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WO2019037222A1 - Utilisation d'un inhibiteur de gpr31 dans la préparation de médicaments - Google Patents

Utilisation d'un inhibiteur de gpr31 dans la préparation de médicaments Download PDF

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WO2019037222A1
WO2019037222A1 PCT/CN2017/106949 CN2017106949W WO2019037222A1 WO 2019037222 A1 WO2019037222 A1 WO 2019037222A1 CN 2017106949 W CN2017106949 W CN 2017106949W WO 2019037222 A1 WO2019037222 A1 WO 2019037222A1
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gpr31
related diseases
ischemia
reperfusion injury
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李红良
张晓晶
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Wuhan University WHU
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biomedical technology, and particularly relates to the use of a GPR31 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, fat Metabolic abnormalities and related diseases.
  • G protein-coupled receptor is a type of membrane protein with 7 transmembrane structures. It has more than 800 family members and is the largest membrane protein in mammalian genome. In humans, GPCRs are widely expressed in tissues and organs such as the cardiovascular system, immune system, and nervous system, and participate in growth and development and various pathophysiological processes. Due to its wide distribution and versatility, GPCR family molecules are regarded as the most potential drug development targets at present, and about 20-30% of FDA-approved marketed drugs use GPCR as a target.
  • the orphan receptor GPR31 is a GPCR family molecule, first discovered by Alessandra Zingoni et al. in 1997 (Zingoni, A. et al. Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor. Genomics 42,519-523 , doi: 10.106/geno. 1997.4754 (1997)). At present, the research on GPR31 is mainly concentrated in the field of cancer, and its function in other pathological processes is still unclear.
  • GPR31 protein contains 319 amino acids with a molecular weight of 35KDa and is highly expressed in platelets, immune cells and various cancer cells, including bladder cancer cells, breast cancer cells, chronic lymphoblastic leukemia cells, etc.
  • the Orphan Receptor GPR31 Is the Platelet and HUVEC Receptor for 12(S)-HETE.Blood 114(2008).
  • Studies have shown that GPR31 expression in clinical patients with colon cancer tissue is significantly higher than adjacent normal tissues, and is positively correlated with cancer metastasis rate, and negatively correlated with 5-year survival rate (Zou, Y. et al.
  • GPR31 binds to RAS family molecules such as KRAS, HRAS, NRAS, etc., and regulates the cell membrane localization of KRAS, thereby participating in the development of cancer (Fehrenbacher N, et al.
  • the G protein-coupled receptor GPR31 promotes membrane association of KRAS [J] .J Cell Biol, 2017: jcb.201609096.).
  • Ischemia-Reperfusion Injury is the first concept proposed by Jennings in 1960. It refers to blood reperfusion after tissue and organ ischemia, which not only fails to restore the function of tissues and organs, but also increases the dysfunction and structure of tissues and organs. damage. Ischemia-reperfusion injury can occur in many important organs including heart, liver, lung, kidney, gastrointestinal tract, and the like.
  • Hepatic Ischemia Reperfusion Injury is a common pathological process in liver surgery. It is more common in pathological and physiological processes such as shock, liver surgery requiring liver blood flow, and liver transplantation.
  • liver transplantation, thrombolytic therapy and hepatic occlusion surgery have been carried out more and more, although liver protection, surgical techniques and intraoperative monitoring are improving, but ischemia and reperfusion
  • the liver injury is still the main cause of postoperative organ dysfunction, graft failure and even patient death.
  • liver tissue cells undergo a series of metabolic, structural and functional damages, which are easy to induce liver failure, which is one of the main factors affecting disease prognosis, surgical success rate and patient survival rate.
  • Acute coronary artery obstructive disease is one of the main causes of death of cardiovascular and cerebrovascular diseases.
  • bypass surgery intervention, and thrombolysis
  • the mortality rate of patients with acute myocardial infarction is still high.
  • Myocardial reperfusion injury occurs in coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, and coronary artery bypass surgery.
  • Myocardial reperfusion injury is mainly caused by free radicals, calcium overload, cell adhesion molecule-mediated neutrophil adhesion, aggregation, exudation, apoptosis, nitric oxide, complement system, renin-angiotensin. , nuclear factor- ⁇ B and so on.
  • the kidney is also a high perfusion organ, sensitive to ischemia and ischemia-reperfusion.
  • Renal ischemia-reperfusion injury is an important injury link of ischemic acute renal failure, and also a limiting factor affecting early recovery of renal function in kidney transplantation.
  • the prevention and treatment strategies for renal ischemia-reperfusion injury mainly include: inhibition of leukocyte activation and leukocyte-endothelial cell interaction, neutralization of reactive oxygen species, and anti-endothelin.
  • the mechanism of ischemic brain injury involves at least the following aspects: excitotoxicity, depolarization around the infarct, inflammation, and apoptosis.
  • Cardiac hypertrophy is an increase in the volume and weight of cardiomyocytes produced by the heart to accommodate various stimuli. His pathological changes include cardiomyocyte hypertrophy, myocardial stromal cell proliferation, and cardiac extracellular matrix alterations, ie, myocardial remodeling. The traditional view is that mature cardiomyocytes end differentiated, lose mitotic ability, and cannot enter the cell cycle; however, there is evidence that there are two processes of cardiomyocyte apoptosis and proliferation in cardiac development and pathology to maintain the steady state of cardiac function. . There are many diseases that cause cardiac hypertrophy in the clinic, such as primary or secondary hypertension, myocardial infarction, valvular disease, and congenital heart disease.
  • cardiac hypertrophy Although early cardiac hypertrophy is conducive to maintaining normal heart function, but cardiac hypertrophy itself can increase myocardial oxygen consumption, reduce myocardial compliance, it will lead to heart failure for a long time, increase the incidence of sudden death.
  • mechanical and neurohumoral factors induce cardiac hypertrophy, including renin-angiotensin system components, catecholamines, insulin-like growth factors, and nitric oxide synthesis systems.
  • the process of cardiomyocyte hypertrophy mainly includes four aspects: the appearance of stimulation signals, transmembrane signaling, immediate activation of early response genes, and expression of functional or structural proteins to the "embryonic" phenotype.
  • the MAPK family signaling pathway, Ca 2+ and its dependent signaling pathway, phosphatidylinositol 3-kinase and its mediated signaling pathway, and JAK/STAT pathway were all involved in the signaling of cardiac hypertrophy, and between the pathways.
  • There are also inextricably linked networks that form intricate signals (Dai Wenjian et al., Advances in Molecular Mechanisms of Cardiac Hypertrophy, Advances in Cardiovascular Diseases, Vol. 30, No. 1, 2009, 47-50).
  • the liver plays an important role in the body's fat metabolism. It participates in many important aspects of lipid metabolism, including fatty acid uptake and synthesis, lipid processing, storage, oxidative decomposition and export. When the amount of fatty acids obtained by the liver exceeds its processing capacity, lipids are deposited in the liver cells in the form of triglycerides, resulting in hepatic steatosis, becoming simple liver steatosis, and then developing into nonalcoholic steatohepatitis. Patients can progress to liver fibrosis, cirrhosis, and even liver cancer (Lu Ran et al., the pathogenesis of non-alcoholic fatty liver disease caused by lipid metabolism disorder, Journal of Clinical Hepatology, 2015, Vol. 31, No.
  • GPR31 Overexpression of GPR31 aggravates the activity of hepatocytes, cardiomyocytes and kidney cells caused by hypoxia and reoxygenation, and promotes the inflammatory response of the corresponding cells, indicating that GPR31 can promote ischemia, reperfusion injury of liver, heart and kidney. And the development of other inflammatory reactions that occur in these organs.
  • the decrease of GPR31 expression in hepatocytes can inhibit cell lipid deposition induced by palmitate and oleic acid stimulation, indicating that GPR31 is expected to be a novel target for regulating hepatocyte fat accumulation, and is applied to abnormal fat metabolism and related diseases. During treatment.
  • GPR31 Overexpression of GPR31 in cardiomyocytes aggravates cardiomyocyte hypertrophy caused by angiotensin II, indicating that GPR31 can promote the development of cardiac hypertrophy-related diseases.
  • GPR31 can be used as a therapeutic target for ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases, inflammatory diseases of the heart, abnormal fat metabolism and related diseases.
  • a first aspect of the present invention provides a use of a GPR31 inhibitor for the preparation of a medicament for treating ischemia-reperfusion injury and a related disease thereof, cardiac hypertrophy and a related disease thereof, an inflammatory disease of the heart, or Abnormal fat metabolism and related diseases.
  • the ischemia-reperfusion injury and related diseases are selected from the group consisting of hepatic ischemia-reperfusion injury and related diseases, cardiac ischemia-reperfusion injury and related diseases, renal ischemia-reperfusion injury and related diseases, And/or cerebral ischemia-reperfusion injury and related diseases.
  • the ischemia-reperfusion injury may be caused by various reasons such as organ transplantation, partial or complete resection of tissue, and tissue ischemia caused by vascular embolization.
  • Inflammatory factors of hepatic ischemia-reperfusion injury and related diseases include, but are not limited to, liver cysts, liver transplantation, thrombolytic therapy, and hepatic occlusion.
  • Inflammatory factors of cardiac ischemia-reperfusion injury and related diseases include but are not limited to: myocardial infarction, myocardial infarction injury, heart transplantation, coronary thrombolysis, percutaneous coronary angioplasty, intracoronary dilatation, coronary Arterial bypass.
  • causes of renal ischemia-reperfusion injury and related diseases include, but are not limited to, kidney transplantation, renal cysts, and renal vascular surgery.
  • the triggering factors of cerebral ischemia-reperfusion injury and related diseases include but are not limited to: stroke, cerebrovascular surgery and the like.
  • the ischemia-reperfusion injury and related diseases are hepatic ischemia-reperfusion injury, cardiac ischemia-reperfusion injury, renal ischemia-reperfusion injury, and/or cerebral ischemia-reperfusion injury.
  • the cardiac hypertrophy and related diseases include, but are not limited to, cardiac hypertrophy, heart failure, arrhythmia, arterial embolism, coronary heart disease, angina pectoris, heart block, and the like.
  • cardiac hypertrophy There are many diseases that cause cardiac hypertrophy, such as primary or secondary hypertension, myocardial infarction, valvular disease, and congenital heart disease.
  • the cardiac hypertrophy and related diseases are cardiac hypertrophy and heart failure.
  • Inflammatory diseases of the heart include, but are not limited to, myocarditis, endocarditis.
  • the abnormalities in fat metabolism and related diseases include, but are not limited to, insulin resistance, metabolic syndrome, nonalcoholic fatty liver disease, obesity, diabetes, hyperglycemia, hyperlipemia, and the like.
  • nonalcoholic fatty liver disease includes: simple liver steatosis, nonalcoholic steatohepatitis, liver fibrosis, cirrhosis, and liver cancer.
  • the abnormal fat metabolism and related diseases are nonalcoholic fatty liver disease, obesity, hyperlipemia, insulin resistance, and more preferably: simple liver steatosis, nonalcoholic steatohepatitis, obesity, hyperlipidemia.
  • the GPR31 inhibitor may be an inhibitor that inhibits GPR31 protein activity or protein level, or an inhibitor that inhibits mRNA levels of GPR31.
  • the inhibitory activity can be reversible or irreversible.
  • Inhibitors that inhibit GPR31 protein activity or protein levels include, but are not limited to, antibodies to GPR31, proteins, polypeptides, enzymes, natural compounds, synthetic compounds, organics, inorganics that inhibit GPR31 protein activity or protein levels.
  • the inhibitor that inhibits GPR31 protein activity or protein level refers to a substance that binds to GPR31 but does not produce a biological response upon binding. The inhibitor can block, inhibit or attenuate the agonist-mediated response and can compete with the agonist for binding to GPR31.
  • the inhibitor that inhibits the mRNA level of GPR31 may be an antisense nucleic acid sequence, siRNA, miRNA, shRNA, dsRNA, or other protein, polypeptide, enzyme, or compound capable of inhibiting the mRNA level of GPR31.
  • the antibodies include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv) (including bispecific) scFv), single chain antibody, Fab fragment, F(ab') fragment, disulfide-linked Fv (sdFv) and any of the above epitope binding Fragment.
  • antibodies useful in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.
  • the immunoglobulin molecule used in the present invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class of immunoglobulin molecules (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) Or subclass.
  • the antibody is a human or humanized monoclonal antibody.
  • a "human" antibody includes an antibody having an amino acid sequence of a human immunoglobulin, and includes an antibody isolated from a human immunoglobulin library or from a mouse or other animal that expresses the antibody from a human gene.
  • the inhibitor is a shRNA of mRNA of GPR31, and the interference targeting sequence is CACTCTCCTGCCTTCAGTTTG.
  • the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'.
  • the medicament further comprises a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable excipients are various excipients commonly used or known in the pharmaceutical field, including but not limited to: diluents, binders, antioxidants, pH adjusters, preservatives, lubricants, disintegrators, etc. .
  • the diluent is, for example, lactose, starch, cellulose derivative, inorganic calcium salt, sorbitol or the like.
  • the binder is, for example, starch, gelatin, sodium carboxymethylcellulose, polyvinylpyrrolidone or the like.
  • the antioxidant is, for example, vitamin E, sodium hydrogen sulfite, sodium sulfite, butylated hydroxyanisole or the like.
  • the pH adjusting agent is, for example, hydrochloric acid, sodium hydroxide, citric acid, tartaric acid, Tris, acetic acid, sodium dihydrogen phosphate, disodium hydrogen phosphate or the like.
  • the preservative is, for example, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, m-cresol, benzalkonium chloride or the like.
  • the lubricant is, for example, magnesium stearate, finely divided silica gel, talc, or the like.
  • the disintegrant is, for example, starch, methyl cellulose, xanthan gum, croscarmellose sodium or the like.
  • the dosage form of the medicament of the present invention may be in the form of an oral preparation, such as a tablet, a capsule, a pill, a powder, a granule, a suspension, a syrup, etc.; or a dosage form for injection administration, such as an injection solution, a powder injection, etc., Intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage form forms used are well known to those of ordinary skill in the pharmaceutical arts.
  • the medicament of the present invention can be administered to a subject by a route known in the art including, but not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal. Cheek, Sublingual, intranasal, transdermal, etc.
  • the dosage administered will depend on the age, health and weight of the recipient, the type of combination, the frequency of treatment, the route of administration, and the like.
  • the drug can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three or four times daily.
  • the dose can be administered one or more times, and the administration time can be from one day to several months or longer.
  • the medicament can also be used in combination with other drugs which can ameliorate or inhibit diseases associated with ischemia-reperfusion injury.
  • the medicament can also be used in combination with other drugs which can ameliorate or inhibit cardiac hypertrophy and related diseases.
  • the medicament can also be used in combination with other drugs which can ameliorate or inhibit inflammatory diseases of the heart.
  • the drug can also be used in combination with other drugs which can ameliorate or inhibit abnormalities in fat metabolism.
  • a second aspect of the present invention provides a use of a vector for expressing a shRNA targeting mRNA of GPR31 for the preparation of a medicament for treating ischemia-reperfusion injury and related diseases, cardiac hypertrophy and related diseases thereof , inflammatory diseases of the heart, or abnormalities in fat metabolism and related diseases.
  • the disease is as defined above.
  • the target sequence for the shRNA interference is CACTCTCCTGCCTTCAGTTTG.
  • the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'.
  • the vector may be an expression vector.
  • a promoter and a transcription termination sequence operably linked to the above shRNA sequence may be included in the expression vector.
  • the expression vector can be a eukaryotic expression vector.
  • the eukaryotic expression vector can be a plasmid expression vector or a viral expression vector.
  • the plasmid expression vector may be, but not limited to, pcDNA3.1+/-, pcDNA4/HisMax B, pSecTag2 A, pVAX1, pBudCE4.1, pTracer CMV2, pcDNA3.1(-)/myc-His A, pcDNA6-Myc/His B, pCEP4, pIRES, pIRESneo, pIRES hyg3, pCMV-myc, pCMV-HA, pIRES-puro3, pIRES-neo3, pCAGGS, pSilencer1.0, pSilencer2.1-U6 hygro, pSilencer3.1-H1hygro , pSilencer3.1-H1neo, pSilencer4.1-CMV neo.
  • the viral expression vector may be a lentiviral vector, an adenoviral vector, an adeno-associated virus expression vector or other types of viral vectors, including but not limited to pLKO.1, pLVX-IRES-ZsGreen1, pCDH-EF1-Luc2-T2A-tdTomato, pCDH-MSCV-MCS-EF1-Puro, pCDH-MSCV-MCS-EF1-copGFP, pLVX-ZsGreen1-C1, pAdEasy-1, pShuttle-CMV, pShuttle, pAdTrack, pAdTrack-CMV, pShuttle-IRES-hrGFP-1, pShuttle-IRES-hrGFP-2, pShuttle-CMV-lacZ, pShuttle-CMV-EGFP-C, pXC1, pBHGE3, pAAV-MCS, pAAV-RC,
  • a third aspect of the present invention provides a use of a lentiviral vector comprising a shRNA targeting mRNA of GPR31 for the preparation of a medicament for treating ischemia-reperfusion injury and a related disease thereof, cardiac hypertrophy and Related diseases, inflammatory diseases of the heart, or abnormalities in fat metabolism and related diseases.
  • the disease is as defined above.
  • the shRNA-interfering targeting sequence is CACTCTCCTGCCTTCAGTTTG or other targeting sequences that can interfere with GPR31 expression.
  • the shRNA sequence is: a forward oligonucleotide: 5'-CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG-3'; a reverse oligonucleotide: 5'-AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG-3'.
  • the lentiviral vector is a pLKO.1 vector.
  • a pharmaceutical carrier which can be used for the preparation of the medicament of the second and third aspects may be an injection vehicle conventionally used in the art, such as an isotonic NaCl solution, an isotonic glucose solution, or isotonicity.
  • a solution containing a buffer system such as a PBS solution or the like. It is also possible to selectively add a protective agent which is inactivated by preventing physical or chemical changes of the lentivirus, such as a divalent cation salt or a surfactant, depending on the needs of the preparation.
  • FIG. 1 Western-blot detection of GPR31 protein expression in liver tissue at different ischemic times.
  • GAPDH is a control standard.
  • FIG. 2 Identification of GPR31 protein expression after L02 cells were transfected with GFP and GPR31 overexpression lentivirus.
  • GAPDH is a control standard.
  • FIG. 3 Statistical analysis of LDH release test results after L02 cells were overexpressed and normally expressed GPR31, respectively (n.s. represents P ⁇ 0.05, ** represents P ⁇ 0.01).
  • Figure 4 Statistical analysis of RT-PCR results of mRNA levels of inflammatory factors Il-6, Tnf- ⁇ , and chemokine Cxcl2 after L02 cells were overexpressed and normally expressed GPR31, respectively. * represents 0.01 ⁇ P ⁇ 0.05, ** represents P ⁇ 0.01).
  • FIG. 5 Identification of GPR31 protein expression in H9C2 cells transfected with GFP and GPR31 overexpression lentivirus.
  • Figure 6 Statistical results of cell viability after hypoxia and reoxygenation in H9C2 cells under overexpression and normal expression of GPR31 (ns represent P ⁇ 0.05, * represents 0.01 ⁇ P ⁇ 0.05, ** represents P ⁇ 0.01).
  • Figure 7 Identification of GPR31 protein expression after HK2 cells transfected with GFP and GPR31 overexpression lentivirus.
  • Figure 8 Statistical analysis of the results of LDH release assay after hypoxia and reoxygenation in HK2 cells under overexpression and normal expression of GPR31 (n.s. represents P ⁇ 0.05, ** represents P ⁇ 0.01).
  • FIG. 9 Identification of GPR31 gene mRNA in L02 cells transfected with shRNA and shGPR31 lentivirus (** represents P ⁇ 0.01).
  • FIG 10 Microscopic examination of hepatocyte oil red O staining after stimulation with palmitate (PA) and oleic acid (OA) (PA 0.5 mM + OA 1 mM) in L02 cells with low expression and normal expression of GPR31, respectively.
  • PA+OA stands for palmitate and oleic acid stimulating groups.
  • FIG. 11A H9C2 cells were treated with angiotensin II in the presence of overexpressed and normally expressed GPR31, and then examined by microscopy.
  • Figure 11B Statistical diagram of cell surface area after treatment with angiotensin II in H9C2 cells overexpressing and normal expression of GPR31 (n.s. represents P ⁇ 0.05, ** represents P ⁇ 0.01).
  • Figure 11C Statistical analysis of RT-PCR results of mRNA expression levels of cell hypertrophic marker genes Anp and Myh7 after treatment with angiotensin II in H9C2 cells overexpressing and normal expression of GPR31 (ns represent P ⁇ 0.05) , ** represents P ⁇ 0.01).
  • mice purchased from Beijing Huakangkang Biotechnology Co., Ltd.
  • mice purchased from Beijing Huakangkang Biotechnology Co., Ltd.
  • background male C57BL/6 strain were selected as experimental subjects.
  • mice All experimental mice were housed in the SPF laboratory animal center of Wuhan University. Breeding conditions: room temperature between 22-24 ° C, humidity between 40-70%, alternating light and dark lighting time is 12h, free to drink water.
  • HEK293T human embryonic kidney cells, purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu43.
  • H9C2 rat cardiomyocytes, purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNR5.
  • the cells were cultured in DMEM high glucose medium (containing 10% FBS, 1% penicillin-streptomycin). Culture environment: 37 ° C, 5% CO 2 .
  • the sample was ground in a -80 ° C pre-cooled grinder adapter with a grinding parameter set to 30 Hz/s for 90 s.
  • the ultrasonic pyrolyzer lysed the sample (5 KHz/time, 1 s each time, interval 1 s, repeated 10 times), and placed on ice for 10 min after completion of the ultrasound.
  • the cells were added to the lysate, and after the completion of the lysis, the supernatant was centrifuged, and the protein sample was quantitatively collected using the BCA Protein Assay Kit.
  • the 2PVDF membrane was immersed in methanol for 15 s before use, and then placed in a transfer solution for use.
  • the film voltage was set to 250V, the current was set to 0.2A, and the transfer was 1.5h.
  • Sealing machine seals the film into the hybrid bag, and adds a primary antibody to seal it.
  • the extracted plasmid can be directly used for GPR31 transient transfer or construction of a lentiviral stable cell line.
  • GPR31 targeting interference sequence is CACTCTCCTGCCTTCAGTTTG, designing oligonucleotides suitable for pLKO.1 vector; forward oligonucleoside Acid: 5'CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTGTTTTTG3'; reverse oligonucleotide: 5'AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGAGCAAACTGAAGGCAGGAGAGTG3'; negative control siRNA sequence: CAACAAGATGAAGAGCACCAA;
  • the resulting plasmid can be used for lentiviral-mediated GPR31 knockdown cell line construction.
  • the 293T cells were digested with trypsin and transferred to a 6-well plate at 1 ⁇ 10 6 293 T/well.
  • PEI 1.6 ⁇ g/ ⁇ l
  • the virus-containing supernatant was harvested 48-72 h after transfection. After centrifugation at 3000 rpm for 10 min, the precipitate was removed and filtered through a 0.45 ⁇ m filter.
  • the filtered virus can be used immediately for infection or storage at -80 °C.
  • the cells were divided into normal control group and H/R experimental group.
  • the control group was changed to complete medium, and cultured at 37 ° C, 5% CO 2 .
  • the experimental group was changed to glucose-free and serum-free DMEM medium, and O 2 /CO was placed.
  • LDH cytotoxic colorimetric test kit G1782, Promega, Madison, WI, USA.
  • Cell viability was measured using a non-radioactive CCK-8 kit (CK04; Dojindo, Kumamoto, Japan). Carry out relevant tests according to the instructions.
  • mice were randomly divided into 6 groups, namely Sham group and operation group (divided into 5 different time points: ischemia 5 min, 10 min, 20 min, 40 min, 60 min).
  • the liver tissues of the mice in the surgery group and the Sham group were taken.
  • Western blot was used to detect the changes of GPR31 protein content in liver tissues of each group (three independent replicates).
  • the primary antibody used for WB was: Anti-GPCR GPR31 antibody (ab75579; Abcam), and the secondary antibody was: Peroxidase AffiniPure goat anti-rabbit-IgG (H+L) (#111-035-003; Jackson Laboratory).
  • a control standard with GAPDH as the expression level was:
  • the results are shown in Fig. 1.
  • the WB results of the Sham group showed almost no GPR31 band, and the GPR31 protein band became more and more obvious in the operation group with the prolongation of ischemia time. This result indicates a positive correlation between the expression of GPR31 protein and the severity of hepatic ischemia-reperfusion injury.
  • L02 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR31H/R group.
  • the adherent L02 cells were transiently transferred to the corresponding plasmids, and H/R treatment was performed 24 hours later (hypoxia 6 h, reoxygenation 6 h). After plasmid transfection was completed, the total protein was extracted, and the overexpression of GPR31 was detected by Western blot (3 independent replicates, 3 replicates each time).
  • the release of LDH in the culture medium was detected after H/R treatment (6 replicates per group) to evaluate the effect of GPR31 overexpression on H/R-induced hepatocyte injury; RNA was extracted for RT-PCR analysis (2 independent Repeat experiments, 3 replicates each time, to detect changes in inflammation-related cytokines and chemokine mRNA levels to evaluate the effect of GPR31 overexpression on H/R-induced hepatocyte inflammatory responses.
  • the LDH release test results were calculated as GFP control group values of 1, and the ratios of the remaining groups were calculated.
  • the primer sequences used in RT-RCR are as follows:
  • H9C2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR31 H/R group.
  • the corresponding recombinant lentiviral virus solution was infected with cultured H9C2 cells, and H/R treatment was performed 24 hours later (anoxia 1 h, reoxygenation 6 h). After plasmid transfection was completed, total cellular protein was extracted, and overexpression of GPR31 was detected by Western blot (3 independent replicates). Cell viability was measured after completion of H/R (6 replicates per group). The test result of the GFP control group was 1, and the ratio of the remaining groups to the group was calculated.
  • the WB test results are shown in Fig. 5. Compared with the GFP group, the GPR31 overexpressing histone band was significantly enhanced, that is, the overexpression of GPR31 was significant in H9C2 cells.
  • HK2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP H/R group, GPR31 H/R group.
  • the corresponding lentiviral solution was infected with HK2 cells, and H/R treatment (hypoxia for 3 h, reoxygenation for 24 h) was performed after 24 h.
  • the total protein was extracted after plasmid transfection, and the overexpression of GPR31 was detected by Western blot. 3 independent replicates).
  • the amount of LDH released from the culture medium (6 replicates per group) was measured after completion of H/R to evaluate the effect of GPR31 overexpression on H/R-induced renal cell injury. LDH release assay in GFP control group The result of the test is 1, and the ratio of the remaining groups to the group is calculated.
  • L02 cells were divided into 4 groups: shRNA control group, shGPR31 control group, shRNA experimental group, and shGPR31 experimental group.
  • Adherent L02 cells were transiently transfected into corresponding plasmids. After 24 h, palmitate (PA) and oleic acid (OA) (PA 0.5 mM + OA 1 mM) were added to the two experimental groups. The same amount of BSA was added to the control group. After 12 h, oil red O staining was performed.
  • the primer sequences used in RT-PCR are as follows:
  • RNA was extracted, and the mRNA content of GPR31 gene was detected by RT-PCR (three independent experiments were repeated twice, each time), and the results are shown in FIG.
  • the mRNA content of the GPR31 gene in the GPR31 knockdown group (shGPR31) was significantly lower than that in the shRNA control group.
  • the results of oil red O staining are shown in Fig. 10.
  • the cells in the control group had no obvious red color, and when stimulated with PA+OA, the cells stained with oil red O were significantly increased compared with the control group, and the GPR31 knockdown group (shGPR31) In the experimental group, the increase in the staining area was smaller than that in the shRNA experimental group. This result indicates that a decrease in GPR31 expression can inhibit lipid deposition of PA-stimulated L02 cells.
  • H9C2 cells were divided into 4 groups: GFP control group, GPR31 control group, GFP AngII group, GPR31 AngII group.
  • the corresponding lentiviral fluids were infected with cultured H9C2 cells, and after 24 hours, they were stimulated with 1 ⁇ M angiotensin II (Ang II) or PBS (control group) for 48 h, and then subjected to immunofluorescence assay. Lentivirus transfection After the formation, the total protein was extracted, and the expression of GPR31 protein in H9C2 cells was detected by Western blot.
  • the cells were harvested for RT-PCR analysis (2 independent replicates, 3 technical replicates each) to detect mRNA levels of the hypertrophic marker genes Anp and Myh7.
  • the cell surface area statistical results were calculated as the GFP control group value of 1, and the ratio of the remaining groups to the group was calculated.
  • the primer sequences used in RT-RCR are as follows:
  • H9C2 cell hypertrophy and cardiac hypertrophy markers were shown in Figure 11.
  • the cell surface area was significantly increased compared with the PBS control group, and the GPR31 overexpression group was significantly larger than GFP AngII.
  • Group Fig. 11A, B
  • mRNA analysis showed that the upregulation of cell hypertrophic marker genes Anp and Myh7 in GPR31 overexpression group was significantly higher than that in the control group after AngII treatment (Fig. 11C).

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Abstract

L'invention concerne l'utilisation de l'inhibiteur de GPR31 dans la préparation d'un médicament pour traiter des lésions d'ischémie-reperfusion et des maladies associées, une hypertrophie cardiaque et des maladies associées, des maladies inflammatoires cardiaques et un métabolisme anormal des graisses et des maladies associées. L'invention concerne également les séquences de ciblage spécifiques perturbées par GPR31 et les séquences de petits ARN en épingle à cheveux (shRNA).
PCT/CN2017/106949 2017-08-21 2017-10-19 Utilisation d'un inhibiteur de gpr31 dans la préparation de médicaments Ceased WO2019037222A1 (fr)

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CN113462769B (zh) * 2021-08-05 2023-06-30 中国医学科学院医药生物技术研究所 抑制剂/CaMKII体系及作为生物标志物中的应用
CN116019914B (zh) * 2021-10-25 2025-08-29 北京大学 Gpr180抑制剂在制备改善糖或脂代谢药物中的用途
CN115287276A (zh) * 2022-03-07 2022-11-04 兰州大学 Sem1蛋白、表达sem1蛋白工程化益生菌在制备治疗和/或预防心脏病药物中的应用
CN114848822A (zh) * 2022-06-01 2022-08-05 合肥工业大学 Gpr31抑制剂在制备预防和治疗血管钙化药物中的应用
CN116236477B (zh) * 2023-01-17 2024-03-26 复旦大学附属中山医院 溶血磷脂酸受体5拮抗剂在制备心脏保护药物中的应用
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