WO2022166839A1 - 银杏叶提取物在制备靶向衰老细胞、抑制肿瘤或延长寿命的药物中的应用 - Google Patents

银杏叶提取物在制备靶向衰老细胞、抑制肿瘤或延长寿命的药物中的应用 Download PDF

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WO2022166839A1
WO2022166839A1 PCT/CN2022/074740 CN2022074740W WO2022166839A1 WO 2022166839 A1 WO2022166839 A1 WO 2022166839A1 CN 2022074740 W CN2022074740 W CN 2022074740W WO 2022166839 A1 WO2022166839 A1 WO 2022166839A1
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ginkgo biloba
biloba extract
cells
tumor
gle
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French (fr)
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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
    • A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
    • 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
    • A61K31/70—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the present invention belongs to the field of cell biology and oncology; the inventors are devoted to research and screening of drugs targeting the tumor microenvironment and helping to enhance the tumor-inhibiting effect of chemotherapeutic drugs, removing senescent cells or inhibiting cell senescence.
  • 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, usually caused by stressful signals such as DNA damage.
  • Replicative senescence of cells refers to the fact that normal cells cease to divide continuously after approximately 30-50 divisions (the "Hayflick limit").
  • Replicative senescence is essentially induced by progressive shortening of telomeres. During each round of DNA replication, telomeres gradually shorten, eventually reaching a critical length that prevents further replication and thus stops cell division. Shorter uncapped telomeres elicit a DNA damage response that directly triggers senescence.
  • 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. In recent years, a large number of studies have shown that SASP plays a core pathological role in this process.
  • SASP factors secreted by senescent cells can also affect surrounding normal cells, and inhibition of SASP can delay the aging of the body.
  • Typical SASP factors include tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1a (IL-1a), matrix metalloproteinases ( MMP), granulocyte-macrophage colony stimulating factor (GM-CSF), and plasminogen activator inhibitor-1 (PAI1), etc., which promote immune system activation, leading to abnormal factors such as senescent cells in the tissue microenvironment It is cleared by the body and exerts tumor suppressor function.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • IL-1a interleukin 1a
  • MMP matrix metalloproteinases
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • PAI1 plasmin
  • SASP can still promote tumor development through specific secreted factors (eg, 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, and this chronic inflammation can also promote the occurrence and development of aging-related tissue damage and degeneration, organ dysfunction, and cancer and other aging-related diseases.
  • SIRT1 is metabolically related, NADH-dependent sirtuins, and SIRT1 has been found to have lifespan-extending effects in various models.
  • SIRT1 inhibits the expression of SASP factors by deacetylating histones H3K9 and H4K16 in the promoter regions of IL-6 and IL-8.
  • SIRT1 is knocked out, the levels of acetylation in these regions during cell senescence are higher than those in control cells .
  • MicroRNAs are a class of highly conserved single-stranded non-coding RNAs, about 20-26 nucleotides in length, that regulate gene expression in eukaryotic cells.
  • the results showed that miR-146, miR-34, miR-21 and miR-183 could regulate the SASP of senescent cells and effectively inhibit the overproduction of inflammatory cytokines.
  • miR-146a/b can reduce the production of IL-1 receptor-related kinase in human umbilical vein endothelial cells; on the contrary, inhibiting miR-146a/b can increase the activity of IL-1 receptor-related kinase, activate the transcription factor NF- ⁇ B, induce IL-6 and IL-8 production.
  • Drugs that delay senescence mainly selectively eliminate senescent cells by temporarily blocking survival pathways (senescent cell anti-apoptotic pathway SCAPs), which protect senescent cells from the regulation of apoptosis-inducing signals in the environment.
  • survival pathways senescent cell anti-apoptotic pathway SCAPs
  • a class of drugs namely Senolytics, may be used in the future to delay, prevent or treat a variety of aging-related diseases.
  • 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.
  • UUVECs senescent human embryonic venous endothelial cells
  • navitoclax targets and kills senescent cells in the culture-adapted IMR90 lung fibroblast-like cell line, but is less effective on senescent human primary 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 application of Ginkgo biloba extract in the preparation of drugs targeting senescent cells, inhibiting tumors or prolonging lifespan.
  • the application of Ginkgo biloba extract is provided for preparing a composition for specifically targeting senescent cells in tumor microenvironment and inhibiting tumor in combination with chemotherapeutic drugs; wherein, the chemotherapeutic drugs are for Chemotherapy drugs that induce senescence-associated secretory phenotype (SASP) in the tumor microenvironment after treatment.
  • chemotherapeutic drugs are for Chemotherapy drugs that induce senescence-associated secretory phenotype (SASP) in the tumor microenvironment after treatment.
  • 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 the senescence-related secretory phenotype caused by DNA damage; preferably, the DNA damage is DNA damage caused by chemotherapeutic drugs.
  • the chemotherapeutic drugs are genotoxic drugs; more preferably, they include: mitoxantrone, doxorubicin, and bleomycin.
  • the Ginkgo biloba extract specifically targets and induces senescent cells in the tumor microenvironment to enter the death program; 7 mediated.
  • the chemotherapeutic drug is mitoxantrone
  • the weight ratio of mitoxantrone to ginkgo biloba extract is 1:20-80; The weight ratio is 1:30-70; more preferably, the weight ratio of mitoxantrone to Ginkgo biloba extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the chemotherapeutic drug is bleomycin
  • the final concentration of bleomycin is 30-70ug/mL, preferably 40-60ug/mL, more preferably 45-55ug/mL
  • Ginkgo biloba extract The final concentration of the substance (low concentration inhibits SASP expression) is 200-550uM, preferably 250-500uM, more preferably 300-420uM (eg 350uM, 400uM).
  • the chemotherapeutic drug is bleomycin
  • the final concentration of bleomycin is 30-70ug/mL, preferably 40-60ug/mL, more preferably 45-55ug/mL
  • Ginkgo biloba extract The final concentration is 700-5000uM, preferably 750-4000uM, more preferably 750-3500uM (such as 780, 800, 900, 1000, 1500, 2000, 2500, 3000uM).
  • the chemotherapeutic drug is doxorubicin
  • the weight ratio of doxorubicin to ginkgo biloba extract is 1:4-16; preferably, the weight ratio of doxorubicin to ginkgo biloba extract is 1:6-14; more preferably, the weight ratio of doxorubicin and Ginkgo biloba extract is 1:8-12 (eg 1:9, 1:10, 1:11).
  • the application of Ginkgo biloba extract for: preparing a composition for inhibiting senescence; or preparing a composition for prolonging lifespan or prolonging survival in later life; or preparing a specific targeted elimination tumor microenvironment
  • a composition of senescent cells, or a composition for inhibiting (reducing) senescence-related secretory phenotype preferably, the Ginkgo biloba extract specifically targets and induces senescent cells in the tumor microenvironment to enter the death program (preferably, Proliferating cells are largely unaffected by it).
  • the concentration of Ginkgo biloba extract is 200-5000uM; preferably 250-4000uM; more preferably 300-3500uM (eg 2500, 3000uM).
  • the preparation method of the Ginkgo biloba extract includes a two-step extraction method: (1) mixed enzyme-catalyzed enzymatic hydrolysis; (2) organic solvent extraction; preferably, in (1), the Enzymes include cellulase, pectinase, ligninase and protease.
  • Ginkgo biloba leaves are crushed and then suspended in water, and mixed with enzyme preparations for 8-20 hours to be fully enzymatically hydrolyzed, and then filtered to obtain an enzymatic hydrolysate; preferably, ( 2), the enzymatic hydrolyzate is mixed with the ethanol solution and then heated to reflux and extracted; preferably, after step (2), it also includes: the extract is concentrated and purified by ultrafiltration based on membrane separation technology, and then subjected to low temperature Vacuum concentration to obtain the final product of Ginkgo biloba extract.
  • a pharmaceutical composition or kit for specifically targeting senescent cells in tumor microenvironment and inhibiting tumors comprising: Ginkgo biloba extract, and chemotherapeutic drugs; wherein, the The chemotherapeutic drugs used are those that induce senescence-related secretory phenotypes in the tumor microenvironment after administration.
  • a method for preparing a pharmaceutical composition or kit for inhibiting tumors comprising: mixing Ginkgo biloba extract with chemotherapeutic drugs; or placing Ginkgo biloba extract and chemotherapeutic drugs in the same kit middle.
  • the chemotherapeutic drug is mitoxantrone
  • the weight ratio of mitoxantrone to ginkgo biloba extract is 1:20-80; The ratio is 1:30-70; more preferably, the weight ratio of mitoxantrone to Ginkgo biloba extract is 1:40-60 (eg 1:45, 1:50, 1:55).
  • the chemotherapeutic drug is bleomycin
  • the final concentration of bleomycin is 30-70ug/mL, preferably 40-60ug/mL, more preferably 45-55ug/mL
  • Ginkgo biloba extract The final concentration of the substance (low concentration inhibits SASP expression) is 200-550uM, preferably 250-500uM, more preferably 300-420uM (eg 350uM, 400uM).
  • the chemotherapeutic drug is bleomycin
  • the final concentration of bleomycin is 30-70ug/mL, preferably 40-60ug/mL, more preferably 45-55ug/mL
  • Ginkgo biloba extract The final concentration is 700-5000uM, preferably 750-4000uM, more preferably 750-3500uM (such as 780, 800, 900, 1000, 1500, 2000, 2500, 3000uM).
  • the chemotherapeutic drug is doxorubicin
  • the weight ratio of doxorubicin to ginkgo biloba extract is 1:4-16; preferably, the weight ratio of doxorubicin to ginkgo biloba extract is 1:6-14; more preferably, the weight ratio of doxorubicin and Ginkgo biloba extract is 1:8-12 (eg 1:9, 1:10, 1:11).
  • the Ginkgo biloba extract is mixed with chemotherapeutic drugs, and divided into unit dosage forms according to the course of treatment.
  • a method for screening potential substances that promote Ginkgo biloba extract to remove senescent cells or inhibit tumors or prolong lifespan in tumor microenvironment comprising: (1) providing a tumor microenvironment system , the system includes tumor cells and stromal cells; (2) the system of (1) treated with chemotherapeutic drugs induces senescence-related secretory phenotypes in the tumor microenvironment, and before and when inducing senescence-related secretory phenotypes in the tumor microenvironment Or after that, it is treated with Ginkgo biloba extract; (3) the candidate substance is added to the system of (2), and its effect on the tumor microenvironment system is observed, if the candidate substance can statistically promote (significantly promote); , such as promoting 10%, 20%, 30%, 50% or more) Ginkgo biloba extract to remove senescent cells in the tumor microenvironment, then this candidate substance can be used in combination with Ginkgo biloba extract to remove tumor microenvironment.
  • apoptosis or senescence-related secretory phenotype is assessed by observing caspase-3/7 activity or SASP factor expression.
  • the SASP factors include but are not limited to: IL6, CXCL8, SPINK1, WNT16B, GM-CSF, MMP3, CXCL1, CXCL3, IL-1 ⁇ , IL-1 ⁇ ; or, by observing the aging marker p16 INK4A in chemotherapy animals. Assessment of apoptosis or senescence-associated secretory phenotypes.
  • 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 Ginkgo biloba 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 combined with Ginkgo biloba extract.
  • a control group is also included, so as to clearly distinguish the difference between the tumor microenvironment system/senescence-related secretory phenotype system in the test group and the control group, or the removal of senescent cells in the tumor microenvironment by Ginkgo biloba extract difference from 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 stromal cells PSC27 (early passages such as p10-20) were stained by SA- ⁇ -Gal on days 7-10 after in vitro treatment with the chemotherapeutic drug bleomycin (BLEO) at a concentration of 50 ⁇ g/ml results after.
  • Top panel representative image, bottom panel, statistical data.
  • FIG. 1 BrdU staining of 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 Immunofluorescence staining of PSC27 cells with ⁇ H2AX after treatment 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.
  • Figure 4 Experimental flow chart for screening natural product drug libraries to obtain plant materials with anti-aging activity.
  • Heatmap shows that the expression of a large number of factors is up-regulated in senescent cells caused by BLEO injury, but many of them are significantly reversed after GLE treatment. Red star logo, typical SASP exogenous factor.
  • FIG. 7 GSEA analysis results show that the expression of SASP or NF- ⁇ B molecular marker-related factors is centrally up-regulated in BLEO-induced senescent cells, but significantly decreased after GLE-treated senescent cells. Left, SASP molecular marker; right, NF- ⁇ B molecular marker.
  • Figure 9 KEGG pathway analysis. Representative pathways on biological process of 100 molecules that GLE caused significant downregulation 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 the 100 molecules that GLE caused significant downregulation in senescent cells. Left Y-axis, percentage. Right Y-axis, log10(p-value).
  • FIG. 11 Real-time 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 GLE. All data are normalized results compared to the CTRL group. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 13 Representative pictures of PSC27 under various conditions after SA- ⁇ -Gal staining. 3 repetitions per set, up and down. Scale bar, 30 ⁇ m.
  • CCK8 detects the survival rate of proliferating cells and senescent cells under increasing concentrations of GLE. P values at each GLE 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 treated with BLEO lesions, followed by the addition of GLE to the medium at day 8.
  • the effect of GLE on cell proliferation potential was determined by comparing the doubling value (PD) of CTRL group, BLEO group, GLE group and BLEO/GLE group. ⁇ , P>0.05; ***, P ⁇ 0.001.
  • Caspase 3/7 activity is induced during GLE treatment of senescent cells.
  • PSC27 cells gradually entered the senescence stage after being treated with BLEO for 12 h. 800 ⁇ M of GLE was added to the medium of senescent cells starting at day 7, NucLight Rapid Red reagent was used to label cells, and caspase 3/7 reagent (IncuCyte) was used for apoptosis detection.
  • Pan-caspase inhibitor (20 ⁇ M QVD-OPh) reverses the senolytic activity of GLE (800 ⁇ M GLE was used in this experiment, while 200 ⁇ M ABT263 was used as a positive control; the latter is a recently reported inducer of apoptosis in senescent cells ). Statistical differences were obtained by two-way ANOVA (Turkey'test).
  • Figure 18 Flow cytometric determination of apoptosis in PSC27 under several conditions. Q2, distribution area of early apoptotic cells; Q3, distribution area of late apoptotic cells.
  • FIG. 20 Schematic diagram of dosing in mice in preclinical trials.
  • 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 the 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 dosing time and mode of administration in preclinical 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 GLE 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 GLE, and the tumor size of each group was compared after the end of the 8th week.
  • Figure 24 Comparison of cellular senescence in lesions of PC3/PSC27 tumor-bearing animals in preclinical trials. Representative pictures after SA- ⁇ -Gal staining. Scale bar, 100 ⁇ m.
  • Figure 25 Parallel analysis of the percentage of SA-beta-Gal staining positive cells in tumor tissue in mice in vivo. ⁇ , P>0.05; **, P ⁇ 0.01: ***, P ⁇ 0.001.
  • FIG 26 Real-time 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 Real-time quantitative PCR (qRT-PCR) assay to analyze the expression status of SASP factor in stromal cells in mouse lesions after vehicle, MIT and MIT/GLE 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 specific isolation of cancer cells from 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 Kaplan Meier data comparison of disease-free survival in NOD/SCID mice after various drug treatments. Animals in Vehicle(V), MIT, GLE and MIT/GLE groups were considered to have severe disease when the tumor volume in vivo exceeded 2000 mm 3 , and the mice needed to be sacrificed 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 counts at the end of the course of treatment under different administration conditions in the pre-clinical setting. WBC, lymphocyte and neutrophil unit volume numbers were compared in parallel. ⁇ , P>0.05.
  • Figure 35 Statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug DOX was administered to mice alone or together with the anti-aging drug GLE, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • FIG. 36 Statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug DOC was administered to mice alone or together with the anti-aging drug GLE, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 37 Statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug VIN alone or together with the anti-aging drug GLE was administered to the mice, and the tumor size of each group was compared after the end of the 8th week.
  • V Vehicle
  • GLE GLE group
  • V Vehicle
  • GLE GLE group
  • Figure 41 Select male mice with the highest lifespan in each group, and perform a comparative analysis of the highest walking speed, endurance and overall lifespan between groups.
  • V Vehicle.
  • N 5/group.
  • Figure 42 Comparative analysis of the disease burden at the end of life for each mouse in the two groups of animals.
  • N 60/group.
  • the inventors are committed to research and screening of drugs that target the tumor microenvironment and remove senescent cells, and reveal that Ginkgo biloba extract (GLE) can target the tumor microenvironment and remove senescent cells. , can promote the inhibition of tumors by chemotherapy drugs by removing senescent stromal cells, and the promoting effect is extremely significant.
  • GLE Ginkgo biloba extract
  • the GLE can also target the clearance of senescent cells, thereby inhibiting the SASP.
  • the GLE can also significantly prolong the lifespan of animals, significantly prolong the survival period of old age, and improve the quality of life of animals.
  • GLE can specifically target and remove senescent cells in the tumor microenvironment, it has no specific inhibitory effect on tumor cells; and although chemotherapeutic drugs can inhibit tumor cells, it has a great impact on the tumor microenvironment. , but it can cause significant side effects, especially the formation and development of SASP, and it is easy to cause cancer cells to develop drug resistance after continuous use.
  • chemotherapeutic drugs can effectively play a benign complementary role in targeting the disease, and achieve unexpected synergistic effects.
  • Ginkgo biloba leaf extract is generally extracted from the leaves of Ginkgo biloba L., a plant in the Ginkgo family.
  • Various methods can be used to extract GLE, such as, but not limited to: enzymatic hydrolysis, water extraction, organic solvent extraction, microwave method, supercritical CO 2 extraction, etc., or a combination thereof.
  • the organic solvent used may include, but is not limited to, ethanol, methanol, acetone, and the like.
  • the crude GLE product can be further purified, and the purification can adopt (but not limited to): solvent extraction method, precipitation method, enzymatic hydrolysis method, ultrafiltration method, macroporous resin method and the like.
  • the Ginkgo biloba extract adopts a two-step preparation process of biocatalysis and chemical extraction: the first step is industrial source mixed enzyme-catalyzed enzymatic hydrolysis, and the second step is chemically pure organic solvent extraction.
  • the selected plant raw material is natural ginkgo biloba, and the selected enzymes include cellulase, pectinase, ligninase and protease for commercial use.
  • the present invention also includes the Ginkgo biloba extract obtained by performing appropriate process changes on the basis of the preferred extraction process.
  • the preferred two-step preparation technique of the present invention differs from most existing phytochemical separation techniques. Compared with traditional methods such as simple solvent extraction, ion precipitation, ultrasonic extraction and microwave extraction, the extraction method of this patent is beneficial to obtain higher purity and proportion of plant polyphenols (white amorphous crystals), including flavanones Classes, anthocyanins, flavonols, anthocyanins, phenolic acids and depsidic acids. Among them, flavanones (mainly catechin compounds) can account for 70-90% of the total amount of polyphenols finally obtained by the method, which generally improves the yield, reduces costs and reduces pollution.
  • flavanones mainly catechin compounds
  • GLE is also commercially available.
  • GLE Ginkgo biloba extract
  • the killing effect of GLE on senescent cells is very ideal at an appropriate concentration.
  • the inventors have found that when GLE reaches a threshold at 2000 [mu]M, senescent cells remain at 20% or less at this point. Therefore, at a certain concentration, GLE is a new type of senolytics and exhibits excellent effects. Target specificity is very good.
  • 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 combination of GLE and genotoxic drugs can rapidly restore the proliferative potential of stromal cells in a short period of time, which is in sharp contrast to the use of genotoxic drugs alone, which is surprising.
  • GLE itself does not affect the PD of proliferating cells, and this data further suggests that GLE is selective and target specific between senescent and normal cells.
  • 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, GLE combined with MIT can significantly reduce the tumor; compared with MIT, the tumor volume was reduced by 55.1%; compared with placebo treatment, the tumor volume was reduced by 74.6%. This inhibitory effect is surprising.
  • the inventors also found that the MIT administration process induced the appearance of a large number of senescent cells in tumor tissue.
  • GLE administration essentially depleted most of the senescent cells within the lesions of these chemotherapy animals.
  • the expression of SASP factors was significantly elevated (mainly in stromal cells); however, this change was largely reversed when GLE was administered.
  • MIT-treated animals were used with GLE, the indices of DNA damage or apoptosis were significantly enhanced, implying enhanced tumor site cytotoxicity in animals treated with these senescent drugs; when GLE was applied therapeutically, cells apoptotic The activity of caspase3/4, a typical marker of apoptosis, was significantly increased.
  • mice treated with the MIT/GLE combination exhibited the longest median survival; survival was greatly prolonged.
  • CONCLUSIONS GLE therapeutically targeting senescent cells can promote tumor suppression and reduce chemoresistance.
  • the inventor's research also found that under the treatment regimen of taking the drug once every two weeks, the GLE group that was administered from the age of 24-27 months (equivalent to the age of 75-90 years in humans), the treatment The post-median survival was 72.8% longer than the Vehicle group with a lower risk of death, indicating that GLE-mediated senescent cell clearance can reduce the risk of death in aged mice and effectively prolong their survival.
  • Intermittent delivery of GLE a biologically active antiaging drug, can significantly reduce the disease burden of aging organisms by removing senescent cells from the microenvironment and increase the lifespan of the organism in the post-treatment phase. This type of treatment, which does not lead to a significant increase in body morbidity, can actually be used safely in the later stages of life.
  • the present invention provides a use of GLE 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 of the substances described can be found in combination with GLE and exert a synergistic effect.
  • 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, when or after inducing the tumor microenvironment to produce a senescence-related secretory phenotype, treating with GLE; in step (3), it further includes: if If the candidate substance can statistically promote GLE 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 GLE 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 Ginkgo biloba on the senescence-related secretory phenotype, the candidate substance can be used in combination with GLE to inhibit senescence.
  • Potential substances associated with secretory phenotypes are examples of potential substances that inhibit the senescence-related secretory phenotype.
  • 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 effective amounts (eg 0.00001-50wt%; preferably 0.0001-20wt%; 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%), and a pharmaceutically acceptable carrier.
  • effective amounts eg 0.00001-50wt%; preferably 0.0001-20wt%; 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier eg 0.00001-50wt%; preferably 0.0001-20wt%; 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier eg 0.00001-50wt%; preferably 0.0001-20wt%; 20wt%; preferably 0.00001-10wt%; more preferably, 0.0001-2wt%
  • a pharmaceutically acceptable carrier eg 0.00001-50wt%; preferably 0.0001-20wt%
  • the present invention also provides a pharmaceutical composition, which contains an effective amount (eg, 0.00001-10wt%; preferably 0.0001-5wt%; more preferably, 0.001-2wt%) of the GLE, and is pharmaceutically acceptable Carrier.
  • an effective amount eg, 0.00001-10wt%; preferably 0.0001-5wt%; more preferably, 0.001-2wt% of the GLE, and is pharmaceutically acceptable Carrier.
  • 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, GLE and chemotherapeutic drugs), the composition may contain minor amounts of minor ingredients and/or impurities that do not affect the active ingredients.
  • sweeteners may be included to improve taste, antioxidants to prevent oxidation, and other additives commonly used in the art.
  • the concentration of the GLE can be 200-550uM, preferably 250-500uM, more preferably 300-420uM; such as 350uM, 400uM.
  • GLE and/or chemotherapeutics 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 GLE 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 GLE 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.
  • 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. Repeated exposure to DNA-damaging cancer therapies or a persistent high-fat diet, for example, may lead to the re-accumulation of senescent cells more rapidly than natural aging. Intermittent use of senolytics reduces the risk of adverse effects in patients and allows senolytics to be used during healthy periods. In addition, intermittent dosing can reduce the side effects of senolytics and reduce the likelihood that patients will develop drug resistance.
  • 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. 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 may 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 GLE 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.
  • the cells in logarithmic growth phase were collected with 0.25% trypsin, centrifuged at 1000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in freshly prepared freezing medium. Aliquot cells into sterile cryovials as indicated. Then, it is cooled by gradient and finally transferred to liquid nitrogen for long-term storage.
  • PSC27-CTRL 50 ⁇ g/ml bleomycin
  • 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 3/7 activity 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 (ie, every two days). 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 with 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)-correctedP value ⁇ 0.05, and downstream analysis was performed with only 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).
  • DESeq2independent 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 previous publications by the inventors (Zhang et al., 2018a).
  • 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);
  • SPINK1 CCTTGGCCCTGTTGAGTCTA (F; SEQ ID NO: 5), GCCCAGATTTTTGAATGAGG (R; SEQ ID NO: 6);
  • WNT16B GCTCCTGTGCTGTGAAAACA (F; SEQ ID NO: 7), TGCATTCTCTGCCTTGTGTC (R; SEQ ID NO: 8);
  • GM-CSF ATGTGAATGCCATCCAGGAG (F; SEQ ID NO: 9), AGGGCAGTGCTGCTTGTAGT (R; SEQ ID NO: 10);
  • MMP3 AGGGAACTTGAGCGTGAATC (F; SEQ ID NO: 11), TCACTTGTCTGTTGCACACG (R; SEQ ID NO: 12);
  • IL-1 ⁇ AATGACGCCCTCAATCAAAG (F; SEQ ID NO: 13), TGGGTATCTCAGGCATCTCC (R; SEQ ID NO: 14);
  • p16 INK4a CTTCCTGGACACGCTGGT (F; SEQ ID NO: 15), ATCTATGCGGGCATGGTTAC (R; SEQ ID NO: 16);
  • IL-1 ⁇ TGGGTATCTCAGGCATCTCC (F; SEQ ID NO: 17), TTCTGCTTGAGAGGTGCTGA (R; SEQ ID NO: 18);
  • AREG AGCTGCCTTTATGTCTGCTG (F; SEQ ID NO: 19), TTTCGTTCCTCAGCTTCTCC (R; SEQ ID NO: 20);
  • CXCL1 CACCCCAAGAACATCCAAAG (F; SEQ ID NO: 21), TAACTATGGGGGATGCAGGA (R; SEQ ID NO: 22);
  • CXCL3 GGAGCACCAACTGACAGGAG (F; SEQ ID NO: 23), CCTTTCCAGCTGTCCCTAGA (R; SEQ ID NO: 24);
  • p21 CIP1 ATGAAAATTCACCCCCTTTCC (F; SEQ ID NO: 25), CCCTAGGCTGTGCTCACTTC (R; SEQ ID NO: 26);
  • BMP6 AAGAAGGCTGGCTGGAATTT (F; SEQ ID NO: 27), GAAGGGCTGCTTGTCGTAAG (R; SEQ ID NO: 28);
  • the senescence-associated beta-galactosidase (SA-beta-Gal) staining method briefly involved washing the cells with PBS in a petri dish and fixing them 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 briefly 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.
  • GLE and ABT263 were added at concentrations of 800 ⁇ 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.
  • GLE Ginkgo biloba leaves are crushed and suspended in water, and mixed with enzyme preparation for 12 hours of full enzymolysis. After filtration, the first step product, namely enzymolysate, is obtained; the enzymolyzed product is mixed with 70% ethanol solution and heated and refluxed. Secondary extraction. Finally, the second-step extract is concentrated and purified by ultrafiltration based on membrane separation technology, and then concentrated in a low-temperature vacuum to obtain the desired final product, namely Ginkgo biloba extract (GLE). Unless otherwise stated, the GLE is used subsequently.
  • GLE Ginkgo biloba extract
  • Immunodeficiency mice NOD-SCID mice, ICR (weight about 25 g) aged 6-8 weeks were used for the relevant animal experiments of the present invention.
  • Stromal cells PSC27 and epithelial cells PC3 were mixed in a predetermined ratio of 1: 4 and each graft contained 1.25 x 106 cells for tissue remodeling.
  • mice were fed a standard experimental diet followed by administration of the chemotherapeutic drugs mitoxantrone (MIT, 0.2 mg/kg dose) and/or Ginkgo biloba extract (GLE) (500 ⁇ l after 2 weeks) , 10mg/kg dose) intraperitoneal administration.
  • 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, 7, and 8.
  • 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 body weight of the drug for MIT and 30 mg/kg body weight for GLE.
  • 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 dose of MIT received throughout the course of treatment was 0.3 mg/kg body weight) to reduce drug-related toxicity.
  • 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.
  • DOX doxorubicin
  • DOC docetaxel
  • VIN vincristine
  • mice 16-month-old male C57BL/6 mice by continuous feeding 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 (GLE) treatment by humans unaware of the design of the preclinical trial.
  • vehicle vehicle
  • GLE drug
  • mice were treated with vehicle or GLE every 2 weeks by oral gavage for 3 consecutive days.
  • some mice were removed from their original cages to try to avoid the animal housing stress that comes with long-term housing in a single cage. 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.
  • 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 bioluminescence 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) x 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 inventors used baseline body weights to assign mice to experimental groups (to achieve similar body weights between groups), so randomization was performed only within weight-matched groups.
  • the sample size was determined based on past experiments, so statistical power analysis was not used. All replicates in the present invention are from different samples, each sample from a different experimental animal.
  • Example 1 can effectively inhibit the expression of SASP when used at low concentrations
  • PSC27 a primary normal human prostate stromal cell line, PSC27, was chosen 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 a specific dose of bleomycin (BLEO), which was optimized in the preliminary experiments, and observed a significant positive rate of senescence-associated ⁇ -galactosidase (SA- ⁇ -Gal) staining increased, the BrdU incorporation rate was greatly reduced, and the DNA damage repair foci (DDR foci) were significantly increased within a few days after drug injury ( Figure 1-3).
  • BLEO bleomycin
  • SA- ⁇ -Gal senescence-associated ⁇ -galactosidase
  • DDR foci DNA damage repair foci
  • the inventors performed RNA-seq sequencing on these cells. Subsequent high-throughput data showed that a botanical raw material, ginkgo leaf extract (GLE), significantly altered the expression profile of senescent cells. Among them, 5455 genes were significantly down-regulated, while 993 genes were up-regulated, where the fold change of each gene in the heatmap was 2.0 (P ⁇ 0.01) ( Figure 5). Importantly, the expression of SASP factors was generally reduced in senescent cells after GLE treatment, and these SASP factors were generally significantly upregulated in senescent cells (Figure 6).
  • GLE ginkgo leaf extract
  • GLE a plant-based natural product, can be used to control the pro-inflammatory phenotype of senescent cells, namely SASP, especially at relatively low concentrations.
  • GLE is a novel senolytics when used at high concentrations
  • the inventors next investigated the potential of population doubling (PD) after genotoxic treatment of stromal cells.
  • the combination treatment group of BLEO and GLE exhibited significantly increased PD capacity compared to the BLEO group of cells that rapidly entered a growth arrest state after injury treatment (Figure 15).
  • GLE itself does not appear to affect the PD of proliferating cells, data that further suggest the selectivity of GLE between senescent and normal cells.
  • Example 3 Therapeutic targeting of senescent cells with GLE promotes tumor regression and effectively reduces 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 promote the development of therapeutic resistance in their surrounding cancer cells 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 with PC3 epithelial cells, which is a typical high-grade prostate cancer cell line.
  • 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/GLE combination exhibited the longest median survival, at least 48.1% longer survival compared to the group treated with MIT alone ( Figure 30, green (4) vs. blue (2) compared to).
  • treating tumor-bearing mice with GLE alone did not result in significant benefit, with only marginal survival extension.
  • GLE a biologically active anti-aging drug
  • Embodiment 5 drug screening
  • 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 Ginkgo biloba extract (GLE) to inhibit aging. Potential substances associated with secretory phenotypes.
  • Screening system The experimental system described in Example 3: PSC27 stromal cells and PC3 epithelial cells were mixed to construct a tissue recombinant; GLE was used for treatment.
  • Test group administer candidate substances to the screening system
  • Control group Candidate substances are not administered to the screening system.
  • the tumor microenvironment system was detected in the test group and the control group respectively; if after adding the candidate substance, the death of senescent cells in the test group was significantly increased compared with that in the control group, then the candidate substance was a potential substance to inhibit tumors.

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Abstract

银杏叶提取物(GLE)在制备靶向衰老细胞、抑制肿瘤或延长寿命的药物中的应用。银杏叶提取物通过靶向作用于肿瘤微环境、清除衰老细胞，在与化疗药物联合应用后，可以通过清除衰老的基质细胞而促进肿瘤的抑制。对于衰老相关分泌表型(SASP)，所述GLE也能靶向清除其中的衰老细胞，从而抑制该SASP。GLE还能显著延长动物寿命，显著延长老年生存期，提高动物生存质量。

Description

银杏叶提取物在制备靶向衰老细胞、抑制肿瘤或延长寿命的药物中的应用 技术领域
本发明属于细胞生物学和肿瘤学领域；本发明人致力于研究筛选靶向作用于肿瘤微环境及有助于增强化疗药物抑瘤效果、清除衰老细胞或抑制细胞衰老的药物，在此揭示了银杏叶提取物在制备抑制细胞衰老、抑制肿瘤或延长寿命的药物中的应用。
背景技术
细胞衰老是指真核细胞的一种相对稳定且通常不可逆的细胞周期停滞的状态，在这种状态下增殖细胞会对促生长刺激产生耐受，通常由DNA损伤等胁迫性信号所引起。细胞的复制性衰老，是指正常细胞在大约30-50次分裂(即“Hayflick极限”)后会停止连续分裂。复制性衰老本质上由端粒渐进缩短所诱导。在每一轮的DNA复制中，端粒都会逐渐缩短，最终达到一个临界长度，阻止进一步复制，从而停止细胞分裂。较短的无帽端粒会引起DNA损伤应答，从而直接触发衰老。
衰老细胞主要通过三种途径参与机体的各种生理和病理过程：(1)衰老细胞基因表达和形态改变逐步累积可影响相应组织的功能；(2)衰老细胞限制干细胞和未分化祖细胞的再生潜能，导致细胞再生能力下降；(3)衰老细胞不仅表现为生长周期停滞，还通过自分泌和旁分泌途径释放大量的细胞因子、趋化因子、生长因子和蛋白酶等，影响邻近细胞和组织的微环境，导致和加速衰老及相关疾病，近年大量研究表明在这一过程中SASP起到核心的病理作用。此外，衰老细胞分泌的这些因子还会影响周围的正常细胞，而抑制SASP则能够延缓机体衰老。典型的SASP因子包括肿瘤坏死因子-α(TNF-α)、白细胞介素6(IL-6)、白细胞介素8(IL-8)、白细胞介素1a(IL-1a)、基质金属蛋白酶(MMP)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)和纤溶酶原激活物抑制因子-1(PAI1)等，这些因子促进免疫系统激活，导致组织微环境中衰老细胞等异常因素被机体清除，发挥肿瘤抑制功能。然而，十分矛盾的是，SASP尚可通过特定分泌因子(如VEGF，ANGPTL4)促进血管生成、细胞外基质重塑或上皮-间质转化(EMT)的因子来促进肿瘤发展。此外，衰老诱导的慢性炎症可引起系统性免疫抑制，这种慢性炎症还可促进衰老相关的组织损伤和变性、器官功能失调和癌症等多种衰老相关疾病的发生和发展。
表观遗传学近年在SASP研究领域取得不少进展。Sirtuins是一种代谢相关、NADH依赖的去乙酰化酶，在不同模型中已发现SIRT1有寿命延长的效应。衰老细胞中SIRT1通过脱乙酰化IL-6和IL-8启动子区组蛋白H3K9和H4K16实现对SASP因子的表达抑制，当敲除SIRT1后，细胞衰老期间这些区域乙酰化水平高于对照组细胞。microRNAs是一类高度保守的单链非编码RNA，长度大约为20～26个核苷酸，在真核细胞中调节基因的表达。研究结果表明，miR-146、miR-34、miR-21和miR-183等可以调节衰老细胞SASP，并能够有效地抑制炎性细胞因子的过度生成。miR-146a/b可以降低人脐静脉内皮细胞中IL-1受体相关激酶的产生；相反抑制miR-146a/b可以提高IL-1受体相关激酶的活性，激活转录因子NF-κB，诱导IL-6和IL-8产生。
表观遗传改变通过影响DNA损伤修复、端粒长度和代谢途径或激活衰老相关基因和miRNAs的表达而影响衰老。多种证据表明染色质状态的改变与细胞衰老的控制密切相关。细胞可以感觉到不同的衰老刺激，这些刺激会激活信号通路，驱动染色质状态的改变。然而，衰老信号引起这种改变的途径仍然很大程度上是未知的。因此，从表观遗传角度揭示细胞衰老及其特定表型发生发展的调控机制，从进而揭示具有靶向价值的关键分子及其信号通路，是将来衰老生物 学和老年医学的一个新兴方向，亟需深入开展相关探索，为临床医学提供重要科学依据和潜在的干预措施。
随着全球人口老龄化的日益加剧，人们对“主动健康、延缓衰老”的兴趣与日俱增，主要是基于一系列靶向衰老的基本机制可以延缓多种衰老相关慢性或非传染性疾病的发生或加剧的科学证据。因此，细胞衰老作为预防或治疗多种衰老相关疾病和提高健康寿命的潜在靶点已获得诸多关注。
延缓衰老的药物主要是通过短暂性阻断生存途径(衰老细胞抗凋亡途径SCAPs)选择性清除衰老细胞，该生存途径可保护衰老细胞免受环境中凋亡诱导信号的调控。临床前研究表明有一类药物，即Senolytics有望将来应用于延缓、预防或治疗多种衰老相关的疾病。
尽管越来越多的实验支持靶向细胞衰老可以同时治疗多种衰老相关疾病，但还有待严谨的人体临床试验以帮助人们更好地评估延缓衰老药物的益处和风险。尽管国际已知的多种SASP抑制剂均可显著减弱SASP，但本质上不会杀死衰老细胞。为了在药理学上减轻衰老细胞的负担，科学家们正在开发“Senolytics”(衰老细胞清除药物)这种性质的小分子、多肽和抗体来选择性地清除衰老细胞。研究者们白2015年发现senolytic药物以来，在鉴别其他小分子senolytic药物及其作用方面取得了相当大的进展。研究发现首个senolytic药物的文章是基于衰老细胞抵抗凋亡的假说，尽管衰老细胞会产生促凋亡SASP因子来触发自身死亡。
事实上，有研究表明在衰老细胞中促凋亡途径确实上调。因此，衰老细胞依赖于衰老相关的抗凋亡途径(SCAPs)来减轻SASP对自身的伤害，这一假说得到了验证。SCAPs是通过生物信息学方法(基于辐射诱导衰老的人前脂肪细胞的表达谱)来鉴定的。有研究通过体外RNA干扰实验发现衰老细胞对SCAPs具有依赖性，并将SCAPs确认为衰老细胞的致命弱点。这一研究发现最终促成了SCAP网络中潜在的senolytic靶点的发现以及第一种senolytic药物的发现，其中senolytic药物包括达沙替尼(dasatinib)(一种FDA批准的酪氨酸激酶抑制剂)和槲皮素(quercetin)(一种存在于许多水果和蔬菜中的黄烷醇)的组合(D+Q)。此外，有研究将BCL-2家族中一种对抗凋亡的蛋白(BCL-XL)鉴定为SASP组分。在这一发现之后，第三种senolytic药物navitoclax也被鉴定出来，它是一种BCL-2家族抑制剂。
衰老细胞存活所需的SCAP在细胞类型之间有所不同。例如，衰老的人类原代脂肪祖细胞存活所需的SCAPs与衰老的人类胚胎静脉内皮细胞(HUVECs)中的SCAPs不同。这种差异意味着靶向单个SCAP的药物可能无法消除多种衰老细胞类型。而且大量研究表明大多数senolytics确实仅对有限的衰老细胞类型有效。例如，navitoclax能够靶向HUVECs，但对衰老的人类脂肪前体细胞无效。有证据表明，即使在一种特定类型的细胞内，senolytics的功效也可能不同。例如，在人肺成纤维细胞中，navitoclax能靶向并杀死适应培养的IMR90肺成纤维细胞样细胞株中的衰老细胞，但对衰老的人肺原代成纤维细胞少有成效。因此，为确定senolytics的广谱作用，仍需要进行针对一系列细胞类型的广泛测试。
发明内容
本发明的目的在于提供银杏叶提取物在制备靶向衰老细胞、抑制肿瘤或延长寿命的药物中的应用。
在本发明的第一方面，提供银杏叶提取物的应用，用于与化疗药物联合制备特异性靶向清除肿瘤微环境中衰老细胞及抑制肿瘤的组合物；其中，所述的化疗药物为给药后诱发肿瘤微环境 发生衰老相关分泌表型(SASP)的化疗药物。
在一个优选例中，所述的肿瘤为在基因毒药物处理后肿瘤微环境中产生衰老相关分泌表型的肿瘤，和/或为在基因毒药物后产生耐药性的肿瘤；较佳地，所述的肿瘤包括(但不限于)：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌，肝癌，胰腺癌，膀胱癌，皮肤癌，肾癌，食管癌、胆管癌、脑癌。
所述衰老相关分泌表型为DNA损伤导致的衰老相关分泌表型；较佳地，所述的DNA损伤为化疗药物造成的DNA损伤。
在另一优选例中，所述的化疗药物为基因毒药物；更佳地包括：米托蒽醌，阿霉素，博莱霉素。
在另一优选例中，所述银杏叶提取物特异性靶向诱导肿瘤微环境中衰老细胞进入死亡程序；较佳地，所述的诱导肿瘤微环境中衰老细胞进入死亡程序由caspase-3/7介导。
在另一优选例中，所述化疗药物为米托蒽醌，米托蒽醌与银杏叶提取物的重量比例为1∶20～80；较佳地，米托蒽醌与银杏叶提取物的重量比例为1∶30～70；更佳地，米托蒽醌与银杏叶提取物的重量比例为1∶40～60(如1∶45，1∶50，1∶55)。
在另一优选例中，所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物(低浓度抑制SASP表达)终浓度200～550uM，较佳地250～500uM，更佳地300～420uM(如350uM，400uM)。
在另一优选例中，所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物(高浓度促使衰老细胞进入死亡程序)终浓度700～5000uM，较佳地750～4000uM，更佳地750～3500uM(如780、800、900、1000、1500、2000、2500、3000uM)。
在另一优选例中，所述化疗药物为阿霉素，阿霉素与银杏叶提取物的重量比例为1∶4～16；较佳地，阿霉素与银杏叶提取物的重量比例为1∶6～14；更佳地，阿霉素与银杏叶提取物的重量比例为1∶8～12(如1∶9，1∶10，1∶11)。
在本发明的另一方面，提供银杏叶提取物的应用，用于：制备抑制衰老的组合物；或制备延长寿命或延长晚年生存期的组合物；或制备特异性靶向清除肿瘤微环境中衰老细胞的组合物，或制备抑制(降低)衰老相关分泌表型的组合物；较佳地，所述银杏叶提取物特异性靶向诱导肿瘤微环境中衰老细胞进入死亡程序(较佳地，增殖态细胞基本不被其影响)。
在一个优选例中，银杏叶提取物的浓度为200～5000uM；较佳地250～4000uM；更佳地300～3500uM(如350、400、500、600、800、900、1000、1500、2000、2500、3000uM)。
在另一优选例中，所述银杏叶提取物的制备方法包括两步提取法：(1)混合酶催化酶解；(2)有机溶剂萃取；较佳地，(1)中，所述的酶包括纤维素酶、果胶酶、木质素酶和蛋白酶，银杏叶粉碎后混悬在水中，加入混合酶制剂进行8～20小时充分酶解，过滤后得到酶解物；较佳地，(2)中，将所述酶解物与乙醇溶液混合后加热回流、提取；较佳地，步骤(2)之后，还包括：对提取物经过基于膜分离技术的超滤浓缩纯化，再经低温真空浓缩，得到银杏叶提取物终产品。
在本发明的另一方面，提供一种用于特异性靶向清除肿瘤微环境中衰老细胞以及抑制肿瘤的药物组合物或药盒，包括：银杏叶提取物，以及化疗药物；其中，所述的化疗药物为给药后诱发肿瘤微环境发生衰老相关分泌表型的化疗药物。
在本发明的另一方面，提供一种制备抑制肿瘤的药物组合物或药盒的方法，包括：将银杏 叶提取物与化疗药物混合；或将银杏叶提取物与化疗药物置于同一药盒中。
在一个优选例中，所述化疗药物为米托蒽醌，米托蒽醌与银杏叶提取物的重量比例为1∶20～80；较佳地，米托蒽醌与银杏叶提取物的重量比例为1∶30～70；更佳地，米托蒽醌与银杏叶提取物的重量比例为1∶40～60(如1∶45，1∶50，1∶55)。
在另一优选例中，所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物(低浓度抑制SASP表达)终浓度200～550uM，较佳地250～500uM，更佳地300～420uM(如350uM，400uM)。
在另一优选例中，所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物(高浓度促使衰老细胞进入死亡程序)终浓度700～5000uM，较佳地750～4000uM，更佳地750～3500uM(如780、800、900、1000、1500、2000、2500、3000uM)。
在另一优选例中，所述化疗药物为阿霉素，阿霉素与银杏叶提取物的重量比例为1∶4～16；较佳地，阿霉素与银杏叶提取物的重量比例为1∶6～14；更佳地，阿霉素与银杏叶提取物的重量比例为1∶8～12(如1∶9，1∶10，1∶11)。
在另一优选例中，将银杏叶提取物与化疗药物混合，根据用药疗程分为单位剂型。
在本发明的另一方面，提供一种筛选促进银杏叶提取物清除肿瘤微环境中衰老细胞或抑制肿瘤或延长寿命的潜在物质的方法，所述方法包括：(1)提供一肿瘤微环境体系，该体系包括肿瘤细胞和基质细胞；(2)利用化疗药物处理(1)的体系、诱发肿瘤微环境发生衰老相关分泌表型，且在诱发肿瘤微环境发生衰老相关分泌表型之前、之时或之后，以银杏叶提取物进行处理；(3)将候选物质加入到(2)的体系中，观测其对肿瘤微环境体系的作用，若所述候选物质在统计学上能够促进(显著促进，如促进10％、20％、30％、50％以上或更高)银杏叶提取物清除肿瘤微环境中衰老细胞，则该候选物质是可与银杏叶提取物联用于清除肿瘤微环境中衰老细胞或抑制肿瘤或延长寿命的潜在物质。
在一个优选例中，通过观测caspase-3/7活性或SASP因子的表达来评估细胞凋亡情况或衰老相关分泌表型的情况。较佳地，所述SASP因子包括但不限于：IL6、CXCL8、SPINK1、WNT16B、GM-CSF、MMP3、CXCL1、CXCL3、IL-1α、IL-1β；或，通过观测化疗动物衰老标记p16 INK4A来评估细胞凋亡情况或衰老相关分泌表型的情况。
在本发明的另一方面，提供一种筛选抑制衰老相关分泌表型的潜在物质的方法，所述方法包括：(1)提供一基质细胞体系，诱导该体系产生衰老相关分泌表型；在诱导该体系产生衰老相关分泌表型之前、之时或之后，以银杏叶提取物进行处理；(2)将候选物质加入到(1)的体系中，观测其对该基质细胞体系的作用，若其能特异性促进(显著促进，如促进10％、20％、30％、50％以上或更高)银杏叶提取物对于衰老相关分泌表型的抑制作用，则该候选物质是可与银杏叶提取物联用、抑制衰老相关分泌表型的潜在物质。
在另一优选例中，还包括设置对照组，从而明确分辨测试组中肿瘤微环境体系/衰老相关分泌表型体系与对照组的差异，或银杏叶提取物对肿瘤微环境中衰老细胞的清除作用与对照组的差异。
在另一优选例中，所述的候选物质包括(但不限于)：针对小分子化合物，混合物(如植物提取物)，生物大分子、信号通路调控试剂等。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1.增殖态人源基质细胞PSC27(早期代数如p10-20)在体外经过化疗药物博来霉素(BLEO)以50μg/ml浓度处理之后第7-10天，通过SA-β-Gal染色之后的结果。上图，代表性图片，下图，统计学数据。CTRL，对照细胞；BLEO，博来霉素处理后细胞。**，P＜0.01。
图2.PSC27细胞经过化疗药物博来霉素(BLEO)处理之后，经过BrdU染色之后的结果。上图，代表性图片，下图，统计学数据。CTRL，对照细胞；BLEO，博来霉素处理后的细胞。***，P＜0.001。
图3.PSC27细胞经过化疗药物博来霉素(BLEO)处理之后，使用γH2AX经过免疫荧光染色(immunofluorescence staining)之后的结果。CTRL，对照细胞；BLEO，博来霉素处理后的细胞。***，P＜0.001。根据细胞核内荧光点的数量，将其分为4类，包括0foci，1～3foci，4～10foci和＞10foci的单个细胞。
图4.筛选天然产物药库以获得具有抗衰老活性植物原料的实验流程图。
图5.RNA-seq数据经软件处理和生信分析之后发现GLE可以使得衰老细胞相比于增殖态细胞显著上调的基因出现明显回落。相比于BLEO组，BLEO/GLE组细胞有5455个基因显著下调，同时有993个基因显著上调(fold change＞2，P＜0.01)。
图6.Heatmap显示BLEO损伤造成的衰老细胞中大量因子表达上调，但经过GLE处理之后有不少出现明显逆转。红星标识，典型SASP外泌因子。
图7.GSEA分析结果显示SASP或NF-κB分子标记相关因子的表达在BLEO造成的衰老细胞中集中上调，但在GLE处理衰老细胞之后发生显著下降。左，SASP分子标记；右，NF-κB分子标记。
图8.蛋白-蛋白相互作用(P PI)生信分析结果显示，GLE显著下调的衰老细胞分子形成一个network，彼此间存在着多种互作关系。
图9.KEGG通路分析GLE在衰老细胞中造成显著下调的100个分子在biological process上的代表性通路。左侧Y轴，percentage。右侧Y轴，log10(p-value)。
图10.KEGG通路分析GLE在衰老细胞中造成显著下调的100个分子在cellular component上的代表性通路。左侧Y轴，percentage。右侧Y轴，log10(p-value)。
图11.荧光定量PCR(qRT-PCR)检测分析一组典型SASP分子在BLEO诱导形成的衰老细胞、被不同浓度的GLE处理条件下的相对表达水平。所有数据均为相比于CTRL组后的规范化结果。*，P＜0.05；**，P＜0.01。
图12.在GLE浓度递增的条件下，用SA-β-Gal染色确定PSC27的衰老与否。^，P＞0.05；**，P＜0.01；****，P＜0.0001。其中，GLE在100μM，200μM，400μM，800μM和1600μM浓度下的P值为这些实验组的细胞阳性比例同0μM时的数据相比得出的统计学显著性。
图13.SA-β-Gal染色后PSC27在各种条件下的代表性图片。每组3个重复，上下排列。标尺，30μm。
图14.CCK8检测增殖态细胞同衰老组细胞在GLE渐增浓度下的存活率。每一GLE浓度下的P值均为CTRL和BLEO组之间相比后的显著性差异。**，P＜0.01；***，P＜0.001；****，P＜0.0001。
图15.PSC27的群体倍增(population doubling)测试。细胞在第10代(p10)时，受到BLEO 损伤性处理，随后GLE在第8天时加入培养基。通过比较分析CTRL组，BLEO组，GLE组和BLEO/GLE组的倍增值(PD)确定GLE对于细胞增殖潜力的影响。^，P＞0.05；***，P＜0.001。
图16.GLE处理衰老细胞过程中诱导出现caspase 3/7活性。PSC27细胞经BLEO在培养条件下处理12h后逐渐进入衰老阶段。800μM的GLE在第7天开始加入衰老细胞的培养基，NucLight Rapid Red试剂用于标记细胞，而caspase 3/7试剂(IncuCyte)用于apoptosis检测。Caspase 3/7活性以每4小时的间隔检测一次(n＝3)。
图17.Pan-caspase抑制剂(20μM QVD-OPh)逆转GLE的senolytic活性(800μM的GLE用于这一实验，而200μM的ABT263作为阳性对照；后者为近年被报道的衰老细胞凋亡诱导剂)。统计学差异通过two-way ANOVA(Turkey’test)获得。
图18.流式细胞仪测定PSC27在几种条件下的细胞凋亡情况。Q2，早期凋亡细胞的分布区域；Q3，晚期凋亡细胞的分布区域。
图19.对比分析细胞经过BLEO和/或GLE处理之后的存活和凋亡数量。***，P＜0.001；****，P＜0.0001。
图20.预临床试验中小鼠给药方式示意图。人源基质细胞PSC27同癌细胞PC3在体外混合(1∶4)之后移植入小鼠皮下形成移植瘤。在单药或组合式给药条件下经过多个治疗周期的处理，最终小鼠处死、病理分析其肿瘤组织有关分子表达变化。
图21.PSC27细胞的CTRL组和BLEO损伤组在体外同PC3混合之后，或者PC3细胞单独移植入小鼠皮下组织形成移植瘤。在第8周结束时解剖并获得肿瘤，检测、对比各组条件下肿瘤的体积。**，P＜0.01；***，P＜0.001；****，P＜0.0001。
图22.预临床试验小鼠给药时间和给药方式示意图。每两周为一次给药周期，在第3/5/7周的第一天分别对小鼠腹腔给药MIT(mitoxantrone，米托蒽醌)。第5周第一天开始对小鼠进行腹腔GLE给药，每周一次。8周疗程结束后，解剖小鼠并进行病理鉴定与表达分析。
图23.肿瘤终端体积统计分析。化疗药物MIT单独或与抗衰老药GLE一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图24.临床前试验中PC3/PSC27荷瘤动物病灶中细胞衰老情况对比。SA-β-Gal染色之后代表性图片。标尺，100μm。
图25.小鼠体内肿瘤组织中SA-β-Gal染色阳性细胞百分比平行分析。^，P＞0.05；**，P＜0.01：***，P＜0.001。
图26.荧光定量PCR(qRT-PCR)检测分析小鼠病灶中上皮癌细胞和基质细胞中SASP典型因子的表达情况。通过LCM技术将基质细胞和癌细胞分别进行特异分离、制备总RNA并用于SASP表达检测。^，P＞0.05；*，P＜0.05；**，P＜0.01；***，P＜0.001。
图27.荧光定量PCR(qRT-PCR)检测分析vehicle、MIT和MIT/GLE给药之后的小鼠病灶中基质细胞SASP因子表达状态。*，P＜0.05；**，P＜0.01；***，P＜0.001。
图28.用LCM技术将病灶中癌细胞进行特异分离之后分析各组小鼠中DNA损伤和凋亡比例。^，P＞0.05；*，P＜0.05；**，P＜0.01。
图29.免疫组化染色(immunohistochemical staining)之后的图片分析。Caspase 3 cleaved(CCL3)在各组小鼠病灶中的信号形成鲜明对比。标尺，200μm。
图30.NOD/SCID小鼠在经过各种给药处理之后，无病生存期的Kaplan Meier数据对比。Vehicle(V)，MIT，GLE和MIT/GLE组动物在体内肿瘤体积超过2000mm 3时，即被认为严重疾 病已经出现，小鼠需要及时处死并检测其荷瘤情况。^，P＞0.05；**，P＜0.01。
图31.各种不同给药处理条件下疗程结束时小鼠体重数据对比分析。^，P＞0.05。
图32.以上不同给药处理条件下疗程结束时小鼠血清学数据对比分析。Creatinine，urine(肾脏指标)，ALP和ALT(肝脏指标)数据平行对比。^，P＞0.05。
图33.各种不同给药处理条件下疗程结束时免疫完整型小鼠(C57BL/6J)体重数据对比分析。^，P＞0.05。
图34.预临床中不同给药处理条件下疗程结束时小鼠血细胞计数对比分析。WBC，lymphocyte和neutrophil单位体积数量平行对比。^，P＞0.05。
图35.肿瘤终端体积统计分析。化疗药物DOX单独或与抗衰老药GLE一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图36.肿瘤终端体积统计分析。化疗药物DOC单独或与抗衰老药GLE一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图37.肿瘤终端体积统计分析。化疗药物VIN单独或与抗衰老药GLE一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图38.预临床阶段小鼠的疗后生存曲线。从24至27月龄时开始，C57BL/6小鼠每两周经受一次Vehicle(V)或GLE腹腔给药(Vehicle组n＝80；GLE组n＝91)。每组动物的中位生存期(median survival)经过计算并予以标明。****，P＜0.0001。
图39.预临床阶段小鼠的总体(终生，或全长)生存曲线。从24至27月龄时开始，C57BL/6小鼠每两周经受一次Vehicle(V)或GLE腹腔给药(Vehicle组n＝80；GLE组n＝91)。每组动物一生中的中位生存期(median survival)经过计算并予以标明。****，P＜0.0001。
图40.选取每组动物中寿命长度位于最高区间的雌性小鼠，进行组间最高步行速度、持久力和总体寿命的比较分析。N＝5。^，P＞0.05；**，P＜0.01。
图41.选取每组动物中寿命长度位于最高区间的雄性小鼠，进行组间最高步行速度、持久力和总体寿命的比较分析。V：Vehicle。N＝5/组。^，P＞0.05；***，P＜0.001。
图42.针对两组动物中每只小鼠在生命终端所罹患的疾病负担进行对比分析。N＝60/组。V：Vehicle。统计结果以box-and-whisker plots显示，每个box展示出median with interquartile range。^，P＞0.05。
图43.针对两组动物中每只小鼠在生命终端所罹患的肿瘤数量进行对比分析。N＝60/组。V：Vehicle。统计结果以box-and-whisker plots显示，每个box展示出median with interquartile range。^，P＞0.05。
具体实施方式
本发明人致力于研究筛选靶向作用于肿瘤微环境、清除衰老细胞的药物，揭示了银杏叶提取物(GLE)通过靶向作用于肿瘤微环境、清除衰老细胞，在与化疗药物联合应用后，可以通过清除衰老的基质细胞而促进化疗药物对肿瘤的抑制，促进效果极其显著。对于衰老相关分泌表型(SASP)，所述GLE也能靶向清除其中的衰老细胞，从而抑制该SASP。并且，所述GLE还能显著延长动物寿命，显著延长老年生存期，提高动物生存质量。
本发明人发现，GLE尽管能够特异性靶向清除肿瘤微环境中衰老细胞，其自身并没有特异性抑制肿瘤细胞的效果；而化疗药物尽管能够抑制肿瘤细胞，其对于肿瘤微环境的影响很大，但 能造成显著副作用尤其是SASP的形成和发展，且持续使用后易于导致癌细胞产生耐药性。而令人意外的是，GLE与一些特定化疗药物的联合应用，则可有效地发挥靶向于疾病的良性互补作用，达到出乎意料的增效效果。
银杏叶提取物
银杏叶提取物(GLE)一般提取自银杏科植物银杏Ginkgo biloba L.的叶。
可以采用多种方法来提取GLE，例如但不限于：酶解法、水提法、有机溶剂提取法、微波法、超临界CO 2提取法等，或它们的组合。例如有机溶剂提取法，所采用的有机溶剂可包括但不限于：乙醇、甲醇、丙酮等。对于GLE粗品，还可以进一步纯化，所述纯化可以采用(但不限于)：溶剂萃取法、沉淀法、酶解法、超滤法、大孔树脂法等。
作为本发明的特别优选的方式，所述的银杏叶提取物采用生物催化与化学提炼两步制备工艺流程：第一步为工业来源混合酶催化酶解，第二步为化学纯有机溶剂萃取。所选植物原料为天然银杏叶，所选的酶包括商业用途的纤维素酶、果胶酶、木质素酶和蛋白酶，银杏叶经粉碎(如机械粉碎)后混悬在水中，加入混合酶制剂进行8～20小时(较佳地10～15小时，如12小时)充分酶解，过滤后得到第一步产物即酶解物；酶解后的银杏叶产物与70％乙醇溶液(优级纯)混合后加热回流进行二次提取。此后，对第二步提取物进行基于膜分离技术的超滤浓缩纯化，再经低温真空浓缩得到所需终产品，即银杏叶提取物。本发明也包括在该优选的提取工艺基础上，进行适当工艺变化而获得的银杏叶提取物。
本发明优选的两步制备技术区别于现有的大多数植物化学分离技术。相比于单纯的溶剂提取、离子沉淀、超声萃取和微波浸提等传统方式，运用该专利的提取方法有利于获取更高纯度和比例的植物多酚(白色无定形结晶)，包括黄烷酮类、花色素类、黄酮醇类，花白素类，酚酸和缩酚酸类。其中黄烷酮类(主要为儿茶素类化合物)可占该方法最终获取多酚总量的70-90％，总体上提高了产率，降低了成本并减少了污染。
此外，GLE也可以通过商购途径获得。
银杏叶提取物剂或其与化疗药物的联合应用
如前所述，本发明人发现，银杏叶提取物(GLE)与一些特定化疗药物的联合应用，可有效地发挥靶向于疾病的良性互补作用，达到极其显著的增效效果。
在抑制SASP表达的药物的筛选中，本发明人发现，尽管SASP因子一般会在衰老细胞中明显上调，但GLE处理之后的衰老细胞中SASP因子的表达普遍降低，这一效应非常明显。
在本发明人的研究中还发现，GLE在适当的浓度下对衰老细胞的杀伤效果非常理想。例如，在一些实施例中，本发明人发现，当GLE在2000μM时达到阈值，衰老细胞此时剩余20％或更低。因此，在一定的浓度下，GLE是一种新型的senolytics，且呈现了优异的效果。靶向特异性非常好。
本发明人的研究中还发现，基质细胞经基因毒药物(实施例中为博莱霉素)处理后群体倍增；与损伤性处理之后迅速进入生长停滞状态的细胞相比，基因毒药物和GLE的联合治疗组表现出显著增高的群体倍增(PD)能力。基质细胞经基因毒性处理后的群体倍增，与损伤性处理之后迅速进入生长停滞状态的细胞相比，基因毒药物和GLE的联合治疗组表现出显著增高的PD能力。GLE与基因毒药物联用可以使得基质细胞在短期内迅速恢复增殖潜力，这与基因毒药物单药使 用形成鲜明对照，这是令人惊讶的。GLE本身不影响增殖细胞的PD，这一数据进一步表明，GLE在衰老细胞与正常细胞之间具有选择性、靶向特异性。
本发明人的研究中还发现，肿瘤移植于动物体后，同由PC3癌细胞和原代PSC27基质细胞组成的移植肿瘤相比，由PC3细胞和衰老PSC27细胞组成的异种移植物(xenograft)体积显著增加。对单独运用MIT治疗后的治疗组相比，GLE联合MIT给药可显著减小肿瘤；与MIT相比，肿瘤体积减少55.1％；与安慰剂治疗相比，肿瘤体积减少74.6％。这一抑制效果令人意外。
本发明人也发现，MIT给药过程诱导了肿瘤组织中大量衰老细胞的出现。然而，GLE给药则将这些化疗动物病灶内的大多数衰老细胞基本耗尽。MIT给药后，SASP因子的表达显著升高(主要发生在基质细胞中)；然而，在使用GLE给药时，这一变化在很大程度上被逆转。当MIT处理的动物与GLE一起使用时，DNA损伤或凋亡的指数明显增强，这意味着这些衰老药物处理条件下的动物体内肿瘤位点细胞毒性增强；当GLE在治疗上应用时，细胞凋亡的典型标志caspase3/4活性显著升高。同时，接受MIT/GLE组合治疗的小鼠表现出最长的中位生存期；存活期大大延长。由此可见，GLE治疗性靶向衰老细胞可促进肿瘤的抑制并降低化疗耐药。
本发明人的研究中还发现，小鼠在每两周服用一次药物的治疗方案下，从24-27个月年龄(相当于人类75-90岁的年龄)开始给药的GLE组，其治疗后中位生存期比Vehicle组延长72.8％，同时具有较低的死亡危险，表明GLE介导的衰老细胞清除可以降低老年小鼠的死亡风险，并有效延长其生存期。间歇性提供GLE这种具有生物活性的抗衰老药物，可以通过清除微环境中衰老细胞的方式，显著减少衰老机体的疾病负担，并可以增加治疗后阶段机体的寿命。这种治疗方式，并不会导致显著上升的机体发病率，在现实中可以在生命的晚期阶段安全使用。
基于本发明人的上述新发现，本发明提供了一种GLE用途，用于制备特异性靶向清除肿瘤微环境中衰老细胞及抑制肿瘤的组合物；或制备抑制衰老相关分泌表型的组合物。
如本发明所用，除非另外说明，所述的“肿瘤”为在基因毒药物处理后肿瘤微环境中产生衰老相关分泌表型的肿瘤，和/或为在基因毒药物后产生耐药性的肿瘤。较佳地包括：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌、肝癌、胰腺癌、膀胱癌、皮肤癌，肾癌，食管癌、胆管癌、脑癌。
如本发明所用，除非另外说明，所述的“化疗药物”为给药后诱发肿瘤微环境发生衰老相关分泌表型(SASP)的化疗药物。
在本发明的一些方式中，所述“衰老相关分泌表型”为DNA损伤情况下发生的衰老相关分泌表型；较佳地，所述的DNA损伤为化疗药物造成的DNA损伤；更佳地，所述化疗药物包括基因毒药物。
药物筛选
在得知了GLE与肿瘤微环境或SASP的密切相关性及其工作机制后，可以基于该特征来筛选进一步优化抑制效果的药物。可从所述的物质中找到靶向作用于肿瘤微环境中的衰老细胞，对于抑制肿瘤、逆转肿瘤耐药性或抑制/延缓衰老相关分泌表型真正有用的药物。或可从所述的物质中找到与GLE联合并发挥增效作用的一或多种物质。
因此，本发明提供一种筛选促进化疗药物抑制肿瘤的潜在物质的方法，所述方法包括：(1)提供一肿瘤微环境体系，该体系包括肿瘤细胞和基质细胞；(2)利用化疗药物处理(1)的体系，诱发肿瘤微环境发生衰老相关分泌表型；(3)将候选物质加入到(2)的体系中，观测其对肿瘤微环境 体系的作用，若其能特异性靶向清除肿瘤微环境中衰老细胞和/或促进基质细胞(为非衰老的细胞)生长(提高基质细胞培增速度)，则其是促进化疗药物抑制肿瘤的潜在物质。在更为优选的方式中，步骤(2)中，还包括：在诱发肿瘤微环境发生衰老相关分泌表型之前、之时或之后，以GLE进行处理；步骤(3)中，还包括：若所述候选物质在统计学上能够促进GLE清除肿瘤微环境中衰老细胞和/或促进基质细胞生长，则该候选物质是可与GLE联用、抑制肿瘤的潜在物质。
本发明也提供了一种筛选抑制衰老相关分泌表型的潜在物质的方法，所述方法包括：(1)提供一基质细胞体系，诱导该体系产生衰老相关分泌表型；(2)将候选物质加入到(1)的体系中，观测其对该基质细胞体系的作用，若其能特异性促进银杏叶对于衰老相关分泌表型的抑制作用，则该候选物质是可与GLE联用、抑制衰老相关分泌表型的潜在物质。
在本发明的优选方式中，在进行筛选时，为了更易于观察到测试组中相应指标的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质、但其它条件与测试组相同的体系。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于抑制肿瘤、逆转肿瘤耐药性或抑制/延缓衰老相关分泌表型真正有用的物质。
另一方面，本发明还提供了采用所述筛选方法获得的抑制肿瘤、逆转肿瘤耐药性或抑制/延缓衰老相关分泌表型的潜在物质。这些初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出真正有用的药物。
药物组合
本发明提供了一种药物组合物，它含有有效量(如0.00001-50wt％；较佳的0.0001-20wt％；更佳的，0.001-10wt％)的所述的GLE、化疗药物(如0.000001-20wt％；较佳的0.00001-10wt％；更佳的，0.0001-2wt％)，以及药学上可接受的载体。此外，应理解，从便于临床给药或根据临床治疗方案所需出发，所述的GLE和化疗药物的混合并非是必需的，它们也可被独立地分置于独立的容器中，置于试剂盒或药盒中，在需要时进行联合应用。
本发明还提供了一种药物组合物，它含有有效量(如0.00001-10wt％；较佳的0.0001-5wt％；更佳的，0.001-2wt％)的所述的GLE，以及药学上可接受的载体。
如本文所用，所述“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，所述“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。适应于注射的药物形式包括：无菌水溶液或分散液和无菌粉(用于临时制备无菌注射溶液或分散液)。在所有情况中，这些形式必须是无菌的且必须是流体以易于注射器排出流体。在制造和储存条件下必须是稳定的，且必须能防止微生物(如细菌和真菌)的污染影响。
如本文所用，术语“含有”或“包括”包括了“包含”、“基本上由……构成”、和“由……构成”。术语“基本上由……构成”指在组合物中，除了含有主要活性成分(如GLE和化疗药物)之外，还可含有少量的且不影响有效成分的次要成分和/或杂质。例如，可以含有甜味 剂以改善口味、抗氧化剂以防止氧化，以及其它本领域常用的添加剂。
本发明人发现，在相对低的浓度下，所述的GLE可以以相对高的效率来抑制SASP的发展。因此，作为本发明的优选方式，当应用于抑制SASP时，所述GLE的浓度可以为200～550uM，较佳地250～500uM，更佳地300～420uM；如350uM，400uM。
本发明人发现，在相对高的浓度下，所述的GLE可以以相对高的效率来清除肿瘤微环境中的衰老细胞。因此，作为本发明的优选方式，当应用于促使衰老细胞进入死亡程序时，银杏叶提取物终浓度可以为700～5000uM，较佳地750～4000uM，更佳地750～3500uM；如780、800、900、1000、1500、2000、2500、3000uM。
应理解，在得知了所述GLE的用途及其在肿瘤微环境或SASP环境中的工作机制后，可以采用本领域熟知的多种方法来使用GLE和/或化疗药物给药于哺乳动物或人。这些方法均可被涵盖于本发明中。
本发明所述的组合物的剂型可以是多种多样的，只要是能够使活性成分有效地到达哺乳动物体内的剂型都是可以的。比如可选自：注射剂、片剂、胶囊剂、粉末、颗粒剂、糖浆、溶液、悬浮液、酊剂、口服液、或气雾剂。
本发明所述的GLE的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的GLE的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。
在特定条件下，senolytic药物的使用频率可能取决于衰老细胞的积累速度，而衰老细胞的积累速度可能会因细胞衰老发生的环境而异。例如，反复接触破坏DNA的癌症疗法或持续的高脂肪饮食，可能会比自然衰老更迅速地导致衰老细胞的重新累积。间歇性使用senolytics可以降低患者产生不良反应的风险，并允许在健康期间使用senolytics。此外，间歇给药还可以减少senolytics产生的副作用并降低患者产生耐药性的可能性。与抗癌药物或抗生素的情况相反，因为衰老细胞不发生分裂，因此机体无法依赖细胞增殖产生senolytics抗性，从而无法获得有利的突变，这可为临床中广泛使用senolytics创造良好的基础。
本发明的具体实施例中，给出了一些针对动物如鼠的给药方案。从动物如鼠的给药剂量换算为适用于人类的给药剂量是本领域技术人员易于作出的，例如可根据Meeh-Rubner公式来进行计算：Meeh-Rubner公式：A＝k×(W 2/3)/10,000。式中A为体表面积，以m 2计算；W为体重，以g计算；K为常数，随动物种类而不同，一般而言，小鼠和大鼠9.1，豚鼠9.8，兔10.1，猫9.9，狗11.2，猴11.8，人10.6。应理解的是，根据药物以及临床情形的不同，根据有经验的药师的评估，给药剂量的换算是可以变化的。
本发明的药物组合物还可被配制成单元剂型的方式，以便于按期、定量地用药。
如本文所用，术语“单元剂型”、“单位剂型”是指为了服用方便，将本发明的组合物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂。所述的单元剂型中含适于单次、单日或单位时间服用量的本发明的组合物。
在本发明的一些优选方式中，所述的组合物为单元剂型。当将组合物制备成单元剂型时，每个几日或几周服用所述单元剂型的组合物1剂。
本发明还提供了含有所述的药物组合物或直接含有所述的GLE和/或化疗药物的药盒。此外，所述的药盒中还可包括说明药盒中药物的使用方法的说明书。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料和方法
1.细胞培养
(1)细胞系维持
正常人源前列腺原代基质细胞系PSC27(获自美国Fred Hutchinson Cancer Research Center)于37℃和5％CO 2条件的培养箱中培养，在PSCC完全培养液中增殖和传代。
(2)细胞冻存与复苏
a.细胞冻存
以0.25％胰蛋白酶收集对数生长期细胞，1000rpm离心2min，弃去上清，重新悬浮细胞于新鲜配置的冻存液中。分装细胞于已标示的无菌冻存管中。然后经梯度降温，最后转入液氮中长期储存。
b.细胞复苏
取出液氮中冻存的细胞，立即放入37℃水浴，使其快速融化。直接加入2ml细胞培养液，使细胞均匀悬浮。待细胞贴壁后，更换新的培养液。
(3)体外实验处理
为造成细胞损伤，PSC27细胞生长至80％(简称PSC27-CTRL)时培养液中加入50μg/ml博来霉素(bleomycin，BLEO)。药物处理12小时后，细胞被PBS简单洗过3次，留置于培养液中7-10天，然后进行后续实验。
2.天然产物库的筛选
针对一个共有41种成分、多为药用植物提取物并有抗衰老潜力的天然产物库(BY-HEALTH)进行药效学分析。将各种产物分别按照一定浓度梯度稀释至96孔板，密度为每孔5000个细胞。培养基使用DMEM，天然产物(或化合物)的工作浓度一般控制在1μM-1mM。药物处理后3-7天，细胞增殖用CCK-8 Cell Counting Kit试剂盒(基于WST-8原理，Vazyme)测定，细胞凋亡活性以Caspase 3/7 activity kit(Promega)确定。
初步确定的候选药物进一步筛选30天。将进入第二轮候选范围的药物稀释到6孔板中，每孔20,000个细胞。培养基和候选药物每隔一天更换一次(即每两天一次)。为确定每种药物对细胞表型和存活率等的影响，项目根据不同浓度的药物进行验证性分析。
3.免疫印记和免疫荧光检测
用NuPAGE 4-12％Bis-Tris gel分离细胞裂解来源蛋白质，并转移到硝化纤维素膜(Life Technologies)上。用5％脱脂牛奶在室温下阻断印迹1h，在4℃下与所需的一抗在制造商协议的浓度下孵育一夜，然后在室温下与辣根过氧化物酶结合二抗(Santa Cruz)孵育1h，用增强化学发光(ECL)检测试剂(Millipore)按照制造商的协议开展膜印迹信号检测，并使用ImageQuant LAS 400 Phospho-Imager(GE Healthcare)。作为一种标准的蛋白质标记，本发明人使用Thermo Fisher Scientific公司提供的PageRuler Plus Prestained Protein Ladder(no.26619)。
对于免疫荧光染色，目标细胞在培养皿中培养之后在coverslip上预种至少24h。在短暂洗涤后，用4％多聚甲醛在PBS中固定8min，用5％正常山羊血清(NGS，Thermo Fisher)阻断30min。小鼠单克隆抗体anti-phospho-Histone H2A.X(Ser139)(clone JBW301，Millipore)和小鼠单克隆抗体anti-BrdU(Cat#347580，BD Biosciences)，及二级抗体Alexa
Figure PCTCN2022074740-appb-000001
488(或594)-conjugated F(ab’)2按顺序加入到覆有固定细胞的载玻片上。细胞核用2μg/ml of 4’，6-diamidino-2-phenylindole(DAPI)进行复染。从3个观察视野中选取最具代表性的一张图像进行数据分析和结果展示。FV1000激光扫描共聚焦显微镜(Olympus)用于获取细胞共聚焦荧光图像。
4.全转录组测序分析(RNA-sequencing)
对不同处理条件下的人源前列腺原代基质细胞系PSC27进行全转录组测序。从基质细胞中获得总RNA样本。其完整性经Bioanalyzer 2100(Agilent)验证，RNA用Illumina HiSeq X10测序，基因表达水平由软件包rsem(https：//deweylab.github.io/rsem/)进行量化。简而言之，以RiboMinus Eukaryote kit(Qiagen，Valencia，CA，USA)消除RNA样品中的rRNA；并根据制造商的指示，在深度测序前用TruSeq Stranded Total RNA preparation kits(Illumina，San Diego，CA，USA)构建链特异性RNA-seq文库。
Paired-end transcriptomic reads取被映射到参考基因组(GRCh38/hg38)，并使用Bowtie工具从Gencode v27进行参考注释。使用picard tools(1.98)脚本标记重复项(https：//github.com/broadinstitute/picard)识别重复读取，只保留非重复读取。Reference splice junctions由参考转录组提供(Ensembl build 73)。用Cufflinks计算FPKM值，用Cufflinks，最大似然估计函数调用差异基因表达。表达显著变化的基因由false discovery rate(FDR)-correctedP value＜0.05定义，仅用状态“Known”和生物型“coding”的ensembl genes 73进行下游分析。
接下来使用Trim Galore(v0.3.0)(http：//www.bioinformatics.babraham.ac.uk/projects/trim galore/)修剪Reads，而质量评估使用FastQC(v0.10.0)(http：//www.bioinformatics.bbsrc.ac.uk/projects/fastqc/)。随后，利用DAVID bioinformatics platform(https：//david.ncifcrf.gov/)、Ingenuity Pathways Analysis(IPA)program(http：//www.ingenuity.com/index.html)。在M ajorbio I-Sanger Cloud Platform(www.i-sanger.com)免费在线平台上对原始数据进行了初步分析，并将原始数据存入NCBI Gene Expression Omnibus(GEO)database数据库，登录代码为GSE156448。
5.蛋白质-蛋白质相互作用网络分析
用STRING3.0进行蛋白质-蛋白质相互作用(P PI)分析。将符合标准的特定蛋白质导入在线分析软件(http：//www.networkanalyst.ca)，选择一个最小交互网络进行进一步的集线器和模块分析。
6.基因集富集分析(GSEA)
基于RNA-seq初步分析所得数据，对于每个差异表达显著基因分析比较，基因是使用从DESeq2获得的“wald statistics”进行排序的，GSEA是在MSigDB(http：//software.broadinstitute.org/gsea/msigdb)中可用的所有规划基因集的这些排序列表上进行的)。DESeq2independent filtering是基于归一化读取计数的平均值，筛选出表达水平很低的基因。SASP和GSEA signature如本发明人过往发表文献所述(Zhang等人，2018a)。
7.定量PCR(RT-PCR)测定基因表达
细胞总RNA的提取、逆转录反应、实时定量PCR反应根据常规技术。其中，所用检测引物序列为(F表示正向引物，R表示反向引物)：
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)；
SPINK1：CCTTGGCCCTGTTGAGTCTA(F；SEQ ID NO：5)，GCCCAGATTTTTGAATGAGG(R；SEQ ID NO：6)；
WNT16B：GCTCCTGTGCTGTGAAAACA(F；SEQ ID NO：7)，TGCATTCTCTGCCTTGTGTC(R；SEQ ID NO：8)；
GM-CSF：ATGTGAATGCCATCCAGGAG(F；SEQ ID NO：9)，AGGGCAGTGCTGCTTGTAGT(R；SEQ ID NO：1O)；
MMP3：AGGGAACTTGAGCGTGAATC(F；SEQ ID NO：11)，TCACTTGTCTGTTGCACACG(R；SEQ ID NO：12)；
IL-1α：AATGACGCCCTCAATCAAAG(F；SEQ ID NO：13)，TGGGTATCTCAGGCATCTCC(R；SEQ ID NO：14)；
p16 INK4a：CTTCCTGGACACGCTGGT(F；SEQ ID NO：15)，ATCTATGCGGGCATGGTTAC(R；SEQ ID NO：16)；
IL-1β：TGGGTATCTCAGGCATCTCC(F；SEQ ID NO：17)，TTCTGCTTGAGAGGTGCTGA(R；SEQ ID NO：18)；
AREG：AGCTGCCTTTATGTCTGCTG(F；SEQ ID NO：19)，TTTCGTTCCTCAGCTTCTCC(R；SEQ ID NO：2O)；
CXCL1：CACCCCAAGAACATCCAAAG(F；SEQ ID NO：21)，TAACTATGGGGGATGCAGGA(R；SEQ ID NO：22)；
CXCL3：GGAGCACCAACTGACAGGAG(F；SEQ ID NO：23)，CCTTTCCAGCTGTCCCTAGA(R；SEQ ID NO：24)；
p21 CIP1：ATGAAATTCACCCCCTTTCC(F；SEQ ID NO：25)，CCCTAGGCTGTGCTCACTTC(R；SEQ ID NO：26)；
BMP6：AAGAAGGCTGGCTGGAATTT(F；SEQ ID NO：27)，GAAGGGCTGCTTGTCGTAAG(R；SEQ ID NO：28)；
8.SA-β-Gal染色
衰老相关β-半乳糖苷酶(SA-β-Gal)染色方法简单地说包括，细胞在培养皿中经PBS洗涤，在室温下固定。在2％甲醛和0.2％戊二醛中作用3min用以固定细胞。然后用新制备的染色液对SA-β-Gal进行染色，在37℃下过夜。第二天拍摄图像并计算单位面积内阳性细胞百分比。
9.克隆扩增实验
单细胞克隆扩增实验简单地说包括：将细胞铺板于明胶涂层的12孔板，密度为2000个细胞/孔。结晶紫染色之后计算细胞克隆数。
10.药物诱导衰老细胞凋亡
将PSC27细胞铺板于96孔皿中，在50μg/ml的BLEO处理下诱导细胞衰老。分别以800μM和1.0μM的浓度加入GLE和ABT263。细胞培养基配以Incucyte Nuclight快速红色试剂(Essen Bioscience)和IncucyteC-3/7细胞凋亡试剂(Essen Bioscience)。选取代表性视野拍照。
GLE：将银杏叶粉碎后混悬在水中，加入混合酶制剂进行12小时充分酶解，过滤后得到第一步产物即酶解物；酶解后的产物与70％乙醇溶液混合后加热回流进行二次提取。最后，对第二步提取物经过基于膜分离技术的超滤浓缩纯化，再经低温真空浓缩得到所需终产品，即银杏叶提取物(GLE)。除非另外说明，后续使用该GLE。
11.小鼠移植瘤接种和预临床治疗试验
所有实验小鼠实验均严格遵循中国科学院上海生命科学研究院实验动物看护和使用委员会(IACUC)的有关规章进行。年龄6-8周的免疫缺陷型小鼠(NOD-SCID mice，ICR)(体重约25g)用于本发明相关动物实验。基质细胞PSC27和上皮细胞PC3以1∶4预先确定的比例混合，而每一移植体包含1.25×10 6细胞，用于组织重构。移植瘤通过皮下移植方式植入小鼠体内，移植手术结束之后8周末动物被执行安乐死。肿瘤体积按照如下公式计算：V＝(π/6)x((1+w)/2) 3(V，体积；1，长度；w，宽度)。
在预临床治疗试验中，经过皮下移植的小鼠被供给标准实验食谱，2周之后实施化疗药物 米托蒽醌(MIT，0.2mg/kg剂量)和/或银杏叶提取物(GLE)(500μl，10mg/kg剂量)腹腔给药。时间点为：前者在第3，5，7周的第一天，后者在第5，7，8周的第一天。整个疗程共进行3次MIT循环给药，每个循环为2周。疗程结束后，小鼠肿瘤被收集用于体积测量和组织学分析。每只小鼠累积性共接受MIT这一药物0.6mg/kg体重，GLE则为30mg/kg体重。
为造成全身范围SASP因子在化疗诱导下表达，MIT按照以上步骤和顺序，经过静脉输注方式对小鼠给药，但剂量下降至0.1mg/kg体重/每次(整个疗程累计接受MIT剂量为0.3mg/kg体重)以减轻药物相关毒性。化疗试验进行到第8周末结束，小鼠处死之后立即解剖，其移植瘤被收集并用于病理系统分析。
使用阿霉素(doxorubicin，DOX)替换MIT时：整个疗程共进行3次DOX循环给药，每次1.0mg/kg剂量；共进行3次给药，总药量3.0mg/kg剂量。
多西紫杉醇(docetaxel，DOC)替换MIT时：整个疗程共进行3次DOC循环给药，每次1.0mg/kg剂量；共进行3次给药，总药量3.0mg/kg剂量。
长春新碱(vincristine，VIN)替换MIT时：整个疗程共进行3次VIN循环给药，每次1.0mg/kg剂量；共进行3次给药，总药量3.0mg/kg剂量。
12.小鼠寿命研究
在细胞移植研究中，本发明人在SPF动物平台通过连续饲养获得了16个月大的雄性C57BL/6小鼠，每个笼子里有4到5只动物。首先按体重从低到高对小鼠进行分类，然后选择了体重相似的小鼠。接下来，衰老(SEN)或对照(CTRL)移植治疗方式，则使用随机数产生器被分配给每间隔一次的小鼠，而中间的小鼠被分配到另一种治疗方式中，从而使衰老和对照移植小鼠的体重匹配。细胞移植1个月后，当小鼠年龄为18个月时，进行身体功能测试。在那之后，除了检查它们的笼子外，没有对这些老鼠进行进一步的测试。最早的死亡发生在上次身体功能测试后大约2个月。19至21个月大的C57BL/6小鼠，每个笼子里安放有3-5只。与移植小鼠一样，小鼠根据体重进行分类，并随机分配给每一组，由不知道预临床试验设计的人进行对照组(vehicle)或药物组(GLE)组处理。从24-27个月龄开始，小鼠每2周用vehicle或GLE治疗一次，每次连续3天口服灌胃。在研究过程中，一些老鼠被从原来的笼子移走，以尽量避免在单一笼子中长期饲养产生的动物居住压力。RotaRod和hanging测试每月进行，因为这些测试是敏感和无创的。试验结束时，对小鼠进行安乐死；如果它们表现出以下几种症状之一，就认为它们已经死亡：(一)不能饮水或吃饭；(二)即使有刺激也不愿意移动；(三)快速减肥；(四)严重的平衡障碍；或(五)机体出血或出现溃疡肿瘤。试验过程中，没有老鼠因为打架、意外死亡或皮炎而被排除在外。进行生物统计时，采用Cox proportional hazard model进行生存分析。
13.预临床动物死后病理检查
研究人员每天对笼子进行检查，并将死鼠从笼子中取出。在动物死亡24小时内，尸体被打开(腹腔、胸腔和颅骨)，并单独保存在10％福尔马林中至少7天。分解或破坏的身体被排除在外。保存的尸体被运到Autopsy专用地点进行病理检查。评估肿瘤负担(每个小鼠不同类型肿瘤的总和)，疾病负担(每个小鼠主要器官不同组织病理学变化的总和)，每个病变的严重程度和炎症(淋巴细胞浸润)。
14.生物发光成像
小鼠腹腔注射3mg荧光素(BioVision，Milpitas，CA)，以体积200μl的PBS递送。小鼠用异氟烷麻醉，使用Xenogen IVIS 200 System(Caliper Life Sciences，Hopkinton，MA)获取生物发 光图像。
15.体能检测
所有检测均在最后一次安慰剂或药物处理后的第5天开始。最大步行速度采用加速RotaRod System(TSE System，Chesterfiled，MO)进行评估。在RotaRod上小鼠被训练3天，速度分别为4，6和8r.p.m，第1、2和3天历时200秒。在测试日，小鼠被放置在RotaRod上，在4r.p.m速度下开始。以5分钟为间隔，转速由4加速到40r.p.m。当老鼠从RotaRod上掉下来时，速度被记录下来。最终结果从3或4个试验中取平均值，并规范为基线速度。在前两个月内训练过的小鼠不再接受训练。
前肢握力(N)使用Grip Strength Meter(Columbus Instruments，Columbus，OH)测定，结果来自超过10个试验的平均值。对于悬挂耐力试验，小鼠被放置在一个2毫米厚的金属线上，后者位于垫子上方35厘米处。小鼠只被允许用前肢抓住电线，悬挂时间根据体重进行规范化，表示为悬挂持续时间(sec)×体重(g)。结果取每只小鼠2到3次实验的平均值。通过Comprehensive Laboratory Animal Monitoring System(CLAMS)监测24小时(12小时光照和12小时黑暗)的日常活动和食物摄入量。CLAMS系统配备了Oxymax Open Circuit Calorimeter System(Columbus Instruments)。对于跑步机性能，小鼠在5°倾斜度下适应在电动跑补机(Columbus Instruments)上跑步，经过3天训练，每天持续5分钟，以5米/分钟的速度开始2分钟，继而加速至到7米/分钟2分钟，然后9米/分钟1分钟。在试验当日，小鼠在跑步机上以5米/分钟的初始速度跑步2分钟，然后每2分钟增加2米/分钟的速度，直到小鼠筋疲力尽。疲劳被定义为即便有轻微的电击刺激和机械刺激，小鼠仍无法回到跑步机上。试验结束后记录距离，用下列公式计算总功(KJ)：质量(kg)×g(9.8m/s 2)×距离(m)×sin(5°)。
16.生物统计学方法
本发明申请中所有涉及细胞增殖率，存活率和SA-β-Gal染色等的体外实验和小鼠移植瘤及预临床药物处理的体内试验均重复3次以上，数据以均值±标准误的形式呈现。统计学分析建立在原始数据的基础上，通过one-way analysis of variance(ANOVA)or a two-tailed Student’s t-test进行计算，而P＜0.05的结果认作具有显著性差异。
因素之间的相关性用Pearson’s correlation coefficients检验。当小鼠在几个队列中获得并分组在笼子中时，采用Cox proportional hazard model进行生存分析。该模型将治疗的性别和年龄作为固定效应，队列和初始笼分配作为随机效应。由于在研究中，一些小鼠被从最初的笼子中移动，以尽量减少来自单笼外壳的压力，本发明人还进行了没有笼效应的分析。这两种分析的结果在方向性或统计意义上没有很大差异，增强了对结果的自信度。生存分析使用statistical software R(version 3.4.1；library‘coxme’)。在大多数实验和结果评估中，研究者对分配采取盲选。本发明人使用基线体重将小鼠分配至实验组(以实现组间相似的体重)，因此只在与体重匹配的组内进行随机化。根据过往的实验确定样本量，因此没有使用statistical power analysis。本发明中的所有重复都来自不同的样本，每个样本来自不同的实验动物。
实施例1.GLE在低浓度下使用时可以有效抑制SASP的表达
为了鉴定能有效调节衰老细胞表型的创新化合物，本发明人利用一个由41种植物衍生物组成的植物化学药库开展了无偏倚性筛选。为了检测这些药物的药效和潜在的生物价值，选择使用原发性正常人前列腺基质细胞系，即PSC27作为体外细胞模型。PSC27主要由成纤维细胞组成， 而非成纤维细胞系(包括内皮细胞和平滑肌细胞)也存在，但比例较小，PSC27在性质上是人源原代基质细胞系，在暴露于基因毒性化疗或电离辐射等胁迫因素后形成典型的SASP。本发明人用预实验中已经优化过的方式，即特定剂量的博莱霉素(BLEO)处理这些细胞，并观察到衰老相关β-半乳糖苷酶(SA-β-Gal)染色阳性率明显升高，BrdU掺入率大幅降低，DNA损伤修复灶(DDR foci)在药物损伤后的数天内显著升高(图1-3)。通过系统筛选的方式来平行比较这些天然药物产品对衰老细胞表达谱的影响(图4)。
本发明人对这些细胞进行了RNA-seq测序。而随后获得的高通量数据表明，一种植物原料，银杏叶提取物(ginkgo leaf extract，GLE)，显著改变了衰老细胞的表达谱。其中5455个基因出现显著下调，同时993个基因发生上调，这里heatmap中每个基因的倍数变化为2.0(P＜0.01)(图5)。重要的是，GLE处理之后的衰老细胞中SASP因子的表达普遍降低，而这些SASP因子一般会在衰老细胞中明显上调(图6)。虽然一些SASP不相关基因的表达情况与那些典型的SASP因子表现出类似的趋势，但GSEA分析的数据进一步揭示了表征SASP表达或NF-KB激活的分子标签的显著抑制，后者是介导促炎SASP发展的主要转录性事件(图7)。基于蛋白质-蛋白质相互作用的生信分析结果显示了一个高度活跃的网络，其涉及多种因素在细胞衰老时显著上调，而一旦细胞处于GLE作用下，则反而呈现下调(图8)。进一步的GO生物信息学数据表明，这些分子在功能上参与了一组重要生物过程，包括信号转导、细胞间通讯、能量调节、细胞代谢和炎症反应(图9)。这些下调基因中的大多数，生化本质上属于表达后即释放至胞外空间的蛋白质，或位于内质网或高尔基体上，总体而言在特征上与这些分子的分泌性质相互呼应(图10)。
为了进一步证实GLE在体外条件下对SASP表达的影响，本发明人在一系列体外浓度梯度下处理了PSC27细胞。数据表明，工作浓度在400μM时的GLE以最大的效率抑制了SASP发生发展(图11)。然而，较低或较高浓度的这种药物的疗效却不理想，尽管后者可能与这种药物的细胞毒性增加引起的细胞应激反应有关(图11)。因此，GLE这一植物性天然产物，可用于控制衰老细胞的促炎表型，即SASP，尤其在相对低浓度下使用能呈现显著的效果。
实施例2.当在高浓度使用时GLE是一种新型的senolytics
鉴于GLE在控制SASP表达方面的显著疗效，接下来探究这种天然产物在较高浓度下杀死衰老细胞的潜力。为此，本发明人测量了随着GLE浓度的增加，体外条件下所处理的(50μg/ml的BLEO处理下诱导细胞衰老)的PSC27衰老细胞的生存百分比。SA-β-Gal染色数据表明，在GLE浓度达到800μM之前，衰老细胞不会被消除(图12)。随着浓度的增加，GLE对衰老细胞(80％染色阳性)的杀伤效果进一步增强，而当GLE在2000μM时达到阈值(衰老细胞此时剩余20％)；当其浓度升高到3000μM时，GLE的杀伤效果没有进一步增强(图12；图13)。
为了进一步剖析这些问题，本发明人做了验证性实验。细胞活力测定表明，与其增殖态对照细胞相比较，GLE从800μM浓度开始诱导衰老细胞显著死亡(图14)。当GLE浓度增加到3000μM时，存活衰老细胞的百分比下降到约10％。然而，即使在GLE的3000μM时，增殖细胞也并未明显减少。这些结果，证实了GLE对衰老细胞高度的选择性和突出的特异性，而这种特征实际是senolytics作为一类独特的抗衰老药的基本技术要求。
本发明人接下来研究了基质细胞经基因毒性处理后群体倍增(population doubling，PD)的潜力。与损伤性处理之后迅速进入生长停滞状态的BLEO这一组细胞相比，BLEO和GLE的联合治疗组表现出显著增高的PD能力(图15)。然而有趣的是，GLE本身似乎不影响增殖细胞的PD， 这一数据进一步表明GLE在衰老细胞与正常细胞之间的选择性。
为了探究GLE是否通过诱导凋亡的方式造成衰老细胞丧失存活能力，本发明人使用GLE在培养条件下分别处理增殖组细胞和衰老组细胞。随后观察到的caspase-3/7活性变化结果，表明GLE引起衰老细胞发生凋亡；从GLE加入之后的第16小时，衰老组开始与对照组之间出现统计学差异(图16)。此外，泛caspase抑制剂QVD可防止GLE对衰老细胞的杀伤，这一过程中的实际效果跟ABT263(一种目前已知的、十分有效的衰老细胞凋亡诱导剂)对衰老细胞的影响非常相似(图17)。上述一系列结果证实，GLE通过诱导凋亡的方式促使衰老细胞进入死亡程序，但增殖态细胞基本不被这一天然药物所靶向或影响。
鉴于GLE对衰老细胞产生的明显影响，本发明人随后分析了GLE诱导细胞凋亡的潜力。流式细胞数据显示衰老PSC27细胞活力显著降低，而其凋亡比例显著升高，但增殖细胞的变化却并不明显(图18；图19)。因此，上述数据一致性支持GLE在体外条件下通过诱导细胞凋亡的方式引起衰老细胞的消除，该天然产物在靶向衰老细胞方面具有突出的潜力。
实施例3.使用GLE治疗性靶向衰老细胞可促进肿瘤消退并能有效降低化疗耐药
鉴于GLE在体外较高浓度条件下清除衰老细胞中的突出选择性，本发明人接下来考虑这种药物是否可以被利用来干预体内与增龄相关的多种疾病。癌症是严重威胁人类寿命和危害健康的主要慢性疾病之一。此外，临床中癌细胞耐药性限制了大多数抗癌治疗的效果，而衰老细胞往往通过在受损肿瘤灶中发展SASP来促进其周边癌细胞治疗性耐药的发生。即便如此，从原发肿瘤中清除衰老细胞以促进癌症治疗指数的可行性与安全性，至今几乎未被科学家们探索过。
首先，本发明人通过将PSC27基质细胞与PC3上皮细胞混合构建成组织重组体，后者是一种典型的高度恶性前列腺癌细胞系。在非肥胖糖尿病和严重联合免疫缺陷(NOD/SCID)实验小鼠大腿后侧皮下植入重组体之前，基质细胞与上皮细胞的数量比例为1∶4。动物在重组体植入体内之后8周结束时，测量肿瘤大小(体积)(图20)。同由PC3癌细胞和原代PSC27基质细胞组成的肿瘤相比，由PC3细胞和衰老PSC27细胞组成的异种移植物(xenograft)体积显著增加(P＜0.001)，这一差异再次证实了衰老细胞在肿瘤进展中的关键促进作用(图21)。
为了更加接近临床条件，本发明人特别设计了一种临床前方案，其中涉及基因毒化疗药物治疗和/或衰老药物干预(图22)。在皮下植入两周后，当观察到体内肿瘤已经稳定被摄取时，本发明人在第3、第5和第7周的第一天分别向实验动物提供单次剂量的MIT(Mitoxantrone，一种化疗剂)或安慰剂，直到8周方案全部结束。同安慰剂治疗组相比，MIT给药可显著延缓肿瘤生长，这证实了MIT作为化疗药物的疗效(肿瘤大小减少43.3％，P＜0.0001)(图23)。值得注意的是，虽然GLE本身并不会引起肿瘤收缩，但对治疗MIT后的小鼠，GLE给药却可显著减小肿瘤；与MIT相比，肿瘤体积减少55.1％，P＜0.001；与安慰剂治疗相比，肿瘤体积减少74.6％，P＜0.0001(图23)。
接下来，本发明人推断细胞衰老是否发生在这些动物的肿瘤灶中。检测结果证明，MIT给药过程诱导了肿瘤组织中大量衰老细胞的出现，尽管这毫不奇怪。然而，GLE给药则将这些化疗动物病灶内的大多数衰老细胞基本耗尽(图24；图25)。激光捕获显微解剖(LCM)和随后的定量PCR结果表明，SASP因子的表达显著升高，包括IL6、CXCL8、SPINK1、WNT16B、GM-CSF、MMP3、IL1α，这一趋势伴随着化疗动物衰老标记p16 INK4A的上调(图26)。有趣的是，这些变化主要发生在基质细胞中，而不是它们邻近的癌细胞，这意味着残留癌细胞再增殖的可能 性，而这些细胞在治疗损伤的TME中产生了获得性耐药。然而，在使用GLE给药时，这一变化在很大程度上被逆转，正如转录水平数据分析结果所展示(图27)。
为了研究直接支持在MIT给药的小鼠中SASP的表达和逆转这种衰老相关模式的机制，本发明人在第一次GLE给药7天后即解剖了这两种药物治疗的动物体内的肿瘤，选择给药7天后这一时间点主要是因为这时病灶中癌细胞耐药克隆尚未形成。与安慰剂相比，MIT给药导致DNA损伤和凋亡程度均显著增加。虽然GLE单独不能诱导DNA损伤或造成凋亡，但化疗药物MIT却可以高度上调这两个指标(图28)。然而，当MIT处理的动物与GLE一起使用时，DNA损伤或凋亡的指数明显增强，这意味着这些衰老药物处理条件下的动物体内肿瘤位点细胞毒性增强。作为支持性证据，当GLE在治疗过程中应用时，caspase 3 cleavage活性升高，这是细胞凋亡的一个典型标志(图29)。
接下来本发明人比较了不同药物处理组动物的生存情况，主要以一种时间延长的方式来评估肿瘤进展的后果。在这一临床前队列中，本发明人对动物进行了肿瘤生长监测，一旦小鼠内体肿瘤负担突出(大小≥2000mm 3)，就会判断为严重疾病已经发生，这是一种用于某些情况下肿瘤等疾病的病情进展的方法。接受MIT/GLE组合治疗的小鼠表现出最长的中位生存期，与仅接受MIT治疗的组相比，存活期延长了至少48.1％(图30，绿色(4)与蓝色(2)相比)。然而，仅用GLE治疗荷瘤小鼠并没有造成显著的好处，只有边际性生存延伸。
值得注意的是，在这些研究中进行的治疗似乎被实验小鼠很好地耐受。本发明人没有观察到尿素、肌酐、肝酶或体重的显著波动(图31；图32)。更重要的是，在本发明设计的各药物剂量下使用的化疗和抗衰老药物不会显著干扰免疫系统的完整性和关键器官的组织稳态，即使在免疫完整型的野生小鼠中也是如此(图33；图34)。这些结果一致证实，抗衰老剂结合常规化疗药物有可能在普遍意义上增强肿瘤反应，而不引起严重的全身毒性。
为了确定GLE在提高化疗治疗效果方面是否具有药物依赖性或特异性，本发明人继而选择使用阿霉素(doxorubicin，DOX)、多西紫杉醇(docetaxel，DOC)与长春新碱(vincristine，VIN)，分别与GLE进行组合并用于预临床试验。结果表明，在这些化疗药物中，只有DOX与GLE联用可以大致呈现MIT与GLE联合治疗所造成的显著效果(图35)，而效果上MIT与GLE的联用显著更为理想。而DOC与VIN尽管在单独使用时可以降低肿瘤体积，但当GLE与其共同给药时并未引起肿瘤进一步收缩，即未能带来更多益处(图36，图37)。因此，GLE在体内条件下提高化疗治疗效果这一特征，仅限于同特定基因毒药物的联用，具有药物类型依赖性。
实施例4.GLE治疗造成的衰老细胞清除可以延长老龄小鼠的晚年生存期，而不增加其在生命晚期阶段的发病率
既然GLE具有在肿瘤小鼠的微环境中清除衰老细胞、降低肿瘤耐药性和提高总体治疗效果的惊人药效，那么对于自然衰老的动物是否也有某种促进健康或延缓疾病的显著益处？为回答这一问题，本发明人首先考虑是否可以使用一种具有潜在转化价值的方法来消除衰老细胞，即：从非常老龄的某一时间点开始进行间歇治疗，能否延长WT小鼠的剩余寿命？对此，一系列体内试验得以相应开展。值得注意、也十分令人惊讶的是，在每两周服用一次药物的治疗方案下，从24-27个月年龄(相当于人类75-90岁的年龄)开始给药的GLE组，其治疗后中位生存期比Vehicle组延长了72.8％，同时具有较低的死亡危险(HR＝0.33，GLE组/Vehicle组；P＜0.0001)(图38，图39)。这一发现，表明GLE介导的衰老细胞清除可以降低老年小鼠的死亡风险，并有效延长其 生存期。
为了进一步检验这种降低老年小鼠死亡率的治疗方案，是否以提高机体的晚期发病率为代价，本发明人评估了这些小鼠的身体功能。尽管GLE组小鼠的剩余寿命较长，但经过GLE每两周一次给药处理的小鼠，在生命的最后2个月的身体功能跟Vehicle处理组的小鼠在雄、和雌两性之间分别比较时，并未出现显著降低((图40，图41)。此外在小鼠尸检中，几种年龄相关疾病的患病率和肿瘤负担，在两组之间也没有出现统计学差异(图42，图43)。因此，间歇性提供GLE这种具有生物活性的抗衰老药物，可以通过清除微环境中衰老细胞的方式，显著减少衰老机体的疾病负担，并可以增加治疗后阶段机体的寿命。这种治疗方式，并不会导致显著上升的机体发病率，在现实中可以在生命的晚期阶段安全使用。
实施例5、药物筛选
1、筛选抑制衰老相关分泌表型的潜在物质
筛选体系：如实施例2中记载的实验体系：PSC27细胞经BLEO(50μg/ml)在培养条件下处理12h后逐渐进入衰老阶段；在诱导该体系产生衰老相关分泌表型之前、之时或之后，以银杏叶提取物(GLE)进行处理。
测试组：向该筛选体系给予候选物质；
对照组：不向该筛选体系给予候选物质。
分别检测测试组和对照组中SASP，测定SASP因子的表达情况，若测试组中SASP因子的表达显著低于对照组，则该候选物质是可与银杏叶提取物(GLE)联合应用于抑制衰老相关分泌表型的潜在物质。
2、筛选抑制肿瘤的潜在物质
筛选体系：如实施例3中记载的实验体系：将PSC27基质细胞与PC3上皮细胞混合构建成组织重组体；以GLE进行处理。
测试组：向该筛选体系给予候选物质；
对照组：不向该筛选体系给予候选物质。
分别检测测试组和对照组中观测肿瘤微环境体系的情况；若是加入候选物质后，测试组衰老细胞死亡比对照组显著增加，则该候选物质是抑制肿瘤的潜在物质。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 银杏叶提取物的应用，用于与化疗药物联合制备特异性靶向清除肿瘤微环境中衰老细胞及抑制肿瘤的组合物；其中，所述的化疗药物为给药后诱发肿瘤微环境发生衰老相关分泌表型的化疗药物。
  2. 如权利要求1所述的应用，其特征在于，所述的肿瘤为在基因毒药物处理后肿瘤微环境中产生衰老相关分泌表型的肿瘤，和/或为在基因毒药物后产生耐药性的肿瘤；较佳地，所述的肿瘤包括：前列腺癌，乳腺癌，肺癌，结直肠癌，胃癌，肝癌，胰腺癌，膀胱癌，皮肤癌，肾癌，食管癌、胆管癌、脑癌；或
    所述衰老相关分泌表型为DNA损伤导致的衰老相关分泌表型；较佳地，所述的DNA损伤为化疗药物造成的DNA损伤。
  3. 如权利要求1所述的应用，其特征在于，所述的化疗药物为基因毒药物；更佳地包括：米托蒽醌，阿霉素，博莱霉素。
  4. 如权利要求3所述的应用，其特征在于，所述化疗药物为米托蒽醌，米托蒽醌与银杏叶提取物的重量比例为1∶20～80；较佳地，米托蒽醌与银杏叶提取物的重量比例为1∶30～70；更佳地，米托蒽醌与银杏叶提取物的重量比例为1∶40～60；或
    所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物终浓度200～550uM，较佳地250～500uM，更佳地300～420uM；或
    所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物终浓度700～5000uM，较佳地750～4000uM，更佳地750～3500uM；
    所述化疗药物为阿霉素，阿霉素与银杏叶提取物的重量比例为1∶4～16；较佳地，阿霉素与银杏叶提取物的重量比例为1∶6～14；更佳地，阿霉素与银杏叶提取物的重量比例为1∶8～12。
  5. 银杏叶提取物的应用，用于：
    制备抑制衰老的组合物；或
    制备延长寿命或延长晚年生存期的组合物；或
    制备特异性靶向清除肿瘤微环境中衰老细胞的组合物，或
    制备抑制衰老相关分泌表型的组合物；较佳地，所述银杏叶提取物特异性靶向诱导肿瘤微环境中衰老细胞进入死亡程序。
  6. 如权利要求5所述的应用，其特征在于，银杏叶提取物的浓度为200～5000uM；较佳地250～4000uM；更佳地300～3500uM；和/或
    所述银杏叶提取物的制备方法包括两步提取法：(1)混合酶催化酶解；(2)有机溶剂萃取；较佳地，(1)中，所述的酶包括纤维素酶、果胶酶、木质素酶和蛋白酶，银杏叶粉碎后混悬在水中，加入混合酶制剂进行8～20小时充分酶解，过滤后得到酶解物；较佳地，(2)中，将所述酶解物与乙醇溶液混合后加热回流、提取；较佳地，步骤(2)之后，还包括：对提取物经过基于膜分离技术的超滤浓缩纯化，再经低温真空浓缩，得到银杏叶提取物终产品。
  7. 一种用于特异性靶向清除肿瘤微环境中衰老细胞以及抑制肿瘤的药物组合物或药盒，包括：银杏叶提取物，以及化疗药物；其中，所述的化疗药物为给药后诱发肿瘤微环境发生衰老相 关分泌表型的化疗药物。
  8. 一种制备抑制肿瘤的药物组合物或药盒的方法，包括：将银杏叶提取物与化疗药物混合；或将银杏叶提取物与化疗药物置于同一药盒中。
  9. 如权利要求7或8所述，其特征在于，所述化疗药物为米托蒽醌，米托蒽醌与银杏叶提取物的重量比例为1∶20～80；较佳地，米托蒽醌与银杏叶提取物的重量比例为1∶30～70；更佳地，米托蒽醌与银杏叶提取物的重量比例为1∶40～60；或
    所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物终浓度200～550uM，较佳地250～500uM，更佳地300～420uM；或
    所述化疗药物为博莱霉素，博莱霉素终浓度30～70ug/mL，较佳地40～60ug/mL更佳地45～55ug/mL；且银杏叶提取物终浓度700～5000uM，较佳地750～4000uM，更佳地750～3500uM；
    所述化疗药物为阿霉素，阿霉素与银杏叶提取物的重量比例为1∶4～16；较佳地，阿霉素与银杏叶提取物的重量比例为1∶6～14；更佳地，阿霉素与银杏叶提取物的重量比例为1∶8～12。
  10. 一种筛选促进银杏叶提取物清除肿瘤微环境中衰老细胞或抑制肿瘤或延长寿命的潜在物质的方法，所述方法包括：
    (1)提供一肿瘤微环境体系，该体系包括肿瘤细胞和基质细胞；
    (2)利用化疗药物处理(1)的体系、诱发肿瘤微环境发生衰老相关分泌表型，且在诱发肿瘤微环境发生衰老相关分泌表型之前、之时或之后，以银杏叶提取物进行处理；
    (3)将候选物质加入到(2)的体系中，观测其对肿瘤微环境体系的作用，若所述候选物质在统计学上能够促进银杏叶提取物清除肿瘤微环境中衰老细胞，则该候选物质是可与银杏叶提取物联用于清除肿瘤微环境中衰老细胞或抑制肿瘤或延长寿命的潜在物质；
    较佳地，通过观测caspase-3/7活性或SASP因子的表达来评估细胞凋亡情况或衰老相关分泌表型的情况；较佳地，所述SASP因子包括但不限于：IL6、CXCL8、SPINK1、WNT16B、GM-CSF、MMP3、CXCL1、CXCL3、IL-1α、IL-1β；或，通过观测化疗动物衰老标记p16 INK4A来评估细胞凋亡情况或衰老相关分泌表型的情况。
  11. 一种筛选抑制衰老相关分泌表型的潜在物质的方法，所述方法包括：
    (1)提供一基质细胞体系，诱导该体系产生衰老相关分泌表型；在诱导该体系产生衰老相关分泌表型之前、之时或之后，以银杏叶提取物进行处理；
    (2)将候选物质加入到(1)的体系中，观测其对该基质细胞体系的作用，若其能特异性促进银杏叶提取物对于衰老相关分泌表型的抑制作用，则该候选物质是可与银杏叶提取物联用、抑制衰老相关分泌表型的潜在物质。
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