WO2024067604A1 - Utilisation d'acide salvianolique a (saa) comme matière première d'un nouveau médicament anti-âge, dans le traitement de la sénescence cellulaire, le traitement des tumeurs et la prolongation de la durée de vie - Google Patents

Utilisation d'acide salvianolique a (saa) comme matière première d'un nouveau médicament anti-âge, dans le traitement de la sénescence cellulaire, le traitement des tumeurs et la prolongation de la durée de vie Download PDF

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WO2024067604A1
WO2024067604A1 PCT/CN2023/121621 CN2023121621W WO2024067604A1 WO 2024067604 A1 WO2024067604 A1 WO 2024067604A1 CN 2023121621 W CN2023121621 W CN 2023121621W WO 2024067604 A1 WO2024067604 A1 WO 2024067604A1
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tumor
cells
cancer
saa
senescent cells
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孙宇
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中国科学院上海营养与健康研究所
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    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • 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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the application of salvianolic acid A (SAA) as a novel anti-aging drug raw material in cell aging, tumor treatment and life extension.
  • SAA salvianolic acid A
  • Cellular senescence refers to a relatively stable and usually irreversible state of cell cycle arrest in eukaryotic cells, in which proliferating cells become resistant to growth-promoting stimuli, usually caused by stress signals such as DNA damage.
  • Replicative senescence refers to the cessation of continuous division of normal cells after approximately 30-50 divisions (the "Hayflick limit").
  • Replicative senescence is essentially induced by the progressive shortening of telomeres. In each round of DNA replication, telomeres gradually shorten and eventually reach a critical length that prevents further replication, thereby stopping cell division. Shorter uncapped telomeres cause a DNA damage response, which directly triggers senescence.
  • Senescent cells are distinct from both quiescent cells, which are able to reenter the cell cycle, and terminally differentiated cells. Senescent cells are characterized by morphological abnormalities, altered metabolic activity, chromatin remodeling, altered gene expression, increased lipofuscin, prominent granularity, severe vacuolation, and a proinflammatory phenotype known as the senescence-associated secretory phenotype (SASP). Disruption of nuclear membrane integrity is observed due to loss of lamin B1 expression in the nuclear lamina. Senescent cells accumulate dysfunctional mitochondria and display elevated levels of reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • Cellular senescence is manifested by nuclear membrane infolding, chromatin condensation, cell volume increase, and activation of multiple downstream signaling pathways including p53, p16INK4A/Rb, PI3K/Akt, FoxO transcription factors, and mitochondrial SIRT1.
  • senescent cells are often associated with many pathological features, including local inflammation. Cellular senescence occurs in damaged cells and prevents them from proliferating in the body. Under the influence of various external stimuli and internal factors, cell damage can lead to obvious signs of cell senescence. When the accumulation of damage reaches a certain limit, various tissue degeneration changes and physiological aging phenotypes that can be discerned by the naked eye appear in the tissue.
  • SASP senescence-associated secretory phenotype
  • 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 the corresponding tissues; (2) Senescent cells limit the regenerative potential of stem cells and undifferentiated progenitor cells, resulting in a decrease in cell regeneration capacity; (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 microenvironment of neighboring cells and tissues, leading to and accelerating aging and related diseases. In recent years, a large number of studies have shown that SASP plays a core pathological role in this process.
  • SASP factors include tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1a (IL-1a), matrix metalloproteinases (MMPs), granulocyte-macrophage colony-stimulating factor (GM-CSF), and plasminogen activator inhibitor-1 (PAI1).
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • IL-1a interleukin 1a
  • MMPs matrix metalloproteinases
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • PAI1 plasminogen activator inhibitor-1
  • SASP can also promote tumor development through specific secretory factors (such as VEGF, ANGPTL4) that promote angiogenesis, extracellular matrix remodeling, or epithelial-mesenchymal transition (EMT).
  • VEGF vascular endothelial growth factor
  • ANGPTL4 vascular endothelial growth factor 4
  • EMT epithelial-mesenchymal transition
  • aging-induced chronic inflammation can cause systemic immunosuppression, which can also promote the occurrence and development of a variety of aging-related diseases such as aging-related tissue damage and degeneration, organ dysfunction, and cancer.
  • Stimuli such as DNA damage, telomere dysfunction, oncogene activation, and oxidative stress can induce SASP in cells, and its mechanism is closely related to transcriptional cascades, autocrine loops, and sustained DNA damage response.
  • overexpression or inhibition of the classic senescence pathways p53 and p16INK4A/Rb cannot affect the expression of SASP, indicating that although the cell cycle arrest and SASP of senescent cells often occur in concert, the regulatory pathways of the two do not completely overlap. It has been reported that the DNA damage response increases the secretion of SASP factors IL-6 and IL-8 by activating the mutated gene of ataxia telangiectasia, Nijmegen breakage syndrome protein 1, and checkpoint kinase 2.
  • the DNA damage response (DDR) is activated immediately after cell damage, and it takes about 1 week or even longer for mature SASP in senescent cells. Moreover, a short-term DNA damage response cannot induce cell senescence or SASP, indicating that there are other mechanisms that induce SASP in addition to the DNA damage response.
  • SIRT1 is a type of metabolism-related, NADH-dependent deacetylase.
  • SIRT1 in senescent cells inhibits the expression of SASP factors by deacetylation of histone H3K9 and H4K16 in the promoter regions of IL-6 and IL-8.
  • SIRT1 is knocked out, the acetylation levels of these regions during cell senescence are higher than those in the control group.
  • microRNAs are a class of highly conserved single-stranded non-coding RNAs with a length of approximately 20 to 26 nucleotides that regulate gene expression in eukaryotic cells.
  • the results of the study showed that miR-146, miR-34, miR-21, and miR-183 can regulate the SASP of senescent cells and effectively inhibit the excessive production of inflammatory cytokines.
  • miR-146a/b can reduce the production of IL-1 receptor-associated kinase in human umbilical vein endothelial cells; conversely, inhibition of miR-146a/b can increase the activity of IL-1 receptor-associated kinase, activate the transcription factor NF- ⁇ B, and induce the production of IL-6 and IL-8.
  • Drugs that delay aging mainly selectively eliminate senescent cells by temporarily blocking a survival pathway (senescent cell anti-apoptotic pathway SCAPs), which protects senescent cells from the regulation of apoptosis-inducing signals in the environment.
  • SCAPs survival pathway
  • a class of drugs, senolytics is expected to be used in the future to delay, prevent or treat a variety of aging-related diseases.
  • SCAPs senescence-associated anti-apoptotic pathways
  • the SCAPs required for senescent cell survival vary between cell types.
  • the SCAPs required for survival of senescent primary human adipocytes are different from those in senescent human embryonic vein endothelial cells (HUVECs). This difference means that drugs targeting a single SCAP may not be able to eliminate multiple senescent cell types.
  • a large number of studies have shown that most senolytics are indeed only effective against a limited number of senescent cell types. For example, navitoclax is able to target HUVECs, but is ineffective against senescent human adipocytes. There is evidence that the efficacy of senolytics may vary even within a specific type of cell.
  • navitoclax can target and kill senescent cells in the culture-adapted IMR-90 lung fibroblast-like cell line, but has little effect on senescent primary human lung fibroblasts. Therefore, extensive testing on a range of cell types is still needed to determine the broad-spectrum effects of senolytics.
  • the frequency of senolytic drug use may depend on the rate of accumulation of senescent cells, which may vary depending on the environment in which cellular senescence occurs. For example, repeated exposure to cancer therapies that damage DNA or a continuous high-fat diet may lead to the re-accumulation of senescent cells more rapidly than natural aging. Intermittent use of senolytics can reduce the risk of adverse reactions in patients and allow senolytics to be used during healthy periods. In addition, intermittent dosing can also reduce the side effects of senolytics and reduce the possibility of patients developing drug resistance.
  • the purpose of the present invention is to provide the application of salvianolic acid A (SAA) as a new anti-aging drug raw material in cell aging, tumor treatment and life extension.
  • SAA salvianolic acid A
  • the present invention provides a novel anti-aging drug raw material which plays an important role in cell aging, tumor treatment and life extension.
  • a use of salvianolic acid A for preparing a preparation or a pharmaceutical composition wherein the preparation or the pharmaceutical composition is used for:
  • SASP senescence-associated secretory phenotype
  • the aging-associated secretory phenotype is an aging-associated secretory phenotype caused by DNA damage; preferably, DNA damage caused by chemotherapeutic drugs or ionizing radiation.
  • the concentration of salvianolic acid A in the preparation or pharmaceutical composition for inhibiting SASP expression is 10-200 ⁇ M, preferably 20-100 ⁇ M, and more preferably 40-60 ⁇ M (eg, 40, 45, 50, 55 or 60 ⁇ M).
  • the anti-aging method includes inducing senescent cells to restore growth and/or proliferation ability.
  • the elimination of senescent cells includes inducing senescent cells to enter a death program.
  • the concentration of salvianolic acid A in the preparation or pharmaceutical composition for removing senescent cells is >100 ⁇ M, preferably 200-3000 ⁇ M, and more preferably 200-2000 ⁇ M (for example, 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ⁇ M).
  • the salvianolic acid A reduces the resistance of the tumor to chemotherapeutic drugs by inducing senescent cells in the tumor microenvironment to enter a death program, preferably by inducing senescent cells in the tumor microenvironment to enter a death program through caspase-3/7 mediation.
  • the chemotherapy drug is a genotoxic drug.
  • the chemotherapy drug is selected from the following group: mitoxantrone, bleomycin, doxorubicin, or a combination thereof.
  • the tumor is selected from the following group: prostate cancer, lung cancer, gastric cancer, liver cancer, kidney tumor, small intestine cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or a combination thereof.
  • the salvianolic acid A has no effect or substantially no effect on proliferating cells.
  • composition or a pharmaceutical combination comprising a therapeutically effective amount of:
  • the chemotherapy drugs can induce the appearance of senescent cells in the tumor microenvironment.
  • the chemotherapy drug can induce the expression of SASP in the tumor microenvironment; or can induce the upregulation of the senescence marker p16 INK4A .
  • the chemotherapy drug is selected from the following group: mitoxantrone, bleomycin, doxorubicin, or a combination thereof.
  • the chemotherapy drug is mitoxantrone
  • the weight ratio of mitoxantrone to salvianolic acid A is 1:10-100; preferably 1:25-75; more preferably 1:40-60 (eg 1:45, 1:50 or 1:55, more preferably 1:50).
  • the chemotherapy drug is bleomycin
  • the concentration of bleomycin is 1-100 ⁇ g/ml; preferably 20-70 ⁇ g/ml; more preferably 40-60 ⁇ g/ml (eg 45, 50 or 55 ⁇ g/ml, more preferably 50 ⁇ g/ml).
  • the chemotherapy drug is doxorubicin
  • the weight ratio of doxorubicin to salvianolic acid A is 1:20-200; preferably 1:50-150; more preferably 1:80-120.
  • the concentration of salvianolic acid A in the pharmaceutical composition is 10-200 ⁇ M, preferably 20-100 ⁇ M, more preferably 40-60 ⁇ M (eg 40, 45, 50, 55 or 60 ⁇ M), wherein salvianolic acid A is used to inhibit SASP expression.
  • the concentration of salvianolic acid A in the pharmaceutical composition is >100 ⁇ M, preferably 200-3000 ⁇ M, and more preferably 200-2000 ⁇ M (for example, 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ⁇ M), wherein salvianolic acid A is used to induce senescent cells to enter a death program.
  • a method for screening a substance that can cooperate with salvianolic acid A in anti-tumor effects comprising the steps of:
  • the anti-tumor effect includes: promoting tumor regression, preferably including clearing senescent cells in the tumor microenvironment.
  • the detection of cell senescence comprises:
  • the substance is considered to be a substance that can synergize with salvianolic acid A in anti-tumor effects.
  • the SASP factors include IL6, CXCL8, SPINK1, WNT16B, GM-CSF, MMP3, and IL1 ⁇ , but are not limited thereto.
  • the tumor tissue contains tumor cells and stromal cells.
  • the detection of cell aging is the detection of stromal cell aging.
  • the method is in vivo screening or in vitro screening.
  • the anti-tumor effect includes: tumor cell killing rate, tumor tissue volume reduction rate, and tumor-bearing animal survival rate, but is not limited thereto.
  • the method further comprises the step of setting up a control group, which may be a no-treatment control group.
  • the substance includes small molecule compounds and biological macromolecules, but is not limited thereto.
  • the candidate substance is a chemotherapeutic drug.
  • the method is a non-diagnostic and non-therapeutic method.
  • the cells are contacted with salvianolic acid A to inhibit the expression of SASP, eliminate the senescent cells, and/or reduce the resistance of the tumor cells to chemotherapy drugs.
  • the tumor is selected from the following group: prostate cancer, lung cancer, gastric cancer, liver cancer, kidney tumor, small intestine cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or a combination thereof.
  • a method for inhibiting SASP expression, resisting aging or removing senescent cells, reducing tumor resistance to chemotherapeutic drugs, or prolonging life span or late-life survival in a subject in need comprising the steps of:
  • Salvianic acid A is administered to subjects in need, thereby inhibiting SASP expression, resisting aging or eliminating senescent cells, reducing tumor resistance to chemotherapy drugs, or prolonging life span or survival in later life.
  • a method for treating a tumor in a subject in need thereof comprising the steps of:
  • the chemotherapeutic drug is a genotoxic drug, and preferably the chemotherapeutic drug can increase the expression of SASP in the tumor microenvironment.
  • Figure 1 shows the results of SA- ⁇ -Gal staining of proliferating human stromal cells PSC27 (early passages such as p10-20) treated with the chemotherapy drug bleomycin (BLEO) at a concentration of 50 ⁇ g/ml in vitro on days 7-10.
  • PSC27 head passages such as p10-20
  • BLEO chemotherapy drug bleomycin
  • Figure 1 shows the results of SA- ⁇ -Gal staining of proliferating human stromal cells PSC27 (early passages such as p10-20) treated with the chemotherapy drug bleomycin (BLEO) at a concentration of 50 ⁇ g/ml in vitro on days 7-10.
  • Upper figure representative images, lower figure, statistical data.
  • CTRL control cells
  • BLEO cells treated with bleomycin.
  • *** P ⁇ 0.001.
  • Figure 2 shows the results of BrdU staining of PSC27 cells after treatment with the chemotherapy drug bleomycin (BLEO).
  • Upper panel representative images, lower panel, statistical data.
  • CTRL control cells;
  • BLEO cells treated with bleomycin. ****, P ⁇ 0.0001.
  • Figure 3 shows the results of immunofluorescence staining of ⁇ H2AX after PSC27 cells were treated with the chemotherapy drug bleomycin (BLEO). Control cells; BLEO, cells treated with bleomycin. ***, P ⁇ 0.001. According to the number of fluorescent spots in the nucleus, they were divided into 4 categories, including single cells with 0 foci, 1-3 foci, 4-10 foci and >10 foci.
  • FIG4 shows an experimental flow chart for large-scale screening of organic chemical drug libraries to obtain small molecule compounds (including natural and synthetic ones) with anti-aging activity.
  • Figure 5 shows a simplified diagram of the chemical molecular structure of SAA.
  • Figure 6 shows the heatmap after RNA-seq data analysis, showing that a large number of factors were upregulated in senescent cells caused by BLEO damage, but many of them were significantly reversed after SAA treatment. * indicates typical SASP exocytosis factors.
  • FIG. 7 shows the results of GSEA analysis indicating that the expression of SASP-specific molecular markers or related factors was centrally upregulated in senescent cells caused by BLEO, but decreased significantly after SAA treatment of senescent cells.
  • FIG8 shows the results of GSEA analysis showing that the expression of NF- ⁇ B molecular markers or related factors was centrally upregulated in senescent cells caused by BLEO, but was significantly decreased after SAA treatment of senescent cells.
  • Figure 9 shows the representative pathways of 100 molecules in biological processes that were significantly downregulated by SAA in aging cells according to KEGG pathway analysis.
  • Figure 10 shows the representative pathways of cellular components of 100 molecules that were significantly downregulated by SAA in aging cells according to KEGG pathway analysis.
  • Figure 11 shows the relative expression levels of a group of typical SASP molecules in senescent cells induced by BLEO and treated with different concentrations of SAA by fluorescence quantitative PCR (qRT-PCR). All data are normalized results compared with the CTRL group. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • Figure 12 shows the senescence of PSC27 determined by SA- ⁇ -Gal staining under conditions of increasing SAA concentrations. ⁇ , P>0.05; **, P ⁇ 0.01; ****, P ⁇ 0.0001. Among them, the P values of SAA at 100 ⁇ M, 200 ⁇ M, 400 ⁇ M, 800 ⁇ M, 1600 ⁇ M, 2000 ⁇ M and 3000 ⁇ M concentrations are the statistical significance of the positive cell ratios of these experimental groups compared with the data at 0 ⁇ M.
  • Figure 13 shows representative images of PSC27 under various conditions after SA- ⁇ -Gal staining. Three replicates per group, arranged up and down. Scale bar, 30 ⁇ m.
  • Figure 14 shows the survival rate of proliferating cells and senescent cells detected by CCK8 under increasing concentrations of SAA.
  • the P value at each SAA concentration is the significant difference between the CTRL and BLEO groups. **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 15 shows the population doubling (PD) test of PSC27.
  • the cells were treated with BLEO at passage 10 (p10), and SAA was then added to the culture medium on day 8.
  • the effect of SAA on cell proliferation potential was determined by comparing the doubling values (PD) of the CTRL group, BLEO group, SAA group, and BLEO/SAA group. ⁇ , P>0.05; ***, P ⁇ 0.001.
  • Figure 16 shows the induction of caspase 3/7 activity during SAA treatment of senescent cells.
  • PSC27 cells gradually entered the senescent stage after being treated with BLEO for 12 hours under culture conditions.
  • 200 ⁇ M SAA was added to the culture medium of senescent cells starting on day 7.
  • NucLight Rapid Red reagent was used to label cells, and caspase 3/7 reagent (IncuCyte) was used for apoptosis detection.
  • FIG 17 shows that the pan-caspase inhibitor (20 ⁇ M QVD-OPh) reversed the senolytic activity of SAA (200 ⁇ M SAA was used in this experiment, and 1.25 ⁇ 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 (Turkey’ test).
  • Figure 18 shows the apoptosis of PSC27 cells under several conditions as determined by flow cytometry: Q2, the distribution area of early apoptotic cells; Q3, the distribution area of late apoptotic cells.
  • FIG19 shows the comparison of the survival and apoptosis of cells after treatment with BLEO and/or SAA. ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 20 shows a schematic diagram of the dosing method for mice in the preclinical trial.
  • Human stromal cells PSC27 and cancer cells PC3 were mixed in vitro (1:4) and then transplanted into mice subcutaneously to form transplanted tumors. After multiple treatment cycles under single-drug or combined drug administration conditions, the mice were finally killed and pathologically analyzed for changes in the expression of related molecules in their tumor tissues.
  • Figure 21 shows that the CTRL group and BLEO injury group of PSC27 cells were mixed with PC3 in vitro, or PC3 cells were transplanted into the subcutaneous tissue of mice to form transplanted tumors. At the end of the 8th week, the tumors were dissected and obtained, and the volume of the tumors under the conditions of each group was detected and compared. **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 22 shows a schematic diagram of the dosing time and method of mice in the pre-clinical trial.
  • the dosing cycle was every two weeks, and MIT (mitoxantrone) was intraperitoneally administered to mice on the first day of the 3rd/5th/7th week, respectively. From the first day of the 5th week, SAA was intraperitoneally administered to mice once a week. After the 8-week treatment, the mice were dissected and pathological identification and expression analysis were performed.
  • Figure 23 shows the statistical analysis of tumor terminal volume.
  • the chemotherapy drug MIT was administered alone or together with the anti-aging drug SAA to mice, and the tumor sizes of each group were compared and analyzed after the 8th week.
  • Figure 24 shows a comparison of cell senescence in lesions of PC3/PSC27 tumor-bearing animals in preclinical trials. Representative images after SA- ⁇ -Gal staining. Scale bar, 100 ⁇ m.
  • Figure 25 shows the parallel analysis of the percentage of SA- ⁇ -Gal positive cells in tumor tissues in mice. ⁇ , P>0.05; *, P ⁇ 0.05; ****, P ⁇ 0.0001.
  • Figure 26 shows the expression of typical SASP factors in epithelial cancer cells and stromal cells in mouse lesions detected by fluorescence quantitative PCR (qRT-PCR). Stromal cells and cancer cells were specifically separated by LCM technology, and total RNA was prepared and used for SASP expression detection. ⁇ , P>0.05; *, P ⁇ 0.05; **, P ⁇ 0.01.
  • FIG27 shows the expression of SASP factors in stromal cells in mouse lesions after administration of vehicle, MIT and MIT/SAA by fluorescence quantitative PCR (qRT-PCR). *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • Figure 28 shows the analysis of DNA damage and apoptosis ratios in each group of mice after specific separation of cancer cells in lesions using LCM technology. ⁇ , P>0.05; *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 29 shows the image analysis after immunohistochemical staining.
  • Scale bar 200 ⁇ m.
  • Figure 30 shows the Kaplan Meier data comparison of disease-free survival of NOD/SCID mice after various drug administrations.
  • MIT, SAA and MIT/SAA groups exceeded 2000 mm 3 , it was considered that serious disease had occurred, and the mice needed to be killed in time and their tumor-bearing status was tested.
  • ⁇ P>0.05; **, P ⁇ 0.01.
  • FIG31 shows the comparative analysis of the weight data of mice at the end of the treatment under various dosing conditions. ⁇ , P>0.05.
  • Figure 32 shows the comparative analysis of the serological data of mice at the end of the treatment under the above different dosing conditions. Creatinine, urine (kidney index), ALP and ALT (liver index) data were compared in parallel. ⁇ , P>0.05.
  • FIG33 shows the comparative analysis of the body weight data of immune intact mice (C57BL/6J) at the end of the treatment course under various dosing treatment conditions. ⁇ , P>0.05.
  • Figure 34 shows the comparative analysis of blood cell counts of mice at the end of the treatment course under different dosing conditions in the pre-clinical trial.
  • the number of WBC, lymphocyte and neutrophil per unit volume was compared in parallel.
  • Figure 35 shows the statistical analysis of tumor terminal volume.
  • the chemotherapy drug DOX was administered alone or together with the anti-aging drug SAA to mice, and the tumor sizes of each group were compared and analyzed after the 8th week.
  • Figure 36 shows the statistical analysis of tumor terminal volume.
  • the chemotherapy drug DOC was administered alone or together with the anti-aging drug SAA to mice, and the tumor sizes of each group were compared and analyzed after the 8th week.
  • Figure 37 shows the statistical analysis of tumor terminal volume.
  • the chemotherapy drug VIN was administered to mice alone or together with the anti-aging drug SAA, and the tumor sizes of each group were compared and analyzed after the 8th week.
  • salvianolic acid A SAA
  • SAA senescence-associated secretory phenotype
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms “comprise”, “include”, and “contain” are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • the term "pharmaceutically acceptable carrier” refers to a substance that is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, has a reasonable benefit/risk ratio.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount that produces a function or activity in humans and/or animals and is acceptable to humans and/or animals. It should be understood by those skilled in the art that the “therapeutically effective amount” may vary depending on the form of the pharmaceutical composition, the route of administration, the adjuvants of the drug used, the severity of the disease, and the combination with other drugs.
  • Salvianolic acid A (SAA) is extracted and refined from the root of Salvia miltiorrhiza Bge., a plant of the Labiatae family. It is a light yellow crystal with a molecular formula of C 26 H 22 O 10 and is soluble in ethanol and ether. The melting point is 315-323°C.
  • SAA can induce senescent cells to enter the death program by inducing apoptosis.
  • the embodiment also verifies that SAA has little effect on proliferating cells at a concentration of 3000 ⁇ M, and has excellent specificity.
  • SAA can significantly enhance the growth arrest after genotoxic treatment (such as BLEO treatment).
  • genotoxic treatment such as BLEO treatment.
  • the population doubling capacity (PD) of stromal cells is improved, and their proliferation capacity is improved, while having little effect on the PD of proliferating cells, showing good selectivity.
  • the present invention has found through research that certain chemotherapy drugs (such as MIT) can induce the appearance of a large number of senescent cells in tumor tissues and the upregulation of SASP factor expression, which mainly occurs in stromal cells. This increase in SASP factors will promote the therapeutic resistance of surrounding cancer cells. SAA can largely reverse this change. Animal experiments have also shown that for transplanted tumors composed of PC3 cancer cells and primary PSC27 stromal cells, the application of SAA alone has no significant effect on tumor volume, but unexpectedly, it can significantly reduce tumor volume in mice treated with MIT. Compared with MIT, the tumor volume was reduced by 55.1%.
  • Prolong life span or late-life survival The present invention has found through experiments that intermittent treatment with administration once every two weeks starting from a certain time point when WT mice are very old (e.g., 24-27 months old) can surprisingly extend the median survival period after treatment by 72.8% compared with the Vehicle group, while having a lower risk of death. This finding indicates that SAA-mediated senescent cell clearance can reduce the risk of death in elderly mice and effectively prolong their survival.
  • the present invention provides the use of SAA for preparing a preparation or a pharmaceutical composition, wherein the preparation or the pharmaceutical composition is used for:
  • SASP senescence-associated secretory phenotype
  • the reduction of tumor resistance to chemotherapy drugs is carried out by removing senescent cells in the tumor microenvironment and/or inhibiting the expression of SASP in the tumor microenvironment.
  • the tumors include: prostate cancer, lung cancer, gastric cancer, liver cancer, kidney tumors, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumors, bladder tumors, brain cancer, endometrial cancer, testicular cancer, thyroid cancer, or a combination thereof.
  • the tumor is a tumor that has been treated with genotoxic chemotherapy drugs, has an increase in senescent cells in the tumor microenvironment and/or high expression of SASP, and such tumors are prone to chemotherapy resistance.
  • the "chemotherapeutic drug” is preferably a chemotherapy drug that can induce an increase in senescent cells and/or high expression of SASP in the tumor microenvironment.
  • compositions and methods of administration are provided.
  • the present invention also provides a pharmaceutical composition or a drug kit, which comprises a therapeutically effective amount of:
  • the chemotherapy drug can induce the appearance of senescent cells in the tumor microenvironment.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • the chemotherapeutic drug used in combination with SAA is preferably a genotoxic drug, which can induce the appearance of senescent cells in the tumor microenvironment, and more preferably can induce increased expression of SASP in the tumor microenvironment or can induce upregulation of the senescence marker p16 INK4A .
  • the pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the dosage form of the pharmaceutical composition of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent.
  • it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the present invention finds that the effective amount of SAA can be different for different purposes.
  • its concentration can be 10-200 ⁇ M, preferably 20-100 ⁇ M, and more preferably 40-60 ⁇ M (e.g., 40, 45, 50, 55 or 60 ⁇ M).
  • SAA when SAA is used to induce senescent cells to enter a death program, its concentration can be >100 ⁇ M, preferably 200-3000 ⁇ M, and more preferably 200-2000 ⁇ M (e.g., 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ⁇ M).
  • concentration can be >100 ⁇ M, preferably 200-3000 ⁇ M, and more preferably 200-2000 ⁇ M (e.g., 200, 300, 400, 500, 600, 7000, 800, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ⁇ M).
  • the effective amount of the active ingredient of the present invention may vary with the mode of administration and the severity of the disease to be treated.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
  • the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, the route of administration, etc.
  • pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.
  • the severity of the disease to be treated by the patient the patient's body weight, the patient's immune status, the route of administration, etc.
  • the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg/kg of animal body weight (preferably 0.0001 mg-10 mg/kg of animal body weight) every two weeks, a satisfactory effect can be obtained.
  • the frequency of senolytic drug use may depend on the rate of accumulation of senescent cells, which may vary depending on the environment in which cellular senescence occurs. For example, repeated exposure to cancer therapies that damage DNA or a continuous high-fat diet may lead to the re-accumulation of senescent cells more rapidly than natural aging. Intermittent use of senolytics can reduce the risk of adverse reactions in patients and allow senolytics to be used during healthy periods. In addition, intermittent dosing can also reduce the side effects of senolytics and reduce the possibility of patients developing drug resistance.
  • the present invention provides a method for screening substances that can cooperate with salvianolic acid A for anti-tumor effects.
  • the method can be a screening for known chemotherapy drugs, or it can be a screening for substances with unknown anti-cancer effects.
  • the method screens whether the candidate substance is suitable for synergistic anti-tumor effects with SAA by detecting the effect of the candidate substance on cell senescence in the tumor microenvironment.
  • the screening method of the present invention comprises the steps of:
  • the detection of cell senescence includes: (i) detecting the number of senescent cells; (ii) detecting the expression of SASP factors; (iii) detecting the senescence marker p16 INK4A .
  • the candidate substance treatment can increase (such as increasing by 10%, 20%, 30%, 40%, 50% or more) the number of senescent cells, SASP factor expression, and/or senescence marker p16 INK4A in tumor tissues, it is considered that salvianolic acid A can play a role in clearing senescent cells in the tumor microenvironment or reducing the expression of SASP factors in the tumor microenvironment when used in combination with the substance, thereby determining that the substance is a substance that can synergize with salvianolic acid A in anti-tumor.
  • the screening method of the present invention can be performed by in vitro experiments or in vivo experiments, but is not limited thereto.
  • Salvianic acid A is a widely used drug in clinical practice and has high safety.
  • 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% CO2, and proliferated and passaged in PSCC complete culture medium.
  • bleomycin 50 ⁇ g/ml bleomycin (BLEO) was added to the culture medium when PSC27 cells grew to 80% (PSC27-CTRL for short). After 12 hours of drug treatment, the cells were simply washed 3 times with PBS and left in the culture medium for 7-10 days before subsequent experiments.
  • the initially identified candidate drugs were further screened for 30 days.
  • the drugs that entered the second round of candidate range were diluted into 6-well plates with 20,000 cells per well.
  • the culture medium and candidate drugs were replaced every other day.
  • the project conducted confirmatory analysis based on different concentrations of drugs.
  • Proteins from cell lysates were separated using NuPAGE 4-12% Bis-Tris gel and transferred to nitrocellulose membranes (Life Technologies). Blots were blocked with 5% skim milk for 1 h at room temperature, incubated with the desired primary antibody at the manufacturer's agreed concentration overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz) for 1 h at room temperature.
  • Membrane blot signal detection was performed using enhanced chemiluminescence (ECL) detection reagent (Millipore) according to the manufacturer's protocol and using ImageQuant LAS 400 Phospho-Imager (GE Healthcare). As a standard protein marker, the inventors used PageRuler Plus Prestained Protein Ladder (no. 26619) provided by Thermo Fisher Scientific.
  • target cells were pre-seeded on coverslips for at least 24 h after culture in culture dishes. After a brief wash, cells 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 antibodies anti-phospho-Histone H2A.X (Ser139) (clone JBW301, Millipore) and mouse monoclonal antibodies anti-BrdU (Cat#347580, BD Biosciences), and secondary antibodies Alexa 488(or 594)-conjugated F(ab')2 was added sequentially to a slide covered with fixed cells.
  • DAPI 4',6-diamidino-2-phenylindole
  • 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 (deweylab.github.io/rsem).
  • rRNA was depleted from RNA samples using RiboMinus Eukaryote kit (Qiagen, Valencia, CA, USA); and strand-specific RNA-seq libraries were constructed using TruSeq Stranded Total RNA preparation kits (Illumina, San Diego, CA, USA) according to the manufacturer's instructions before deep sequencing.
  • Paired-end transcriptomic reads were mapped to the reference genome (GRCh38/hg38) and annotated with the reference from Gencode v27 using the Bowtie tool. Duplicate reads were identified using the picard tools (1.98) script to mark duplicates (github.com/broadinstitute/picard), and only non-duplicate reads were retained. 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 the Cufflinks, maximum likelihood estimation function. Genes with significantly changed expression were defined by false discovery rate (FDR)-corrected P value ⁇ 0.05, and only Ensembl genes 73 with status “Known” and biotype “coding” were used for downstream analysis.
  • FDR false discovery rate
  • Trim Galore (v0.3.0) (www.bioinformatics.babraham.ac.uk/projects/trim_galore) was used to trim reads, and FastQC (v0.10.0) (www.bioinformatics.bbsrc.ac.uk/projects/fastqc) was used for quality assessment.
  • DAVID bioinformatics platform (david.ncifcrf.gov), Ingenuity Pathways Analysis (IPA) program (www.ingenuity.com/index.html) were used.
  • the raw data were preliminarily analyzed on the Majorbio I-Sanger Cloud Platform (www.i-sanger.com) free online platform and stored in the NCBI Gene Expression Omnibus (GEO) database with the accession code GSE156448.
  • PPI Protein-protein interaction
  • GSEA Gene Set Enrichment Analysis
  • RNA from cells in the growth or stasis phase Extract total RNA from cells in the growth or stasis phase with Trizol reagent. Add 1 ml Trizol to each T25 culture flask cell, scrape the cell layer with a cell scraper and transfer it to a centrifuge tube, mix thoroughly until it is not sticky.
  • the cDNA of the reverse transcription reaction product was diluted 50 times as a template.
  • Samples were added according to the above standards, and the reaction conditions were: pre-denaturation at 95°C for 15 seconds, then 95°C for 5 seconds, 60°C for 31 seconds, and 40 cycles; the melting curve conditions were 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds.
  • the samples were reacted on an ABI ViiA7 (ABI) instrument.
  • the expression of ⁇ -actin was used as an internal reference.
  • the amplification of each gene was checked by software analysis, the corresponding threshold cycle number was derived, and the relative expression of each gene was calculated using the 2- ⁇ Ct method.
  • the peak and waveform of the melting curve were analyzed to determine whether the amplified product obtained was a specific single target fragment.
  • Senescence-associated ⁇ -galactosidase (SA- ⁇ -Gal) staining was performed as previously reported (Debacq-Chainiaux et al., 2009). Briefly, cells were washed in PBS and fixed at room temperature. Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 3 min. SA- ⁇ -Gal staining was then performed with freshly prepared staining solution at 37°C overnight. Images were taken the next day and the percentage of positive cells per unit area was calculated.
  • PSC27 cells were plated in 96-well dishes and induced to senescence by treatment with 50 ⁇ g/ml BLEO.
  • SAA and ABT263 were added at concentrations of 200 ⁇ M and 1.25 ⁇ M, respectively.
  • the cell culture medium was supplemented with Incucyte Nuclight Fast Red Reagent (Essen Bioscience) and Incucyte C-3/7 Apoptosis Reagent (Essen Bioscience). Representative fields were selected for photography.
  • mice All experiments on experimental mice were conducted in strict accordance with the relevant regulations of the Laboratory Animal Care and Use Committee (IACUC) of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
  • Immunodeficient mice NOD-SCID mice, ICR
  • Stromal cells PSC27 and epithelial cells PC3 were mixed in a predetermined ratio of 1:4, and each transplant contained 1.25 ⁇ 10 6 cells for tissue reconstruction.
  • the transplanted tumors were implanted into mice by subcutaneous transplantation, and the animals were euthanized 8 weeks after the transplantation surgery.
  • mice with subcutaneous transplantation were fed a standard experimental diet, and two weeks later, the chemotherapy drugs mitoxantrone (MIT, 0.2 mg/kg dose) and/or SAA (500 ⁇ l, 10 mg/kg dose) were intraperitoneally administered.
  • the time points were: the former was on the first day of the 3rd, 5th, and 7th week, and the latter was on the first day of the 5th and 7th week.
  • a total of three cycles of MIT were administered throughout the treatment course, each cycle lasting 2 weeks.
  • the mouse tumors were collected for volume measurement and histological analysis. Each mouse received a total of 0.6 mg/kg body weight of MIT and 30 mg/kg body weight of SAA.
  • MIT was administered to mice by intravenous infusion according to the above steps and sequence, but the dose was reduced to 0.1 mg/kg body weight/each time (the cumulative MIT dose received during the entire treatment course was 0.3 mg/kg body weight) to reduce drug-related toxicity.
  • the chemotherapy trial ended at the end of the 8th week, and the mice were killed and dissected immediately, and their transplanted tumors were collected And used for pathological system analysis.
  • the inventors obtained 16-month-old male C57BL/6 mice by continuous breeding in the SPF animal platform, with 4 to 5 animals in each cage.
  • the inventors first sorted the mice from low to high by weight, and then selected mice of similar weight.
  • the aging (SEN) or control (CTRL) transplantation treatment was assigned to mice at intervals using a random number generator, while the middle mice were assigned to another treatment, so that the weight of the aging and control transplanted mice was matched.
  • One month after the cell transplantation when the mice were 18 months old, physical function tests were performed. After that, no further tests were performed on these mice except for checking their cages. The earliest death occurred about 2 months after the last physical function test.
  • mice C57BL/6 mice aged 19 to 21 months were placed in 3-5 mice per cage. As with the transplanted mice, the mice were sorted according to weight and randomly assigned to each group, and the control group (vehicle) or drug group (SAA) group treatment was performed by people who did not know the design of the pre-clinical trial. Starting from 24-27 months of age, mice were treated with vehicle or SAA once every 2 weeks by oral gavage for 3 consecutive days each time. During the study, some mice were removed from their original cages to minimize animal housing stress caused by long-term housing in a single cage. RotaRod and hanging tests were performed monthly because these tests are sensitive and non-invasive.
  • vehicle vehicle
  • SAA drug group
  • mice At the end of the experiment, the inventors euthanized the mice; if they showed one of the following symptoms, the inventors considered them dead: (a) unable to drink water or eat; (b) unwilling to move even with stimulation; (c) rapid weight loss; (d) severe balance disorders; or (v) bleeding in the body or the appearance of ulcerated tumors.
  • no mice were excluded due to fighting, accidental death or dermatitis.
  • the inventors used the Cox proportional hazard model for survival analysis.
  • Cages were checked daily and dead mice were removed from cages. Within 24 hours of death, carcasses were opened (abdomen, thorax, and skull) and individually preserved in 10% formalin for at least 7 days. Decomposed or damaged bodies were excluded. Preserved carcasses were transported to a dedicated Autopsy location for pathological examination. Tumor burden (the sum of different types of tumors per mouse), disease burden (the sum of different histopathological changes in major organs of each mouse), severity of each lesion, and inflammation (lymphocyte infiltration) were assessed.
  • Tumor burden the sum of different types of tumors per mouse
  • disease burden the 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 luciferin (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 measured using a Grip Strength Meter (Columbus Instruments, Columbus, OH), and the results were averaged from more than 10 trials.
  • For the hanging endurance test mice were placed on a 2 mm thick metal wire, which was located 35 cm above the mat. Mice were only allowed to grasp the wire with their forelimbs, and the hanging time was normalized according to body weight and expressed as hanging duration (sec) ⁇ body weight (g). The 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 the 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, with 3 days of training, lasting 5 minutes per day, starting at 5 m/min for 2 minutes, then accelerating to 7 m/min for 2 minutes, and then 9 m/min for 1 minute.
  • mice were treadmilled at an initial speed of 5 m/min for 2 min, and then the speed was increased by 2 m/min every 2 min until the mice were exhausted. Fatigue was defined as the inability of the mice to return to the treadmill even with mild electric shock and mechanical stimulation.
  • the distance was recorded and the total work (KJ) was calculated using the following formula: mass (kg) ⁇ g (9.8 m/s 2 ) ⁇ distance (m) ⁇ sin (5°).
  • Example 1 SAA can effectively inhibit the expression of SASP when used at low concentrations
  • the present invention has carried out unbiased screening of an organic chemical drug library composed of small molecules.
  • the inventors chose to use a primary normal human prostate stromal cell line, namely PSC27, as an in vitro cell model.
  • PSC27 is mainly composed of fibroblasts, while non-fibroblast cell lines (including endothelial cells and smooth muscle cells) also exist, but in a smaller proportion.
  • PSC27 is a human primary stromal cell line in nature, and forms a typical SASP after exposure to stress factors such as genotoxic chemotherapy or ionizing radiation.
  • the inventors treated these cells with a specific dose of bleomycin (BLEO) in a way that has been optimized in the preliminary experiment, and observed that the positive rate of senescence-associated ⁇ -galactosidase (SA- ⁇ -Gal) staining was significantly increased, the BrdU incorporation rate was greatly reduced, and the DNA damage repair foci (DDRfoci) increased significantly within a few days after drug damage ( Figures 1-3).
  • the inventors compared the effects of these natural drug products on the expression profile of senescent cells in parallel by high-throughput high-content system screening (Figure 4).
  • the inventors performed RNA-seq sequencing on these cells.
  • the high-throughput data subsequently obtained showed that a small molecule compound, salvianolic acid A (SAA) (Figure 5), significantly changed the expression profile of senescent cells.
  • SAA salvianolic acid A
  • thousands of genes were significantly downregulated, while multiple genes were upregulated.
  • the expression of SASP factors in senescent cells after SAA treatment was generally reduced, while these SASP factors were generally significantly upregulated in senescent cells (Figure 6).
  • Example 2 SAA is a novel senolytic when used at high concentrations
  • the inventors next investigated the potential for population doubling (PD) of stromal cells after genotoxic treatment. Compared with the BLEO group of cells that quickly entered a growth arrest state after damaging treatment, the combined treatment group of BLEO and SAA showed a significantly increased PD capacity (Figure 15). Interestingly, however, SAA itself did not seem to affect the PD of proliferating cells, which further suggests the selectivity of SAA between senescent cells and normal cells.
  • Example 3 Therapeutic targeting of senescent cells using SAA can promote tumor regression and effectively reduce chemotherapy resistance
  • cancer is one of the major chronic diseases that seriously threatens human life span and health.
  • cancer cell resistance limits the effectiveness of most anti-cancer treatments, and senescent cells often promote the occurrence of therapeutic resistance in surrounding cancer cells by developing SASP in damaged tumor foci. Even so, the feasibility and safety of removing senescent cells from primary tumors to promote the cancer therapeutic index has hardly been explored by scientists so far.
  • the inventors constructed tissue reconstructive cells by mixing PSC27 stromal cells with PC3 epithelial cells.
  • the latter is a typical highly malignant prostate cancer cell line.
  • the ratio of stromal cells to epithelial cells was 1:4.
  • the tumor size (volume) was measured ( Figure 20).
  • the inventors then compared the survival of animals in the different drug treatment groups, primarily evaluating the consequences of tumor progression in a time-extended manner.
  • the inventors performed Tumor growth monitoring, once the mouse has a significant tumor burden (size ⁇ 2000 mm 3 ), is considered severe disease, which is a method used to monitor the progression of certain diseases such as tumors.
  • Mice treated with the MIT/SAA combination showed the longest median survival, extending survival by at least 48.1% compared to the group treated with MIT alone ( FIG. 30 ).
  • treating tumor-bearing mice with SAA alone did not result in a significant benefit, with only a marginal extension of survival.
  • Example 4 Elimination of senescent cells caused by SAA treatment can prolong the late-life survival of experimental mice
  • the present invention further studies whether it also has some significant benefits for promoting health or delaying disease in naturally aged animals.
  • the inventors first considered whether a method with potential translational value could be used to eliminate senescent cells, namely: intermittent treatment from a very old time point to explore whether the remaining life span of WT mice could be extended. Based on this, a series of in vivo experiments were carried out.

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Abstract

La présente invention concerne l'utilisation de l'acide salvianolique A (SAA) comme matière première d'un nouveau médicament anti-âge, dans le traitement de la sénescence cellulaire, le traitement des tumeurs et la prolongation de la durée de vie. Selon la présente invention, il a été découvert que le SAA peut cibler spécifiquement des cellules sénescentes et qu'il présente un effet d'inhibition de l'expression du phénotype sécrétoire associé à la sénescence (SASP), d'élimination des cellules sénescentes et de récupération de l'activité de prolifération des cellules sénescentes. En outre, le SAA peut éliminer les cellules stromales sénescentes et inverser la tendance d'expression du SASP des cellules stromales sénescentes dans un micro-environnement tumoral après un traitement par médicaments chimiothérapeutiques génotoxiques, ce qui réduit la résistance des tumeurs aux médicaments chimiothérapeutiques et améliore de façon significative les effets antitumoraux réels des médicaments chimiothérapeutiques. De plus, le SAA peut prolonger de façon significative la survie d'animaux de laboratoire à un stade avancé de leur vie.
PCT/CN2023/121621 2022-09-26 2023-09-26 Utilisation d'acide salvianolique a (saa) comme matière première d'un nouveau médicament anti-âge, dans le traitement de la sénescence cellulaire, le traitement des tumeurs et la prolongation de la durée de vie WO2024067604A1 (fr)

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CN202211176616.5A CN117205193B (zh) 2022-09-26 2022-09-26 丹参酚酸a(saa)作为新型抗衰老药物原料在细胞衰老、肿瘤治疗与延长寿命中的应用
CN202211176616.5 2022-09-26

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