WO2018137701A1 - Composition pharmaceutique ciblant cxcr7 et méthode - Google Patents

Composition pharmaceutique ciblant cxcr7 et méthode Download PDF

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WO2018137701A1
WO2018137701A1 PCT/CN2018/074264 CN2018074264W WO2018137701A1 WO 2018137701 A1 WO2018137701 A1 WO 2018137701A1 CN 2018074264 W CN2018074264 W CN 2018074264W WO 2018137701 A1 WO2018137701 A1 WO 2018137701A1
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cxcr7
cxcr4
protein
expression
drug
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PCT/CN2018/074264
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Chinese (zh)
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王淼
郝会峰
陈虹
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中国医学科学院阜外医院
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Priority to CN201880008458.8A priority Critical patent/CN110225768A/zh
Publication of WO2018137701A1 publication Critical patent/WO2018137701A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A61L29/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/606—Coatings

Definitions

  • the present invention relates to pharmaceutical compositions and methods for treating cardiovascular diseases.
  • the present invention relates to a method and a pharmaceutical composition for vascular endothelial injury disease in a subject to be treated with CXCR7 or to improve cardiac remodeling after myocardial infarction in a subject.
  • GWA genome-wide association study
  • CXCL12 locus which encodes the chemokine CXCL12, also known as stromal cell-derived factor-1, SDF1
  • CAD coronary artery disease
  • MI myocardial infarction
  • Higher plasma CXCL12 is associated with myocardial infarction (MI) and death events in patients with chronic kidney disease (European heart journal. 2014; 35: 2115-2122), and a prospective study of the Framingham Heart Study, which is also associated with heart failure Associated with total mortality (Arteriosclerosis, thrombosis, and vascular biology. 2014; 34: 2100-2105).
  • CXCL12 has two receptors: CXCR4, a classical G-protein coupled receptor (GPCR), and CXCR7, which was discovered in 2005 as the second receptor for CXCL12 (The Journal of biological chemistry .2005;280:35760-35766).
  • CXCR4 is thought to be involved in vascular remodeling (Circulation. 2003; 108: 2491-2497; Circulation research. 2005; 96: 784-791; Arteriosclerosis, thrombosis, and vascular biology. 2014; 34: 1209-1220; Thrombosis and haemostasis. 2012 ; 107: 356-368), atherosclerosis (Circulation research.
  • CXCR7 myocardial infarction
  • a decrease in CXCR4 (rather than CXCR7) expression is associated with all-cause death and/or MI joint end points (Journal of thrombosis and haemostasis: JTH. 2015; 13: 719-728).
  • CXCR7 is closely related to chemokine receptors in phylogeny, binds to CXCL12 with higher affinity than CXCR4, transmits signals through ⁇ -arrestin (rather than classical G-protein), but cannot be coupled with G protein to induce A typical chemokine receptor-mediated cellular response (J Exp Med. 2006; 203: 2201-2213).
  • CXCR7 was previously thought to act as a scavenging receptor for CXCL12, mediating efficient endocytosis and degradation (PLoS One. 2010; 5: e9175; Cell. 2008; 132: 463-473; Proc Natl Acad Sci USA. 2010; 107: 628-632).
  • CXCR7 also has signaling activity beyond ligand clearance, including signaling activity in tumor cell growth and organ regeneration (Proc Natl Acad Sci USA. 2007; 104: 15735-15740; J Biol Chem. 2008 ; 283: 4283-4294; Mol Cancer. 2014; 13: 198; Nature medicine. 2016; 22: 154-162).
  • CXCR7 is expressed in brain, heart, kidney, endothelial and tumor cells (J Exp Med. 2006; 203: 2201-2213; PLoS One. 2011; 6: e20680). It is widely expressed in tumor vascular endothelium (Proc Natl Acad Sci USA. 2007; 104: 15735-15740) and is induced by hypoxia (PLoS One. 2013; 8: e55290). Platelets express both CXCR4 and CXCR7 (European heart journal. 2014; 35: 386-394), but CXCR7 protein is not expressed on leukocytes in human or mouse blood (Journal of Immunology. 2010; 185: 5130-5139).
  • CXCR7-deficient mice die from abnormal heart valves before and after birth (Proc Natl Acad Sci USA. 2007; 104: 14759-14764). Weber et al. used hyperlipidemia Apoe -/- mice to confirm that the overall knockout of CXCR7 exacerbates atherosclerosis due to defects in cholesterol uptake in adipose tissue (Circulation. 2014; 129: 1244-1253) .
  • the invention provides a method of treating or preventing a cardiovascular disease in a subject comprising administering to the subject an effective amount of a first drug that increases the expression and/or activity of a CXCR7 protein.
  • the present invention provides a method of treating a vascular endothelial injury disease in a subject or ameliorating cardiac remodeling after myocardial infarction in a subject, comprising administering to the subject an effective amount of a first drug that increases expression and/or activity of a CXCR7 protein. .
  • the method of the present invention for treating or preventing cardiovascular disease in a subject further comprises administering to the subject an effective amount of a second drug that reduces expression and/or activity of the CXCR4 protein.
  • the methods of the invention further comprise administering to the subject a drug that inhibits platelet activation and/or aggregation.
  • the method of the invention further comprises administering to the subject a drug that stabilizes the plaque.
  • the invention also provides a pharmaceutical composition for treating or preventing a cardiovascular disease in a subject, comprising an effective amount of a first drug for increasing the expression and/or activity of a CXCR7 protein.
  • a pharmaceutical composition for treating a vascular endothelial injury disease in a subject or ameliorating cardiac remodeling after myocardial infarction in a subject comprising an effective amount of an amount for increasing the expression and/or activity of the CXCR7 protein. a drug.
  • the pharmaceutical composition further comprises an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of the CXCR4 protein.
  • the pharmaceutical composition is for use in combination with an effective amount of a selective antagonist comprising CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of a CXCR4 protein.
  • the pharmaceutical composition is for use in combination with a drug that inhibits platelet activation and/or aggregation.
  • the pharmaceutical composition is for use in combination with a drug that stabilizes the plaque.
  • the present invention also provides the use of a first medicament for increasing the expression and/or activity of a CXCR7 protein for the preparation of a pharmaceutical composition for treating or preventing a cardiovascular disease in a subject.
  • the pharmaceutical composition further comprises an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of the CXCR4 protein.
  • the pharmaceutical composition is used in combination with an effective amount of a selective antagonist comprising CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of a CXCR4 protein.
  • the pharmaceutical composition is for use in combination with a medicament for inhibiting platelet activation and/or aggregation.
  • the pharmaceutical composition is for use in combination with a drug that stabilizes the plaque.
  • the disease is selected from the group consisting of thrombosis, thromboembolism, vascular wall injury, vascular stenosis after injury, vascular restenosis after PCI and Bypass, coronary heart disease, myocardial deficiency Blood, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, and any combination thereof.
  • the invention also provides a vascular stent or catheter with a balloon, wherein the surface of the stent or balloon is coated with an effective amount of a first drug for increasing the expression and/or activity of the CXCR7 protein.
  • the surface of the stent or balloon is further coated with an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule or expression that inhibits expression of the CXCR4 protein.
  • Carrier In one embodiment of the vascular stent or balloon with a balloon of the invention, the surface of the stent or balloon is further coated with an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule or expression that inhibits expression of the CXCR4 protein. Carrier.
  • the vascular stent or the balloon-equipped catheter of the present invention is for treating or preventing vascular injury and/or myocardial ischemia-related disease in a subject selected from the group consisting of thrombosis, thromboembolism, blood vessel wall damage, Vascular stenosis after injury, vascular restenosis after PCI and Bypass, coronary heart disease, myocardial ischemia, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, and any combination thereof.
  • the present invention also provides a method of treating or preventing a thrombosis-related disease in a subject, comprising administering to the subject an effective amount of a drug that reduces the level or activity of circulating CXCL12, or a selective antagonist of CXCR4, or inhibiting CXCR4 protein expression.
  • a drug that reduces the level or activity of circulating CXCL12, or a selective antagonist of CXCR4, or inhibiting CXCR4 protein expression Nucleic acid molecule or expression vector, or a combination thereof.
  • the present invention also provides a pharmaceutical composition for treating or preventing a thrombosis-related disease in a subject, which comprises an effective amount of a drug which lowers the level or activity of CXCL12 in circulation, or a selective antagonist of CXCR4, or inhibits CXCR4 protein expression.
  • a pharmaceutical composition for treating or preventing a thrombosis-related disease in a subject which comprises an effective amount of a drug which lowers the level or activity of CXCL12 in circulation, or a selective antagonist of CXCR4, or inhibits CXCR4 protein expression.
  • Nucleic acid molecule or expression vector, or a combination thereof are examples of nucleic acid molecule or expression vector, or a combination thereof.
  • the present invention also provides a medicament for reducing the level or activity of CXCL12 in circulation, or a selective antagonist of CXCR4, or a nucleic acid molecule or expression vector for inhibiting expression of CXCR4 protein, or a combination thereof for preparing thrombus for treating or preventing a subject Use in a pharmaceutical composition of a related disease.
  • the invention also provides a pharmaceutical composition for treating cancer comprising a CXCR7 inhibitor, wherein the CXCR7 inhibitor does not increase blood CXCL12 levels when administered to a subject.
  • the invention also provides a method of screening for a medicament for treating cancer with high cardiovascular safety, comprising:
  • the activity of the CXCR7 protein in the tissue sample of the animal is decreased relative to the activity of the CXCR7 protein in the same tissue sample of the control animal to which the drug candidate is not administered, and the content of CXCL12 protein in the blood sample of the animal is not administered.
  • the level of CXCL12 protein in the blood sample of the control animal of the drug candidate is comparable or lower, suggesting that the drug candidate is a drug for treating cancer with high cardiovascular safety.
  • the invention also provides methods of screening for a medicament useful for treating or preventing a cardiovascular disease, comprising:
  • the expression level and/or activity of the CXCR7 protein in the tissue sample of the animal relative to the expression level and/or activity of the CXCR7 protein in the same tissue sample of the control animal to which the drug candidate is not administered is indicative of the candidate drug Can treat or prevent cardiovascular disease.
  • AD Proliferative femoral artery (B) 28 days after injury in the healthy femoral artery (A) and guidewire in mice, and CXCR7 in aortic sections (C, D) from patients with aortic dissection Red) and vWF (endothelial cell marker, green) were immunofluorescently stained and DAPI stained with nuclei (blue). At the far right is the combination of the three colors.
  • the gray photo in C1 shows the staining position in Figure 1C at a lower magnification. The arrow points to a severe injury. Representative sections from three independent stains are shown.
  • L lumen
  • P plaque
  • Bar 50 ⁇ m.
  • A-F CXCR7 mRNA expression (A) in mouse lung endothelial cells (MLEC) isolated from endothelial CXCR7 conditional knockout mice (cKO) and littermate control (Ctl) was detected by RT-PCR.
  • DAPI stained with nuclei blue.
  • C neointimal formation
  • D intima to media ratio
  • E median thickness remained unchanged
  • IL-1 ⁇ (10 ng/mL) treatment increased CXCR7 mRNA (A) and protein (B) levels in cultured mouse lung endothelial cells (MLEC).
  • IL-1 ⁇ (10 ng/mL) promotes cell growth.
  • Drug inhibition (CCX771) or gene deletion CXCR7 inhibits cell proliferation in MLEC (C) and mouse aortic endothelial cells (MAEC) (D).
  • MAEC immunoblot analysis indicated that IL-1 ⁇ increased the ERK signaling pathway, which is inhibited by CXCR7 inhibition or deletion (E, F).
  • CCX771 (G) or CXCR7 (H) knockdown reduces cell proliferation when stimulated with IL-1 ⁇ .
  • Each experiment was conducted no less than 3 times. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • CXCR4 and CXCL12 in cultured mouse lung endothelial cells (MLEC) treated with IL-1 ⁇ .
  • CXCR4 (A) and CXCL12 (B) mRNA were detected by RT-PCR before and 6 hours after stimulation with IL-1 ⁇ (10 ng/mL).
  • CXCR4 protein levels (C) of cells incubated with IL-1 ⁇ (10 ng/mL) for 0, 6, 12 and 24 hours were detected by immunoblotting.
  • J Effect of drug inhibition of CXCR7 on proliferation of unstimulated endothelial cells. Proliferation studies were performed in mouse lung endothelial cells (MLEC; A) and mouse aortic endothelial cells (MAEC; B) that were not stimulated by IL-1 ⁇ . No statistical significance.
  • CXCR7 inhibition reduces TNF ⁇ -induced endothelial cell proliferation and angiogenesis.
  • TNF ⁇ -induced CXCR7 protein expression A
  • B endothelial cell proliferation
  • D silencing
  • Bar 200 ⁇ m.
  • HUVEC HUVEC
  • HAEC human aortic endothelial cells
  • si-CXCR7 si-CXCR7 or negative si-RNA (si-Neg)
  • Bar 500 ⁇ m.
  • vascular density (H) in the muscle space (IS) of the gastrocnemius muscle was measured by immunostaining of vWF.
  • vWF green
  • DAPI blue
  • EF ejection fraction
  • FAC left ventricular sectional area change score
  • E/A ratio of early mitral peak to end-diastolic filling rate
  • LVAWd Left ventricular anterior wall thickness
  • CXCR7 is expressed in Ctl mouse endothelial cells after MI, but not in cKO endothelial cells (G).
  • MI increases CXCL12 plasma levels in Ctl and cKO mice. Compared to Ctl, cKO showed higher plasma CXCL12 levels before MI (defined as "0") and after MI. *, p ⁇ 0.05vs.Ctl; 0 in p ⁇ 0.05 vs. cKO; 0 in p ⁇ 0.05 vs. Ctl.
  • A-F Expression of CXCR7 was confirmed in a 293T cell line transfected with adenovirus expressing CXCR7 (Ad-CXCR7) (A).
  • EF ejection fraction
  • LVAWd diastolic left ventricular anterior wall thickness
  • D, F; n 10 Ad-Neg, 11 Ad-CXCR7.
  • A-F Conditional knockout (cKO) of endothelial CXCR7 induced by tamoxifen treatment is described in detail in the method and is used to determine plasma CXCL12 and photochemically induced thrombosis.
  • Wild type mice (C57BL/6) were treated by subcutaneous injection of 20 mg/kg CCX771 or vehicle, and plasma CXCL12 and photochemically induced thrombosis were examined two hours later. Representative carotid blood flow during induced thrombosis is shown in the accompanying drawings (A&C).
  • A-F Intravenous infusion of CXCL12 increases circulating CXCL12 levels (A&B) and enhances collagen-induced ex vivo whole blood accumulation (C&D).
  • CXCL12 infusion accelerates photochemically induced thrombosis (E&F) and AMD3100 (CXCR4-specific inhibitor) eliminates accelerated thrombosis.
  • E&F photochemically induced thrombosis
  • AMD3100 CXCR4-specific inhibitor
  • A-B AMD3100 treatment abolishes the prothrombotic response caused by loss of endothelial CXCR7.
  • A shows representative carotid blood flow during induced thrombosis.
  • B represents the results of statistical analysis of occlusion time in each group.
  • Platelets significantly contribute to the increase in circulating CXCL12 after endothelial CXCR7 removal
  • A-H Platelet agonist U46619 (A) or collagen (B) triggers CXCL12 release when whole blood is treated ex vivo.
  • Intravenous injection of U46619 (20 ⁇ g/mouse) reduced platelet counts in 3 minutes (Fig. 4C) and increased plasma CXCL12 (Fig. 4D).
  • Platelet depletion by anti-CD41 antibodies reduced circulating CXCL12 in cKO and Ctl, but elevated CXCL12 levels were maintained in cKO mice (Fig. 4G).
  • the proportion of CXCL12 decreased per million platelets in cKO was higher than that of control (H).
  • Flow cytometric analysis of P-selectin (CD62P) expression in platelets showed higher CD62P expression and activated platelets (E&F) in cKO compared to Ctl.
  • I Platelet depletion and recovery time.
  • Figure 15A-B Correlation of CXCL12 levels in human blood circulation with platelet reactivity.
  • CXCR7 plays a key role in maintaining endothelial integrity. Specifically, the present inventors have found that CXCR7 is expressed in damaged blood vessels, which can be induced by inflammatory factors released by vascular injury, and promotes proliferation of inflammation-associated endothelial cells; whereas deletion of endothelial CXCR7 promotes repair of endothelial cells Reduced blood vessels caused by stenosis. The inventors have also discovered that endothelial CXCR7 also plays a key role in ischemia-induced angiogenesis.
  • Angiogenesis is a process of endothelial cell dependence, which results in the formation of new blood vessels, which is necessary for the regeneration of vascular regeneration after cardiomyocytes and the promotion of tissue regeneration after ischemia injury. More importantly, the inventors found that delivery of the CXCR7 gene to the myocardium by the adenovirus improves cardiac function after MI and reduces myocardial infarct size. Therefore, activation of CXCR7 will be able to treat vascular endothelial injury or improve cardiac remodeling after myocardial infarction.
  • the invention provides a method of treating or preventing a cardiovascular disease in a subject comprising administering to the subject an effective amount of a first drug that increases the expression and/or activity of a CXCR7 protein.
  • the present invention provides a method of treating a vascular endothelial injury disease in a subject or ameliorating cardiac remodeling after myocardial infarction in a subject, comprising administering to the subject an effective amount of a first drug that increases expression and/or activity of a CXCR7 protein. .
  • the term "subject” refers to a mammal, preferably a primate, more preferably a human.
  • the first medicament comprises a selective agonist of CXCR7, an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof, or a combination thereof.
  • the selective agonist of CXCR7 is selected from the group consisting of an activated antibody of CXCR7, an activated ligand of CXCR7, TC14012 or a functional analog thereof, and combinations thereof.
  • the method of treating or preventing cardiovascular disease in a subject of the present invention further comprises administering to the subject an effective amount of a second drug that reduces expression and/or activity of the CXCR4 protein.
  • the second drug comprises a selective antagonist of CXCR4, a nucleic acid molecule or expression vector that inhibits expression of a CXCR4 protein, or a combination thereof.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, TC14012 or a functional analog thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene. In another specific embodiment, the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • the methods of the invention further comprise administering to the subject a drug that inhibits platelet activation and/or aggregation. In another embodiment, the methods of the invention further comprise administering to the subject a drug that stabilizes the plaque, such as a statin.
  • the subject is administered to the subject by oral administration, buccal administration, inhalation, intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intraperitoneal injection or topical administration. And / or a second drug.
  • the administration is achieved by intracoronary administration or by coating the first and/or second medicament on a balloon stent or a balloon of a catheter with a balloon. Topical application.
  • the invention also provides a pharmaceutical composition for treating or preventing a cardiovascular disease in a subject, comprising an effective amount of a first drug for increasing the expression and/or activity of a CXCR7 protein.
  • a pharmaceutical composition for treating a vascular endothelial injury disease in a subject or ameliorating cardiac remodeling after myocardial infarction in a subject comprising an effective amount of an amount for increasing the expression and/or activity of the CXCR7 protein. a drug.
  • the first drug comprises a selective agonist of CXCR7, an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof, or a combination thereof.
  • the selective agonist of CXCR7 is selected from the group consisting of an activated antibody of CXCR7, an activated ligand of CXCR7, TC14012 or a functional analog thereof, and combinations thereof.
  • the pharmaceutical composition further comprises an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of the CXCR4 protein.
  • the pharmaceutical composition is for use in combination with an effective amount of a selective antagonist comprising CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of a CXCR4 protein.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, TC14012 or a functional analog thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene. In another specific embodiment, the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • the pharmaceutical composition is for use in combination with a drug that inhibits platelet activation and/or aggregation. In another embodiment, the pharmaceutical composition is for use in combination with a drug that stabilizes the plaque, such as a statin.
  • the pharmaceutical composition is for administration to a subject by oral, buccal, inhalation, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal or topical administration.
  • the pharmaceutical composition is for intracoronary administration.
  • the present invention also provides the use of a first medicament for increasing the expression and/or activity of a CXCR7 protein for the preparation of a pharmaceutical composition for treating or preventing a cardiovascular disease in a subject.
  • the first medicament comprises an effective amount of a selective agonist of CXCR7, an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof, or a combination thereof.
  • the selective agonist of CXCR7 is selected from the group consisting of an activated antibody of CXCR7, an activated ligand of CXCR7, TC14012 or a functional analog thereof, and combinations thereof.
  • the pharmaceutical composition further comprises an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of the CXCR4 protein.
  • the pharmaceutical composition is used in combination with an effective amount of a selective antagonist comprising CXCR4 or a nucleic acid molecule or expression vector that inhibits expression of a CXCR4 protein.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, TC14012 or a functional analog thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene. In another specific embodiment, the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • the pharmaceutical composition is for use in combination with a medicament for inhibiting platelet activation and/or aggregation.
  • the pharmaceutical composition is for use in combination with a drug that stabilizes the plaque, such as a statin.
  • the pharmaceutical composition is for administration to a subject by oral, buccal, inhalation, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal or topical administration.
  • the pharmaceutical composition is formulated in a form for intracoronary administration.
  • the disease is selected from the group consisting of thrombosis, thromboembolism, vascular wall injury, vascular stenosis after injury, vascular restenosis after PCI and Bypass, coronary heart disease, myocardial deficiency Blood, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, and any combination thereof.
  • the invention also provides a vascular stent or catheter with a balloon, wherein the surface of the stent or balloon is coated with an effective amount of a first drug for increasing the expression and/or activity of the CXCR7 protein.
  • the first drug comprises a selective agonist of CXCR7, an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof, or combination.
  • the selective agonist of CXCR7 is selected from the group consisting of an activated antibody of CXCR7, an activated ligand of CXCR7, TC14012 or a functional analog thereof, and combinations thereof.
  • the surface of the stent or balloon is further coated with an effective amount of a selective antagonist of CXCR4 or a nucleic acid molecule that inhibits expression of the CXCR4 protein or Expression vector.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • the vascular stent or the balloon-equipped catheter of the present invention is for treating or preventing vascular injury and/or myocardial ischemia-related disease in a subject selected from the group consisting of thrombosis, thromboembolism, blood vessel wall damage, Vascular stenosis after injury, vascular restenosis after PCI and Bypass, coronary heart disease, myocardial ischemia, myocardial infarction, heart failure after myocardial infarction, arrhythmia after myocardial infarction, and any combination thereof.
  • the vascular injury-related disease is coronary plaque and stenosis.
  • the myocardial ischemia related disease is myocardial infarction.
  • CXCL12 causes a tendency to thrombosis at a concentration having pathophysiological significance.
  • the inventors have also surprisingly found that the sensitizing effect of platelet activation is caused by circulating CXCL12, rather than the vessel wall CXCL12.
  • the present inventors have further found that the trapped receptor endothelial CXCR7 of CXCL12 is essential for maintaining the physiological level of CXCL12, while inhibiting CXCR7 leads to an increase in CXCL12, which acts on platelet CXCR4, thereby promoting thrombus formation.
  • the present invention also provides a method of treating or preventing a thrombosis-related disease in a subject, comprising administering to the subject an effective amount of a drug that reduces the level or activity of CXCL12 in circulation, or a selective antagonist of CXCR4, or inhibiting CXCR4 A nucleic acid molecule or expression vector, or a combination thereof, expressed by a protein.
  • the drug that reduces the level of circulating CXCL12 is a drug that increases the expression and/or activity of a CXCR7 protein, such as an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof.
  • the drug that reduces the level or activity of CXCL12 in the circulation is an anti-CXCL12 antibody.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • the present invention also provides a pharmaceutical composition for treating or preventing a thrombosis-related disease in a subject, which comprises an effective amount of a drug which lowers the level or activity of CXCL12 in circulation, or a selective antagonist of CXCR4, or inhibits CXCR4 protein expression.
  • the drug that reduces the level of circulating CXCL12 is a drug that increases the expression and/or activity of a CXCR7 protein, such as an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof.
  • the drug that reduces the level or activity of CXCL12 in the circulation is an anti-CXCL12 antibody.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • the present invention also provides a medicament for reducing the level or activity of CXCL12 in circulation, or a selective antagonist of CXCR4, or a nucleic acid molecule or expression vector for inhibiting expression of CXCR4 protein, or a combination thereof for preparing thrombus for treating or preventing a subject Use in a pharmaceutical composition of a related disease.
  • the drug that reduces the level of circulating CXCL12 is a drug that increases the expression and/or activity of a CXCR7 protein, such as an expression vector comprising a polynucleotide encoding a CXCR7 protein or a functional fragment thereof.
  • the drug that reduces the level or activity of CXCL12 in the circulation is an anti-CXCL12 antibody.
  • the selective antagonist of CXCR4 is selected from the group consisting of AMD3100 or a functional analog thereof, a blocking antibody to CXCR4 or an antigen binding fragment thereof, and combinations thereof.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an siRNA or a precursor thereof that targets a transcription product of the CXCR4 gene.
  • the nucleic acid molecule that inhibits expression of a CXCR4 protein is an antisense RNA that targets a transcription product of the CXCR4 gene.
  • CXCR7 inhibitors have been proposed for the treatment of diseases such as cancer.
  • an inhibitor such as CCX771
  • CCX771 increases the risk of cardiovascular thrombosis by increasing the level of CXCL12 in the blood. Therefore, in any treatment using a CXCR7 inhibitor such as cancer treatment, it is preferred to use a CXCR7 inhibitor that does not increase the level of CXCL12.
  • the invention also provides a pharmaceutical composition for treating cancer comprising a CXCR7 inhibitor, wherein the CXCR7 inhibitor does not increase blood CXCL12 levels when administered to a subject.
  • the cancer includes, but is not limited to, liver cancer, breast cancer, and the like.
  • the invention also provides a method of screening for a medicament for treating cancer with high cardiovascular safety, comprising:
  • the activity of the CXCR7 protein in the tissue sample of the animal is decreased relative to the activity of the CXCR7 protein in the same tissue sample of the control animal to which the drug candidate is not administered, and the content of CXCL12 protein in the blood sample of the animal is not administered.
  • the level of CXCL12 protein in the blood sample of the control animal of the drug candidate is comparable or lower, suggesting that the drug candidate is a drug for treating cancer with high cardiovascular safety.
  • the invention also provides methods of screening for a medicament useful for treating or preventing a cardiovascular disease, comprising:
  • the expression level and/or activity of the CXCR7 protein in the tissue sample of the animal relative to the expression level and/or activity of the CXCR7 protein in the same tissue sample of the control animal to which the drug candidate is not administered is indicative of the candidate drug Can treat or prevent cardiovascular disease.
  • the activity of the CXCR7 protein is selected from the group consisting of promoting vascular endothelial proliferation, promoting angiogenesis, promoting damage to blood vessel repair, and reducing Myocardial infarct size, improved myocardial remodeling after infarction, and improved cardiac function after infarction.
  • the above pharmaceutical or pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an undesirable, allergic or other untoward reaction when administered to an animal, such as a human, as desired.
  • the preparation of suitable pharmaceutical compositions is known to those skilled in the art in light of the present disclosure and is exemplified in "Remington: The Science and Practice of Pharmacy," 21st Edition, 2005, which is incorporated herein by reference.
  • human administration it should be understood that the preparation should also meet the criteria for sterility, pyrogenicity, overall safety, and purity required by the drug approval authority.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, antioxidants, salts, coatings, surfactants, preservatives (eg, methyl or propyl paraben, sorbic acid, antibacterial).
  • Agent antifungal agent
  • isotonic agent such as paraffin
  • adsorbent for example, kaolin, bentonite
  • drug stabilizer for example, sodium lauryl sulfate
  • gel adhesive
  • adhesive eg, syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, carboxymethylcellulose, alginate
  • excipients eg, lactose, polyethylene glycol
  • disintegrants eg Agar, starch, lactose, calcium phosphate, calcium carbonate, alginic acid, sorbitol, glycine
  • wetting agents eg, cetyl alcohol, glyceryl monostearate
  • lubricants eg, quarters) Ammonium salt
  • edible oil eg, almond oil, coconut oil, oily ester or propylene glycol
  • sweeteners eg, flavoring agents, coloring agents, fillers (eg, starch, lac
  • the composition can comprise a plurality of antioxidants to retard oxidation of one or more components.
  • antioxidants include ascorbic acid, cysteine hydrochloride, sodium sulfite, sodium hydrogensulfite, sodium metabisulfite, ascorbyl palmitate, butylated hydroxytoluene, butylated hydroxyanisole, lecithin, propyl gallate, and tocopherol.
  • microorganisms can be achieved by the use of preservatives such as various antibacterial and antifungal agents including, but not limited to, parabens (for example, methylparaben, p-hydroxyl Propyl benzoate), chlorobutanol, phenol, sorbic acid, thimerosal or a combination thereof.
  • preservatives such as various antibacterial and antifungal agents including, but not limited to, parabens (for example, methylparaben, p-hydroxyl Propyl benzoate), chlorobutanol, phenol, sorbic acid, thimerosal or a combination thereof.
  • the carrier can be a solvent or dispersion medium including, but not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), liquid ( For example, triglycerides, vegetable oils, liposomes, and combinations thereof.
  • a coating such as lecithin
  • a surfactant For example, hydroxypropyl cellulose
  • prolonged absorption of the injectable compositions can be brought about by the use of agents that delay absorption (e.g., aluminum monostearate, gelatin, or a combination thereof) in the compositions.
  • agents that delay absorption e.g., aluminum monostearate, gelatin, or a combination thereof
  • an effective amount refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially arrest the symptoms of a disease or condition.
  • the actual dosage of a composition of the invention administered to a patient can be determined according to the following physical and physiological factors: body weight, sex, severity of symptoms, type of disease being treated, prior or current therapeutic intervention, unknown etiology of the patient, time of administration, The excretion rate of the specific compound and the route of administration. In any event, the concentration of the active ingredient in the composition and the appropriate dosage for the subject will be determined by the medical personnel responsible for administration.
  • Endothelial CXCR7 deleted mice were constructed using the tamoxifen-CreERT2 strategy. Briefly, as described previously (Nature cell biology. 2015; 17: 123-136) mice bearing loxP-site flanking CXCR7 (CXCR7 f/f ) and Cdh5-promoter driven by Ralf Adams CreERT2 (Cdh5(PAC)-CreERT2+) mice (Nature. 2010; 465: 483-486) were crossed.
  • Ctl The resulting progeny CXCR7 f/f Cdh5-CreERT2+ male mice were crossed with CXCR7 f/f Cdh5-CreERT2-male to produce endothelial CXCR7 conditional knockout animals (CXCR7 f/f Cdh5-CreERT2+, cKO for short) and littermate control (CXCR7 f/f Cdh5-CreERT2-, referred to as Ctl).
  • mice and littermates were injected intraperitoneally with tamoxifen (Alfa Aesar, Heysham, England) (37.5 mg/ml in sunflower seed oil) at a dose of 150 mg/ Kg weight per day for three consecutive days. The mice were then allowed to rest for three days before another three days of injection.
  • C57BL/6 mice were purchased from the China Food and Drug Administration and used to evaluate the effect of adenovirus overexpressing CXCR7 in MI. All animal programs were approved by the National Cardiovascular Disease Center, Fuwai Hospital, Laboratory Animal Center, and the Institutional Animal Care and Use Committee.
  • Femoral artery injury models were prepared as previously described (Circulation. 2011; 123:631-639). Briefly, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg/kg). A one-sided inguinal incision exposes the femoral artery and separates the accompanying nerves and veins. The proximal and distal ends of the femoral artery were tied using a 6-0 suture thread to temporarily control blood flow. A small section of the artery between the rectus femoris and the medial femoral muscle was isolated, and the proximal end was restrained with a 6-0 suture thread, and the artery was laterally cut in the section.
  • a flexible guide wire (diameter 0.35 mm, Cook Inc., IN, USA) was then inserted from the segment into the femoral artery and inserted 5 mm or more into the radial artery.
  • the guidewire was left there for 3 minutes to exfoliate and dilate the artery.
  • the guidewire is then removed and the suture thread that binds the proximal end of the artery is tightened.
  • the suture thread used to interrupt the blood flow is relaxed to restore blood flow to the femoral artery.
  • the skin incision was closed using a 5-0 suture thread.
  • Femoral arteries were collected, paraffin-embedded, and transverse sections of 10-13 layers of injured arteries were continuously made from the distal end of the femoral artery segment at 150 ⁇ m intervals for morphological or histological analysis.
  • Arterial sections on day 28 of injury were subjected to H&E staining to observe the severity of hyperplasia, and the most severely propagated sections were used for comparison. Images were acquired using a CCD camera mounted on an inverted microscope (DM6000B; Leica), and images were measured using Image-Pro Plus 6.0 software (Media Cybernetics). The area of the official cavity, the inner area of the inner elastic layer and the inner area of the outer elastic layer were collected and analyzed. To assess endothelial regeneration and leukocyte infiltration/migration, immunostaining analysis was performed on the middle of the artery on day 7 of injury using the corresponding antibodies.
  • LAD left anterior descending coronary artery
  • a recombinant adenovirus expressing mouse CXCR7 (Ad-CXCR7) was injected into the left ventricle of each mouse 1 minute prior to coronary artery ligation (using 418-1506 of mouse CXCR7 mRNA (NM_001271607.1))
  • the nucleotide sequence of the position is used to construct a recombinant adenovirus or an empty vector at a dose of 1 x 10 9 plaque forming units per mouse.
  • the success of the ligation was confirmed by left ventricular color change and electrocardiogram (ECG) ST-segment elevation.
  • cardiac function and left ventricular structure were measured by echocardiography (VisualSonics VeVo 2100 Imaging System), and the measured indicators were left ventricular ejection fraction (EF), left ventricular partial area change (FAC), and left ventricular diameter shortening score. (FS), mitral valve diastolic early/late blood flow peak velocity ratio (E/A), left ventricular anterior wall thickness (LVAW), left ventricular posterior wall thickness (LVPW), and left ventricular volume, and left ventricular size were measured.
  • E/A mitral valve diastolic early/late blood flow peak velocity ratio
  • LVAW left ventricular anterior wall thickness
  • LVPW left ventricular posterior wall thickness
  • left ventricular volume and left ventricular size
  • Cardiac infarction was assessed by Masson staining (Circulation. 2015; 132: 47-58). Briefly, a series of parasternal short-axis slices (thickness 5 ⁇ m) were obtained at 200 ⁇ m intervals. Representative midsection sections were stained using Masson's trichrome reagent (Leagene Biotec. Co, Ltd) and photographed using a Zeiss optical microscope (AXI0; Zeiss). Infarct size was measured and calculated using Image-Pro Plus 6.0 software (Media Cybernetics).
  • Hind limb ischemia is caused by ligation of the left femoral artery, and the ligation site is bifurcated at the distal end of the saphenous artery (Arteriosclerosis, thrombosis, and vascular biology. 2014; 34: 408-418). Blood flow to the hind limbs was measured before and after ligation using a laser Doppler flow meter (LDF; PeriCam PSI). Mice in which the blood flow of hind limbs was reduced by not less than 50% after ligation were included in the experiment. For mice that successfully ligated the artery, blood flow was measured three times on days 4, 7, and 14, respectively. Blood flow was measured and analyzed blindly.
  • LDF laser Doppler flow meter
  • mice were sacrificed by administration of an excess of sedative.
  • the gastrocnemius muscle was divided, fixed and paraffin embedded for analysis of vascular density. Briefly, the largest transverse section of each gastrocnemius muscle was stained, and three muscle gaps were randomly photographed in each section. The blood vessels in the photo are then counted and analyzed.
  • MLEC Mouse lung endothelial cells
  • mice were perfused through the right ventricle with sterile PBS to remove blood cells.
  • the lung lobes were isolated, minced, and digested with collagenase (180-200 U/mL; Worthington) at 37 ° C (40 minutes).
  • collagenase 180-200 U/mL; Worthington
  • the cells were incubated with Dynabeads (Dynal Biotech) coated with anti-mouse CD31 (BD Biosciences).
  • the cells on the beads were separated using a magnetic separator (Dynal) and then cultured for 3 days in a collagen I (Worthington) coated Petri dish/culture flask containing 20% fetal bovine serum (FBS), 1% AA ( GIBCO), and DMEM of 100 mg/L endothelial cell growth additive (ECGS; ScienCell).
  • FBS fetal bovine serum
  • GIBCO fetal bovine serum
  • DMEM 100 mg/L endothelial cell growth additive
  • the cells were dissociated and selected with Dynabeads coated with rat anti-mouse CD102 (ICAM-2; Pharmingen).
  • IAM-2 rat anti-mouse CD102
  • Mouse aortic endothelial cells were isolated as previously described (Cell metabolism. 2011; 13: 592-600). Briefly, the aorta was collected and the fat and connective tissue surrounding the adventitia were removed and cut into small pieces of 1-2 mm 2 . The aortic fragments were cultured in medium for 5-7 days to grow endothelial cells. The endothelial cells are then passaged and cultured. To isolate human aortic endothelial cells (HAEC), human aortic samples from an external hospital were collected in DMEM. The aortic intimal layer was exfoliated and treated in the same manner as MAEC culture.
  • HAEC human aortic endothelial cells
  • Myocardial fibroblasts were isolated as previously described (Journal of cellular and molecular medicine. 2014; 18: 2266-2274). Briefly, the ventricles of newborn Wistar rats were isolated, washed and minced in PBS. The tissue was then digested in PBS containing 0.06% collagenase (Worthington) at 37 °C. The collected cell suspension was centrifuged and resuspended in 10% DMEM. The suspension was placed in a culture flask and incubated for 90 minutes. Fibroblasts tend to adhere to the bottom. Remove non-adherent cells. Adherent cardiac fibroblasts are cultured and subsequently passaged with trypsin.
  • siRNA gene silencing was performed in order to knock down CXCR7, CXCR4, ⁇ -arrestin1 or ⁇ -arrestin2 proteins in endothelial cells. Briefly, endothelial cells were seeded in 12-well plates. Before transfection, 40pmol siRNA mixed with 2.0 ⁇ L Hieff Trans TM Liposomal Transfection Reagent ( Yeasen, China) in 200 ⁇ L DMEM for 20 minutes. The medium was changed to DMEM. After 20 minutes, the siRNA transfection reagent mixture was added to the wells (200 ⁇ L/well). Transfection lasted for 6 hours. The transfection medium was then discarded and the cells were cultured in medium containing 20% FBS for not less than 6 hours prior to further analysis.
  • the siRNA used in this study is as follows:
  • si-CXCR4 (Arteriosclerosis, thrombosis, and vascular biology. 2014; 34: 1716-1722):
  • Cell proliferation studies Cell growth was measured using Cell Counting Kit-8 (CCK-8; Yeasen, Shanghai, China). Briefly, cells were planted in a 9-well flat bottom plate. After the cells were completely attached to the bottom, the cells were starved for 6-8 h in medium containing 3% FBS but no ECGS. The medium was then replaced with a medium-CCK-8 mixture (10:1 by volume). After 4 hours, the absorbance at 450 nm was measured as a background. The cells were then incubated with the indicated reagents for 48 hours. Finally, the medium was replaced again with the medium-CCK-8 mixture. After 4 hours, the absorbance at 450 nm was measured to show cell growth.
  • CCK-8 Cell Counting Kit-8
  • reagent concentrations used in the cell studies were as follows: 10 ng/mL IL-1 ⁇ , 1 ⁇ M CCX771 or CCX704 (both compounds were supplied by ChemoCentryx, Inc., Mountain View, CA, USA).
  • Tubule formation assay hypoxic-conditioned media for endothelial tubule formation assays. Briefly, cardiac fibroblasts in DMEM containing 3% FBS received hypoxia for 12 hours by AnaeroPack-Anaero (MGC, Japan). Conditioned medium was collected. For tubelet formation assays, Matrigel (Corning, NY, USA) with reduced growth factors was dispersed in 96-well plates (40 ⁇ L/well) using a cold pipette. Matrigel was then polymerized at 37 ° C for 1 hour. Endothelial cells were then trypsinized, resuspended in conditioned medium, and plated in plates at a concentration of 2 x 10 4 cells per well.
  • IL-1 ⁇ 10 ng/mL
  • TNF ⁇ 25 ng/mL
  • FBS medium containing 20% FBS
  • Microtubule formation was observed and photographed every 2 hours using an inverted phase contrast imaging microscope (Leica, Germany) with a 5x objective. Results from the 6th hour were used for analysis. Record and analyze at least 5 images from different cells.
  • Calcium ion response assay Cells with negative siRNA or si-CXCR7 were plated in 96-well plates and grown overnight in medium containing 20% FBS and 10 ng/mL IL-1 ⁇ .
  • For Calcium Fluorescence staining cell-like solution (AAT Bioquest, Sunnyvale, CA) with a Dye Cal-520 TM incubated for 2 hours. Fluorescence was monitored using excitation and emission at wavelengths of 490 and 525 nm, respectively, using a FlexStation 3 Microplate Reader (Molecular Devices, USA). After a 17 second baseline measurement, 100 ng/mL CXCL12 was added and the resulting calcium ion response was additionally measured for 78 seconds. The CXCR4 inhibitor AMD3100 (1 ⁇ g/mL) was added 2 hours before CXCL12 stimulation.
  • Imaging flow cytometric assay Cell surface expression of CXCR4 and CXCR7 was analyzed in HUVEC using ImageStreamX Mark II Imaging Flow Cytometer (Merck, Darmstadt, Germany). Briefly, HUVECs transfected with si-RNA or si-CXCR7 in 12-well plates were treated with IL-1 ⁇ (10 ng/mL) for 6-8 hours. It was then trypsinized, centrifuged and resuspended in 100 ⁇ L of FACS buffer (HBSS containing 0.6 mg/mL bovine serum albumin and 0.3 mM EDTA). The antibody was added to the cell suspension and placed on ice for 30 minutes for staining.
  • FACS buffer HBSS containing 0.6 mg/mL bovine serum albumin and 0.3 mM EDTA
  • CXCR7 was stained with anti-CXCR7 mAb (11G8, 1:100) and Alexa Fluor-594-conjugated secondary antibody.
  • CXCR4 was labeled with CXCR4 antibody (polyclonal antibody, 1:200, Sigma) and Alexa Fluor-488-conjugated secondary antibody. Cells were then analyzed and photographed using the ImageStreamX Mark II Imaging Flow Cytometer.
  • RNA isolation cells were lysed in TRIzol (Invitrogen). The solution was then mixed with chloroform (5:1, by volume) and centrifuged (12,000 g; 15 min; 4 °C). The aqueous phase was collected, mixed with isopropanol and centrifuged (12,000 g; 10 min; 4 ° C). After washing with 75% alcohol, the resulting RNA can be used. use Select Master Mix (Invitrogen) for quantitative RT-PCR.
  • the primers used in this study are as follows:
  • CXCR4 forward CTCTACAGCAGCGTTCTC;
  • eNOS forward GGCATCACCAGGAAGAAG;
  • Sections (5 ⁇ m) from paraffin-embedded tissues were dewaxed, rehydrated, and subjected to antigen retrieval by boiling in EDTA antigen-repairing water (pH 9.0; ZSGB-BIO, Beijing, China) for 2 minutes.
  • HUVEC was plated on coverslips (NUNC) and fixed in 4% paraformaldehyde for 20 minutes. The samples were then incubated with goat serum containing 0.3% Triton X-100 for blocking and membrane disruption. After incubation, the antibody was incubated overnight at 4 °C with the primary antibody and the samples were incubated with Alexa Fluor-594 coupled and/or Alexa Fluor-488 conjugated secondary antibody for 3 hours at room temperature.
  • the coverslips were covered with VectaShield medium containing DAPI to stain the nuclei.
  • Sections were imaged using a Zeiss inverted fluorescence microscope (AXI0; Zeiss) equipped with Zen software or a laser scanning confocal microscope (SP8; Leica) equipped with a 20x water immersion objective. Images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA).
  • the CXCL12 concentration was determined using a human CXCL12 ELISA kit (R&D Systems, Minnesota, USA).
  • PDGF-BB concentrations were determined using a human PDGF-BB Quantikine ELISA kit (R&D Systems, Minnesota, USA).
  • Plasma glucose (GLU), total cholesterol (CHOL, triglyceride (TG), free fatty acid (FFA), low density lipoprotein cholesterol (LDL-C) using an automated biochemical analyzer (AU5421, Beckman Coulter, California, USA) ), high density lipoprotein cholesterol (HDL-C) and alanine aminotransferase (ALT) were measured.
  • MAEC was pre-starved for 6-8 h in medium containing 3% FBS and added to the indicated reagent temperature for 12 hours.
  • the cells were then lysed in RIPA buffer containing protease inhibitors (Roche, Basel, Switherland), centrifuged (15800 g, 10 min), cell lysates were mixed with loading buffer and separated by 10% SDS-PAGE, then transferred to On the PVDF membrane. Hybridization of the membrane with the designated antibody. Some membranes re-hybridize with actin antibodies after decolorization.
  • mice were intravenously injected with rose bengal (50 mg/kg).
  • the left common carotid artery was exposed to a 2.5-mW green laser (540 nm; Melles Griot Inc).
  • Blood flow was continuously detected by pulsed Doppler (Transonic, Sidney, Australia) from the onset of injury until a stable blockage (defined as no blood flow within 2 minutes) or 90 minutes without occlusion occurred.
  • the occlusion time is defined as the time between the onset of vascular injury and the occurrence of stable occlusion. To calculate the average occlusion time, the obstruction time for animals that did not occlude was classified as 90 minutes.
  • ACD anticoagulated whole blood (ACD: blood volume ratio 1:9) was mixed with the same volume of pre-warmed physiological saline, and collagen (2 ⁇ g/mL) was added to induce aggregation.
  • the sample was equilibrated at 37 ° C for 7 minutes before the measurement. Platelet aggregation was then carried out in a Chronolog 710 aggregometer (Chronolog, Havertown, PA, USA) under constant agitation (1200 rpm) at 37 °C. The results were recorded and analyzed using Aggro/Link5 software (Chronolog, Havertown, PA, USA).
  • platelets were activated in vivo and in vitro, respectively.
  • the platelet activation method is the same as that used to measure platelet aggregation. Briefly, ACD anticoagulated whole blood was mixed with the same volume of pre-warmed physiological saline. The samples were equilibrated at 37 °C for 7 minutes and then collagen (2 ⁇ g/mL) or U46619 (2 mM; Sigma) was added to activate platelets. After 30 minutes, centrifugation (6000 g, 5 min, 4 ° C) and plasma were collected for detection of CXCL12. In vivo, anesthetized mice were intravenously injected with U46619 (20 ug/mouse). EDTA anticoagulated whole blood was collected within 10 minutes.
  • Platelets were depleted by intravenous injection of CD41 antibody (20 ug/mouse; Ebioscience, USA). EDTA anticoagulated whole blood samples were collected before injection, 2 hours and 5 days after injection, respectively.
  • the patients in this study were 95 patients who were diagnosed with acute myocardial infarction (AMI) and who underwent PCI immediately from August 2014 to January 2015.
  • AMI acute myocardial infarction
  • Example 1 Endothelial expression of CXCR7 in injured arteries of mice and humans
  • CXCR7 The vascular expression of CXCR7 in mice was first examined.
  • CXCR7 is expressed at low levels in healthy mouse dispersed endothelial cells (Fig. 1A). However, in damaged arteries, CXCR7 expression is upregulated and is mainly found in endothelial cells of the neointimal, colocalizing with the endothelial cell marker vWF (Fig. 1B).
  • Fig. 1C the atherosclerotic plaque shoulder
  • Fig. 1D microvessels in the plaque
  • mice The baseline arterial expression of CXCR7 in cKO mice and littermates was similar before tamoxifen induction (Fig. 1E).
  • mice After tamoxifen treatment, mice underwent endothelium exfoliation injury by angioplasty in the femoral artery. This injury induces vascular hyperplasia and mimics clinical restenosis after percutaneous coronary intervention. All mice were not subjected to genetic manipulation related to lipid metabolism and fed a normal diet.
  • endothelial CXCR7 The mRNA expression of endothelial CXCR7 in endothelial cells isolated from cKO mice was essentially zero (Fig. 2A), which was further confirmed by immunostaining of damaged arteries (Fig. 2B). Deletion of CXCR7 did not alter the expression of CXCR4 and CXCL12 (Fig. 2G). Endothelial exfoliation damage leads to neointimal hyperplasia. Deletion of endothelial CXCR7 significantly increased the ratio of the neointimal zone and neointimal to medial membrane, but did not alter the medial thickness (Fig. 2C-F). Deletion of endothelial CXCR7 did not alter the body weight or plasma lipids of these normal blood lipid mice.
  • Example 3 CXCR7 increases IL-1 ⁇ -treated endothelial cell proliferation and promotes endothelial regeneration after endothelial exfoliation injury
  • IL-1 ⁇ stimulation up-regulated CXCR7 (Figs. 4A and 4B), CXCR4 and CXCL12 expression (Fig. 4I).
  • IL-1 ⁇ promotes proliferation of CXCR7-functioning endothelial cells but does not promote proliferation of CXCR7-deficient cells (Fig. 4C and 4D).
  • endothelial cells were treated with the CXCR7-specific antagonist CCX771 (which has an IC50 of about 5.3 nM, which does not affect the binding of CXCL12 to CXCR4) (Journal of immunology. 2009; 183:3204-3211) and the control compound CCX704.
  • CCX771 inhibits proliferation of endothelial cells from the lung source (Fig. 4C) and aortic source (Fig. 4D). CCX771 treatment reduced ERK phosphorylation but did not affect JNK or p38 phosphorylation ( Figures 4E and 4F). This is consistent with the role of CXCR7 in promoting endothelial cell proliferation. This growth-promoting effect was not significant in the absence of IL-1 ⁇ treatment (Fig. 4J). In addition, in HUVEC, CCX771 or siRNA knockdown CXCR7 inhibited cell proliferation in the presence of IL-1 ⁇ (Figs. 4G and 4H). Similar results were observed when TNF ⁇ was used (Fig. 4K).
  • CXCR7 can promote endothelial regeneration of damaged blood vessels, promote endothelial cell repair, and reduce blood vessel stenosis caused by injury.
  • CXCR4 siRNA or AMD3100 (a CXCR4 antagonist with an IC50 of about 44 nM (Journal of immunology. 2009; 183:3204-3211) does not affect the binding of CXCL2 to CXCR7 (J Exp Med. 2006; 203: 2201-2213) Or with the weak binding of CXCR7, there was no significant difference in the proliferation of endothelial cells treated with Ki of about 34.5 ⁇ M (Molecular pharmacology. 2009; 75: 1240-1247) (C and D of Fig. 4L). It can be seen that inhibition of CXCR4 does not affect endothelial cell proliferation.
  • Tubule formation was used to examine the role of CXCR7 in endothelial cell neovascularization responses.
  • Blocking CXCR7 by siRNA significantly inhibited angiogenesis in HUVEC and HAEC as well as mouse EC ( Figures AA-D and A and B of Figure 4L).
  • endothelial CXCR7 deletion significantly reduced blood flow recovery after femoral artery ligation, which was detected by laser Doppler imaging (Fig. 5E-G). Further histochemical staining of the endothelium showed a decrease in the number of blood vessels in the ischemic gastrocnemius muscle ( Figures 5H and 5I).
  • CXCR7 or CXCR4 Knockdown of CXCR7 or CXCR4 by small RNA (si-RNA) transfection.
  • Transfection control si-Neg
  • si-CXCR7 si-CXCR7 1, si-CXCR7 2, and si-CXCR7 3
  • si-CXCR4 si-CXCR4 1, si
  • MAEC mouse arterial endothelial cells
  • CXCR7 (A) and CXCR4 (C) protein expression was determined.
  • a (CXCR4 immunoblot picture) and B show that CXCR7 knockdown has no effect on CXCR4 expression. *, p ⁇ 0.05 vs. si-Neg.
  • endothelial cell CXCR7 plays a key role in promoting ischemia-induced angiogenesis, whereas ischemia-induced angiogenesis was previously thought to be mediated only by the interaction of CXCL12 with CXCR4 (Trends Immunol. 2007; 28:299 -307).
  • Example 5 Deletion of endothelial CXCR7 impairs cardiac function after MI and increases mortality and infarct size
  • endothelial CXCR7 deletion significantly shortened survival and reduced cumulative survival within 30 days after MI compared to control mice (Ctl) (Fig. 6A).
  • cKO showed significant disruption of cardiac function and remodeling after MI, including decreased EF, E/A, and LVAWd, although there was no change in baseline cardiac function of cKO ( Figure 6B-F, Tables 2 and 3).
  • n 12 in both groups.
  • n 7Ctl, 6cKO.
  • Example 6 Gene delivery of CXCR7 to infarcted hearts improves cardiac function and reduces infarct size after MI
  • Ad-CXCR7 a recombinant adenovirus expressing CXCR7 expressing CXCR7
  • Left ventricular cavity see Materials and General Methods for details.
  • the results showed that Ad-CXCR7 delivery improved cardiac function after MI and reduced infarct size compared to the control vehicle ( Figure 8, Table 4).
  • n 10Ad-Neg.11Ad-CXCR7.
  • Example 7 Activation of CXCR7 promotes endothelial cell proliferation and reduces infarct size
  • Mouse aortic endothelial cells were stimulated with IL-1 ⁇ and treated with CXCR7 selective agonist TC14012 (https://www.rndsystems.com/cn/products/tc-14012_4300) (Cayman Chemical, Michigan, USA) Hours were then measured for cell growth using Cell Counting Kit-8 (CCK-8; Yeasen, Shanghai, China). The results showed that TC14012 significantly promoted the growth of mouse aortic endothelial cells at 100 ng/mL compared to the control (0 ng/mL) (p ⁇ 0.05) (Fig. 9).
  • CXCR7 selective agonist such as TC14012 to activate CXCR7 can reduce the range of myocardial infarction caused by ischemia, and has a protective effect on ischemic myocardium.
  • the reason and activation of CXCR7 directly promote vascular endothelial cells in myocardial tissue of infarcted area. Proliferation is associated with angiogenesis, which facilitates myocardial remodeling and recovery of cardiac function after MI.
  • Example 8 Deletion of endothelial CXCR7 and pharmacological blockade of CXCR7 raises the level of CXCL12 in the circulation
  • CXCR7 conditional knockout mice were constructed and photochemically induced in mice as described in the literature (The Journal of clinical investigation. 2006; 116: 1391-1399). Thrombosis assessment. The time to complete occlusion of cKO mice was significantly shorter compared to control mice born in littermates (Ctl) (Fig. 11A & B). Deletion of CXCR7 by endothelial cells leads to an increase in the level of CXCL12 in the circulation (Fig. 11E), which is consistent with the function of the ligand scavenger of CXCR7 (PLoS One. 2010; 5: e9175; Blood.
  • Example 9 Elevated levels of CXCL12 in the circulation promote thrombosis through the CXCL12-CXCR4 signaling pathway
  • CXCL12 promotes platelet activation via its cognate receptor CXCR4.
  • CXCL12 receptor for CXCL12
  • a venous access was established in mice to directly infuse CXCL12 (infusion of CXCL12 or physiological saline in saline at 0.25 or 0.5 ng/min) In the bloodstream (Figure 12A).
  • CXCL12 was confirmed to be successfully delivered by measuring the plasma concentration of CXCL12 (Fig. 12B).
  • Moderate elevation of CXCL12 in the post-infusion cycle promoted ex vivo platelet aggregation in a concentration dependent manner (Figure 12C&D).
  • the CXCL12 infusion also accelerated thrombus formation in vivo (Figure 12E&F).
  • This prothrombotic effect of CXCL12 was abolished by pretreatment with the CXCR4-specific antagonist AMD3100 (Fig. 12E & F).
  • AMD3100 treatment also abolished the prothrombotic effect caused by endothelial cell CXCR7 knockout (Fig. 13A & B).
  • a physiologically elevated CXCL12 in the circulation causes a tendency to thrombosis.
  • Example 10 Platelet promotes and responds to elevated CXCL12 following endothelial cell CXCR7 deletion
  • Platelets contain CXCL12 and release CXCL12 upon activation (The Journal of experimental medicine. 2006; 203: 1221-1233), but it is unclear whether platelets in vivo are the source of CXCL12 and whether platelets contribute to the elevation of CXCL12 in cKO.
  • Fig. 14I platelets were depleted by injection of anti-CD41 antibodies to mice, which resulted in substantial depletion of platelets 2 hours after injection and recovered on day 5 (Fig. 14I).
  • Platelet depletion resulted in a decrease in circulating CXCL12 in both cKO and Ctl, but cKO mice maintained higher CXCL12 levels compared to both (Fig. 14G).
  • CXCL12 in the circulation also includes CXCL12 from other sources than platelets, and circulating CXCL12 levels are regulated by endothelial cell CXCR7. It is worth noting that platelets contribute more to circulating CXCL12 in cKO than Ctl ( Figure 14H).
  • CD62P p-selectin
  • Example 11 Correlation between CXCL12 levels and platelet reactivity in human blood circulation
  • the inventors further investigated the association of circulating CXCL12 with platelet reactivity in patients with acute myocardial infarction.
  • Peripheral venous blood from consecutively enrolled patients was collected for measurement of plasma CXCL12 and platelet reactivity was measured in situ using the VerifyNow method (see Materials and General Methods section for details).
  • CXCL12 levels were positively correlated with ADP-induced platelet reactivity (Fig. 15A), which supports a direct effect of CXCL12 in the circulation on platelet activation.
  • CXCL12 is not associated with VerifyNow-AA (Fig. 15B), which may be related to the use of aspirin in these patients (Table 1), as TxB 2 release triggered by CXCL12 mediates platelet aggregation in vitro (Blood. 2000; 96:50- 57).

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Abstract

La présente invention concerne une composition pharmaceutique et une méthode servant au traitement d'une maladie cardiovasculaire. En particulier, la présente invention concerne une méthode de ciblage de CXCR7 et une composition pharmaceutique pour traiter une lésion endothéliale vasculaire chez un sujet ou pour améliorer un remodelage cardiaque chez un sujet après un infarctus du myocarde.
PCT/CN2018/074264 2017-01-26 2018-01-26 Composition pharmaceutique ciblant cxcr7 et méthode WO2018137701A1 (fr)

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Citations (4)

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WO2009120186A1 (fr) * 2008-03-24 2009-10-01 Carolus Therapeutics, Inc. Procédés et compositions pour traiter l'athérosclérose et des états associés
CN102088993A (zh) * 2008-03-20 2011-06-08 卡罗勒斯治疗公司 炎症的治疗方法
WO2012047339A2 (fr) * 2010-06-28 2012-04-12 The General Hospital Corporation Anti-cxcr4 en tant que sensibilisant à des agents de traitement d'un cancer
CN106065401A (zh) * 2015-04-20 2016-11-02 成都医学院 慢病毒介导cxcr7高表达工程化内皮祖细胞在缺血性疾病中的治疗应用

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CN103397052A (zh) * 2013-08-15 2013-11-20 温州医科大学 高表达人cxcr7基因的内皮祖细胞及制备方法和应用

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CN102088993A (zh) * 2008-03-20 2011-06-08 卡罗勒斯治疗公司 炎症的治疗方法
WO2009120186A1 (fr) * 2008-03-24 2009-10-01 Carolus Therapeutics, Inc. Procédés et compositions pour traiter l'athérosclérose et des états associés
WO2012047339A2 (fr) * 2010-06-28 2012-04-12 The General Hospital Corporation Anti-cxcr4 en tant que sensibilisant à des agents de traitement d'un cancer
CN106065401A (zh) * 2015-04-20 2016-11-02 成都医学院 慢病毒介导cxcr7高表达工程化内皮祖细胞在缺血性疾病中的治疗应用

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