promotes the expression level of PD-L1 in CD11b+F4/80+ macrophages in the spinal cord of EAE mice.
  • FIG. 2 e shows that IGF-2 does not affect the expression levels of MHC-I, MHC-II, CD80, and CD86 in thioglycollate medium-induced peritoneal macrophages.
  • FIG. 2 f shows that IGF-2 does not affect the expression levels of TNF- ⁇ , IL-6 and TGF- ⁇ in peritoneal macrophages under lipopolysaccharide (LPS) stimulation and thioglycollate medium-induction.
  • LPS lipopolysaccharide
  • FIG. 2 g shows that IGF-2 inhibits the mRNA levels of inducible nitric oxide synthase (iNOS) and the production of nitrite regulated by the enzyme, in the peritoneal macrophages under lipopolysaccharide (LPS) stimulation and thioglycollate medium-induction.
  • iNOS inducible nitric oxide synthase
  • LPS lipopolysaccharide
  • FIG. 3 shows that insulin-like growth factor-2 (IGF-2) treated macrophages are effective in treating EAE.
  • IGF-2 insulin-like growth factor-2
  • FIG. 3 a shows that after IGF-2 pretreatment, under 100 ng/ml LPS stimulation, the ability of peritoneal macrophages induced by thioglycollate medium to express messenger RNA and precursor proteins of IL-1 ⁇ is significantly decreased.
  • FIG. 3 b shows that after IGF-2 pretreatment, under 100 ng/ml LPS stimulation, the IL-1 ⁇ protein level secreted by peritoneal macrophages induced by thioglycollate medium is significantly decreased.
  • FIG. 3 c shows that after IGF-2 pretreatment, under 100 ng/ml LPS stimulation, the PD-L1 protein level expressed by peritoneal macrophages induced by thioglycollate medium is significantly increased.
  • FIGS. 3 d and 3 e show that IGF-2 treated macrophages are able to promote Treg differentiation depend on PD-L1.
  • FIG. 3 f shows that IGF-2 treated macrophages significantly inhibits EAE.
  • FIG. 3 g shows that IGF-2 treated macrophages can effectively promote the proportion of Tregs in EAE mice.
  • FIG. 4 shows that bone marrow derived macrophages treated with insulin-like growth factor-2 (IGF-2) to promote the differentiation ability of regulatory T cell.
  • IGF-2 insulin-like growth factor-2
  • FIG. 4 a shows that bone marrow derived macrophages treated with IGF-2 express significant higher level of PD-L1 under LPS stimulation.
  • FIGS. 4 b and 4 c show that bone marrow derived macrophages treated with IGF-2 promote Treg through PD-L1.
  • FIG. 4 d shows no change in the expression levels of MHC-I, MHC-II, CD80, and CD86 in bone marrow derived macrophages treated with IGF-2.
  • FIG. 5 shows that bone marrow derived macrophages treated with insulin-like growth factor-2 (IGF-2) are effectively in alleviating experimental autoimmune encephalomyelitis (EAE) and inflammatory bowel disease (IBD).
  • IGF-2 insulin-like growth factor-2
  • FIG. 5 a shows that bone marrow derived macrophages treated with IGF-2 are effective in treating EAE.
  • FIGS. 5 b and 5 c show that bone marrow derived macrophages treated with IGF-2 effectively increase the proportion of Treg in EAE mice.
  • FIG. 5 d shows that bone marrow derived macrophages treated with IGF-2 relieved weight loss in IBD mice. Macrophages treated with IGF-2 shows the ability to effectively treat IBD.
  • FIG. 5 e shows that bone marrow derived macrophages treated with IGF-2 can effectively prolong the survival time of mice with IBD.
  • FIGS. 5 f and 5 g show that bone marrow derived macrophages treated with IGF-2 effectively increase the proportion of Treg cells in spleen in the IBD mice.
  • FIG. 6 shows that insulin-like factor-2 (IGF-2) affects the anti-inflammatory prosperities of macrophages through regulating their glycometabolism.
  • IGF-2 insulin-like factor-2
  • FIG. 6 a shows that glucose consumption was significantly reduced in macrophages after IGF-2 treated.
  • FIG. 6 b shows that lactic acid accumulation was significantly reduced in macrophages after IGF-2 treated.
  • FIG. 6 c shows that NAD+/NADH ratio was significantly reduced in macrophages after IGF-2 treated.
  • FIG. 6 d shows that the ability of oxidative phosphorylation and oxygen consumption was significantly increased in macrophages after IGF-2 treated.
  • FIG. 6 e shows that extracellular acidification was significantly reduced in macrophages after IGF-2 treated.
  • FIG. 6 f shows that macrophages preferred to conduct oxidative phosphorylation after IGF-2 treated.
  • FIG. 6 g shows that oligomycin could inhibit the improvement of PD-L1 expression in peritoneal macrophages treated with IGF-2 and induced by thioglycollate medium.
  • FIG. 6 h shows that oligomycin inhibits oxidative phosphorylation, and demonstrates that macrophages treated with IGF-2 have high expression of PD-L1 depended on aerobic respiration and the ability to promote the production of Treg.
  • FIG. 7 shows that insulin-like growth facor-2 (IGF-2) and IGF-2 treated macrophages are effective in treating experimental bowel disease.
  • IGF-2 insulin-like growth facor-2
  • FIG. 7 a shows that IGF-2 protects IBD mice from body weight loss.
  • FIG. 7 b shows that IBD mice received IGF-2 treatment have significant longer colon length.
  • FIG. 7 c shows that numbers of mononuclear cells infiltration significantly decrease in colon after IGF-2 treatment.
  • FIGS. 7 d and 7 e show that the proportion of Treg cells in the lamina intestinal of colon significantly increase in the IBD mice after IGF-2 treatment.
  • FIG. 7 f shows that the expression of PD-L1 significantly increases in CD11b+F4/80+ macrophages in the lamina intestinal of the IBD mice after IGF-2 treatment.
  • FIG. 7 g shows that the expression level of IL-1 ⁇ significantly decrease in CD11b+F4/80+ macrophages in the lamina intestinal of the IBD mice after IGF-2 treatment.
  • FIG. 7 h shows that IGF-2 treated macrophage relieves the body weight loss of experimental bowel disease mice.
  • FIG. 7 i shows that IGF-2 treated macrophage effectively prolongs the survival time of the IBD mice.
  • FIG. 7 j shows that the proportion of Treg cells in spleen significantly increase in the IBD mice injected with IGF-2 treated macrophage.
  • FIG. 8 shows that insulin-like growth factor-2 (IGF-2) regulates immune response in the spleen and peritoneal lymph nodes of inflammatory bowel disease (IBD) mice.
  • IGF-2 insulin-like growth factor-2
  • FIGS. 8 a and 8 b show that IGF-2 significantly up regulates the proportion of Tregs in the peritoneal lymph nodes of mice with IBD.
  • FIG. 8 c shows that IGF-2 does not affect the proportion of spleen macrophages in mice with IBD.
  • FIG. 8 d shows that IGF-2 upregulates the proportion of CD11b+F4/80+ macrophages in the colon lamina intestinal of mice with IBD.
  • FIG. 8 e shows that IGF-2 significantly downregulates the expression level of IL-1 ⁇ in spleen macrophages in mice with IBD.
  • FIG. 8 f shows that IGF-2 significantly up regulates the expression level of PD-L1 in spleen macrophages in mice with IBD.
  • FIG. 9 shows that peritoneal macrophages treated with insulin-like growth factor-2 (IGF-2) and induced by thioglycollate medium can upregulate Tregs in lamina intestinal of colon and peritoneal lymph nodes of IBD mice.
  • IGF-2 insulin-like growth factor-2
  • FIG. 9 a shows that IGF-2 treated macrophages can significantly increase the proportion of Tregs in peritoneal lymph nodes of IBD mice.
  • FIG. 9 b shows that IGF-2 pre-treated macrophages can significantly increase the proportion of Tregs in lamina intestinal of colon of IBD mice.
  • FIG. 10 shows that separate injection of insulin-like growth factor-2 (IGF-2) and clodronate liposome can significantly inhibit the progress of EAE and play an effective therapeutic role when compared with that of control mice group. Importantly, combined injection of IGF-2 and clodronate liposome has significantly better therapeutic effects on EAE than other groups.
  • IGF-2 insulin-like growth factor-2
  • clodronate liposome has significantly better therapeutic effects on EAE than other groups.
  • FIG. 11 shows the tertiary structure of insulin-like growth factor-2 (IGF-2).
  • FIG. 12 shows the key binding sites of insulin-like growth factor-2 (IGF-2) to the receptor.
  • IGF-2 insulin-like growth factor-2
  • FIG. 13A shows the results of glucose tolerance test.
  • FIG. 13B shows the results of insulin tolerance test.
  • IGF-2 applied in the reference and description of FIGS. 1-13 is active fragment of IGF-2 containing amino acids from position 25 to position 91. (IGF 25-91 )
  • the inventors After extensive and thorough studies, the inventors have firstly established a pharmaceutical composition containing insulin-like growth factor-2 and use thereof.
  • This invention provides the use of IGF-2 and its active fragment for preparing a pharmaceutical composition.
  • the pharmaceutical composition can be used for (i) enhancing PD-L1 expression and/or (ii) inhibiting IL-1 ⁇ expression in macrophages.
  • the invention also provides a pharmaceutical composition containing insulin-like growth factor-2 as an active ingredient.
  • IGF-2 insulin-like growth factor-2
  • IGF 25-91 insulin-like growth factor-2
  • IGF 25-91 insulin-like growth factor-2
  • IGF 25-91 insulin-like growth factor-2
  • IGF 25-91 insulin-like growth factor-2
  • IGF 25-91 the active fragment having amino acids from position 25 to position 91 of IGF-2
  • IGF 25-91 indirectly upregulates regulatory T cells (Treg) by inducing macrophages with low IL-1 expression and high PD-L1 expression, thereby promoting the recovery of autoimmune diseases.
  • IGF 25-91 mainly promotes the reprogramming of macrophages into macrophages with immunosuppressive function by affecting the aerobic glycolysis and oxidative phosphorylation pathways of macrophages, and such macrophages treated with IGF 25-91 have a very significant effect on the treatment of autoimmune diseases.
  • the present invention clarifies the regulatory mechanism of IGF 25-91 on macrophages, and it is found that IGF 25-91 induces the application potential of macrophages with immunosuppressive function in the treatment of autoimmune diseases, thus forming a new technique for macrophage immunotherapy.
  • IGF-2 Insulin-like growth factor-2
  • IGF-2 precursor protein which is translated from mRNA, consists of 180 amino acids, as shown in the sequence SEQ ID NO.: 1 (mgipmgksmlvlltflafascciaayrpset lcggelvdtlqfvcgdrgfyfsrpasrvsrrsrgiveeccfrscdlalletycatpakserdvstpptvlpdnfprypvgkffqydtwkqst qrlrrglpallrarrghvlakeleafreakrhrplialptqdpahggappemasnrk);
  • the active form produced after modification after translation consists of 67 amino acids, as shown in the sequence SEQ ID NO.: 1 (mgipmgksmlvlltflafascciaayrpset lcggelvdtlqfv
  • the tertiary structure of IGF-2 is composed of three ⁇ -helixes and two ⁇ -sheets as shown in FIG. 11 , wherein amino acids 11G-21C forms a helix, 25G-27Y forms the first sheet, the second helix is 42I-49R, the third helix is 53L-58T and the second sheet is 59Y-61A. It is predicted that T16 and F19 on the first helix and L63 on the third helix are sites where IGF-2 binds to receptor of IGF-2 as shown in FIG. 12 .
  • IGF-2 has an important role in embryo development and can promote embryo development and organ formation. It has also been reported to be linked to memory and reproduction, and studies in genetically deficient mice have shown that lack of insulin-like growth factor-2 signaling can lead to poor brain development.
  • the present invention has found that administration of IGF-2 or active fragment having the amino acids sequence from position 25 to position 91 of IGF-2 (IGF 25-91 ) alone can effectively treat experimental autoimmune encephalomyelitis or inflammatory bowel disease, which are two autoimmune diseases.
  • IGF 25-91 IGF 25-91
  • the proportion of regulatory T cells in the IGF 25-91 treatment group was significantly increased, and the proportions of TH1 and TH17 cells were significantly decreased.
  • IGF 25-91 could not directly affect the efficiency of T cell differentiation into Th1, Th17 and Treg.
  • IGF 25-91 has an indirect effect on the proportion change 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. It was found in the study that the expression of IL-1 ⁇ in macrophages at the injury site of mice treated with IGF 25-91 was significantly decreased, while the expression of PD-L1 was significantly increased, suggesting that IGF 25-91 played a role in inhibiting autoimmune diseases through promoting the proportion of macrophages with anti-inflammatory effects.
  • IGF 25-91 does not directly induce the expression of anti-inflammatory genes in macrophages.
  • the bone marrow or enterocoelia derived mature macrophages treated with IGF 25-91 exhibit significantly decrease of IL-1 ⁇ expression and significantly increase of PD-L1 expression in response to lipopolysaccharide stimulation.
  • Macrophages play an important role in the occurrence and development of autoimmune diseases.
  • a series of studies have shown that macrophages can exert strong proinflammatory ability under the stimulation of inflammatory factors such as interferon and lipopolysaccharide, which aggravate the process of autoimmune diseases.
  • 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 the condition of proinflammatory stimulation. Therefore, the treatment of autoimmune diseases by IGF 25-91 is likely to be achieved by reprogramming macrophages.
  • macrophages induced by IGF 25-91 were co-cultured with T cells after LPS pre-stimulation. It was found that macrophages treated with IGF 25-91 could significantly promote the differentiation of Treg, and the promotion depended on the high expression of PD-L1. Further, the studies showed that macrophages treated with IGF 25-91 could also be directly used to treat autoreactive encephalomyelitis and inflammatory bowel disease. During the treatment process, the infusion of these macrophages also promoted the increase of Treg proportion in mice.
  • IGF 25-91 affects the expression of PD-L1 in macrophages
  • control cells a detailed comparison was made between macrophages treated with IGF 25-91 and control cells. It was found that macrophages treated with IGF 25-91 can significantly change the status of glucose metabolism pathway of macrophages, making them more apt to oxidative phosphorylation metabolic pathway. When blocking macrophages mitochondrial oxidative phosphorylation, the high expression of PD-L1 and the ability to promote Treg differentiation induced by insulin-like growth factor ⁇ 2 in macrophages also disappeared. These studies illustrate that IGF 25-91 endows macrophages with the ability of promoting Treg generation and treating autoimmune diseases by changing the metabolism tendencies of macrophages.
  • IGF 25-91 plays a therapeutic role in multiple sclerosis and inflammatory bowel disease by inducing immunosuppressive macrophages and up-regulating regulatory T cells. Macrophages treated with IGF 25-91 may play a therapeutic role in autoimmune diseases. In addition, the targeted regulation of aerobic glycolysis and oxidative phosphorylation in macrophages can induce the production of immunosuppressant macrophages and play a role in the treatment of various autoimmune diseases.
  • This invention provides a pharmaceutical composition for (i) promoting PD-L1 expression and/or (ii) inhibiting IL-1 ⁇ expression in macrophages. It contains a safe and effective dose of IGF-2 (or its active fragments) or contains IGF-2 treated macrophages.
  • the pharmaceutical composition of the invention can be used for (i) promoting PD-L1 expression and/or (ii) inhibiting IL-1 ⁇ expression in macrophages.
  • the pharmaceutical composition can be used together with other autoimmune diseases therapies.
  • the pharmaceutical composition of the invention can also contain macrophage scavenger which is an agent for removing macrophages or inhibiting macrophage migration.
  • macrophage scavenger which is an agent for removing macrophages or inhibiting macrophage migration.
  • the invention also provides a medicine kit comprising:
  • a first container which contains an active ingredient (i) IGF-2 or a medicine containing the active ingredient (a);
  • a second container which contains an active ingredients
  • macrophage scavenger or a medicine containing the active ingredient (b), wherein the macrophage scavenger is agent for removing macrophage or inhibiting macrophage migration
  • the pharmaceutical composition of the invention contains a safe and effective dose of IGF-2 and a pharmaceutically acceptable carrier.
  • the carrier includes (not limited to): saline, buffer solution, glucose, water, glycerin, alcohol, powder and the combination thereof.
  • the formulation of the medicine should be compatible with the method of administration.
  • the pharmaceutical composition of the invention can be prepared in the form of an injection, for example, by a conventional method using saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules may be prepared by conventional methods.
  • Pharmaceutical composition such as injection, solution, tablet and capsule should be manufactured under sterile conditions.
  • the pharmaceutical composition of the invention can also be made into powder for atomizing inhalation.
  • the dose of the active ingredient is an effective dose, such as about 1 ⁇ g/kg-50 mg/kg, about 5 ⁇ g/kg-10 mg/kg, about 10 ⁇ g/kg-5 mg/kg of body weight per day.
  • the pharmaceutical composition can be administered together with other therapies.
  • the pharmaceutical composition of the invention can be administered to a subject (such as human and non-human mammal) in routine manner.
  • a subject such as human and non-human mammal
  • the representative way for administration includes (but not limit to): oral administration, injection and aerosol inhalation.
  • the pharmaceutical composition should be administered to a mammal in a safe and effective dose.
  • the safe and effective dose is usually at least 10 ⁇ g/kg of body weight and no more than 50 mg/kg of body weight under most conditions.
  • the preferred dose should range from 10 ⁇ g/kg to 20 mg/kg of body weight.
  • the exact dose should be determined according to the administration route and the patient health conditions which are all within the expertise of a skilled physician.
  • IGF-2 and the active fragment thereof can (i) promote PD-L1 expression and/or (ii) inhibit IL-1 ⁇ expression in macrophages.
  • Macrophages treated with IGF-2 or the active fragment thereof can promote Treg differentiation and inhibit Th1 cell and/or Th17 cell differentiation so as to treat autoimmune diseases.
  • Combination therapy of IGF-2 or active fragment thereof with macrophage scavenger can be used for treating autoimmune diseases.
  • Emulsification of antigen Two glass syringes 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 into the syringes, and the bubbles in the syringes were removed and the syringes were pushed back and forth to obtain an emulsion. After about 500 pushes, the push resistance would gradually increase indicating the ingredients were fully mixed into an emulsified state.
  • pertussis toxin PT solution: pertussis toxin was dissolve in PBS at a working concentration of 1 ng/ ⁇ L and 200 ⁇ L per mouse was injected through the tail vein.
  • insulin-like growth factor 2 was used for treatment via intraperitoneal injection at a dose of 5 ng/mouse everyday.
  • 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. Centrifugation was carried out at 2000 rpm for 20 minutes at room temperature under zero acceleration. After centrifugation, cells were aspirated from the liquid junction layer of high-low density and washed twice with PBS to obtain lymphocytes.
  • mice After the EAE mice were sacrificed, the spleen of the mice was removed and a single cell suspension was obtained.
  • the splenocytes were plated in a U-bottom 96-well plate at a number of 3-5 ⁇ 10 5 per well, and MOG was added to 20 ug/ml.
  • H3 labeled thymidine was added. After 6 hours, the well plate was freeze-thawed repeatedly and then vacuum-adsorbed onto a special filter. The scintillation solution was added and then detected on the machine.
  • Dextran sodium sulfate was formulated into a solution at a mass to volume ratio of 2.5:100, filtered with a 22 ⁇ m strainer to ensure sterility, and sealed with a parafilm.
  • insulin-like growth factor-2 was injected intraperitoneally at a dose of 50 ng/mouse everyday.
  • the saline treated group served as a positive control.
  • the colon was removed from the IBD mice, the feces were removed 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 conditions of 250 rpm for 20 min at 37° C. The washed colon tissue was cut as much as possible with scissors and added into HBSS containing DNAase I, dispase and type VIII collegenase under the conditions of 250 rpm for 30 min at 37° C. 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. Centrifugation was carried out at 2000 rpm for 20 minutes at room temperature under zero acceleration. After centrifugation, cells were aspirated from the liquid junction layer of high-low density and washed twice with PBS to obtain lymphocytes.
  • Cells were implanted on the hippocampal test plate at a density of 1 ⁇ 10 5 cells per well.
  • XF culture was supplemented with 10 mM glucose, 2 mM glutamine and 2 mM pyruvate to prepare a test medium.
  • the cells were washed twice with the test medium before testing on the machine.
  • Metabolic inhibitors were prepared using test medium: 1 ⁇ M oligomycin, 0.75 ⁇ M FCCP, 100 ⁇ M rotenone and 1 ⁇ M Mantimycin.
  • the consumption rate of oxygen and the concentration of extracellular hydrogen ion at basic status and under drug stimulation conditions were measured.
  • the instrument used was Extracellular flux analyzer.
  • the preparation process of bone marrow derived macrophages was as follows: mouse bone marrow was blown out with 1 ml syringe. After being full dispersed, the cells were centrifuged according to the condition of 400 g for 5 min. The supernatant was discarded, the cells were blown into single cell suspension, and the cells were laid on an uncoated culture dish in DMEM/F12 completely medium (containing 10% FBS, 20% L929 supernatant, 100 U/ml Penicillin, 100 U/ml Streptomycin, 2 mm L—Glutamine). At the fourth day of culture, 5 ml solution was added, and the cells were further cultured for 6 days, and mature macrophages could be obtained. IGF-2 stimulation was added on Day 1, 3 and 5, respectively.
  • peritoneal derived macrophages The preparation process of peritoneal derived macrophages was as follows: 2 ml of 4% thioglycollate solution was intraperitoneally injected into 8-12 weeks aged C57BL/6 mice, cytokines or PBS were intraperitoneally injected daily every day. On the third day, mice were sacrificed. Macrophages in abdominal cavity were blown out by using a 10 ml syringe with 10 ml volume of PBS. After centrifugation, the cells were scattered to form a single cell suspension, and were then inoculated in an uncoated cell dish or a 6-well plate, for following experiments.
  • Macrophages derived from mature bone marrow or peritoneal were stimulated with LPS for 24 hours.
  • the stimulation solution was aspirated and cells were washed with PBS for three times to fully remove the residual LPS.
  • the macrophages (2.5 ⁇ 10 5 ) and selected CD4 + CD62L + T cells (1 ⁇ 10 6 ) were laid on a 96-well plate. After 72 hours of co-culture, the cells were blown out for flow cytometry staining and analysis.
  • mice purchased from slake Shanghai experimental animal center of Chinese academy of sciences
  • a high-fat diet for 20 weeks, starting from the 5th week.
  • mice were starved overnight, and 1 g glucose/kg body weight was intraperitoneally injected. At different time points, a drop of tail venous blood of each mouse was siphoned into the blood glucose test paper, and blood glucose was measured with a blood glucose meter.
  • mice were starved overnight for about 16 hours, and 0.75U insulin/kg body weight was intraperitoneally injected.
  • a drop of tail venous blood of each mouse was siphoned into the blood glucose test paper, and blood glucose was measured with a blood glucose meter, and accurate timing was conducted with a timer.
  • IGF 25-91 When treated with IGF 25-91 in mice induced EAE, it was observed that IGF 25-91 effectively alleviated the pathogenesis and severity of EAE, which was manifested by decreased clinical score, decreased number of infiltrating mononuclear cells in the spinal cord, and decreased proliferation of MOG-specific T cells in EAE mice ( FIGS. 1 a - c ). Meanwhile, flow cytometry analysis showed that the use of IGF 25-91 could effectively reduce the proportions of Th1 and Th17 in the spinal cord of EAE mice and increase the proportion of Treg ( FIGS. 1 d - g ).
  • IGF 25-91 can be used in the treatment of EAE and can influence the ratio of different T cell subsets and the gene expression of macrophages in diseased mice.
  • Macrophages Treated with IGF 25-91 can Promote the Production of Treg and Treat EAE
  • the treated macrophages (including bone marrow derived macrophages and peritoneal derived macrophages) were used in the treatment of EAE.
  • the results showed that macrophages pretreated with IGF 25-91 could effectively alleviate the clinical scores of EAE, and promote the proportion of Treg in the spleen of EAE mice ( FIGS. 3 f - g ; FIGS. 5 a - c ).
  • IGF 25-91 When tracking the oxygen consumption and acid production in macrophages, it was found that IGF 25-91 could effectively improve the efficiency of the oxygen consumption in macrophages, and at the same time reduce the acid production in macrophages in the background state ( FIGS. 6 d - f ).This suggests that IGF 25-91 can tilt macrophage metabolism toward the oxidative phosphorylation pathway.
  • IGF 25-91 and IGF 25-91 Pre-Treated Macrophages are Effective in Treating Inflammatory Bowel Diseases (IBD).
  • IGF 25-91 was used to treat IBD, an autoimmune disease dominated by the innate immune system.
  • the weight loss of IBD mice was alleviated, colon injury and infiltration of monocytes in the colon were significantly reduced ( FIGS. 7 a - c ).
  • Flow cytometry was used to analyze T cell populations and macrophage gene expression, and it was found that IGF 25-91 could increase Treg percentages in the colon and mesenteric lymph nodes ( FIGS. 7 d - e ; FIGS. 8 a - b ).
  • IGF 25-91 can significantly inhibit inflammatory diseases, such as the above-mentioned multiple sclerosis and inflammatory bowel diseases. Moreover, this effect is closely related to the IGF 25-91 regulation on macrophages which are reprogrammed into macrophages with immunosuppressive function, thus inhibiting over-strong inflammatory response.
  • inflammatory diseases such as the above-mentioned multiple sclerosis and inflammatory bowel diseases.
  • this effect is closely related to the IGF 25-91 regulation on macrophages which are reprogrammed into macrophages with immunosuppressive function, thus inhibiting over-strong inflammatory response.
  • systemic and local inflammation is often accompanied by a large amount of proinflammatory macrophage infiltration, and the macrophage infiltration in inflammatory site often depends on an influx of peripheral monocytes and its evolution into proinflammatory macrophages. Therefore, we investigated whether a combination of clearance of resident proinflammatory macrophages and the reprogramming function of IGF 25-91 on macrophages can optimize the treatment
  • mice in different experimental groups received IGF 25-91 , Clodronate liposome (for removing macrophage) or a combined injection of IGF 25-91 and Clodronate liposome.
  • the progress of EAE in mice was observed. It was found that single injection of IGF 25-91 and Clodronate liposome could significantly inhibit the progress of EAE and had an effective therapeutic effect compared with the control group. More importantly, a combined injection of IGF 25-91 and Clodronate liposome had a more significant therapeutic effect on EAE compared with other groups ( FIG. 10 ). Therefore, the combination therapy of IGF 25-91 and macrophage scavenger has a more optimized effect in the treatment of inflammatory diseases by reprogramming cells into macrophages with immunosuppressive function.
  • IGF 25-91 Pre-Treated Macrophages can be Used to Treat Obesity-Induced Insulin Resistance.
  • mice were fed with high fat diet for 20 weeks and then injected with PBS, macrophages (5 ⁇ 10 6 ) or IGF 25-91 pre-treated macrophages (5 ⁇ 10 6 ). Two weeks after the treatment, glucose tolerance test and insulin tolerance test were conducted, and glucose changes in each group were detected. The results showed that IGF 25-91 pre-treated macrophages could significant improve the ability of glucose tolerance in the insulin resistance mice ( FIGS. 13A and 13B ). These results demonstrated that IGF 25-91 pre-treated macrophages were effective in treating insulin resistance and diabetes.

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