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WO2015025956A1 - Composition pharmaceutique pour le traitement des lésions du myocarde, composition pharmaceutique pour la prévention des lésions du myocarde, composition pharmaceutique pour le traitement de l'insuffisance cardiaque, composition pharmaceutique pour la prévention de l'insuffisance cardiaque, procédé de traitement ou de prévention des lésions du myocarde ou de l'insuffisance cardiaque, mfg-e8, utilisations de mfg-e8 et procédé de criblage de composés permettant de traiter ou de prévenir les lésions du myocarde ou l'insuffisance cardiaque - Google Patents

Composition pharmaceutique pour le traitement des lésions du myocarde, composition pharmaceutique pour la prévention des lésions du myocarde, composition pharmaceutique pour le traitement de l'insuffisance cardiaque, composition pharmaceutique pour la prévention de l'insuffisance cardiaque, procédé de traitement ou de prévention des lésions du myocarde ou de l'insuffisance cardiaque, mfg-e8, utilisations de mfg-e8 et procédé de criblage de composés permettant de traiter ou de prévenir les lésions du myocarde ou l'insuffisance cardiaque Download PDF

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WO2015025956A1
WO2015025956A1 PCT/JP2014/072028 JP2014072028W WO2015025956A1 WO 2015025956 A1 WO2015025956 A1 WO 2015025956A1 JP 2014072028 W JP2014072028 W JP 2014072028W WO 2015025956 A1 WO2015025956 A1 WO 2015025956A1
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mfg
myocardial infarction
heart failure
cells
treating
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PCT/JP2014/072028
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English (en)
Japanese (ja)
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道雄 仲矢
等 黒瀬
雅彦 黒田
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国立大学法人九州大学
学校法人東京医科大学
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Publication of WO2015025956A1 publication Critical patent/WO2015025956A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure

Definitions

  • the present invention relates to a pharmaceutical composition for treating or preventing myocardial injury or heart failure, a method for treating or preventing myocardial injury or heart failure, MFG-E8, MFG for the manufacture of a medicament for treating or preventing myocardial injury or heart failure.
  • MFG-E8 MFG for the manufacture of a medicament for treating or preventing myocardial injury or heart failure.
  • the heart contributes to blood circulation throughout the body by constantly contracting and expanding. In addition, the heart supplies itself with blood through the coronary arteries.
  • Myocardial infarction is a state in which blood is not supplied downstream because the coronary artery is occluded by arteriosclerosis or the like. Lifestyle-related diseases such as diabetes and hyperlipidemia are known as risk factors for myocardial infarction, and the number of patients is expected to increase with an aging society in the future.
  • the first treatment of myocardial infarction is reopening of the coronary artery by surgery, and at the same time, a method of removing the thrombus caused by the thrombolytic agent is used. Patients will continue to take antiplatelet drugs to prevent reocclusion.
  • an inflammatory reaction is first caused with myocardial necrosis.
  • events such as leukocyte migration, extracellular matrix protein degradation, and dead cell removal occur.
  • the period from the onset of myocardial infarction to about the fourth day is regarded as the inflammatory stage.
  • the repair reaction is promoted as the inflammatory response is attenuated.
  • events such as fibroblast development, tissue fibrosis, and angiogenesis occur.
  • the repair period is about 2 weeks after the onset of myocardial infarction in humans.
  • Non-Patent Document 2 Cardiac rupture is thought to occur frequently 1 to 4 days after the onset of myocardial infarction, and there are many reports of relationships with inflammatory and repair responses, so understanding of inflammatory and repair responses after myocardial infarction is important. It is considered important in treatment (see Non-Patent Document 2).
  • TSP-1 Thrombopondin1
  • TSP-1 Thrombopondin1
  • Periostin produced from the early stage to the late stage after myocardial infarction has a delayed development of myofibroblasts after infarction and a high mortality in the deficient mice compared to wild-type mice (see Non-Patent Documents 5 and 6).
  • Periostin contributes to the repair reaction via myofibroblasts.
  • various proteins involved in inflammation and repair are produced after myocardial infarction, but how each protein is involved in inflammation or the response to which protein is fibrosis or angiogenesis There is still insufficient understanding as to whether this is causing a repair response.
  • Milk fat global epidermal growth factor 8 (hereinafter referred to as “MFG-E8”) is a secreted protein that is produced by macrophages of several tissues (see Non-Patent Document 7). Recently, it has been reported that MFG-E8 is an important molecule that promotes phagocytosis of apoptotic cells by macrophages (see Non-Patent Document 8). It is known that apoptotic cells expose phosphatidylserine (PS) on their surface (see Non-Patent Documents 9 and 10). MFG-E8 is considered to promote phagocytosis by binding this PS to integrin ⁇ v ⁇ 3 or ⁇ v ⁇ 5 present on the surface of phagocytic cells and bridging them (see Non-Patent Document 8).
  • PS phosphatidylserine
  • Apoptotic cells are normally phagocytically removed. However, if it remains without being removed for some reason, the membrane structure that has been maintained until then collapses and shifts to a state called late necrosis. In late-stage necrotic cells, various factors present in the cells are released to the outside of the cells to cause inflammation. From these, antibodies against self are produced (see Non-Patent Documents 9 to 11). Very recently, not only immunocompetent cells such as macrophages and microglia, but also cells that were previously thought to have no phagocytic ability, are responsible for the phagocytosis of dead cells under certain circumstances. It has become clear that it contributes to maintaining homeostasis.
  • Non-Patent Document 12 it has been reported that Sertoli cells that exist as a support for sperm cells in the testis phagocytose sperm cells that have undergone apoptosis. Inhibition of phagocytosis of dead cells by Sertoli cells was observed to cause abnormal spermatogenesis, and phagocytosis by Sertoli cells was considered important in the testis. It has also been reported that phagocytosis of apoptotic neuron progenitor cells by neuronal progenitor cells is involved in the progression of neurogenesis in the brain (see Non-Patent Document 13).
  • the present invention has been made in view of the above circumstances, and is a pharmaceutical composition for treating or preventing myocardial injury or heart failure, a method for treating or preventing myocardial injury or heart failure, MFG-E8, treating or preventing myocardial injury or heart failure. It is an object of the present invention to provide a method for screening a compound for treating or preventing myocardial injury or heart failure, and the use of MFG-E8 for the manufacture of a medicament for prevention.
  • MFG-E8 which has been reported to be involved in the mechanism of removing phagocytosis of apoptotic cells, and involved the involvement of MFG-E8 in phagocytosis of dead cells during myocardial infarction.
  • MFG-E8 expressed by resident fibroblast-derived myofibroblasts is involved in phagocytosis of dead cells. I found it.
  • administration of MFG-E8 contributes to the improvement of cardiac function after myocardial infarction, and the present invention has been completed.
  • the pharmaceutical composition for treating or preventing myocardial injury or heart failure according to the present invention, the method for treating or preventing myocardial injury or heart failure, MFG-E8, and the manufacture of a medicament for treating or preventing myocardial injury or heart failure
  • the method of screening for a compound for treating or preventing myocardial injury or heart failure and the use of MFG-E8 are [1] to [11] below.
  • a pharmaceutical composition for preventing myocardial injury, comprising MFG-E8 as an active ingredient [3] A pharmaceutical composition for treating heart failure, comprising MFG-E8 as an active ingredient.
  • a pharmaceutical composition for preventing heart failure comprising MFG-E8 as an active ingredient.
  • a method for treating or preventing myocardial injury or heart failure comprising a step of administering MFG-E8.
  • MFG-E8 for use as a therapeutic or prophylactic agent for myocardial injury or heart failure.
  • a method for screening a compound for preventing myocardial injury or heart failure using MFG-E8 expression induction as an index is provided.
  • [10] A method for screening a compound for treating myocardial injury or heart failure using the MFG-E8 bioassay system.
  • [11] A method for screening a compound for preventing myocardial injury or heart failure using the MFG-E8 bioassay system.
  • MFG-E8 can be used to effectively treat or prevent myocardial injury such as myocardial infarction or heart failure caused by myocardial infarction.
  • Experimental Example 1 it is the figure which showed the expression level of MFG-E8 protein computed from the result of the Western blot.
  • Experimental Example 2 it is the fluorescence-staining photograph figure which examined the expression of MFG-E8 in an immune cell in a mouse
  • Experimental Example 2 it is the fluorescence-staining photograph figure which confirmed the expression of MFG-E8 in the fibroblast (vimentin positive) in a mouse
  • Experimental Example 2 it is the fluorescence-staining photograph figure which confirmed the expression of MFG-E8 in the myofibroblast in a mouse
  • FIG. 4 it is a photograph figure which shows the fluorescence staining result of MFG-E8 in the infarct area
  • Experimental Example 5 it is a photograph figure which shows the fluorescence dyeing
  • Experimental Example 6 it is a figure which shows the result of having measured the expression level of MFG-E8 etc. after TGF- ⁇ 1 stimulation over time in rat fibroblasts.
  • Experimental Example 6 it is a figure which shows the result of having measured the expression level of MFG-E8 etc. when TGF- ⁇ 1 and CCG1423 are used in rat fibroblasts.
  • Experimental Example 6 it is a figure which shows the result of having measured the expression level of MFG-E8 etc. when TGF- ⁇ 2 and siSRF are used in a human cell line.
  • Experimental Example 7 it is a figure which shows the result of having measured the expression level of MFG-E8 etc. in a mouse
  • Experimental Example 7 it is a figure which shows the result of having measured the expression level of MFG-E8 etc. in a mouse
  • Experimental Example 8 it is a figure which shows the result of having confirmed the peripheral blood component after bone marrow transplantation.
  • Experimental Example 8 it is a figure which shows the result of having examined the expression of MFG-E8 in the bone marrow origin myofibroblast by fluorescent staining.
  • Experimental Example 8 it is a figure which shows the result of having confirmed the peripheral blood component after bone marrow transplantation.
  • Experimental Example 8 it is a figure which shows the result of having examined the expression of MFG-E8 in the bone marrow origin myofibroblast by fluorescent staining.
  • Experimental Example 8 it is a figure which shows the result of having examined the expression of MFG-E8 in a FSP-1 (S100A4) positive myofibroblast by fluorescent staining.
  • Experimental example 9 it is a figure which shows the 28-day survival rate in the wild type mouse
  • Experimental example 10 it is a figure which shows the HE dyeing
  • FIG. 10 is a graph showing the thickness of the left ventricular wall after myocardial infarction in MFG-E8-deficient mice in Experimental Example 10.
  • Experimental example 10 it is a figure which shows the picrosirius red dyeing
  • Experimental Example 10 it is a figure which shows the amount of collagen deposition in the heart tissue after myocardial infarction in an MFG-E8 deficient mouse.
  • Experimental Example 11 it is a figure which shows the fluorescence dyeing
  • Experimental Example 11 it is a figure which shows the amount of myofibroblasts of the heart tissue after myocardial infarction in the MFG-E8 deficient mouse.
  • Experimental example 12 it is a figure which shows the echocardiographic evaluation result of the 3rd day after myocardial infarction operation.
  • Experimental Example 12 it is a figure which shows the echocardiographic evaluation result of the 28th day after myocardial infarction operation.
  • Experimental example 12 it is a figure which shows the cardiac hemodynamic evaluation result on the 3rd day after myocardial infarction operation.
  • Experimental Example 12 it is a figure which shows the cardiac hemodynamic evaluation result of the 28th day after myocardial infarction operation.
  • Experimental example 12 it is a figure which shows the organ weight and the body weight measurement result on the 3rd day after myocardial infarction operation.
  • Experimental example 12 it is a figure which shows the organ weight and the body weight measurement result on the 28th day after myocardial infarction operation.
  • Experimental example 13 it is the figure which carried out the fluorescence observation of the apoptotic cell taken in into the myofibroblast of a wild type mouse after myocardial infarction.
  • fluorescence observation was performed on apoptotic cells incorporated into myofibroblasts of MFG-E8-deficient mice after myocardial infarction.
  • Experimental Example 13 it is a figure which shows the relationship examination result of recombinant MFG-E8 addition amount and myofibroblast phagocytic ability.
  • Experimental example 14 it is a figure which shows the fraction of a macrophage or a non-white blood cell.
  • it is the result of having confirmed the integrin alpha v expression in a macrophage or a non-white blood cell.
  • it is the fluorescence-staining photograph figure which confirmed the integrin (beta) 5 expression in a macrophage.
  • experimental example 14 it is the result of confirming integrin ⁇ v ⁇ 3 expression in the CD11b positive fraction.
  • mice 14 it is the result of having confirmed the integrin ⁇ v ⁇ 3 expression in the CD11b negative fraction. In Experimental example 14, it is the result of confirming integrin ⁇ v ⁇ 3 expression in the F4 / 80 positive fraction. In Experimental example 14, it is the result of confirming integrin ⁇ v ⁇ 3 expression in the ⁇ SMA positive fraction.
  • the ratio of macrophages was confirmed in wild-type mice or MFG-E8-deficient mice after myocardial infarction. In Experimental Example 15, the ratio of leukocytes, granulocytes, monocytes or macrophages was confirmed in wild-type mice or MFG-E8-deficient mice after myocardial infarction.
  • mice In Experimental Example 15, the ratios of macrophages and neutrophils were confirmed in wild-type mice or MFG-E8-deficient mice after myocardial infarction.
  • Experimental Example 16 it is a figure which shows the result of having investigated the expression change of the inflammation related factor after a myocardial infarction treatment, and the fibrosis related factor in a wild type mouse
  • Experimental example 17 it is a figure which shows the IL-6 production amount change of a myofibroblast by an apoptotic cell phagocytosis.
  • Experimental Example 18 MFG-E8 positive myofibroblasts were confirmed in the infarct region of a human myocardial infarction patient.
  • Experimental Example 19 it is a figure which shows the administration site
  • Experimental Example 19 it is a figure which shows the infarct area
  • the pharmaceutical composition for treating myocardial injury the pharmaceutical composition for preventing myocardial injury
  • the pharmaceutical composition for treating heart failure the pharmaceutical composition for preventing heart failure
  • treatment of myocardial injury or heart failure sometimes referred to as “preventive pharmaceutical composition”.
  • the pharmaceutical composition for treating or preventing myocardial injury or heart failure of the present invention contains MFG-E8 as an active ingredient. That is, the pharmaceutical composition of the present invention containing MFG-E8 as an active ingredient can be administered orally or parenterally (eg, nasal, pulmonary, intestinal, transdermal, subcutaneous, intravenous, intramuscular). Or myocardial injury or heart failure can be treated or prevented by administration to animals other than humans.
  • the amount of MFG-E8 contained in the pharmaceutical composition as an active ingredient is not particularly limited as long as it is an effective amount capable of producing an effect, and the target disease type and condition; target animal species and their age , Sex, body weight; can be determined appropriately according to the dosage form described below. For example, in the range of 0.001 to 10 mg / kg, preferably 0.05 to 1 mg / kg, more preferably 0.05 to 0.5 mg / kg, this is divided into once or several times a day. Can be appropriately determined according to the use form and dosage form.
  • a therapeutic effect can be achieved.
  • a prophylactic effect can be produced by using the pharmaceutical composition of the present invention for a subject who has not developed myocardial injury or heart failure, particularly a subject who has a risk of myocardial injury or heart failure.
  • the pharmaceutical composition of the present invention can be used after preparing an appropriate dosage form according to the administration route.
  • Specific examples of the dosage form include injections, transdermal preparations, suppositories, powders, tablets, capsules, granules, capsules, patches, ointments, haptics, aerosols, and the like.
  • the pharmaceutical composition of the present invention can contain MFG-E8, which is an active ingredient, and other additives and drugs.
  • the other additive include one or more known and commonly used excipients, binders, disintegrants, lubricants and the like.
  • the other drug is not particularly limited as long as it does not inhibit the action of the active ingredient of the present invention.
  • the method for treating or preventing myocardial injury or heart failure of the present invention comprises the step of administering MFG-E8.
  • the subject to which MFG-E8 is administered is not particularly limited as long as it is a subject requiring treatment or prevention. Specifically, humans or non-human animals can be mentioned.
  • the timing of administration of MFG-E8 is not particularly limited, and if it is intended for therapeutic effect, it is preferably performed after the onset of myocardial injury or heart failure, with the aim of preventing effect. If so, it is preferably performed at any time before the onset of myocardial injury or heart failure.
  • MFG-E8 contributes to the recovery of cardiac function by contributing to phagocytosis of dead cells after myocardial infarction, and therefore, as a treatment after myocardial injury such as myocardial infarction, and prevention of heart failure related to myocardial infarction. It is preferably used after the onset of myocardial infarction.
  • the method for administering MFG-E8 is not particularly limited and may be oral or parenteral (for example, nasal, pulmonary, enteral, transdermal, subcutaneous, intravenous) , Intramuscular).
  • MFG-E8 may be administered alone or in any dosage form together with other additives and drugs.
  • the additive, drug, and dosage form include those described above.
  • the dose of MFG-E8 is not particularly limited as long as it is an effective amount capable of exerting an effect.
  • the MFG-E8 of the present invention is for use as a therapeutic or prophylactic agent for myocardial injury or heart failure.
  • the method of using (administering) MFG-E8 is the same as in the above embodiment.
  • MFG-E8 of the present invention is for the manufacture of a medicament for treating or preventing myocardial injury or heart failure.
  • examples of the medicament for treating or preventing myocardial injury or heart failure include the aforementioned pharmaceutical composition.
  • the compound screening method for treating or preventing myocardial injury or heart failure uses a system for examining the induction of MFG-E8 expression or a bioassay system for MFG-E8. There are no particular limitations on the system for examining the induction of MFG-E8 expression or the bioassay system for MFG-E8, and the methods used in the examples described later can be used.
  • an assay relating to cardiac findings, cardiac function or prognosis after the occurrence of myocardial infarction an assay relating to cardiac findings, cardiac function or prognosis after the occurrence of myocardial infarction; Assay for changes in apoptotic cell phagocytosis of myofibroblasts when a purified or artificially produced candidate substance is added; in a myocardial infarction animal model, cardiac findings after administration of a candidate substance after the occurrence of myocardial infarction; Assay for cardiac function or prognosis; Binding assay between candidate substance and apoptotic cells; Binding assay between candidate substance and cells expressing ⁇ v ⁇ 3 integrin; Promoter region of MFG-E8 gene was isolated, and Luciferase gene was linked downstream Examples include a reporter and a Luciferase assay using the reporter.
  • Wild-type C57BL / 6J mouse obtained from Nippon Claire Co., Ltd.
  • MFG-E8 gene knockout (deficient) mice Individuals assigned by Professor Shigekazu Nagata (Kyoto University graduate School of Medicine, Department of Biomedical Sciences, Department of Biomedical Chemistry) were bred in the animal breeding room (SPF). A thing was used.
  • Anti-MFG-E8 antibody MBL Anti-GAPDH antibody: Santacruz Anti- ⁇ SMA antibody: Thermo Fisher Scientific Anti-CD68 antibody: Serotec Anti-Gr-1 antibody: BioLegend Anti-vimentin antibody: BD Biosciences PE-conjugated anti-integrin ⁇ v antibody: BioLegend FITC-conjugated anti-integrin ⁇ 3 antibody: BioLegend FITC-conjugated anti-integrin ⁇ 5 antibody: eBioscience Anti-CD45 antibody: BioLegend FITC-conjugated anti-CD45.1 antibody: BioLegend APC-conjugated anti-CD45.2 antibody: BioLegend Alexa fluor 488-conjugated anti-CD45.2 antibody: BioLegend APC-conjugated anti-CD11b antibobdy: BioLegend FITC-conjugated anti-CD11b antibobdy: BioLegend PerCP / Cy5.5-conjugated anti-F4
  • results are based on experiments conducted under the same independent condition of at least 3 cases, and all are expressed as mean value ⁇ standard error.
  • a comparison between two groups is performed using unpaired t test, and a comparison between multiple groups is performed using Student-Newman-Keuls test by One way-ANOVA. There is a significant difference when the P value is less than 5%. It was judged.
  • the myocardial infarction model mouse used in the present specification was prepared as follows. First, 8-10 week old male wild-type C57BL / 6J mice or MFG-E8 gene knockout mice (hereinafter referred to as “MFG-E8 deficient mice”, “MFG-E8 KO mice”, and “KO mice”) Prepared.) To this mouse, somnopentyl injection (50 mg / kg sodium pentobarbital) was intraperitoneally administered and then fixed to the operating table in the supine position. After shaving the neck and chest, the midline of the neck was incised under a surgical microscope to expose the trachea.
  • the cannula was inserted into the trachea, and artificial respiration was performed at a tidal volume of 0.5 cc and a respiration rate of 120 times / min.
  • the myocardial infarction was performed by exposing the heart through an incision between the second ribs on the left side of the radius and ligating the left anterior descending coronary artery with a 6 mm silk blade suture. Thereafter, the incision site was sutured with a suture.
  • Mice subjected to the above treatment were designated as a myocardial infarction (MI) group, and mice treated similarly except for ligation of the coronary artery were designated as a sham treatment (control) group.
  • MI myocardial infarction
  • mice treated similarly except for ligation of the coronary artery were designated as a sham treatment (control) group.
  • mice heart tissue and the like were collected after a predetermined number of days, and various examinations shown below were performed.
  • the supernatant obtained by centrifuging the homogenized solution was subjected to protein quantification using BIO-RAD Protein Assay (manufactured by BIO-RAD), followed by 2 ⁇ SDS sample buffer (100 mM Tris-Cl pH 6.8, 20%). Glycerol, 2% SDS, 0.04% Bromophenol Blue) and boiled at 95 ° C. for 5 minutes.
  • the obtained sample was electrophoresed by SDS PAGE Gel at 20-30 ⁇ g per well, and then transferred to a PVDF membrane (Amersham Hybond-P).
  • Example 2 Identification of cells producing MFG-E8 Using mouse heart tissue collected 3 days after myocardial infarction, identification of cells producing MFG-E8 was attempted by fluorescent staining. Specifically, the extracted mouse heart was embedded in Surgipath FSC 22 (manufactured by Leica) and frozen in liquid nitrogen to obtain a frozen specimen. The frozen specimen was sliced to 4 ⁇ m with a cryostat (model CM1100; manufactured by Leica) and air-dried for 1 hour. The frozen sections were fixed with -20 ° C Cold-acetone or 4% PFA for 10 minutes and incubated with 5% BSA / PBS for 1 hour. Then, it was left overnight at 4 ° C.
  • Surgipath FSC 22 manufactured by Leica
  • FIG. 2 shows the results of examining the expression of MFG-E8 in immune cells by co-staining of immune cell markers (CD68, Gr-1) and MFG-E8.
  • CD68 is a marker for monocytes or macrophages
  • Gr-1 is a marker for granulocytes such as neutrophils.
  • FIG. 3A and 3B show the results of examining the expression of MFG-E8 in myofibroblasts by co-staining with myofibroblast markers and MFG-E8.
  • Vimentin (FIG. 3A) or ⁇ SMA ( ⁇ -smooth muscle actin; FIG. 3B) was used as a fibroblast marker.
  • FIG. 3A Vimentin
  • ⁇ SMA ⁇ -smooth muscle actin
  • Example 3 Examination of the origin of myofibroblasts producing MFG-E8 Using mouse heart tissue collected on the first day after myocardial infarction, myofibroblasts producing MFG-E8 by fluorescence staining The origin was examined. Specifically, the frozen section was fluorescently stained in the same manner as in Experimental Example 2 except that the tissue on the first day after the procedure was used and the antibody was changed, and observation and imaging were performed with a fluorescent microscope. The results are shown in FIG. In FIG. 4, the upper row shows the results of co-staining with Vimentin and MFG-E8, which are fibroblast markers including undifferentiated fibroblasts, and the lower row shows ⁇ SMA and MFG, which are markers of differentiated myofibroblasts.
  • Example 4 Examination of expression distribution of MFG-E8 around the infarct site Examination of MFG-E8 expression distribution around the myocardial infarction site using mouse heart tissue collected 3 days after myocardial infarction went. Specifically, the frozen section was fluorescently stained and observed and imaged with a fluorescence microscope in the same manner as in Experimental Example 2 except that the preparation method of the specimen and the section was changed. In this examination, the cut surface of the section is as shown in FIG. FIG. 6 shows the result of co-staining of MFG-E8 and nucleus, and FIG. 7 shows the result of staining or co-staining of MFG-E8, ⁇ SMA, and nucleus. From the results of FIGS. 6 to 7, it was confirmed that MFG-E8 was highly expressed in myofibroblasts in the infarct region.
  • Example 5 Examination of expression distribution of MFG-E8 before and after myocardial infarction operation Using mouse heart tissue before myocardial infarction operation and mouse heart tissue collected on the 3rd day after myocardial infarction operation, The expression distribution was examined. Specifically, in the same manner as in Experimental Example 2, frozen sections were prepared and MFG-E8 and ⁇ SMA were fluorescently stained, and observed and imaged with a fluorescence microscope. The results are shown in FIG. In FIG. 8, the upper part shows the result in the mouse heart tissue before the myocardial infarction operation, and the lower part shows the result in the mouse heart tissue on the third day after the myocardial infarction operation. As is clear from the results of FIG.
  • MFG-E8 is hardly expressed in the mouse heart tissue before the myocardial infarction, whereas MFG-E8 is highly expressed in the mouse heart tissue after the myocardial infarction. I found out. In the mouse heart tissue after myocardial infarction, high expression of MFG-E8 was observed particularly at the infarct site.
  • serum response factor SRF
  • MRTF myocardin-related transcription factor
  • ⁇ SMA myofibroblasts
  • CCG1423 an inhibitor of SRF and MRTF, changes in the expression level of MFG-E8 and the like by TGF- ⁇ stimulation, and expression of MFG-E8 and the like by TGF- ⁇ stimulation when siRNA for SRF is used. The amount change was also examined.
  • TGF- ⁇ 1 Induction of expression of MFG-E8 and the like in rat cardiac fibroblasts by stimulation with TGF- ⁇ 1 Specifically, 10 ng / mL TGF was isolated from resident fibroblasts isolated from rat neonatal heart. Stimulation was performed by adding - ⁇ 1 to the medium. After stimulation, fibroblasts at 24 hours, 48 hours, and 72 hours were collected. In some samples, CCG1423 was added to the medium at a concentration of 1 ⁇ M or 10 ⁇ M immediately before stimulation with 10 ng / mL of TGF- ⁇ 1, and collected by culturing for 24 hours.
  • the primers and TaqMan probe used for quantification of the sample mRNA were those designed by Primer Express Software (manufactured by Applied Biosystems). For the measurement, based on the protocol of One-Step PrimeScript TM RT-PCR Kit (manufactured by TAKARA), a reverse transcription reaction was performed at 42 ° C. for 10 minutes. After denaturation of the reverse transcriptase at 95 ° C. for 10 seconds, the reaction was performed at 60 ° C. for 35 seconds. The extension reaction was performed 40 cycles. Analysis was performed using 18S rRNA as an internal standard.
  • FIG. 9A shows the results of examining the temporal changes in the expression levels of Acta2, Adam12 and Mfg-e8 by TGF- ⁇ 1.
  • FIG. 9B shows the results relating to the expression level of MFG-E8 when CCG1423 was used in combination with TGF- ⁇ 1 expression induction for 24 hours (24 hours).
  • the result shown in the figure is each expression level corrected by 18S rRNA which is an internal standard.
  • Acta2 is Smooth Muscle Actin
  • Adam12 is a protease having a disintegrin domain and a metalloprotease domain, and is a protease that is considered to be involved in the TGF- ⁇ signal, and is a marker molecule for myofibroblasts.
  • MFG-E8 and the like is enhanced by differentiating fibroblasts into myofibroblasts by the action of TGF- ⁇ , and MFG-E8 and the like in myofibroblasts and human cell lines. It was suggested that expression is dependent on downstream factors and / or cofactors of TGF- ⁇ .
  • Myofibroblasts were isolated. The isolated cells were cultured overnight in DMEM (10% FBS, 50 U / ml penicillin / streptomycin), and those stuck to the plate were used as isolated myofibroblasts. The isolated myofibroblasts were collected for 24 hours in the presence or absence of CCG1423 and then collected by a conventional method. Using the recovered myofibroblasts, the expression levels of Acta2 and Mfg-e8 were measured by real-time PCR in the same manner as in Experimental Example 6. The results are shown in FIG. 10A.
  • mice having haplotypes different from Ly5 (CD45) antigen were used (obtained from Sankyo Lab Service).
  • ⁇ -ray irradiation (10 Gy) was performed using a gamma cell 40 on MFG-E8 KO mice having a haplotype of Ly5.2, and this was used as a recipient.
  • a wild-type mouse having a Ly5.1 haplotype was used as a donor, and bone marrow from which bone marrow fluid was collected from the tibia and femur of the donor mouse was subjected to bone marrow transplantation by intravenous injection through the recipient's fundus vein.
  • FIG. 11A shows the result of co-staining using mouse heart tissue collected on the third day after myocardial infarction in the same manner as in Experimental Example 2.
  • bone marrow-derived cells expressing CD45 (CD45.1) were found in the mouse heart tissue 3 days after myocardial infarction. However, almost no expression of MFG-E8 was observed in these bone marrow-derived cells expressing CD45.
  • the bone marrow of MFG-E8 KO mice having Ly5.2 haplotype was transplanted to wild-type mice having Ly5.1 haplotype by a conventional method, and myocardial infarction was performed.
  • bone marrow-derived cells were confirmed using peripheral blood of bone marrow transplanted mice, 99.61% of all bone marrow cells present in peripheral blood vessels were occupied by donor-derived cells in bone marrow transplanted mice. This was confirmed (FIG. 12A).
  • Co-staining was performed in the same manner as in Experimental Example 2 using mouse heart tissue collected on the third day after myocardial infarction. The result is shown in FIG. 12B.
  • bone marrow-derived cells expressing CD45 (CD45.2) were found in the mouse heart tissue 3 days after myocardial infarction.
  • CD45 expression and MFG-E8 expression were not colocalized.
  • myofibroblasts expressing vimentin express MFG-E8.
  • myofibroblasts derived from endothelial-mesenchymal transition cells that express FSP-1 did not express MFG-E8.
  • FIG. 15A the left ventricular wall of the MFG-E8 KO mouse (KO MI) after the myocardial infarction was compared with the left ventricular wall of the wild type mouse. Its thickness was significantly reduced.
  • FIG. 15B is a graph in which the thickness of the left ventricular wall is digitized. As is clear from FIG.
  • the left ventricular wall thickness is significantly thinner in the myocardial infarction group (MI) than in the non-surgery group (sham), and the MFG-E8 KO mouse is treated.
  • the group had thinner walls than the wild-type mouse treatment group.
  • the transducer was moved and an M-mode image was recorded from a short-axis image with the largest left ventricular cavity.
  • the left ventricular end diastolic diameter (LVIDd) and end systolic diameter (LVIDs) were measured according to the M-mode method according to the American College of Echocardiography leading-edge to leading-edge.
  • the end diastole was when the left ventricular expansion diameter was the largest, and the end systolic diameter was when the movement of the left ventricular rear wall was the largest.
  • the ejection fraction (EF) was calculated using the Pombo method.
  • FIG. 20A The result of the wild type mouse is shown in FIG. 20A, and the result of the MFG-E8 KO mouse is shown in FIG. 20B.
  • FIGS. 20A and 20B in the wild type, TUNEL-positive apoptotic cells were incorporated into ⁇ SMA-positive myofibroblasts (FIG. 20A).
  • ANTI-FLAG M2-Affinity Gel was buffer-substituted 3 times with PBS and then packed in a Poly-Prep chromatography column (BIO-RAD) to prepare a column bed. After washing the column with PBS, the collected cell supernatant was added to adsorb FLAG-MFG-E8. After washing with PBS, an eluate (10 mM Tris pH 7.5, 5M LiCl) was added to elute FLAG-MFG-E8. In order to concentrate the eluted fraction, it was added to Amicon Ultra-4 (MILLIPORE), centrifuged at 3500 rpm at 4 ° C., PBS was added for buffer exchange, and the mixture was centrifuged again. The concentrated protein solution was finally sterilized by filtration using ULTRA FREE-MC (MILLIPORE).
  • MILLIPORE Amicon Ultra-4
  • F4 / 80-positive macrophage cells (FIG. 24A, upper part) and CD11b-negative non-white blood cells (FIG. 24A, lower part) were fractionated, and integrin expression was confirmed. As a result, it was confirmed that integrin ⁇ v was expressed in both macrophages and non-white blood cells (FIG. 24B).
  • the integrin expression of the F4 / 80 positive fraction which is a macrophage marker, was observed, but the integrin ⁇ v expression was also observed in the macrophage, but the integrin ⁇ 3 and ⁇ 5 expression could not be confirmed.
  • FIG. 25C Examples of CD11b negative cells present in the heart after myocardial infarction include myofibroblasts.
  • the action point of MFG-E8 is not leukocytes such as macrophages but myofibroblasts.
  • fibroblasts collected from the heart were induced to differentiate by culturing for 48 hours in the presence of TGF- ⁇ 1 (1 ng / ml) to obtain myofibroblasts.
  • the obtained myofibroblasts were seeded on a 6-well plate, apoptotic thymocytes were added at a ratio of 10 to myofibroblasts 1, and incubated at 37 ° C. for 2 hours. Thereafter, the cells were washed with PBS to remove apoptotic cells that were not phagocytosed.
  • this myofibroblast is more likely to produce anti-inflammatory cytokine TGF- ⁇ by phagocytosis of apoptotic cells (FIG. 28B).
  • TNF- ⁇ , IL-1 ⁇ and IL-6 which are inflammatory cytokines, are more difficult to produce (FIG. 28B; the results of TNF- ⁇ and IL-1 ⁇ are not shown).
  • MFG-E8 used for administration was obtained by sterilizing the recombinant MFG-E8 produced in Experimental Example 13 using ULTRA FREE-MC (manufactured by Millipore).
  • ULTRA FREE-MC manufactured by Millipore
  • a 29G needle (BD Rhodos TM; 326631) was used. After administration, it was confirmed that there was no bleeding or solution leakage, and the incision was sutured with a suture.
  • FIG. 30A shows a schematic diagram of the treatment. The ligation point is ⁇ , and the MFG-E8 administration point is ⁇ .
  • FIG. 30B shows a photograph of the heart after TTC staining.
  • the Evans blue non-stained part corresponds to a risk area (AAR), and the TTC stained part corresponds to an infarcted area.
  • FIG. 30C shows the result of measuring the size of the infarct region and the size of the risk region, and quantifying the ratio of the infarct region to the risk region or the ratio of the risk region to the left ventricle. As a result of FIGS. 30B to 30C, no difference was observed between the MFG-E8 administration group and the non-administration group.
  • FIG. 31A shows the results of the expression levels of IL-1 ⁇ and MIP-2 when 1.6 ⁇ g of MFG-E8 was administered, corrected by GAPDH. As a result, increased expression levels of IL-1 ⁇ and MIP-2 were observed at the infarct site. It was also found that this increase in expression was significantly suppressed in the MFG-E8 administration group.
  • FIG. 31B shows the results of the expression levels of IL-1 ⁇ and MIP-2 when 1.6 ⁇ g or 3.2 ⁇ g of MFG-E8 was corrected with 18S rRNA. As a result, it became clear that the inflammation after the infarction was suppressed depending on the concentration of MFG-E8 to be administered.
  • HW heart weight
  • BW body weight
  • MFG-E8 is considered to have a high possibility of becoming a new treatment method including the combined use with an existing treatment method.
  • the preventive pharmaceutical composition and the method for treating or preventing myocardial injury or heart failure according to the present invention are very useful for the prevention or treatment of myocardial injury or heart failure, and can be used in the pharmaceutical or medical industry.

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Abstract

Le MFG-E8 est produit par les myofibroblastes qui apparaissent dans la région d'un infarctus. La production de MFG-E8 est régulée par les signaux TGF-β/SRF. Les myofibroblastes ingèrent les cellules mortes par l'intermédiaire de MFG-E8, induisant une réponse anti-inflammatoire due à la phagocytose réalisée par ces macrophages. En conséquence, chez les souris déficientes en MFG-E8, il reste dans la région d'un infarctus beaucoup plus de cellules apoptotiques qui n'ont pas subi de phagocytose, entraînant une inflammation excessive dans ladite région. Ainsi, le taux de survie après un infarctus du myocarde diminue considérablement chez les souris déficientes en MFG-E8. Inversement, l'injection de MFG-E8 dans la région de l'infarctus facilite l'élimination des cellules apoptotiques, ce qui soulage l'inflammation dans ladite région et entraîne une amélioration marquée de la fonction cardiaque après un infarctus du myocarde. Ces résultats indiquent que le MFG-E8 peut constituer une nouvelle cible de traitement pour le traitement des infarctus du myocarde.
PCT/JP2014/072028 2013-08-22 2014-08-22 Composition pharmaceutique pour le traitement des lésions du myocarde, composition pharmaceutique pour la prévention des lésions du myocarde, composition pharmaceutique pour le traitement de l'insuffisance cardiaque, composition pharmaceutique pour la prévention de l'insuffisance cardiaque, procédé de traitement ou de prévention des lésions du myocarde ou de l'insuffisance cardiaque, mfg-e8, utilisations de mfg-e8 et procédé de criblage de composés permettant de traiter ou de prévenir les lésions du myocarde ou l'insuffisance cardiaque WO2015025956A1 (fr)

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CN106555002A (zh) * 2016-11-16 2017-04-05 武汉大学 乳脂肪球表皮生长因子8在心脏重构和心力衰竭的诊断及治疗中的应用
WO2018212372A1 (fr) * 2017-05-17 2018-11-22 (주)넥셀 Protéine recombinante pour la prévention ou le traitement de la fibrose tissulaire et composition pour la prévention ou le traitement de la fibrose tissulaire comprenant celle-ci
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CN111447950A (zh) * 2017-11-30 2020-07-24 国立大学法人筑波大学 活性调节剂
WO2019107445A1 (fr) * 2017-11-30 2019-06-06 国立大学法人筑波大学 Modulateur d'activité
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CN111447950B (zh) * 2017-11-30 2023-05-23 国立大学法人筑波大学 活性调节剂
TWI842689B (zh) * 2017-11-30 2024-05-21 國立大學法人筑波大學 活性調節劑
WO2020085545A1 (fr) * 2018-10-25 2020-04-30 (주)넥셀 Protéine recombinante pour la prévention ou le traitement de l'infarctus du myocarde et composition la comprenant pour la prévention ou le traitement de l'infarctus du myocarde
JP2022509445A (ja) * 2018-10-25 2022-01-20 ネクセル カンパニー，リミテッド 線維症を治療又は予防するための組成物及び方法
US12139518B2 (en) 2018-10-25 2024-11-12 Nexel Co., Ltd. Compositions and methods for treating or preventing fibrosis
WO2022196747A1 (fr) * 2021-03-18 2022-09-22 国立大学法人東京大学 Thérapie de l'insuffisance cardiaque et de ses maladies concomitantes, agent thérapeutique et méthode de diagnostic

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