WO2022083748A1 - Utilisation d'un extrait de pépins de raisin dans la préparation de médicaments pour l'élimination ciblée de cellules sénescentes dans un microenvironnement tumoral et pour l'inhibition tumorale - Google Patents

Utilisation d'un extrait de pépins de raisin dans la préparation de médicaments pour l'élimination ciblée de cellules sénescentes dans un microenvironnement tumoral et pour l'inhibition tumorale Download PDF

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WO2022083748A1
WO2022083748A1 PCT/CN2021/125776 CN2021125776W WO2022083748A1 WO 2022083748 A1 WO2022083748 A1 WO 2022083748A1 CN 2021125776 W CN2021125776 W CN 2021125776W WO 2022083748 A1 WO2022083748 A1 WO 2022083748A1
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grape seed
seed extract
weight ratio
cells
tumor
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孙宇
许奇霞
张旭光
贺瑞坤
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汤臣倍健股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • 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
    • A61K31/28—Compounds containing heavy metals
    • A61K31/282—Platinum compounds
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N25/00—Investigating or analyzing materials by the use of thermal means
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention belongs to the field of biomedicine, and more particularly, the invention relates to the use of grape seed extract in the preparation of medicines for targeting and eliminating senescent cells in tumor microenvironment and inhibiting tumors.
  • Cellular senescence refers to a relatively stable and often irreversible state of cell cycle arrest in eukaryotic cells in which proliferating cells become resistant to growth-promoting stimuli.
  • Cellular senescence is usually caused by stress signals such as DNA damage.
  • Senescent cells are distinct from both resting and terminally differentiated cells, in which resting cells are able to re-enter the cell cycle. Senescent cells are characterized by morphological abnormalities, changes in metabolic activity, chromatin remodeling, altered gene expression, increased lipofuscin, prominent granularity, severe vacuolization, and the development of a senescence-associated secretory phenotype (SASP). Proinflammatory phenotype. As nuclear lamina lamin B1 tends to disappear, disruption of nuclear envelope integrity can be observed. Senescent cells accumulate dysfunctional mitochondria and exhibit elevated levels of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • Cell senescence is manifested by infolding of the nuclear membrane, chromatin pyknosis, and increased cell volume, which activates multiple downstream signaling pathways including p53, p16 INK4A /Rb, PI3K/Akt, FoxO transcription factors, and mitochondrial SIRT1.
  • senescent cells are often associated with a number of pathological features, including local inflammation. Cellular senescence occurs in damaged cells and prevents them from proliferating in an organism. Under the influence of various external stimuli and internal factors, cell damage can lead to obvious signs of cellular senescence. When the accumulation of damage reaches a certain limit, various degenerative changes and physiological aging phenotypes can be seen in the tissue.
  • Senescent cells participate in various physiological and pathological processes of the body mainly through three pathways: (1) The gradual accumulation of gene expression and morphological changes in senescent cells can affect the function of corresponding tissues; (2) Senescent cells restrict the regeneration of stem cells and undifferentiated progenitor cells. (3) Senescent cells not only show growth cycle arrest, but also release a large number of cytokines, chemokines, growth factors and proteases through autocrine and paracrine pathways, affecting the regeneration of adjacent cells and tissues. The microenvironment causes and accelerates aging and related diseases.
  • SASP tumor necrosis factor- ⁇
  • IL-6 interleukin 6
  • CXCL8 interleukin 8
  • IL-1 ⁇ interleukin 1A
  • MMPs matrix metalloproteinases
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • PAI1 plasminogen activator inhibitory factor-1
  • VEGF vascular endothelial growth factor
  • ANGPTL4 vascular endothelial growth factor receptor 4
  • EMT epithelial-mesenchymal transition
  • Stimuli such as DNA damage, telomere dysfunction, oncogene activation, and oxidative stress can induce SASP in cells, and the mechanisms are closely related to transcriptional cascades, autocrine loops, and persistent DNA damage responses.
  • overexpression or inhibition of the canonical senescence pathways p53 and p16 INK4A /Rb did not affect the expression of SASP, indicating that although cycle arrest and SASP in senescent cells often co-occur, the regulatory pathways of the two do not completely overlap.
  • the DNA damage response has been reported to increase the secretion of SASP factors IL-6 and IL-8 by activating the telangiectatic ataxia mutant gene, Nijmegen break syndrome protein 1, and checkpoint kinase 2.
  • DNA damage response is activated immediately after cell damage, and it takes about 1 week or even longer for senescent cells to develop mature SASP, and the transient DNA damage response does not induce cellular senescence or SASP, indicating that in addition to In addition to the DNA damage response, there are other mechanisms co-inducing SASP.
  • NF- ⁇ B and c/EBP ⁇ transcription factors are all involved in the regulation of SASP in senescent cells.
  • NF- ⁇ B and c/EBP ⁇ transcription factors are increased in activity upon cellular senescence and are involved in the expression of cytokines that regulate cellular stress and inflammatory signaling.
  • cytokines that regulate cellular stress and inflammatory signaling.
  • the phosphorylated NF- ⁇ B/RelA subunit enters the nucleus, binds to the SASP promoter, and regulates the expression of SASP factors. Therefore, NF- ⁇ B is often called the master regulator of SASP.
  • GATA4 zinc finger transcription factor 4
  • GATA4 can affect SASP-related genes IL-6, IL-8, Expression of CXCL1.
  • p38MAPK is a member of the serine/threonine protein kinase family and is an important signal transduction molecule. Activating or blocking p38MAPK is sufficient to affect the formation of SASP in senescent cells.
  • p38 MAPK is activated a few days after the onset of the senescence program and indirectly activates NF- ⁇ B by activating mitogen and stress-activated protein kinases MSK1 and MSK2, resulting in the accumulation of p65 and p50 in the nucleus, which is consistent with the early development of SASP Consistent.
  • Senescent cells do not directly secrete the pro-inflammatory factor IL-1 ⁇ , but a large amount of IL-1 ⁇ is distributed on the surface of senescent cells, which together with NF- ⁇ B forms a positive feed-forward loop to promote the encoding and transcription of inflammatory factors, and establish and maintain SASP.
  • mTOR promotes the secretion of SASP factors by regulating the level of IL-1 ⁇ , while rapamycin does not affect the level of IL-1 ⁇ mRNA, but significantly reduces the expression of IL-1 ⁇ protein on the surface of senescent cells.
  • mTOR can also regulate p38MAPK downstream signal MAPKAPK2 to affect SASP factor secretion.
  • MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1, thereby limiting its ability to degrade SASP factor transcripts.
  • Transcription factor c/EBP ⁇ is related to cell senescence induced by tumor gene activation.
  • HMGB2 targets c/EBP ⁇ to regulate SASP, and promotes the expression of SASP genes by inhibiting the spread of heterochromatin.
  • SAHF senescence-associated heterochromatin loci
  • SASP inhibitors are known to significantly attenuate SASP, they do not inherently kill senescent cells.
  • SCAPs senescence-associated anti-apoptotic pathways
  • the SCAP required for senescent cell survival varies between cell types.
  • the SCAPs required for survival of senescent human primary adipose progenitor cells differ from those in senescent human embryonic venous endothelial cells (HUVECs). This difference means that drugs targeting a single SCAP may not be able to eliminate multiple senescent cell types.
  • HUVECs senescent human embryonic venous endothelial cells
  • senolytics are indeed only effective against a limited number of senescent cell types. For example, navitoclax was able to target HUVECs but was ineffective against senescent human adipose progenitor cells.
  • Evidence suggests that the efficacy of senolytics may vary even within one specific type of cell.
  • navitoclax targets and kills senescent cells in the culture-adapted IMR90 lung fibroblast-like cell line, but is less effective on senescent primary human lung fibroblasts. Therefore, extensive testing on a range of cell types is still required to determine the broad-spectrum effects of senolytics.
  • the purpose of the present invention is to provide the use of grape seed extract in the preparation of a drug for targeting and eliminating senescent cells in tumor microenvironment and inhibiting tumor.
  • the application of grape seed extract which is used in combination with chemotherapeutic drugs to prepare a composition for specifically targeting senescent cells in tumor microenvironment and inhibiting tumors; wherein, the chemotherapeutic drugs It is a chemotherapeutic drug that induces the senescence-associated secretory phenotype (SASP) in the tumor microenvironment after administration.
  • SASP senescence-associated secretory phenotype
  • the tumor is a tumor that produces a senescence-related secretory phenotype in the tumor microenvironment after genotoxic drug treatment, and/or a tumor that develops drug resistance after genotoxic drug treatment;
  • the tumors include (but are not limited to): prostate cancer, breast cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, esophageal cancer, bile duct cancer, and brain cancer.
  • the senescence-related secretory phenotype is a senescence-related secretory phenotype caused by DNA damage; preferably, the DNA damage is DNA damage caused by a chemotherapeutic drug.
  • the chemotherapeutic drugs are genotoxic drugs; more preferably, they include: bleomycin, mitoxantrone, cisplatin, carboplatin, saplatin, and cyclophosphamide.
  • the grape seed extract specifically targets and induces senescent cells in the tumor microenvironment to enter the death program, and promotes the growth of stromal cells (which are non-senescent cells) (improves the growth rate of stromal cells).
  • the chemotherapeutic drug is bleomycin.
  • the final concentration of bleomycin is 30-70 ⁇ g/mL, and the final concentration of grape seed extract is 5-5 ⁇ g/mL. 60 ⁇ M; preferably, the final concentration of bleomycin is 40 to 60 ⁇ g/mL, and the final concentration of grape seed extract is 10 to 50 ⁇ M; more preferably, the final concentration of bleomycin is 45 to 55 ⁇ g/mL, and the final concentration of grape seed extract is 45 to 55 ⁇ g/mL.
  • the final concentration is 10-35 ⁇ M (eg 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M).
  • the chemotherapeutic drug is mitoxantrone
  • the weight ratio of mitoxantrone to grape seed extract is 1:20-80; preferably Preferably, the weight ratio of mitoxantrone to grape seed extract is 1:30-70; more preferably, the weight ratio of mitoxantrone to grape seed extract is 1:40-60 (eg :50, 1:55).
  • the chemotherapeutic drug is cisplatin, and when cisplatin and grape seed extract are combined, the weight ratio of cisplatin and grape seed extract is 1:20-80; The weight ratio of the seed extract is 1:30-70; more preferably, the weight ratio of cisplatin and grape seed extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the chemotherapeutic drug is carboplatin, and when carboplatin and grape seed extract are combined, the weight ratio of carboplatin and grape seed extract is 1:20-80; The weight ratio of the seed extract is 1:30-70; more preferably, the weight ratio of carboplatin and grape seed extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the chemotherapeutic drug is satraplatin, and when satraplatin and grape seed extract are combined, the weight ratio of satraplatin and grape seed extract is 1:20-80; The weight ratio of the seed extract is 1:30-70; more preferably, the weight ratio of satraplatin and grape seed extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the chemotherapeutic drug is cyclophosphamide
  • the weight ratio of cyclophosphamide and grape seed extract is 1:20-80
  • the weight ratio of phosphoramide to grape seed extract is 1:30 to 70; more preferably, the weight ratio of cyclophosphamide to grape seed extract is 1:40 to 60 (eg 1:45, 1:50, 1:45, 1:50, 1:40). 55).
  • a pharmaceutical composition or kit for specifically targeting senescent cells in tumor microenvironment and inhibiting tumors comprising: grape seed extract, and chemotherapeutic drugs; wherein, the The chemotherapeutic drugs used are those that induce senescence-related secretory phenotypes in the tumor microenvironment after administration.
  • the grape seed extract is obtained by using grape seed as a raw material, and is obtained by extraction by one or more methods including the following group: solvent extraction method, membrane separation technology, extraction technology, and countercurrent extraction technology.
  • the final concentration of bleomycin when bleomycin is combined with grape seed extract, is 30-70 ⁇ g/mL, and the final concentration of grape seed extract is 5-60 ⁇ M;
  • the final concentration of bleomycin is 40-60 ⁇ g/mL, and the final concentration of grape seed extract is 10-50 ⁇ M; more preferably, the final concentration of bleomycin is 45-55 ⁇ g/mL, and the final concentration of grape seed extract is 10 to 35 ⁇ M (eg 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M).
  • the weight ratio of mitoxantrone to grape seed extract is 1:20-80; preferably, The weight ratio of mitoxantrone to grape seed extract is 1:30-70; more preferably, the weight ratio of mitoxantrone to grape seed extract is 1:40-60 (such as 1:45, 1:50 , 1:55).
  • the weight ratio of cisplatin and grape seed extract is 1:20-80;
  • the weight ratio of the seed extract is 1:30-70; more preferably, the weight ratio of cisplatin and grape seed extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the weight ratio of carboplatin and grape seed extract is 1:20-80;
  • the weight ratio of the seed extract is 1:30-70; more preferably, the weight ratio of carboplatin and grape seed extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the weight ratio of satraplatin and grape seed extract is 1:20-80;
  • the weight ratio of the seed extract is 1:30-70; more preferably, the weight ratio of satraplatin and grape seed extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the weight ratio of cyclophosphamide and grape seed extract is 1:20-80;
  • the weight ratio of amide to grape seed extract is 1:30 ⁇ 70; more preferably, the weight ratio of cyclophosphamide to grape seed extract is 1:40 ⁇ 60 (such as 1:45, 1:50, 1:55 ).
  • a method for preparing a pharmaceutical composition or kit for inhibiting tumors comprising: mixing grape seed extract and chemotherapeutic drugs; or placing the grape seed extract and chemotherapeutic drugs in the same kit middle.
  • the grape seed extract is mixed with a chemotherapeutic drug, and divided into unit dosage forms according to the course of treatment.
  • the use of grape seed extract is provided for preparing a composition for specifically targeting senescent cells in the tumor microenvironment, or for preparing a composition for inhibiting (reducing) senescence-related secretory phenotypes
  • the grape seed extract specifically targets and induces senescent cells in the tumor microenvironment to enter the death program, and promotes the growth of stromal cells (which are non-senescent cells) (improves the growth rate of stromal cells).
  • the composition is also used for: alleviating body dysfunction (including but not limited to: enhancing exercise ability and enhancing endurance).
  • composition is also used for: prolonging survival in later life.
  • the concentration of grape seed extract is 0.5-3 ⁇ M; preferably 0.8-2 ⁇ M; more preferably 1-1.5 ⁇ M (eg 1.25 ⁇ M).
  • a method for screening potential substances that promote chemotherapeutic drugs to inhibit tumors comprising: (1) providing a tumor microenvironment system, the system including tumor cells and stromal cells; (2) The system of (1) is treated with chemotherapeutic drugs to induce senescence-related secretory phenotypes in the tumor microenvironment; (3) the candidate substances are added to the system of (2) to observe their effects on the tumor microenvironment system.
  • step (2) it further includes: before, during or after inducing the tumor microenvironment to produce an aging-related secretory phenotype, treating with grape seed extract; in step (3), it also includes: If the candidate substance can statistically promote the grape seed extract to clear senescent cells in the tumor microenvironment and/or promote the growth of stromal cells, the candidate substance is a potential substance that can be used in combination with the grape seed extract to inhibit tumors.
  • a method for screening potential substances that inhibit the senescence-related secretory phenotype comprising: (1) providing a stromal cell system, inducing the system to produce the senescence-related secretory phenotype; The system was treated with grape seed extract before, during or after the senescence-related secretory phenotype; (2) the candidate substance was added to the system of (1), and its effect on the stromal cell system was observed.
  • the candidate substance can be extracted with grape seed Potential substances used in combination to inhibit aging-related secretory phenotypes.
  • apoptosis or senescence-related secretory phenotype is assessed by observing caspase 3 cleavage activity or SASP factor expression.
  • the SASP factors include but are not limited to: IL6, CXCL8, SPINK1, WNT16B, GM-CSF, MMP3, IL1 ⁇ .
  • the apoptosis or senescence-related secretory phenotype is assessed by observing the senescence marker p16 INK4A in the chemotherapy animals.
  • a control group is also included, so as to clearly distinguish the difference between the tumor microenvironment system in the test group and the control group, or the difference in the clearance effect of grape seed extract on senescent cells in the tumor microenvironment and the control group.
  • the candidate substances include (but are not limited to): small molecule compounds, mixtures (such as plant extracts), biological macromolecules, signaling pathway regulatory reagents, and the like.
  • FIG. 1 Proliferating human-derived stromal cells PSC27 (early passages such as p10-20) were treated with the chemotherapeutic drug bleomycin (BLEO) at a concentration of 50 ⁇ g/ml in vitro on days 7-10, and were stained by SA- ⁇ -Gal results after. Top panel, representative image, bottom panel, statistical data. CTRL, control cells; BLEO, cells after bleomycin treatment. **, P ⁇ 0.01.
  • BLEO chemotherapeutic drug bleomycin
  • FIG. 1 The results of BrdU staining in PSC27 cells treated with the chemotherapeutic drug bleomycin (BLEO). Top panel, representative image, bottom panel, statistical data. CTRL, control cells; BLEO, cells after bleomycin treatment. ***, P ⁇ 0.001.
  • FIG. 3 The results of immunofluorescence staining with ⁇ H2AX after treatment of PSC27 cells with the chemotherapeutic drug bleomycin (BLEO). CTRL, control cells; BLEO, cells after bleomycin treatment. ***, P ⁇ 0.001. According to the number of fluorescent spots in the nucleus, they were divided into 4 categories, including 0 foci, 1-3 foci, 4-10 foci and single cells >10 foci.
  • HBF1203 is a human breast stromal cell line
  • WI38 is a human embryonic lung cell
  • BJ is a human skin fibroblast.
  • Heatmap shows that the expression of a large number of factors in senescent cells caused by BLEO injury is up-regulated, but many of them are significantly reversed after GSE treatment. Red star logo, typical SASP exogenous factor.
  • FIG. 7 The results of GSEA analysis showed that the expression of SASP or NF- ⁇ B molecular marker-related factors was centrally up-regulated in senescent cells caused by BLEO, but significantly decreased after GSE treatment of senescent cells. Left, SASP molecular marker; right, NF- ⁇ B molecular marker.
  • Figure 9 KEGG pathway analysis. Representative pathways in biological process of 100 molecules that GSE significantly down-regulated in senescent cells. Left Y-axis, percentage. Right Y-axis, log10(p-value).
  • Figure 10 KEGG pathway analysis. Representative pathways on the cellular component of 100 molecules that GSE significantly down-regulated in senescent cells. Left Y-axis, percentage. Right Y-axis, log10(p-value).
  • FIG. 11 Fluorescence quantitative PCR (qRT-PCR) detection and analysis of the relative expression levels of a group of typical SASP molecules in BLEO-induced senescent cells treated with different concentrations of GSE. All data are normalized results compared to the CTRL group. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • FIG. 12 The senescence of PSC27 was determined by SA- ⁇ -Gal staining under the condition of increasing GSE concentration. ⁇ , P>0.05; **, P ⁇ 0.01; ****, P ⁇ 0.0001. Among them, the P values of GSE at the concentrations of 5 ⁇ M, 12.5 ⁇ M, 25 ⁇ M and 50 ⁇ M were statistically significant compared with the data at 0 ⁇ M for the positive proportion of cells in these experimental groups.
  • Figure 13 Representative pictures of PSC27 under various conditions after SA- ⁇ -Gal staining. 3 repetitions per set, up and down. Scale bar, 20 ⁇ m.
  • CCK8 detects the survival rate of proliferating cells and senescent cells under increasing concentrations of GSE. P values at each GSE concentration are significant differences between the CTRL and BLEO groups after comparison. **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 15 Population doubling test for PSC27.
  • Cells at passage 10 (p10) were BLEO-injuriously treated, followed by the addition of GSE to the medium at day 8.
  • the effect of GSE on cell proliferation potential was determined by comparing the doubling value (PD) of CTRL group, BLEO group, GSE group and BLEO/GSE group. ⁇ , P>0.05; ***, P ⁇ 0.001.
  • FIG. 1 Pan-caspase inhibitor (20 ⁇ M QVD-OPh) reversed the senolytic activity of GSE (5 ⁇ M GSE was used in this experiment, while 1 ⁇ M ABT263 was used as a positive control; the latter is a senescent cell apoptosis inducer reported in recent years ). Statistical differences were obtained by two-way ANOVA (Tukey's test).
  • Figure 18 Flow cytometry to determine the apoptosis of PSC27 under several conditions. Q2, distribution area of early apoptotic cells; Q3, distribution area of late apoptotic cells.
  • FIG. 20 Schematic diagram of the administration method in mice in the pre-clinical trial.
  • Human stromal cells PSC27 and cancer cells PC3 were mixed in vitro (1:4) and then transplanted into mice subcutaneously to form xenografts. After multiple treatment cycles under the condition of single-drug or combined administration, the mice were finally sacrificed, and the expression changes of related molecules in tumor tissue were analyzed pathologically.
  • FIG. 21 The CTRL group and the BLEO injury group of PSC27 cells were mixed with PC3 in vitro, or PC3 cells were transplanted into the subcutaneous tissue of mice alone to form xenografts. Tumors were dissected and obtained at the end of the 8th week, and the volume of the tumors under the conditions of each group was measured and compared. **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • FIG 22 Schematic diagram of administration time and administration method in pre-clinical trial mice. Every two weeks was a dosing cycle, and MIT (mitoxantrone, mitoxantrone) was intraperitoneally administered to the mice on the first day of the 3rd/5th/7th week respectively. Mice were dosed with intraperitoneal GSE starting on the first day of week 5, once a week. After the 8-week course of treatment, the mice were dissected for pathological identification and expression analysis.
  • MIT mitoxantrone, mitoxantrone
  • Figure 23 Statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug MIT was administered to mice alone or together with the anti-aging drug GSE, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 24 Comparison of cell senescence in lesions of PC3/PSC27 tumor-bearing animals in preclinical experiments. Representative pictures after SA- ⁇ -Gal staining. Scale bar, 100 ⁇ m.
  • Figure 25 Parallel analysis of the percentage of SA- ⁇ -Gal staining positive cells in tumor tissue in mice. ⁇ , P>0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • FIG. 26 Fluorescence quantitative PCR (qRT-PCR) detection and analysis of the expression of SASP typical factors in epithelial cancer cells and stromal cells in mouse lesions.
  • the stromal cells and cancer cells were specifically isolated by LCM technology, and total RNA was prepared and used for the detection of SASP expression.
  • ⁇ P>0.05; *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • FIG. 27 Fluorescence quantitative PCR (qRT-PCR) detection and analysis of the expression status of SASP factors in stromal cells in mouse lesions after vehicle, MIT and MIT/GSE administration. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • Figure 28 Analysis of DNA damage and apoptosis ratios in each group of mice after the specific separation of cancer cells in the lesions by LCM technology. ⁇ , P>0.05; *, P ⁇ 0.05; **, P ⁇ 0.01.
  • FIG. 29 Image analysis after immunohistochemical staining.
  • Scale bar 200 ⁇ m.
  • Figure 30 Comparison of Kaplan Meier data of disease-free survival in NOD/SCID mice after various drug treatments. When the tumor volume of the animals in Vehicle, MIT, GSE and MIT/GSE groups exceeds 2000mm 3 , it is considered that a serious disease has appeared, and the mice need to be killed in time and their tumor bearing status detected. ⁇ , P>0.05; **, P ⁇ 0.01.
  • Figure 31 Comparative analysis of mouse body weight data at the end of the course of treatment under various drug treatment conditions. ⁇ , p>0.05.
  • Figure 32 Comparative analysis of mouse serological data at the end of the course of treatment under the above different administration conditions. Creatinine, urine (renal index), ALP and ALT (liver index) data were compared in parallel. ⁇ , p>0.05.
  • Figure 33 Comparative analysis of body weight data of immune intact mice (C57BL/6J) at the end of the course of treatment under various administration conditions. ⁇ , p>0.05.
  • Figure 34 Comparative analysis of mouse blood cell counts at the end of the course of treatment under different administration conditions in pre-clinical treatment. WBC, lymphocyte and neutrophil unit volume numbers were compared in parallel. ⁇ , p>0.05.
  • FIG 35 Bioluminescence (BLI) images in animals showing the location and signal intensity of reporter cells.
  • PSC27 cells that continue to express luciferase and enter the senescence stage induced by BLEO were transplanted into mice in advance by intraperitoneal injection; 2 days after the last vehicle or GSE administration, they were obtained using Berthold LB983 (BERTHOLD Technologies) small animal in vivo molecular imaging system Luciferase signaling in mice. Ruler, 15mm.
  • Figure 36 In vivo bioluminescence (BLI) images of animals showing the location and signal intensity of reporter cells. Proliferating PSC27 cells that continuously express luciferase were transplanted into mice in advance by intraperitoneal injection; 2 days after the last vehicle or GSE administration, Berthold LB983 (BERTHOLD Technologies) small animal in vivo molecular imaging system was used to obtain luciferase signals in mice. Ruler, 15mm.
  • Figure 37 The test flow for testing the physical fitness of experimental mice in the pre-clinical process. After 20-month-old mice were dosed with Vehicle or GSE every two weeks, relevant physical fitness tests were performed at the end of the 4th month.
  • Figure 38 A series of physical fitness measurements were performed on experimental mice, including quantitative detection of maximum walking speed, endurance, grip strength, treadmill endurance, daily activities, body weight and food intake. ⁇ , P>0.05; *, P ⁇ 0.05.
  • Figure 39 Relative mRNA expression levels of a representative panel of SASP factors in stromal cells in solid organ microenvironments. Mice were sacrificed at the age of 24 months and then dissected to obtain their solid organs such as lung, prostate and colorectum, and extracted total RNA from their stromal tissues for quantitative analysis by qRT-PCR. 6-month-old (6M) mice served as controls, and the signals of the other two groups of 24-month-old (24M) mice were normalized and plotted. ⁇ , P>0.05; *, P ⁇ 0.05.
  • Figure 40 Mouse lifespan analysis experimental design. Mice aged 24 to 27 months were dosed biweekly with Vehicle or GSE, and their survival was continuously monitored and their maximal lifespan recorded.
  • Figure 47 Statistical analysis of tumor terminal volume; the chemotherapeutic drug doxorubicin DOX alone or together with the anti-aging drug GSE was administered to mice, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 48 Statistical analysis of tumor terminal volume; the chemotherapeutic drug taxane DOC alone or together with the anti-aging drug GSE was administered to mice, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 49 Statistical analysis of tumor terminal volume; the chemotherapeutic drug vincristine VIN alone or together with the anti-aging drug GSE was administered to mice, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • GSE grape seed extract
  • SASP senescence-associated secretory phenotype
  • GSE can specifically target and remove senescent cells in the tumor microenvironment, it has no specific inhibitory effect on tumor cells. It is very large, causing the formation and development of SASP at the tissue level, and it is easy to lead to drug resistance of cancer cells after continuous use.
  • the combined application of GSE and some specific chemotherapeutic drugs can effectively exert the benign complementary effect of targeted lesions and achieve surprising synergistic effects.
  • Grape Seed Extract is a potent active nutrient extracted from the seeds of grapes, known to be naturally derived antioxidants.
  • GSE GSE
  • Various methods can be used to extract GSE, such as but not limited to: water extraction, organic solvent extraction, microwave method, supercritical CO 2 extraction, etc. It is preferably an organic solvent extraction method, and the organic solvent used includes but is not limited to: ethanol, methanol, acetone, and the like. Since the crude GSE contains many impurities, it can be further purified. The purification of the crude GSE can be carried out (but not limited to): solvent extraction method, precipitation method, enzymatic hydrolysis method, ultrafiltration method, macroporous resin method and the like.
  • the GSE is prepared by the following method: extracted by one or more methods including the following group: solvent extraction method, membrane separation technology, extraction technology, countercurrent extraction technology.
  • the GSE obtained by the above method has high content of active ingredients and controllable quality, the effect of targeting on removing senescent cells is extremely significant, the synergistic effect with chemotherapeutic drugs is ideal, and it is suitable for industrialized large-scale mass production.
  • GSE as a known product, is also commercially available.
  • Grape seed extract or its combined application with chemotherapeutic drugs Grape seed extract or its combined application with chemotherapeutic drugs
  • GSE grape seed extract
  • the killing effect of GSE on senescent cells is very ideal at an appropriate concentration.
  • the inventors have found that when GSE reaches a threshold at 25 [mu]M, senescent cells remain at 20% or less at this point. Therefore, at a certain concentration, GSE is a new type of senolytics and exhibits excellent effects.
  • the inventors also found that the population of stromal cells doubled after being treated with a genotoxic drug (bleomycin in the example); The combination treatment group exhibited significantly higher population doubling (PD) capacity.
  • the population of stromal cells doubled after genotoxic treatment, and the combination treatment group of genotoxic drugs and GSE exhibited significantly increased PD capacity compared with cells that rapidly entered growth arrest after the injury treatment.
  • the combination of GSE and genotoxic drugs can rapidly restore the proliferative potential of stromal cells in the short term, which is in sharp contrast to the use of genotoxic drugs alone, which is surprising. What is more interesting is that GSE promotes senescent cells to enter the death program by inducing apoptosis, but proliferating cells are basically not targeted by this natural drug, which reflects the good targeting specificity and safety of GSE.
  • the inventors' research also found that, after tumor transplantation into animals, the volume of xenografts composed of PC3 cells and senescent PSC27 cells was higher than that of the transplanted tumors composed of PC3 cancer cells and primary PSC27 stromal cells. A significant increase. Compared with the treatment group treated with MIT alone, GSE combined with MIT can significantly reduce the tumor; compared with MIT, tumor volume decreased by 55.2%, P ⁇ 0.001; compared with placebo treatment, tumor volume decreased by 74.9% , P ⁇ 0.0001. This inhibitory effect was very unexpected.
  • the inventors also found that the MIT administration process induced the appearance of a large number of senescent cells in the tumor tissue.
  • GSE administration essentially depleted most of the senescent cells within the lesions of these chemotherapy animals.
  • the expression of SASP factors was significantly increased (mainly in stromal cells); however, this change was largely reversed when GSE was administered.
  • MIT-treated animals were treated with GSE, the indices of DNA damage or apoptosis were significantly enhanced, implying enhanced tumor site cytotoxicity in animals treated with these senescent drugs; when GSE was applied therapeutically, cell apoptosis
  • the activity of caspase 3 cleavage, a typical marker of apoptosis was significantly increased.
  • mice treated with the MIT/GSE combination exhibited the longest median survival; survival was extended by at least 48.1%.
  • therapeutic targeting of senescent cells with GSE can promote tumor regression and reduce chemoresistance.
  • GSE can selectively kill senescent cells in the tissue microenvironment.
  • GSE alleviated physical impairment in mice, manifested in significantly improved maximum walking speed, suspension endurance, grip strength, treadmill endurance, and ability to perform daily activities.
  • the expression of several important SASP components in stromal cells in the visceral tissue microenvironment of aged mice in the GSE-treated group was generally reduced.
  • GSE-mediated depletion of senescent cells reduces mortality risk and effectively prolongs survival in aged mice. Therefore, intermittent provision of GSE, a biologically active anti-aging drug, can significantly reduce the disease burden of aging organisms by removing senescent cells in the microenvironment, and can increase the lifespan of the organism in the post-treatment period.
  • the present invention provides a use of GSE for preparing a composition for specifically targeting senescent cells in tumor microenvironment and inhibiting tumor; or preparing a composition for inhibiting senescence-related secretory phenotype .
  • a "tumor” is one that develops a senescence-associated secretory phenotype in the tumor microenvironment following genotoxic drug treatment, and/or one that develops drug resistance following genotoxic drug treatment . It preferably includes prostate cancer, breast cancer, lung cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer, kidney cancer, esophageal cancer, bile duct cancer, and brain cancer.
  • the "chemotherapeutic drug” is a chemotherapeutic drug that induces a senescence-associated secretory phenotype (SASP) in the tumor microenvironment after administration.
  • SASP senescence-associated secretory phenotype
  • the "senescence-related secretory phenotype" is a senescence-related secretory phenotype that occurs in the presence of DNA damage; preferably, the DNA damage is DNA damage caused by chemotherapeutic drugs; more preferably , the chemotherapeutic drugs include genotoxic drugs.
  • drugs that further optimize the inhibitory effect can be screened based on this feature. From the substances described, drugs that target senescent cells in the tumor microenvironment can be found, and are truly useful for inhibiting tumors, reversing tumor drug resistance, or inhibiting/delaying senescence-related secretory phenotypes. Alternatively, one or more substances that combine and synergize with GSE can be found from the substances described.
  • the present invention provides a method for screening potential substances that promote chemotherapeutic drugs to inhibit tumors, the method comprising: (1) providing a tumor microenvironment system including tumor cells and stromal cells; (2) treating with chemotherapeutic drugs The system of (1) induces a senescence-related secretory phenotype in the tumor microenvironment; (3) the candidate substance is added to the system of (2) to observe its effect on the tumor microenvironment system. If it can be specifically targeted for clearance Senescent cells in the tumor microenvironment and/or promoting the growth of stromal cells (which are non-senescent cells) (increasing the growth rate of stromal cells) are potential substances that promote chemotherapeutic drugs to inhibit tumors.
  • step (2) it further includes: before, during or after inducing the tumor microenvironment to produce a senescence-related secretory phenotype, treating with GSE; in step (3), it further includes: if If the candidate substance can statistically promote GSE to clear senescent cells in the tumor microenvironment and/or promote the growth of stromal cells, the candidate substance is a potential substance that can be used in combination with GSE to inhibit tumors.
  • the present invention also provides a method for screening potential substances that inhibit the senescence-related secretory phenotype, the method comprising: (1) providing a stromal cell system, inducing the system to produce the senescence-related secretory phenotype; (2) adding the candidate substances Add it to the system of (1), and observe its effect on the stromal cell system. If it can specifically promote the inhibitory effect of grape seed on the senescence-related secretory phenotype, the candidate substance can be used in combination with GSE to inhibit senescence. Potential substances associated with secretory phenotypes.
  • a control group may also be set, and the control group may not add the candidate substance, but other conditions and The same system as the test group.
  • the method further includes: further cell experiments and/or animal experiments are performed on the obtained potential substances, so as to further select and determine the relevant factors for inhibiting tumor, reversing tumor drug resistance or inhibiting/delaying aging. Secreted phenotype really useful substances.
  • the present invention also provides potential substances obtained by the screening method for inhibiting tumors, reversing tumor drug resistance, or inhibiting/delaying senescence-related secretory phenotypes.
  • These initially screened substances can form a screening library, so that people can finally screen out really useful drugs.
  • the present invention provides a pharmaceutical composition, which contains the GSE, chemotherapeutic drugs (such as 0.000001- 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%), and a pharmaceutically acceptable carrier.
  • chemotherapeutic drugs such as 0.000001- 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier such as 0.000001- 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier such as 0.000001- 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier such as 0.000001- 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier such as 0.000001- 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2
  • the present invention also provides a pharmaceutical composition, which contains an effective amount (such as 0.00001-10wt%; preferably 0.0001-5wt%; more preferably, 0.001-2wt%) of the GSE, and is pharmaceutically acceptable a.
  • an effective amount such as 0.00001-10wt%; preferably 0.0001-5wt%; more preferably, 0.001-2wt% of the GSE, and is pharmaceutically acceptable a.
  • the "effective amount” refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • the "pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, buffers.
  • auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the carrier may also contain cell transfection reagents.
  • the pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, these forms must be sterile and must be fluid for easy syringe expelling. It must be stable under the conditions of manufacture and storage and must be resistant to the contaminating influence of microorganisms such as bacteria and fungi.
  • the terms “comprising” or “including” include “comprising,” “consisting essentially of,” and “consisting of.”
  • the term “consisting essentially of” means that in addition to the main active ingredients (eg GSE and chemotherapeutics), the composition may contain minor amounts of minor ingredients and/or impurities that do not affect the active ingredients. For example, sweeteners to improve taste, antioxidants to prevent oxidation, and other additives commonly used in the art may be included.
  • the concentration of the GSE when applied to inhibit SASP, can be 0.5-3 ⁇ M; preferably 0.8-2 ⁇ M; more preferably 1-1.5 ⁇ M (eg 1.25 ⁇ M).
  • the GSE can clear senescent cells in the tumor microenvironment with relatively high efficiency. Therefore, as a preferred mode of the present invention, when the chemotherapeutic drug is bleomycin, when bleomycin is combined with GSE, the final concentration of bleomycin is 30-70 ⁇ g/mL, and the final concentration of GSE is 5-60 ⁇ M; Preferably, the final concentration of bleomycin is 40-60 ⁇ g/mL, and the final concentration of GSE is 10-50 ⁇ M; more preferably, the final concentration of bleomycin is 45-55 ⁇ g/mL, and the final concentration of GSE is 10-35 ⁇ M (such as 15 ⁇ M, 20 ⁇ M, 25 ⁇ M, 30 ⁇ M).
  • the chemotherapeutic drug is mitoxantrone, and when mitoxantrone is combined with GSE, the weight ratio of mitoxantrone to GSE is 1:20-80; The weight ratio to GSE is 1:30-70; more preferably, the weight ratio of mitoxantrone to GSE is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the chemotherapeutic drugs cisplatin, carboplatin, satraplatin or cyclophosphamide are administered in a similar manner to mitoxantrone.
  • GSE and/or chemotherapeutic drugs can be administered to mammals or mammals using a variety of methods well known in the art. people. These methods are all encompassed by the present invention.
  • the dosage form of the composition of the present invention can be various, as long as the dosage form can effectively reach the body of the mammal.
  • it can be selected from injections, tablets, capsules, powders, granules, syrups, solutions, suspensions, tinctures, oral liquids, or aerosols.
  • the effective amount of GSE described in the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the GSE such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the administration way etc.
  • A is the body surface area, calculated in m2 ;
  • W is the body weight, calculated in g;
  • K is a constant, which varies with animal species, generally speaking, mice and rats are 9.1, guinea pigs are 9.8, rabbits are 10.1, cats are 9.9, Dog 11.2, Monkey 11.8, Human 10.6. It will be understood that, depending on the drug and the clinical situation, the conversion of the administered dose may vary according to the assessment of an experienced pharmacist.
  • compositions of the present invention can also be formulated in unit dosage form for scheduled and metered administration.
  • unit dosage form and “unit dosage form” refer to the preparation of the composition of the present invention into a dosage form required for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid agent.
  • Said unit dosage form contains the composition of the present invention in an amount suitable for single, single day or unit time administration.
  • the composition is in unit dosage form.
  • one dose of the composition in the unit dosage form is administered every few days or weeks.
  • the present invention also provides a kit containing the pharmaceutical composition or directly containing the GSE and/or chemotherapeutic drugs.
  • the medicine box may further include instructions for using the medicine in the medicine box.
  • the normal human prostate primary stromal cell line PSC27 obtained from Fred Hutchinson Cancer Research Center, USA was cultured in an incubator at 37° C. and 5% CO 2 , and propagated and passaged in PSCC complete culture medium.
  • Cell cryopreservation collect cells in logarithmic growth phase with 0.25% trypsin, centrifuge at 1000 rpm for 2 min, discard the supernatant, and resuspend the cells in freshly prepared freezing medium. Aliquot cells into sterile cryovials as indicated. Then, it was cooled by gradient and finally transferred to liquid nitrogen for long-term storage.
  • Cell recovery Take out the cells frozen in liquid nitrogen and put them in a 37°C water bath immediately to thaw them quickly. Add 2ml of cell culture medium directly to suspend the cells evenly. After the cells adhered, replace with a new medium.
  • Pharmacodynamic analysis was performed on a natural product library (BY-HEALTH) with a total of 41 components, mostly medicinal plant extracts and anti-aging potential. Each product was diluted to a 96-well plate according to a certain concentration gradient, and the density was 5000 cells per well. The medium uses DMEM, and the working concentration of natural products (or compounds) is generally controlled at 1 ⁇ M-1 mM. 3-7 days after drug treatment, cell proliferation was determined with CCK-8 Cell Counting Kit (based on WST-8 principle, Vazyme), and apoptosis activity was determined with Caspase 3/7activity kit (Promega).
  • the preliminary identified drug candidates are further screened for 30 days. Drugs entering the second round of candidates were diluted into 6-well plates at 20,000 cells per well. Medium and drug candidates were changed every other day. To determine the effect of each drug on cell phenotype, viability, etc., the project conducted a confirmatory analysis based on different concentrations of the drug.
  • target cells were pre-seeded on coverslips for at least 24 h after culturing in petri dishes. After a brief wash, they were fixed with 4% paraformaldehyde in PBS for 8 min and blocked with 5% normal goat serum (NGS, Thermo Fisher) for 30 min.
  • Mouse monoclonal antibody anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) and mouse monoclonal antibody anti-BrdU (Cat#347580, BD Biosciences), and secondary antibody Alexa 488(or 594)-conjugated F(ab')2 was added sequentially to slides covered with fixed cells.
  • Nuclei were counterstained with 2 ⁇ g/ml of 4',6-diamidino-2-phenylindole (DAPI). Select the most representative image from the three observation fields for data analysis and result display.
  • a FV1000 laser scanning confocal microscope (Olympus) was used to acquire confocal fluorescence images of cells.
  • RNA samples were obtained from stromal cells. Its integrity was verified by Bioanalyzer 2100 (Agilent), RNA was sequenced by Illumina HiSeq X10, and gene expression levels were quantified by the software package rsem (https://deweylab.github.io/rsem/).
  • RNA samples were depleted of rRNA with the RiboMinus Eukaryote kit (Qiagen, Valencia, CA, USA); and prior to deep sequencing with TruSeq Stranded Total RNA preparation kits (Illumina, San Diego, CA) according to the manufacturer's instructions , USA) to construct strand-specific RNA-seq libraries.
  • Paired-end transcriptomic reads were mapped to the reference genome (GRCh38/hg38) and reference annotated from Gencode v27 using the Bowtie tool. Use the picard tools (1.98) script to mark duplicates (https://github.com/broadinstitute/picard) to identify duplicate reads and keep only non-duplicate reads.
  • Reference splice junctions were provided by the reference transcriptome (Ensembl build 73).
  • FPKM values were calculated using Cufflinks, and differential gene expression was called using Cufflinks, the maximum likelihood estimation function. Genes with significantly altered expression were defined by false discovery rate (FDR)-corrected P value ⁇ 0.05, and downstream analysis was performed only with ensembl genes 73 with status "Known” and biotype "coding".
  • PPI Protein-protein interaction
  • GSEA Gene Set Enrichment Analysis
  • genes were ranked using "wald statistics" obtained from DESeq2, GSEA in MSigDB (http://software.broadinstitute.org/gsea) based on data obtained from preliminary RNA-seq analysis /msigdb) on these sorted lists of all planned gene sets available).
  • DESeq2 independent filtering is based on the mean of normalized read counts to screen out genes with very low expression levels.
  • SASP and GSEA signatures are as described in the inventor's literature (Zhang et al., Nature Communications, 9(1): 1723.2018).
  • the used detection primer sequence is (F represents forward primer, R represents reverse primer):
  • IL6 TTCTGCGCAGCTTTAAGGAG (F; SEQ ID NO: 1), AGGTGCCCATGCTACATTTG (R; SEQ ID NO: 2);
  • CXCL8 ATGACTTCCAAGCTGGCCGTG (F; SEQ ID NO: 3), TGTGTTGGCGCAGTGTGGTC (R; SEQ ID NO: 4);
  • IL-1 ⁇ AATGACGCCCTCAATCAAAG (F; SEQ ID NO: 5), TGGGTATCTCAGGCATCTCC (R; SEQ ID NO: 6);
  • IL-1 ⁇ TGGGTATCTCAGGCATCTCC (F; SEQ ID NO: 7), TTCTGCTTGAGAGGTGCTGA (R; SEQ ID NO: 8);
  • GM-CSF ATGTGAATGCCATCCAGGAG (F; SEQ ID NO: 9), AGGGCAGTGCTGCTTGTAGT (R; SEQ ID NO: 10);
  • AREG AGCTGCCTTTATGTCTGCTG (F; SEQ ID NO: 11), TTTCGTTCCTCAGCTTCTCC (R; SEQ ID NO: 12);
  • CXCL1 CACCCCAAGAACATCCAAAG (F; SEQ ID NO: 13), TAACTATGGGGGATGCAGGA (R; SEQ ID NO: 14);
  • CXCL3 GGAGCACCAACTGACAGGAG (F; SEQ ID NO: 15), CCTTTCCAGCTGTCCCTAGA (R; SEQ ID NO: 16);
  • SPINK1 CCTTGGCCCTGTTGAGTCTA (F; SEQ ID NO: 17), GCCCAGATTTTTGAATGAGG (R; SEQ ID NO: 18);
  • WNT16B GCTCCTGTGCTGTGAAAACA (F; SEQ ID NO: 19), TGCATTCTCTGCCTTGTGTC (R; SEQ ID NO: 20);
  • MMP3 AGGGAACTTGAGCGTGAATC (F; SEQ ID NO: 21), TCACTTGTCTGTTGCACACG (R; SEQ ID NO: 22);
  • p16 INK4a CTTCCTGGACACGCTGGT (F; SEQ ID NO: 23), ATCTATGCGGGCATGGTTAC (R; SEQ ID NO: 24);
  • p21 CIP1 ATGAAAATTCACCCCCTTTCC (F; SEQ ID NO: 25), CCCTAGGCTGTGCTCACTTC (R; SEQ ID NO: 26).
  • Senescence-associated beta-galactosidase (SA-beta-Gal) staining can include: cell culture dishes washed with PBS and fixed at room temperature. Cells were fixed in 2% formaldehyde and 0.2 glutaraldehyde for 3 min. SA- ⁇ -Gal was then stained with freshly prepared staining solution overnight at 37°C. Images were taken the next day and the percentage of positive cells per unit area was calculated.
  • Single-cell clonal expansion experiments consisted of plating cells in gelatin-coated 12-well plates at a density of 2000 cells/well. Cell clones were counted after crystal violet staining.
  • PSC27 cells were plated in 96-well dishes and cell senescence was induced under BLEO treatment at 50 ⁇ g/ml. Grape seed extract (GSE) and ABT263 were added at concentrations of 5.0 ⁇ m and 1.0 ⁇ m, respectively. Cell culture medium was supplemented with Incucyte Nuclight Fast Red Reagent (Essen Bioscience) and Incucyte C-3/7 Apoptosis Reagent (Essen Bioscience). Select a representative field of view to take pictures.
  • GSE Grape seed extract
  • ABT263 were added at concentrations of 5.0 ⁇ m and 1.0 ⁇ m, respectively.
  • Cell culture medium was supplemented with Incucyte Nuclight Fast Red Reagent (Essen Bioscience) and Incucyte C-3/7 Apoptosis Reagent (Essen Bioscience). Select a representative field of view to take pictures.
  • GSE obtained from By-Health Company, batch number C01201911260006.
  • ABT263 purchased from Selleckchem Company.
  • mice were fed a standard experimental diet followed by the chemotherapeutic drug mitoxantrone (MIT, 0.2 mg/kg dose) and/or grape seed extract (GSE) (500 ⁇ l, 10 mg/kg dose) intraperitoneally.
  • MIT chemotherapeutic drug mitoxantrone
  • GSE grape seed extract
  • the time points are: the former is on the first day of weeks 3, 5, and 7, and the latter is on the first day of weeks 5 and 7.
  • a total of 3 cycles of MIT were administered throughout the course of treatment, and each cycle was 2 weeks.
  • mouse tumors were collected for volume measurement and histological analysis. Each mouse received a cumulative total of 0.6 mg/kg of MIT and 30 mg/kg of GSE.
  • MIT was administered to mice by intravenous infusion according to the above steps and sequence (detection of the expression of SASP factors in the whole body under the induction of chemotherapy and the effect of GSE by eliminating senescent cells in the body).
  • the overall effect of controlling the broad-spectrum expression of SASP but the dose was reduced to 0.1 mg/kg body weight per session (the cumulative MIT dose received throughout the course of treatment was 0.3 mg/kg body weight) to reduce drug-related toxicity.
  • the dose of grape seed extract (GSE) was reduced to 5 mg/kg, administered in combination with MIT, or alone, by intravenous infusion. Chemotherapy experiments were carried out until the end of the eighth week, and the mice were dissected immediately after sacrifice, and their xenografts were collected and used for pathological system analysis.
  • mice 16-month-old male C57BL/6 mice were obtained by continuous rearing on the SPF animal platform, with 4 to 5 animals per cage. Mice were first sorted by weight from low to high, then mice of similar weight were selected. Next, senescence (SEN) or control (CTRL) transplantation treatments, using a random number generator, were assigned to mice at each interval, while mice in the middle were assigned to the other treatment modality, resulting in senescence and the body weight of control transplanted mice. One month after cell transplantation, when the mice were 18 months old, physical function tests were performed. After that, no further tests were performed on the mice, except to examine their cages. The earliest death occurred approximately 2 months after the last physical function test.
  • mice C57BL/6 mice aged 19 to 21 months were housed 3-5 per cage.
  • the mice were classified according to body weight and randomly assigned to each group to receive control (vehicle) or drug (GSE) treatment by humans unaware of the design of the preclinical trial. From 24 to 27 months of age, mice were treated with vehicle or GSE every 2 weeks, orally for 3 consecutive days each time.
  • vehicle or GSE every 2 weeks, orally for 3 consecutive days each time.
  • RotaRod and hanging tests are performed monthly as these tests are sensitive and non-invasive.
  • mice were euthanized; they were considered dead if they exhibited one of the following symptoms: (1) unable to drink or eat; (2) unwilling to move even when stimulated; (3) fast Weight loss; (4) severe balance disorders; or (5) bleeding or ulceration of the body.
  • no mice were excluded due to fights, accidental death, or dermatitis.
  • Cox proportional hazard model was used for survival analysis.
  • Cages were inspected daily and dead mice were removed from cages.
  • Carcasses were opened (abdominal, thoracic and skull) within 24 hours of animal death and kept individually in 10% formalin for at least 7 days. Decomposed or destroyed bodies are excluded. Preserved bodies were transported to a dedicated Autopsy site for pathological examination. Tumor burden (sum of different tumor types per mouse), disease burden (sum of different histopathological changes in major organs of each mouse), severity of each lesion and inflammation (lymphocyte infiltration) were assessed.
  • mice were injected intraperitoneally with 3 mg of fluorescein (BioVision, Milpitas, CA), delivered in a volume of 200 ⁇ l of PBS. Mice were anesthetized with isoflurane and bioluminescent images were acquired using the Xenogen IVIS 200 System (Caliper Life Sciences, Hopkinton, MA).
  • Forelimb grip strength was determined using the Grip Strength Meter (Columbus Instruments, Columbus, OH) and results were averaged over 10 trials.
  • For the hanging endurance test mice were placed on a 2 mm thick metal wire 35 cm above the mat. Mice were only allowed to grasp the wire with their forelimbs, and hanging time was normalized to body weight and expressed as hanging duration (sec) ⁇ body weight (g). Results were averaged from 2 to 3 experiments per mouse. Daily activity and food intake were monitored for 24 hours (12 hours light and 12 hours dark) by Comprehensive Laboratory Animal Monitoring System (CLAMS). The CLAMS system was equipped with an Oxymax Open Circuit Calorimeter System (Columbus Instruments).
  • mice were acclimated to running on an electric treadmill (Columbus Instruments) at a 5° incline for 3 days for 5 min per day, starting at 5 m/min for 2 min and then accelerating to to 7 m/min for 2 minutes, then 9 m/min for 1 minute.
  • mice ran on a treadmill at an initial speed of 5 m/min for 2 minutes, and then increased the speed by 2 m/min every 2 minutes until the mice were exhausted.
  • Fatigue was defined as the inability of mice to return to the treadmill despite mild electrical and mechanical stimulation.
  • the distance was recorded, and the total work (KJ) was calculated by the following formula: mass (kg) ⁇ g (9.8m/s 2 ) ⁇ distance (m) ⁇ sin (5°).
  • the present invention uses baseline body weights to assign mice to experimental groups (to achieve similar body weights between groups), so randomization is performed only within weight-matched groups.
  • the present invention determines the sample size based on past experiments, so statistical power analysis is not used. All replicates in the present invention are from different samples, each sample from a different experimental animal.
  • PSC27 is a primary normal human prostate stromal cell line, namely PSC27, as an in vitro cell model.
  • PSC27 is mainly composed of fibroblasts, but non-fibroblast cell lines (including endothelial cells and smooth muscle cells) are also present, but in smaller proportions, PSC27 is a primary human stromal cell line in nature, and it is A typical SASP is formed after stress factors such as ionizing radiation.
  • the inventors treated these cells with bleomycin (BLEO) at a specific dose (50 ⁇ g/ml) in a manner optimized in the preliminary experiments and observed senescence-associated ⁇ -galactosidase (SA- ⁇ -GAL) staining
  • BLEO bleomycin
  • SA- ⁇ -GAL senescence-associated ⁇ -galactosidase
  • the inventors performed RNA-seq sequencing on these cells. Subsequent high-throughput data showed that a plant material, Grape Seed Extract (GSE), significantly altered the expression profile of senescent cells. Among them, 2644 genes were significantly down-regulated, while 1472 genes were up-regulated, and the fold change of each gene in the heatmap here was 2.0 (P ⁇ 0.01) ( Figure 5). Importantly, the expression of SASP factors was generally reduced in senescent cells following GSE treatment (50 ⁇ g/ml), whereas these SASP factors were generally significantly upregulated in senescent cells (Figure 6).
  • GSE Grape Seed Extract
  • GSE can effectively inhibit the expression of SASP when used at a low concentration (eg, 1.25 ⁇ M).
  • the inventors next investigated the potential of Population Doubling (PD) after genotoxic treatment of stromal cells.
  • the combined treatment group of BLEO and GSE exhibited significantly increased PD capacity compared to the BLEO group cells that rapidly entered a growth arrest state after the invasive treatment ( Figure 15).
  • GSE itself does not appear to affect the PD of proliferating cells, a data that further suggests the selectivity of GSE between senescent versus normal cells.
  • GSE causes senescent cells to lose their viability by inducing apoptosis
  • the inventors used GSE to treat proliferation group cells and senescence group cells respectively under culture conditions.
  • PSC27 cells were treated with BLEO (50 ⁇ g/ml) for 12h and gradually entered the senescence stage; 5 ⁇ M GSE was added to the medium of senescent cells on the 7th day.
  • the subsequent observed changes in caspase-3/7 activity indicated that GSE induced apoptosis of senescent cells; from the 16th hour after the addition of GSE, there was a statistical difference between the senescent group and the control group ( Figure 16).
  • pan-caspase inhibitor QVD prevented GSE killing of senescent cells in a process very similar to the effect of ABT263 (positive control, a known inducer of apoptosis in senescent cells) on senescent cells (Fig. 17).
  • ABT263 positive control, a known inducer of apoptosis in senescent cells
  • Fig. 17 a known inducer of apoptosis in senescent cells
  • Example 3 Therapeutic targeting of senescent cells by GSE can promote tumor regression and reduce chemoresistance
  • Cancer is one of the major chronic diseases that seriously threaten human lifespan and endanger health.
  • cancer cell drug resistance limits the efficacy of most anticancer treatments in the clinic, and senescent cells often contribute to the development of therapeutic resistance by developing SASP in damaged tumor foci. Even so, the feasibility and safety of removing senescent cells from primary tumors to boost the cancer therapeutic index has so far been little explored by scientists.
  • the inventors constructed a tissue recombinant by mixing PSC27 stromal cells (primary cells (CTRL group) or senescent cells (SEN group, ie BLEO injury group)) with PC3 epithelial cells, the latter is a typical highly Malignant prostate cancer cell lines.
  • the ratio of stromal cells to epithelial cells was 1:4 before the recombinants were implanted subcutaneously in the posterior thigh of non-obese diabetic and severe combined immunodeficiency (NOD/SCID) mice.
  • Tumor size (volume) was measured in animals at the end of 8 weeks after recombinant implantation ( Figure 20).
  • mice treated with the MIT/GSE combination exhibited the longest median survival with at least 48.1% longer survival compared to the group treated with MIT alone ( Figure 30, green vs. blue).
  • treating tumor-bearing mice with GSE alone did not result in significant benefit, with only marginal survival extension.
  • the inventors also compared the effects of MIT, BLEO and some other chemotherapeutic drugs in combination with GSE, and the results showed that the efficacy of doxorubicin+grapeseed was significantly lower than that of MIT+grapeseed and BLEO+grapeseed ( Figure 47).
  • the inventors also used taxane (docetaxel, DOC) and vincristine (VIN) to be administered alone or separately Mice were dosed in combination with grape seed extract (GSE), and the terminal volume of the mice was measured.
  • genotoxic drugs such as mitoxantrone and bleomycin are preferred when used in combination with GSE.
  • Example 4 Depletion of senescent cells by GSE treatment can reduce body dysfunction and prolong the later life of aging mice without increasing their morbidity in the later stages of life
  • GSE has the potency to clear senescent cells in the tumor microenvironment in mice, reduce tumor resistance, and improve overall therapeutic efficacy, is there some significant health-promoting or disease-delaying benefit in naturally aging animals as well?
  • the inventors first tested the potential of GSE to deplete senescent or control cells expressing luciferase (LUC) and injected intraperitoneally into wild-type (WT) mice. Compared with Vehicle treatment, the bioluminescence intensity of GSE-treated LUC senescent cell-transplanted mice was significantly reduced in the corresponding parts of the body (Fig. 35).
  • Fig. 43, Fig. 44 mice treated with GSE every two weeks had similar physical function in the last 2 months of life between males and females as Vehicle-treated mice.
  • GSE a biologically active anti-aging drug
  • Embodiment 5 drug screening
  • Screening system The experimental system described in Example 2: PSC27 cells were treated with BLEO (50 ⁇ g/ml) for 12 h under culture conditions and gradually entered the senescence stage; 5 ⁇ M GSE was added to the senescent cell culture medium on the 7th day. Treatment with grape seed extract was performed before, during or after induction of the senescence-associated secretory phenotype in the system.
  • Test group administer candidate substances to the screening system
  • Control group Candidate substances are not administered to the screening system.
  • the SASP in the test group and the control group were detected respectively, and the expression of SASP factors was determined. If the expression of SASP factors in the test group was significantly lower than that in the control group, the candidate substance could be used in combination with grape seed extract to inhibit aging-related secretion. type of potential substances.
  • Screening system The experimental system described in Example 3: PSC27 stromal cells and PC3 epithelial cells were mixed to construct tissue recombinants.
  • Test group administer candidate substances to the screening system
  • Control group Candidate substances are not administered to the screening system.
  • the candidate substance is an inhibitor of tumor. potential substances.

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Abstract

L'invention concerne une utilisation d'extrait de pépins de raisin (GSE) dans la préparation de médicaments pour l'élimination ciblée de cellules sénescentes dans un microenvironnement tumoral et pour l'inhibition tumorale. Le GSE permet d'obtenir une élimination ciblée des cellules sénescentes dans le microenvironnement tumoral, et l'application conjointe du GSE avec des médicaments chimiothérapeutiques peut favoriser l'inhibition tumorale par élimination des cellules stromales sénescentes. Pour un phénotype sécrétoire associé à la sénescence (SASP), le GSE peut également obtenir une élimination ciblée de cellules sénescentes dans le SASP de manière à inhiber le SASP.
PCT/CN2021/125776 2020-10-22 2021-10-22 Utilisation d'un extrait de pépins de raisin dans la préparation de médicaments pour l'élimination ciblée de cellules sénescentes dans un microenvironnement tumoral et pour l'inhibition tumorale WO2022083748A1 (fr)

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