WO2022165868A1 - 一种下调衰老相关分泌表型的抗衰老植物多酚类药物及其应用 - Google Patents

一种下调衰老相关分泌表型的抗衰老植物多酚类药物及其应用 Download PDF

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WO2022165868A1
WO2022165868A1 PCT/CN2021/077495 CN2021077495W WO2022165868A1 WO 2022165868 A1 WO2022165868 A1 WO 2022165868A1 CN 2021077495 W CN2021077495 W CN 2021077495W WO 2022165868 A1 WO2022165868 A1 WO 2022165868A1
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cells
cancer
pharmaceutically acceptable
senescence
tumor
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PCT/CN2021/077495
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English (en)
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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Priority to JP2023547195A priority Critical patent/JP2024508644A/ja
Priority to US18/262,319 priority patent/US20240091193A1/en
Priority to KR1020237029731A priority patent/KR20230142758A/ko
Priority to AU2021425528A priority patent/AU2021425528A1/en
Priority to EP21923878.9A priority patent/EP4212155A1/en
Publication of WO2022165868A1 publication Critical patent/WO2022165868A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the invention belongs to the field of biomedicine, and more particularly, the invention relates to an anti-aging drug for down-regulating or eliminating senescent cells and its application.
  • 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.
  • Senescent cells are characterized by morphological abnormalities, changes in metabolic activity, chromatin remodeling, altered gene expression, increased lipofuscin, pronounced granularity, severe vacuolation, and the emergence of a senescence-associated secretory phenotype (SASP) pro-inflammatory phenotype.
  • SASP senescence-associated secretory phenotype
  • aging may have evolved as a mechanism to avoid malignant transformation of damaged cells
  • the occurrence of aging may lead to many age-related pathologies, including cancer, cardiovascular and cerebrovascular diseases, osteoporosis, arthritis, metabolic diseases, neurological
  • a series of clinical problems such as degenerative symptoms.
  • Cell senescence is manifested by infolding of the nuclear membrane, chromatin pyknosis, and increased cell volume, which activates multiple downstream signaling pathways including p53, p16 INK4A /Rb, PI3K/Akt, FoxO transcription factors, and mitochondrial SIRT1.
  • senescent cells are often associated with a number of pathological features, including local inflammation. Cellular senescence occurs in damaged cells and prevents them from proliferating in an organism. Under the influence of various external stimuli and internal factors, cell damage can lead to obvious signs of cellular senescence. When the accumulation of damage reaches a certain limit, various degenerative changes and physiological aging phenotypes can be seen in the tissue.
  • SASP senescence-associated secretory phenotype
  • SASP inhibitors Although a variety of SASP inhibitors known internationally can significantly attenuate SASP, they do not intrinsically kill senescent cells. To pharmacologically reduce the burden on senescent cells, scientists are developing small molecules, peptides and antibodies of the nature of "senolytics" (senescent cell-eliminating drugs) to selectively eliminate senescent cells. Since the discovery of senolytic drugs in 2015, researchers have made considerable progress in identifying other small molecule senolytic drugs and their effects. Numerous studies have shown that most senolytics are only effective against a limited number of senescent cell types. For example, navitoclax was able to target HUVECs but was ineffective against senescent human adipocytes.
  • senolytics may vary even within one specific type of cell.
  • navitoclax targets and kills senescent cells in the culture-adapted IMR90 lung fibroblast-like cell line, but is less effective on senescent primary human lung fibroblasts.
  • a first aspect of the present invention provides a pharmaceutical composition, comprising proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and optional pharmaceutically acceptable excipients
  • the procyanidins are oligomeric procyanidins, preferably including procyanidin C1.
  • the final concentration of procyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof in the pharmaceutical composition is at least 1 ⁇ M, such as at least 1 ⁇ M, at least 10 ⁇ M, at least 20 ⁇ M, at least 30 ⁇ M, at least 40 ⁇ M, at least 50 ⁇ M, at least 100 ⁇ M, at least 200 ⁇ M, at least 500 ⁇ M, at least 1 mM, or a range between any two of the above.
  • the pharmaceutical composition further comprises an agent capable of inducing the generation of senescent cells in the subject.
  • the agent induces the production of senescent cells in tumor tissue.
  • the agent is capable of causing DNA damage and/or apoptosis, eg, DNA double-strand breaks.
  • the agent is MIT or DOX.
  • the present invention also provides the use of proanthocyanidins or pharmaceutically acceptable salts, hydrates or prodrugs thereof in the preparation of medicaments or preparations for: down-regulating senescence-associated secretory phenotype (SASP), reducing the expression of SASP factors expression or activity, reducing the expression or activity of markers of cellular senescence, inducing apoptosis in non-proliferative cells, reducing or eliminating non-proliferative cells, delaying aging, prolonging the lifespan of a subject, reducing the burden of age-related disease in a subject, preventing, ameliorating and Treat diseases that benefit from reduction or elimination of non-proliferating cells, reduce resistance to cancer therapy, enhance the efficacy of agents that induce cellular senescence, promote tumor regression, reduce tumor size, prevent or treat cancer, or prolong cancer survival Expect.
  • SASP senescence-associated secretory phenotype
  • the procyanidins are oligomeric procyanidins, preferably including procyanidin C1.
  • the SASP factors include extracellular matrix proteins, inflammatory cytokines, and cancer cell growth factors.
  • the SASP factors include the factors shown in FIG. 6 .
  • the SASP factor is selected from the group consisting of: IL6, CXCL8, MCP2, CXCL1, GM-CSF, MMP3, AREG, SFRP2, ANGPTL4, IL1a.
  • the cellular senescence marker factor is selected from the group consisting of: p16 INK4a , p21 CIP1 .
  • the non-proliferating cells are senescent cells, such as naturally senescent cells or damaged cells.
  • the damaged cells include damaged cells in the tissue microenvironment, preferably damaged cells caused by chemotherapy or radiation therapy.
  • the radiation therapy comprises: ionizing radiation, alpha, beta or gamma radiation therapy.
  • the disease that benefits from the reduction or elimination of non-proliferative cells is an age-related disease, including but not limited to cancer, cardiovascular and cerebrovascular disease, osteoporosis, age-related degenerative joint disease (such as arthritis), metabolic diseases, neurodegenerative diseases.
  • the cancer is prostate cancer; the tumor is a prostate tumor.
  • the agents that induce cellular senescence include agents that cause DNA damage and/or apoptosis, such as chemotherapeutic agents or radiation.
  • the agent comprises MIT or DOX.
  • the subject is an elderly subject.
  • the elderly subject is a subject corresponding to a mouse of at least 20 months of age or a human of at least 60 years of age.
  • the elderly subject is a subject corresponding to a mouse of at least 24 months of age or a human of at least 75 years of age. More preferably, the elderly subject is a subject corresponding to a 24-27 month old mouse or a 75-90 year old human.
  • the cancer therapy comprises chemotherapy or radiation therapy, eg, MIT, DOX therapy, ionizing radiation, alpha, beta or gamma radiation therapy.
  • chemotherapy or radiation therapy eg, MIT, DOX therapy, ionizing radiation, alpha, beta or gamma radiation therapy.
  • the present invention also provides the use of (a) proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof and (b) an agent capable of inducing senescent cells in a subject in the preparation of a medicament or a formulation for use in : Promote tumor regression, reduce tumor volume, prevent or treat cancer, and prolong cancer survival.
  • the procyanidins are oligomeric procyanidins, preferably including procyanidin C1.
  • the agent induces the production of senescent cells in tumor tissue.
  • the agent is capable of causing DNA damage and/or apoptosis, eg, DNA double-strand breaks.
  • the agent is MIT or DOX.
  • (a) is capable of eliminating senescent cells.
  • the tumor is a prostate tumor; the cancer is prostate cancer.
  • kits or kit comprising the pharmaceutical composition described in the first aspect herein and optionally an agent capable of inducing the production of senescent cells in a subject.
  • the kit or kit comprises container 1 and container 2, containing respectively (a) a procyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof and optionally a pharmaceutically acceptable acceptable excipients, and (b) the agent capable of inducing senescent cells in a subject and optional pharmaceutically acceptable excipients.
  • compositions, kit or kit in the pharmaceutical composition, kit or kit, (a) and optional (b) are the active ingredients, and other ingredients are pharmaceutically acceptable excipients and the like.
  • the dosage form of the pharmaceutical composition comprises: oral preparation, injection, infusion preparation, tablet, powder, capsule, and pill; preferably, the dosage form is oral preparation.
  • a method of altering a non-proliferating cell or object comprising treating the non-proliferating cell or subjecting it to procyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof
  • the changes include one or more selected from the group consisting of down-regulating the senescence-associated secretory phenotype (SASP), reducing the expression or activity of SASP factors, reducing the expression or activity of markers of cellular senescence, inducing a non-proliferative state Apoptosis, reduction or elimination of non-proliferating cells, delaying senescence, prolonging the lifespan of a subject, reducing the burden of age-related disease in a subject, preventing, alleviating and treating diseases that benefit from the reduction or elimination of non-proliferating cells, enhancing cellular senescence-inducing The cytotoxicity of the agent, or the reduction of resistance to cancer therapy.
  • SASP senescence-associated secretory phenotype
  • the final concentration of procyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof is at least 1 ⁇ M, such as at least 10 ⁇ M, at least 20 ⁇ M, at least 30 ⁇ M, at least 40 ⁇ M, at least 50 ⁇ M, at least 100 ⁇ M, At least 200 ⁇ M, at least 500 ⁇ M, at least 1 mM, or a range between any two of the above.
  • the procyanidins are oligomeric procyanidins, preferably including procyanidin C1.
  • a method of enhancing the cytotoxicity of an agent capable of inducing cellular senescence, promoting tumor regression, reducing tumor volume, preventing or treating cancer, or prolonging cancer survival comprising using (a ) a proanthocyanidin, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) an agent capable of inducing senescent cells in a subject to treat or administer to the subject.
  • the procyanidins are oligomeric procyanidins, preferably including procyanidin C1.
  • the agent induces the production of senescent cells in tumor tissue.
  • the agent is capable of causing DNA damage and/or apoptosis, eg, DNA double-strand breaks.
  • the agent is MIT or DOX.
  • the tumor is a prostate tumor; the cancer is prostate cancer.
  • the final concentration of procyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof is at least 10 ⁇ M, such as at least 20 ⁇ M, at least 30 ⁇ M, at least 40 ⁇ M, at least 50 ⁇ M, at least 100 ⁇ M, at least 200 ⁇ M, At least 500 ⁇ M, at least 1 mM, or a range between any two of the above.
  • the uses or methods described in any of the embodiments herein are not directed towards clinical disease treatment.
  • Figure 1 shows that proliferating human stromal cells PSC27 (early passage such as p10-20) were treated with the chemotherapeutic drug bleomycin (BLEO) at a concentration of 50 ⁇ g/ml on days 7-10 by SA- ⁇ -Gal staining in vitro results after.
  • Top panel representative image, bottom panel, statistical data.
  • CTRL control cells;
  • BLEO cells after bleomycin treatment. **, P ⁇ 0.01.
  • Figure 2 shows the results of 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.
  • Figure 3 shows the results of 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 shows the experimental flow chart of screening natural product drug libraries to obtain plant materials with anti-aging activity.
  • FIG. 5 shows that after software processing and bioinformatics analysis of RNA-seq data, it was found that PCC1 can significantly reduce genes that were significantly up-regulated in senescent cells compared to proliferating cells. Compared with BLEO group, 4406 genes were significantly down-regulated and 2766 genes were significantly up-regulated in BLEO/PCC1 group cells (fold change>2, P ⁇ 0.01).
  • the Heatmap in Figure 6 shows that the expression of a large number of factors was up-regulated in senescent cells caused by BLEO injury, but many of them were significantly reversed after PCC1 treatment. Red star logo, typical SASP exogenous factor.
  • Figure 7 shows the results of GSEA analysis showing that the expression of SASP or NF- ⁇ B molecular marker-related factors was centrally up-regulated in BLEO-induced senescent cells, but significantly decreased after PCC1 treatment of senescent cells.
  • SASP molecular marker Left, SASP molecular marker; right, NF- ⁇ B molecular marker.
  • FIG 8 shows the results of protein-protein interaction (PPI) bioinformatics analysis, showing that senescent cell molecules significantly down-regulated by PCC1 form a network, and there are various interactions between them.
  • PPI protein-protein interaction
  • Figure 9 shows a representative pathway of 100 molecules on biological processes that cause significant downregulation of PCC1 in senescent cells by KEGG pathway analysis. Left Y-axis, percentage. Right Y-axis, log10 (p-value).
  • Figure 10 shows a representative pathway of 100 molecules on the cellular component of KEGG pathway analysis of PCC1 causing significant downregulation in senescent cells.
  • Left Y-axis percentage.
  • Right Y-axis log10 (p-value).
  • Figure 11 shows quantitative PCR (qRT-PCR) assay to analyze the relative expression levels of a group of typical SASP molecules in BLEO-induced senescent cells treated with different concentrations of PCC1. All data are normalized results compared to the CTRL group. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 12 shows the determination of senescence of PSC27 by SA- ⁇ -Gal staining under the condition of increasing PCC1 concentration.
  • ⁇ P>0.05; **, P ⁇ 0.01; ****, P ⁇ 0.0001.
  • the P values of PCC1 at the concentrations of 1 ⁇ M, 10 ⁇ M, 20 ⁇ M, 50 ⁇ M, 100 ⁇ M, 150 ⁇ M and 200 ⁇ M were statistically significant compared with the data at 0 ⁇ M for the positive proportion of cells in these experimental groups.
  • Figure 13 shows representative pictures of PSC27 under various conditions after SA- ⁇ -Gal staining. 3 repetitions per set, up and down. Scale bar, 30 ⁇ m.
  • Figure 14 shows the survival rate of proliferating cells and senescent cells detected by CCK8 under increasing concentrations of PCC1. P values at each PCC1 concentration are significant differences between the CTRL and BLEO groups after comparison. **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 15 shows a population doubling test for PSC27.
  • Cells were treated with BLEO injury at passage 10 (p10) and then PCC1 was added to the medium on day 8.
  • the effect of PCC1 on cell proliferation potential was determined by comparing the doubling proliferation (PD) of CTRL group, BLEO group, PCC1 group and BLEO/PCC1 group. ⁇ , P>0.05; ***, P ⁇ 0.001.
  • Figure 16 shows the induction of caspase 3/7 activity during PCC1 treatment of senescent cells.
  • PSC27 cells gradually entered the senescence stage after being treated with BLEO for 12 h.
  • 50 ⁇ M PCC1 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.
  • Figure 17 shows senolytic activity reversed by a Pan-caspase inhibitor (20 cM QVD-OPh) (50 ⁇ M PCC1 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 shows apoptosis of PSC27 under several conditions determined by flow cytometry. Q2, distribution area of early apoptotic cells; Q3, distribution area of late apoptotic cells.
  • Figure 19 shows a comparative analysis of the number of viable and apoptotic cells after BLEO and/or PCCl treatment. ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • Figure 20 shows a schematic diagram of the dosing regimen in mice in the pre-clinical trial.
  • Human stromal cells PSC27 and cancer cells PC3 were mixed in vitro (1:4) and then transplanted into mice subcutaneously to form xenografts. After multiple treatment cycles under the condition of single-drug or combined administration, the mice were finally sacrificed, and the expression changes of related molecules in tumor tissue were analyzed pathologically.
  • Figure 21 shows that 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.
  • Figure 22 shows a schematic diagram of the 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 PCC1 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 shows a statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug MIT was administered to mice alone or together with the anti-aging drug PCC1, and the tumor size of each group was compared after the end of the 8th week.
  • Figure 24 shows a 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 shows a 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.
  • Figure 26 shows the expression of SASP typical factors in epithelial cancer cells and stromal cells in mouse lesions analyzed by real-time quantitative PCR (qRT-PCR).
  • 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.
  • Figure 27 shows quantitative PCR (qRT-PCR) assays to analyze the expression status of SASP factors in stromal cells in mouse lesions following vehicle, MIT and MIT/PCCl administration. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • Figure 28 shows the analysis of DNA damage and apoptosis ratios in each group of mice after the specific isolation of cancer cells in the lesions by LCM technology. ⁇ , P>0.05; *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 29 shows picture analysis after immunohistochemical staining.
  • Scale bar 200 ⁇ m.
  • Figure 30 shows the Kaplan Meier data comparison of disease-free survival in NOD/SCID mice after various drug treatments.
  • Vehicle, MIT, PCC1 and MIT/PCC1 animals 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.
  • Figure 31 shows a comparative analysis of mouse body weight data at the end of a course of treatment under various drug treatment conditions. ⁇ , P>0.05.
  • Figure 32 shows the comparative analysis of mouse serological data at the end of the course of treatment under the above different administration conditions. Creatinine, urea (renal index), ALP and ALT (liver index) data were compared in parallel. ⁇ , P>0.05.
  • Figure 33 shows a comparative analysis of the body weight data of immunized intact mice (C57BL/6J) at the end of the course of treatment under various dosing conditions. ⁇ , P>0.05.
  • Figure 34 shows a comparative analysis of mouse blood cell counts at the end of a course of treatment under different dosing treatment conditions in the pre-clinical setting. The number of WBC, lymphocytes and neutrophils per unit volume was compared in parallel. ⁇ , P>0.05.
  • Figure 35 shows statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug DOX alone or together with the anti-aging drug PCC1 was administered to the mice, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 36 shows statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug DOC was administered to mice alone or together with the anti-aging drug PCC1, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 37 shows statistical analysis of tumor terminal volume.
  • the chemotherapeutic drug VIN alone or together with the anti-aging drug PCC1 was administered to the mice, and the tumor size of each group was compared and analyzed after the end of the 8th week.
  • Figure 40 shows the selection of female mice with the highest lifespan in each group of animals, and a comparative analysis of the highest walking speed, endurance and overall lifespan between groups.
  • N 5.
  • Figure 41 shows that the male mice with the highest life span in each group were selected for comparative analysis of the highest walking speed, endurance and overall life span between groups.
  • N 5/group.
  • proanthocyanidins have extremely excellent effects on down-regulating or removing senescent cells in the body, so that they can be applied to remove damaged cells in the tissue microenvironment, and can also be used to remove cells that naturally age with age.
  • a “proliferating cell” refers to a cell capable of maintaining a state of continuous, active division and continuous proliferation.
  • Non-proliferating cells in the narrow sense are senescent cells, such as naturally senescent cells or damaged cells, including damaged cells in the tissue microenvironment, preferably damaged cells caused by chemotherapy or radiation therapy.
  • senescent cells refer to cells whose ability to proliferate and divide is reduced and their physiological functions decline.
  • Procyanidins as used herein is a general term for polyphenolic compounds, including oligomeric procyanidins.
  • Anthocyanins are an important family of plant polyphenols. Most of this type of flavanol compounds are connected by catechin or epicatechin through C4-C6 and C4-C8 of flavan bonds.
  • the compound is gallic acid ester; their commonality is that they are oligomers, mostly dimers and trimers, which are composed of anthocyanin-containing monomers in their chemical nature.
  • Exemplary oligomeric proanthocyanidins have units of formula I with 1-6 more units, where wavy lines indicate connections to other units.
  • the procyanidin is procyanidin C1 (PCC1) of the formula
  • PCC1 is a trimer in the plant polyphenol family, which not only has antioxidant, anti-inflammatory, anti-cancer and other effects, but also has the function of targeting and removing senescent cells.
  • “compounds” may be compounds in pure form, or more than 85% pure (preferably more than 90%, such as 95%, 98%, 99%) compound of.
  • the compounds of the present invention can be obtained by various methods well-known in the art and using well-known raw materials, such as chemical synthesis or from organisms (such as microorganisms) Extraction methods, these methods are all included in the present invention.
  • proanthocyanidins are also commercialized drugs, so their finished products are easily obtained by those skilled in the art.
  • pharmaceutically acceptable salts of procyanidins are also included, which also retain the chemical activity of procyanidins.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or animals without undue adverse side effects (eg, toxicity, irritation, and allergy), ie, with a reasonable benefit/risk ratio.
  • the "pharmaceutically acceptable salts” can be acid salts and basic salts of procyanidins.
  • “Pharmaceutically acceptable acid salts” refers to salts that retain the biological activity and properties of the free base without exhibiting undesirable biological activity or other changes. Such salts may be composed of inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and similar acids.
  • Such salts may also be composed of organic acids such as, but not limited to, acetic acid, dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, Camphorsulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclonic acid, dodecylsulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, isethionic acid , formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid , glycolic acid, hippuric acid, isobutyric acid, lactic
  • “Pharmaceutically acceptable base salts” refers to salts that retain the biological activity and properties of the free acid without exhibiting undesirable biological activity or other changes. These salts are prepared by adding an inorganic or organic base to the free acid. Salts obtained with inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts.
  • Salts obtained from organic bases include, but are not limited to, primary, secondary, and tertiary ammonium salts, and substituted amines include natural substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, dimethine Ethylamine, Triethylamine, Tripropylamine, Diethanolamine, Ethanolamine, Danol, 2-Dimethylaminoethanol, 2-Diethylaminoethanol, Dicyclohexylamine, Lysine, Arginine, Histidine, Coffee In, Procaine, Haramine, Choline, Betaine, Phenylbenzylamine, N,N'-Bisbenzylethylenediamine, Ethylenediamine, Glucosamine, Meglucamine, Theobromine, Tris Ethanolamine, Tromethamine, Purine, Piperazine, Piperidine, N-Ethylpiperidine, Polyamide Res
  • the compounds disclosed in this patent may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and similar structures.
  • prodrugs of procyanidins are also included, and the "prodrug” refers to a compound that undergoes metabolism or chemical reaction in the subject's body to convert the procyanidins into desired procyanidins when taken in an appropriate manner.
  • PCC1 proanthocyanidins
  • the present invention provides the use of proanthocyanidins in the preparation of medicines or preparations for: down-regulating senescence-associated secretory phenotype (SASP), reducing the expression or activity of SASP factors, reducing the expression of cellular senescence markers, or activity, induce apoptosis of non-proliferative cells, reduce or eliminate non-proliferative cells, delay aging, prolong lifespan of subjects, reduce age-related disease burden in subjects, prevent, alleviate and treat diseases that benefit from reduction or elimination of non-proliferative cells, Reduce resistance to cancer therapy, promote tumor regression, reduce tumor size, prevent or treat cancer, or prolong cancer survival.
  • SASP senescence-associated secretory phenotype
  • reducing the expression or activity of SASP factors reducing the expression of cellular senescence markers, or activity
  • induce apoptosis of non-proliferative cells reduce or eliminate non-proliferative cells, delay aging, prolong lifespan of subjects, reduce age-related disease burden in subjects, prevent,
  • a substance eg, PCCl
  • PCCl a substance that can eliminate or eliminate non-proliferating cells by inducing apoptosis.
  • SASP factors include extracellular matrix proteins, inflammatory cytokines, and cancer cell growth factors.
  • the SASP factors may include the factors shown in Figure 6 or selected from one or more of: IL6, CXCL8, MCP2, CXCL1, GM-CSF, MMP3, AREG, SFRP2, ANGPTL4, IL1a.
  • the “diseases that benefit from the reduction or elimination of non-proliferative cells” as described herein are generally age-related diseases, including but not limited to cancer, cardiovascular and cerebrovascular diseases, osteoporosis, age-related degenerative joint diseases (such as arthritis), Metabolic and neurodegenerative diseases.
  • the cancer is prostate cancer.
  • procyanidins can also be used to extend the lifespan of a subject and reduce the age-related disease burden in a subject.
  • the subject is an elderly subject, eg, a subject corresponding to a mouse of at least 20 months of age or a human of at least 60 years of age.
  • the elderly subject is a subject corresponding to a mouse of at least 24 months of age or a human of at least 75 years of age. More preferably, the elderly subject is a subject corresponding to a 24-27 month old mouse or a 75-90 year old human.
  • an elderly subject is used as the research subject in the specific embodiment, this is only an example to facilitate the analysis of the results (eg, elderly subjects have more age-related diseases).
  • proanthocyanidins in eliminating senescent cells found in the present invention, those skilled in the art should know that they can be used in subjects of any age to eliminate senescent cells, prolong lifespan, and reduce the burden of age-related diseases.
  • procyanidins can also be used to reduce patient resistance to cancer therapy.
  • the cancer therapy includes chemotherapy or radiation therapy; chemotherapy such as cytotoxic therapy such as MIT or DOX, radiation therapy such as ionizing radiation, mainly including ⁇ , ⁇ , ⁇ and X-rays as well as proton, neutron flow therapy and the like.
  • procyanidins can enhance the cytotoxicity of agents that induce cellular senescence when used in combination with certain agents.
  • the agent that induces cellular senescence may be an agent that induces senescent cells by causing DNA damage and/or apoptosis, such as chemotherapeutic agents or radiation.
  • the present invention also provides the use of procyanidins in enhancing the efficacy of agents that induce cellular senescence, and the combined use of procyanidins and agents inducing cellular senescence in promoting tumor regression, reducing tumor volume, preventing or treating cancer, and prolonging cancer survival.
  • the cell is a tumor cell; the tumor is a prostate tumor; the cancer is prostate cancer.
  • a method of achieving the above use comprising using (a) a procyanidin as described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and optionally (b) An agent capable of inducing the production of senescent cells in a subject treats the senescent cells or administers them to a subject in need thereof.
  • administering or “administering” refers to providing a compound or pharmaceutical composition of the present invention to a subject having or at risk for the disease or disorder to be treated or prevented.
  • composition of the present invention uses substances such as proanthocyanidins or its salts as active components.
  • containing procyanidins eg PCC1 can down-regulate senescence-associated secretory phenotype (SASP), reduce the expression or activity of SASP factors, reduce the expression or activity of markers of cellular senescence, induce apoptosis in non-proliferating cells (senescent cells) death, reduction or elimination of non-proliferating cells (senescent cells), delaying aging, prolonging the lifespan of a subject, reducing the age-related disease burden in a subject, preventing, alleviating and treating diseases that benefit from the reduction or elimination of non-proliferating cells, reducing the risk of cancer therapy of drug resistance.
  • SASP senescence-associated secretory phenotype
  • the composition when the composition further comprises a cellular senescence-inducing agent (eg, chemotherapeutic agent or radiation) as an active ingredient, the composition can promote tumor regression, reduce tumor volume, prevent or treat cancer, prolong cancer survival.
  • a cellular senescence-inducing agent eg, chemotherapeutic agent or radiation
  • composition described herein when used as a medicine, it also includes a pharmaceutically acceptable adjuvant.
  • pharmaceutically acceptable adjuvants are pharmaceutically or food acceptable carriers, solvents for delivering the active ingredients (eg, procyanidins and optional cellular senescence-inducing agents) in the compositions of the present invention to animals or humans , suspending agents or excipients.
  • Exemplary excipients can be liquid or solid, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, Sweeteners, dyes/colorants, flavor enhancers, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gas, or other suitable Inactive ingredients used in combination with pharmacologically active compounds. More specifically, suitable excipients can be those commonly used in the art for the administration of small molecule compounds.
  • excipients include various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as white Proteins, and detergents such as Tween 80, monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.
  • buffers such as PBS, saline
  • polyethylene glycol glycerol
  • polypropylene glycol dimethyl sulfoxide
  • amides such as dimethylacetamide
  • proteins such as white Proteins
  • detergents such as Tween 80
  • monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.
  • compositions will contain a therapeutically effective amount of the active ingredients described herein.
  • a therapeutically effective amount refers to a dose that will effect treatment, prevention, alleviation and/or amelioration of a disease or disorder in a subject.
  • the therapeutically effective amount can be determined according to factors such as the patient's age, sex, the condition and its severity, and other physical conditions of the patient.
  • a therapeutically effective amount may be administered as a single dose, or may be administered in multiple doses according to an effective therapeutic regimen.
  • a subject or patient generally refers to a mammal, especially a human.
  • the composition contains, for example, 0.001-50%, preferably 0.01-30%, more preferably 0.05-10% by weight of active ingredients (eg, procyanidins and optional cellular senescence-inducing agents).
  • the pharmaceutical compositions or mixtures of the present invention can be prepared in any conventional formulation by conventional methods.
  • the dosage form may be various, as long as the dosage form can effectively reach the mammalian body of the active ingredient.
  • it can be selected from: injections, infusions, tablets, capsules, and pills.
  • the active ingredients eg, procyanidins and optional cellular senescence-inducing agents
  • the active ingredient mixtures or pharmaceutical compositions of the present invention may also be stored in sterile devices suitable for injection or instillation.
  • the effective dosage of the active ingredients in the composition may vary with the mode of administration and the severity of the disease to be treated, as can be based on the experience and advice of the clinician.
  • mice are also used as experimental animals, and it is easy for those skilled in the art to convert the dosage of mice to the dosage suitable for human beings. For example, it can be calculated according to the Meeh-Rubner formula:
  • 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, 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, and 11.2 for monkeys. 11.8, people 10.6.
  • Proanthocyanidins and optional cellular senescence-inducing agents or pharmaceutical compositions can be administered orally, as well as intravenously, intramuscularly, or subcutaneously. It may preferably be administered orally.
  • Pharmaceutical forms suitable for oral administration include, but are not limited to, tablets, powders, capsules, sustained release formulations, and the like.
  • the pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders. In all cases, these forms must be sterile and must be fluid for easy syringe expelling.
  • procyanidins and optional cellular senescence-inducing agents can also be administered in combination with other active ingredients or drugs.
  • the present invention also provides a kit or kit for down-regulating or eliminating senescent cells, or prolonging the lifespan of a body, the kit or kit containing the pharmaceutical composition described in any of the embodiments herein.
  • the kit or kit contains a mixture of the procyanidins described herein and an optional cellular senescence-inducing agent.
  • the kit or kit contains: container 1, and a procyanidin described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, placed in container 1; and container 2, and placed in container 2 Cellular Senescence Inducing Reagents in .
  • the kit or kit may also contain some adjuvant materials, such as measuring instruments, containers such as syringes, etc., required for the use or administration of the compositions in various dosage forms.
  • the kit or kit may also contain instructions for use, explaining the method of treating down-regulating or eliminating senescent cells or prolonging the survival period of the body.
  • a pharmaceutical composition comprising (a) a proanthocyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) an agent capable of inducing a subject to produce senescent cells, and optionally a pharmaceutically acceptable accessories,
  • the procyanidins are oligomeric procyanidins.
  • the final concentration of the procyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof in the pharmaceutical composition is at least 1 ⁇ M, and/or
  • the procyanidin is procyanidin C1, and/or
  • Such agents include agents that cause DNA damage and/or apoptosis.
  • SASP senescence-associated secretory phenotype
  • the procyanidins are oligomeric procyanidins.
  • the procyanidin is procyanidin C1, and/or
  • the SASP factors include extracellular matrix proteins, inflammatory cytokines, and cancer cell growth factors, and/or
  • the non-proliferating cells are senescent cells, preferably naturally senescent or damaged cells, and/or
  • the diseases that benefit from the reduction or elimination of non-proliferative cells are age-related diseases, preferably cancer, cardiovascular and cerebrovascular diseases, osteoporosis, age-related degenerative joint diseases, metabolic diseases, neurodegenerative diseases, and/ or
  • the agents capable of inducing cellular senescence include agents that cause DNA damage and/or apoptosis, and/or
  • the subject is an elderly subject, and/or
  • the cancer therapy includes chemotherapy or radiation therapy.
  • a substance comprising (a) proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) an agent capable of inducing the production of senescent cells in a subject, in the preparation of a medicament or a preparation, the Drugs or preparations for: promoting tumor regression, reducing tumor size, preventing or treating cancer, or prolonging cancer survival,
  • the proanthocyanidins are oligomeric proanthocyanidins, more preferably procyanidin C1,
  • the agent capable of inducing senescent cells in the subject comprises an agent that causes DNA damage and/or apoptosis.
  • kits or test kit comprising the pharmaceutical composition of item 1 or 2,
  • the kit or kit comprises container 1 and container 2, respectively containing (a) proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof and optional pharmaceutically acceptable excipients, and (b) an agent and optional pharmaceutically acceptable excipients capable of inducing the production of senescent cells in a subject,
  • the procyanidins are oligomeric procyanidins, more preferably procyanidin C1.
  • a method of altering non-proliferating cells comprising treating the non-proliferating cells with proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof, the alteration comprising one selected from the group consisting of or more: down-regulates senescence-associated secretory phenotype (SASP), reduces the expression or activity of SASP factors, reduces the expression or activity of markers of cellular senescence, induces apoptosis in non-proliferative cells, reduces or eliminates non-proliferative cells, or reduce the resistance of cells to treatment with cancer therapy,
  • SASP down-regulates senescence-associated secretory phenotype
  • the procyanidins are oligomeric procyanidins, more preferably procyanidin C1, and/or
  • the final concentration of proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof is at least 1 ⁇ M.
  • a method of enhancing the cytotoxicity of an agent capable of inducing cellular senescence comprising using (a) a proanthocyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) an agent capable of inducing cellular senescence.
  • reagents to treat cells comprising using (a) a proanthocyanidin or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) an agent capable of inducing cellular senescence.
  • the procyanidins are oligomeric procyanidins, more preferably procyanidin C1, and/or
  • the agent capable of inducing cellular senescence causes DNA damage and/or apoptosis, and/or
  • the final concentration of proanthocyanidins or a pharmaceutically acceptable salt, hydrate or prodrug thereof is at least 10 ⁇ M.
  • 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. 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 preseeded on coverslips for at least 24 h after culture 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)-European F(ab')2 was added sequentially to slides covered with fixed cells. Nuclei were counterstained with 2 ⁇ g/m 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 Bowtie tools. 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 with Cufflinks and differential gene expression was called with the Cufflinks maximum likelihood estimation function. Genes with significantly altered expression were defined by false discovery rate (FDR)-corrected P-values ⁇ 0.05, and downstream analyses were performed only with Ensembl Genes 73 with status "Known” and biotype "coding".
  • PPI Protein-protein interaction
  • GSEA Gene Set Enrichment Analysis
  • genes were ranked using "wald statistics" obtained from DESeq2, GSEA in MSigDB (http://software.broadinstitute.org/gsea) based on data obtained from preliminary RNA-seq analysis /msigdb) on these sorted lists of all planned gene sets available).
  • DESeq2 independent filtering is based on the mean of normalized read counts to screen for genes with very low expression levels.
  • the GSEA signature of SASP is as described in our previous publications (Zhang et al., 2018a).
  • Trizol reagent Extract the total RNA of cells in growth phase or arrest phase with Trizol reagent, add 1ml Trizol to each T25 culture flask cell, scrape the cell layer with a cell scraper, transfer it to a centrifuge tube, and mix well until it is not viscous.
  • Oligomeric dT 23 VN (50uM), 1ul; total RNA, 1-2ug; RNase-free ddH2O was added to 8ul . Heated at 65°C for 5 min, quickly placed on ice and quenched, and let stand for 2 min.
  • First-strand cDNA synthesis was performed under the following conditions: 25°C for 5 min, 50°C for 45 min, and 85°C for 5 min.
  • the reverse transcription reaction product cDNA was diluted 50-fold as a template.
  • the PCR reaction solution was configured as follows: AceQ SYBR Green Master Mix, 10ul; Primer 1 (10uM), 0.4ul; Primer 2 (10uM), 0.4ul; Rox Reference Dye, 0.4ul; Template, 2ul; ddH 2 O was added to 20ul.
  • reaction conditions are: pre-denaturation at 95°C for 15sec, then 95°C for 5sec, 60°C for 31sec, 40 cycles; melting curve conditions are 95°C for 15sec, 60°C for 30sec, and 95°C for 15sec.
  • the samples were reacted on an ABI ViiA7 (ABI) instrument.
  • the expression of ⁇ -actin was used as an internal reference.
  • the amplification of each gene was analyzed by software, the corresponding threshold cycle number was derived, and the 2- ⁇ Ct method was used to calculate the relative expression level of each gene. The peaks and waveforms of the melting curve were analyzed to determine whether the resulting amplification product was a specific single target fragment.
  • the sequences of the detection primers used are as follows, F represents the forward primer, and R represents the reverse primer:
  • IL6 (F: SEQ ID NO: 1, R: SEQ ID NO: 2); CXCL8 (F: SEQ ID NO: 3, R: SEQ ID NO: 4); SPINK1 (F: SEQ ID NO: 5, R: SEQ ID NO: 6); WNT16B (F: SEQ ID NO: 7, R: SEQ ID NO: 8); GM-CSF (F: SEQ ID NO: 9, R: SEQ ID NO: 10); MMP3 (F : SEQ ID NO: 11, R: SEQ ID NO: 12); IL-1 ⁇ (F: SEQ ID NO: 13, R: SEQ ID NO: 14); p16INK4a (F: SEQ ID NO: 15, R: SEQ ID NO: 16); IL-1 ⁇ (F: SEQ ID NO: 17, R: SEQ ID NO: 18); AREG (F: SEQ ID NO: 19, R: SEQ ID NO: 20); CXCL1 (F: SEQ ID NO: 21, R: SEQ ID NO: 22); CXCL3 (F: SEQ ID
  • Senescence-associated beta-galactosidase (SA-beta-Gal) staining was performed using previously reported procedures (Debacq-Chainiaux et al., 2009). Briefly, cell culture dishes were washed with PBS and fixed at room temperature. Cells were fixed in 2% formaldehyde and 0.2% glutaraldehyde for 3 min. SA- ⁇ -Gal was then stained with freshly prepared staining solution overnight at 37°C. Images were taken the next day and the percentage of positive cells per unit area was calculated.
  • PSC27 cells were plated in 96-well dishes and cell senescence was induced under BLEO treatment at 50 ⁇ g/ml.
  • PCC1 and ABT263 were added at concentrations of 50 ⁇ 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.
  • mice All experimental mouse experiments were carried out in strict accordance with the relevant regulations of the Laboratory Animal Care and Use Committee (IACUC) of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
  • Immunodeficient mice NOD-SCID mice, ICR (weight about 25 g) aged 6-8 weeks were used for the animal experiments related to this patent.
  • 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.
  • the xenografts were implanted into mice by subcutaneous transplantation, and the animals were euthanized 8 weeks after the transplantation.
  • mice were fed a standard experimental diet followed by administration of the chemotherapeutic drugs mitoxantrone (MIT, 0.2 mg/kg dose) and/or procyanidin C1 (PCC1) (500 ⁇ l, 10 mg after 2 weeks) /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 and 7.
  • a total of 3 cycles of MIT were administered throughout the course of treatment, and each cycle was 2 weeks.
  • mouse tumors were collected for volume measurement and histological analysis. Each mouse received a cumulative total of 0.6 mg/kg of MIT and 30 mg/kg of PCC1.
  • 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.
  • mice 16-month-old male C57BL/6 mice by continuous rearing on the SPF animal platform with 4 to 5 animals per cage.
  • 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.
  • mice were classified according to body weight and randomly assigned to each group to receive control (vehicle) or drug (PCC1) treatment by humans blinded to the design of the preclinical trial. Starting at 24-27 months of age, mice were treated with vehicle or PCC1 every 2 weeks by oral gavage for 3 consecutive days.
  • vehicle or PCC1 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 we euthanized the mice; we considered them dead if they exhibited one of the following symptoms: (1) unable to drink or eat; (2) unwilling to move even when stimulated; (3) ) Rapid weight loss; (4) Severe balance disorder; or (5) Bleeding or ulceration of the body.
  • no mice were excluded due to fights, accidental death, or dermatitis.
  • 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 cadavers were transported to a dedicated autopsy site for pathological examination. Tumor burden (sum of different tumor types per mouse), disease burden (sum of different histopathological changes in major organs of each mouse), severity of each lesion and inflammation (lymphocyte infiltration) were assessed.
  • mice were injected intraperitoneally with 3 mg of fluorescein (BioVision, Milpitas, CA), delivered in a volume of 200 ⁇ l of PBS. Mice were anesthetized with isoflurane and bioluminescent images were acquired using the Xenogen IVIS 200 System (Caliper Life Sciences, Hopkinton, MA).
  • Forelimb grip strength was determined using the Grip Strength Meter (Columbus Instruments, Columbus, OH) and results were averaged over 10 trials.
  • For the hanging endurance test mice were placed on a 2 mm thick metal wire 35 cm above the mat. Mice were only allowed to grasp the wire with their forelimbs, and hanging time was normalized to body weight and expressed as hanging duration (sec) 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 a Comprehensive Laboratory Animal Monitoring System (CLAMS). The CLAMS system was equipped with an Oxymax Open Circuit Calorimeter System (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 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°).
  • PCC1 can effectively inhibit the expression of SASP when used at low concentrations
  • PSC27 a primary normal human prostate stromal cell line
  • non-fibroblast cell lines including endothelial cells and smooth muscle cells
  • 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.
  • PCC1 a plant-based natural product, can be used to control the pro-inflammatory phenotype of senescent cells, namely SASP, especially at relatively low concentrations.
  • PCC1 is a novel senolytics when used at high concentrations
  • PCC1 causes senescent cells to lose their viability by inducing apoptosis
  • PCC1 to treat proliferation group cells and senescence group cells separately under culture conditions.
  • the subsequent observed changes in caspase-3/7 activity indicated that PCC1 caused senescent cells to undergo apoptosis; from the 16th hour after the addition of PCC1, there was a statistical difference between the senescent group and the control group (Fig. 16).
  • the pan-caspase inhibitor QVD prevented PCC1 from killing senescent cells, a process whose actual effect was similar to that of ABT263, a currently known and highly potent inducer of senescent apoptosis, on senescent cells very similar (Figure 17).
  • the above series of results confirmed that PCC1 promotes senescent cells to enter the death program by inducing apoptosis, but proliferating cells are basically not targeted or affected by this natural drug.
  • Example 3 Therapeutic targeting of senescent cells using PCC1 promotes tumor regression and effectively reduces chemoresistance
  • tissue recombinants by mixing PSC27 stromal cells with PC3 epithelial cells, a typical highly malignant 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/PCC1 combination exhibited the longest median survival, extending survival by at least 48.1% compared to the MIT-only group (FIG. 30, green vs. blue).
  • PCC1 MIT-only group
  • MIT and DOX are genotoxic drugs that can cause typical DNA double-strand breaks, which in turn cause cellular senescence.
  • the mechanism of action of VIN is to attach to microtubules and inhibit the process of mitosis. Therefore, the feature that PCC1 can improve the therapeutic effect of chemotherapy under in vivo conditions can be used in combination with drugs that induce the body to produce senescent cells, which is drug-type-dependent.
  • PCC1 a biologically active anti-aging drug

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Abstract

一种药物组合物，其包含(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导对象产生衰老细胞的试剂，和任选的药学上可接受的辅料。原花青素或其药学上可接受的盐、水合物或前药及该药物组合物在制备用于下述的药物或制剂中的用途：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、降低对癌症疗法的耐药性、增强能诱导细胞衰老的试剂的功效、促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期，尤其是前列腺癌或肿瘤。

Description

一种下调衰老相关分泌表型的抗衰老植物多酚类药物及其应用 技术领域
本发明属于生物医学领域，更具体地，本发明涉及下调或清除衰老细胞的抗衰老药物及其应用。
背景技术
细胞衰老是指真核细胞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。衰老细胞(senescent cell)的特征是形态学异常、代谢活性变化、染色质重构、基因表达改变、脂褐素增加、颗粒性明显、空泡化严重以及出现一种称为衰老相关分泌表型(SASP)的促炎症表型。衰老的生物学作用比较复杂，衰老细胞的保护作用和有害作用均已有描述，主要取决于病理生理学环境。例如，尽管衰老可能作为避免受损细胞恶性转化的机制进化而来，但衰老的发生可能会导致许多年龄相关病变，包括癌症、心脑血管疾病、骨质疏松、关节炎、代谢性疾病、神经退行性症状等一系列临床问题。
细胞衰老表现为核膜内折，染色质固缩，细胞体积增大，激活下游包括p53、p16 INK4A/Rb、PI3K/Akt、FoxO转录因子和线粒体SIRT1等在内的多条信号通路。除了进入永久性增殖停滞，衰老细胞常关系到许多病理学特征，包括局部炎症。细胞衰老发生于受损细胞，并防止其在生物体内增殖。在各种外界刺激和内部因素影响下，细胞损伤可以导致明显的细胞衰老迹象。当损伤累积达到一定的限度，组织中呈现各种肉眼可辨的组织退行变化和生理上的衰老表型。
尤其值得注意的是，衰老细胞中炎症性细胞因子的表达水平显著升高，这一现象被称为衰老相关分泌表型(senescence-associated secretory phenotype，SASP)。SASP这一概念是由Coppe等人于2008年首次提出。他们发现衰老细胞能通过分泌胞外基质蛋白、炎症相关因子及癌细胞生长因子促进邻近癌前细胞发生癌变或癌细胞的恶性程度上升，并统称这些蛋白为SASP因子。
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(eliminate)衰老细胞。研究者们自2015年发现senolytic药物以来，在鉴别其它小分子senolytic药物及其作用方面取得了相当大的进展。大量研究表明多数senolytics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，但对衰老的原代人肺成纤维细胞少有成效。
因此，本领域仍需寻找更有效、适用范围更广的抗衰老药物。
发明内容
本发明第一方面提供一种药物组合物，包含原花青素或其药学上可接受的盐、水合物或前药，和任选的药学上可接受的辅料
在一个或多个实施方案中，原花青素是低聚原花青素，优选包括原花青素C1。
在一个或多个实施方案中，原花青素或其药学上可接受的盐、水合物或前药在药物组合物中的终浓度为至少1μM，如至少1μM、至少10μM、至少20μM、至少30μM、至少40μM、至少50μM、至少100μM、至少200μM、至少500μM、至少1mM或上述任意两个数值之间的范围。
在一个或多个实施方案中，所述药物组合物还包含能诱导对象产生衰老细胞的试剂。
在一个或多个实施方案中，所述试剂能诱导肿瘤组织中产生衰老细胞。
在一个或多个实施方案中，所述试剂能导致DNA损伤和/或细胞凋亡，例如DNA双链断裂。
在一个或多个实施方案中，所述试剂是MIT或DOX。
本发明还提供原花青素或其药学上可接受的盐、水合物或前药在制备药物或制剂中的用途，所述药物或制剂用于：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、降低对癌症疗法的耐药性、增强能诱导细胞衰老的试剂的功效、促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期。
在一个或多个实施方案中，原花青素是低聚原花青素，优选包括原花青素C1。
在一个或多个实施方案中，所述SASP因子包括胞外基质蛋白、炎症性细胞因子及癌细胞生长因子。
在一个或多个实施方案中，所述SASP因子包括图6所示的因子。
在一个或多个实施方案中，所述SASP因子选自：IL6、CXCL8、MCP2、CXCL1、GM-CSF、MMP3、AREG、SFRP2、ANGPTL4、IL1a。
在一个或多个实施方案中，所述细胞衰老标志性因子选自：p16 INK4a、p21 CIP1。
在一个或多个实施方案中，非增殖态细胞是衰老细胞，例如自然衰老细胞或受损细胞。所述受损细胞包括组织微环境中的受损细胞，优选由化疗或辐射治疗引起的受损细胞。在一个或多个实施方案中，所述的辐射治疗包括：电离辐射，α、β或γ射线治疗。
在一个或多个实施方案中，所述受益于非增殖态细胞减少或消除的疾病是年龄相关疾病，包括但不限于癌症、心脑血管疾病、骨质疏松、年龄相关的退行性关节疾病(如关节炎)、代谢性疾病、神经退行性疾病。优选地，所述癌症是前列腺癌；所述肿瘤是前列腺肿瘤。
在一个或多个实施方案中，所述能诱导细胞衰老的试剂包括导致DNA损伤和/或细胞凋亡的试剂，例如化疗剂或辐射。优选地，所述试剂包括MIT或DOX。
在一个或多个实施方案中，所述对象是年长对象。在具体实施方案中，所述年长对象是对应于至少20月龄小鼠或至少60岁人类的对象。优选地，所述年长对象是对应于至少24月龄小鼠或至少75岁人类的对象。更优选地，所述年长对象是对应于24-27月龄小鼠或75-90岁人类的对象。
在一个或多个实施方案中，所述癌症疗法包括化疗或辐射治疗，例如MIT、DOX疗法、电离辐射，α、β或γ射线治疗。
本发明还提供(a)原花青素或其药学上可接受的盐、水合物或前药和(b)能诱导对象产生衰老细胞的试剂在制备药物或制剂中的用途，所述药物或制剂用于：促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、延长癌症存活期。
在一个或多个实施方案中，原花青素是低聚原花青素，优选包括原花青素C1。
在一个或多个实施方案中，所述试剂能诱导肿瘤组织中产生衰老细胞。
在一个或多个实施方案中，所述试剂能导致DNA损伤和/或细胞凋亡，例如DNA双链断裂。
在一个或多个实施方案中，所述试剂是MIT或DOX。
在一个或多个实施方案中，(a)能消除衰老细胞。
在一个或多个实施方案中，所述肿瘤是前列腺肿瘤；所述癌症是前列腺癌。
本发明另一方面提供药盒或试剂盒，其包括本文第一方面所述的药物组合物和任选的能诱导对象产生衰老细胞的试剂。
在一个或多个实施方案中，所述药盒或试剂盒包括容器1及容器2，分别装有(a)原花青素或其药学上可接受的盐、水合物或前药和任选的药学上可接受的辅料，和(b)所述能诱导对象产生衰老细胞的试剂和任选的药学上可接受的辅料。
在一个或多个实施方案中，所述药物组合物、药盒或试剂盒中，以(a)和任选的(b)为活性成分，其它成分为药学上可接受的辅料等。
在一个或多个实施方案中，所述的药物组合物的剂型包含：口服剂，注射剂，输液剂，片剂，粉剂，胶囊剂，丸剂；优选剂型为口服剂。
在本发明的另一方面提供一种使非增殖态细胞或对象发生变化的方法，所述方法包括用原花青素或其药学上可接受的盐、水合物或前药处理非增殖态细胞或将其给予对象，所述变化包括选自以下的一种或多种：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、增强能诱导细胞衰老的试剂的细胞毒性、或降低对癌症疗法的耐药性。
在一个或多个实施方案中，原花青素或其药学上可接受的盐、水合物或前药的终浓度为至少1μM，如至少10μM、至少20μM、至少30μM、至少40μM、至少50μM、至少100μM、至少200μM、至少500μM、至少1mM或上述任意两个数值之间的范围。
在一个或多个实施方案中，原花青素是低聚原花青素，优选包括原花青素C1。
在本发明的另一方面提供一种增强能诱导细胞衰老的试剂的细胞毒性、促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期的方法，所述方法包括用(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导对象产生衰老细胞的试剂处理细胞或将其给予对象。
在一个或多个实施方案中，原花青素是低聚原花青素，优选包括原花青素C1。
在一个或多个实施方案中，所述试剂能诱导肿瘤组织中产生衰老细胞。
在一个或多个实施方案中，所述试剂能导致DNA损伤和/或细胞凋亡，例如DNA 双链断裂。
在一个或多个实施方案中，所述试剂是MIT或DOX。
在一个或多个实施方案中，所述肿瘤是前列腺肿瘤；所述癌症是前列腺癌。
在一个或多个实施方案中，原花青素或其药学上可接受的盐、水合物或前药的终浓度为至少10μM，如至少20μM、至少30μM、至少40μM、至少50μM、至少100μM、至少200μM、至少500μM、至少1mM或上述任意两个数值之间的范围。
在一个或多个实施方案中，本文任一实施方案所述用途或方法不以临床疾病治疗为直接目的。
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显示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显示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显示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显示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显示RNA-seq数据经软件处理和生信分析之后发现PCC1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/PCC1组细胞有4406个基因显著下调，同时有2766个基因显著上调(倍数变化＞2，P＜0.01)。
图6的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PCC1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显示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PCC1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显示蛋白-蛋白相互作用(PPI)生信分析结果显示，PCC1显著下调的衰老细胞分子形成网络，彼此间存在着多种互作关系。
图9显示KEGG通路分析PCC1在衰老细胞中造成显著下调的100个分子在生物过程(biological process)上的代表性通路。左侧Y轴，百分比。右侧Y轴，log10(p-值)。
图10显示KEGG通路分析PCC1在衰老细胞中造成显著下调的100个分子在细胞组分(cellular component)上的代表性通路。左侧Y轴，百分比。右侧Y轴，log10(p-值)。
图11显示荧光定量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PCC1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显示在PCC1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，PCC1在1μM，10μM，20μM，50μM，100μM，150μM和200μM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显示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显示CCK8检测增殖态细胞同衰老组细胞在PCC1渐增浓度下的存活率。每一PCC1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显示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PCC1在第8天时加入培养基。通过比较分析CTRL组，BLEO组，PCC1组和BLEO/PCC1组的倍增殖(PD)确定PCC1对于细胞增殖潜力的影响。^，P＞0.05；***，P＜0.001。
图16显示PCC1处理衰老细胞过程中诱导出现半胱天冬酶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。50μM的PCC1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，而半胱天冬酶3/7试剂(IncuCyte)用于细胞凋亡检测。半胱天冬酶3/7活性以每4小时的间隔检测一次(n＝3)。
图17显示Pan-caspase抑制剂(20cM QVD-OPh)逆转的senolytic活性(50μM的PCC1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)。统计学差异通过双因素(two-way)ANOVA(Turkey’test)获得。
图18显示流式细胞仪测定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显示对比分析细胞经过BLEO和/或PCC1处理之后的存活和凋亡数量。***，P＜0.001；****，P＜0.0001。
图20显示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显示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显示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PCC1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显示肿瘤终端体积统计分析。化疗药物MIT单独或与抗衰老药PCC1一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图24显示临床前试验中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显示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显示荧光定量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显示荧光定量PCR(qRT-PCR)检测分析载剂、MIT和MIT/PCC1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显示用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显示免疫组化染色之后的图片分析。切割后的半胱天冬酶3(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显示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。载剂，MIT，PCC1和MIT/PCC1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显示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显示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。肌酸酐，尿素(肾脏指标)，ALP和ALT(肝脏指标)数据平行对比。^，P＞0.05。
图33显示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显示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，淋巴细胞和中性粒细胞单位体积数量平行对比。^，P＞0.05。
图35显示肿瘤终端体积统计分析。化疗药物DOX单独或与抗衰老药PCC1一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图36显示肿瘤终端体积统计分析。化疗药物DOC单独或与抗衰老药PCC1一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图37显示肿瘤终端体积统计分析。化疗药物VIN单独或与抗衰老药PCC1一起用于对小鼠给药，第8周结束之后对比分析各组肿瘤大小。
图38显示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载剂或PCC1腹腔给药(载剂组n＝80；PCC1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显示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载剂或PCC1腹腔给药(载剂组n＝80；PCC1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显示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显示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。N＝5/组。^，P＞0.05；***，P＜0.001。
图42显示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。统计结果以箱线图(box-and-whisker plots)显示，每个箱(box)展示出含四分位范围的中位数(median with interquartile range)。^，P＞0.05。
图43显示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。统计结果以箱线图(box-and-whisker plots)显示，每个箱(box)展示出含四分位范围的中位数(median with interquartile range)。^，P＞0.05。
具体实施方式
本发明人发现原花青素对于下调或清除机体衰老细胞具有极其优异的效果，从而可应用于清除组织微环境中的受损细胞，也可应用于清除随年龄增长而自然衰老的细胞。
如本发明所用，“增殖态细胞”是指能够保持连续、活跃的分裂和不断增殖的状态的细胞。狭义的“非增殖态细胞”是衰老细胞，例如自然衰老细胞或受损细胞，所述受损细胞包括组织微环境中的受损细胞，优选由化疗或辐射治疗引起的受损细胞。如本发明所用，“衰老细胞”是指增殖与分裂的能力降低、生理功能发生衰退的细胞。
本文所述“原花青素”是多酚类化合物的总称，包括低聚原花青素。花青素是植物性多酚家族中的重要一类，这一类黄烷醇化合物多是通过黄烷键的C4-C6和C4-C8将由儿茶素或表儿茶素连接而成，部分化合物则是没食子酸脂；它们的共性是化学本质上均为含有花青素单体聚合而成的寡聚体，多为二聚体和三聚体。示例性的低聚原花青素具有式I所示的单元，单元数量多1-6，其中波浪线表示与其它单元的连接。
Figure PCTCN2021077495-appb-000001
在一些具体实施方案中，原花青素是下式所示的原花青素C1(PCC1)
Figure PCTCN2021077495-appb-000002
PCC1是植物性多酚家族中的三聚体，它不仅具有抗氧化、抗炎、抗癌等作用，还有靶向清除衰老细胞的功能。
在本发明中，“化合物”(包括原花青素、其盐或前药等)可以是纯净形式存在的化合物，或纯度大于85％(较佳地大于90％，例如95％，98％，99％)的化合物。
本领域人员应理解，在得知了本发明化合物的结构以后，可通过多种本领域熟知的方法、利用公知的原料，来获得本发明的化合物，比如化学合成或从生物(如微生物)中提取的方法，这些方法均包含在本发明中。此外，原花青素还是商品化的药物，因此其成品是本领域技术人员易于获得的。
在本发明中，还包括原花青素的药学上可接受的盐，其也保留有原花青素的化学活性。本发明中，“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)即有合理的效益/风险比的物质。所述的“药学上可接受的盐”可以是原花青素的酸式盐和碱式盐。
“药学上可接受的酸式盐”是指可保持游离碱的生物活性和性质的盐，该类盐不会出现不理想的生物活性或其它方面的变化。该类盐可由无机酸构成，例如但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸及类似的酸。该类盐还可由有机酸构成，例如但不限于乙酸、二氯乙酸、己二酸、褐藻酸、抗坏血酸、天冬氨酸、苯磺酸、苯甲酸、4-乙酰氨基苯甲酸、樟脑酸、樟脑磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、柠檬酸、环拉酸、十二烷基磺酸、1，2-乙二磺酸、乙烷磺酸、羟乙基磺酸、蚁酸、延胡索酸(fumaric acid)、半乳糖二酸、龙胆酸、葡庚糖酸、葡萄糖酸、葡糖醛酸、谷氨酸、戊二酸、2-氧代戊二酸、甘油磷酸、羟基乙酸、马尿酸、异丁酸、乳酸、乳糖酸、月桂酸、顺丁烯二酸、苹果酸、丙二酸、苦杏仁酸、甲烷磺酸、粘酸、萘-1，5-二磺酸、2-萘磺酸、1-萘酚-2-甲酸、烟酸、油酸、乳清酸、草酸、棕榈酸、双羟萘酸、丙酸、焦谷氨酸、丙酮酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、酒石酸、硫氰酸、对甲苯磺酸、三氟乙酸、十一烯酸及类似酸。
“药学上可接受的碱式盐”是指可保持游离酸的生物活性和性质的盐，该类盐不会出现不理想的生物活性或其它方面的变化。这些盐通过向游离酸中加入无机碱或有机碱制成。通过无机碱得到的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐及类似盐。优选的无机盐为铵盐、钠盐、钾盐、钙盐以及镁盐。通过有机碱得到的盐包括但不限于一级、二级、三级铵盐，取代的胺包括天然取代的胺、环胺以及碱性离子交换树脂，例如氨气、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、丹醇、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、哈胺、胆碱、甜菜碱、苯乙苄胺、N，N′-双苄基乙撑二胺、乙二胺、葡萄糖胺、甲葡糖胺、可可碱、三乙醇胺、缓血酸胺、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚酰胺树脂以及类似结构。优选的有机碱为异丙胺、二乙胺、乙醇胺、三甲胺、二环己胺、胆碱和咖啡因。
本专利公开的化合物可以作为水合物存在，包括单水合物、二水合物、半水合 物、倍半水合物、三水合物、四水合物及类似结构。在本发明中，还包括原花青素的前药，所述的“前药”指当用适当的方法服用后，该前药在对象体内进行代谢或化学反应而转变成所需原花青素的一种化合物。
原花青素
发明人发现原花青素(本文称为PCC1)可以有效抑制SASP的表达，并显著降低衰老细胞存活率。
因此，本发明提供原花青素在制备药物或制剂中的用途，所述药物或制剂用于：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、降低对癌症疗法的耐药性、促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期。本文中，“个体”、“对象”或“患者”指哺乳动物，尤其指人。
本文中，“消除”、“清除”和互换使用，表示物质利用细胞自身机制选择性破坏非增殖态细胞(衰老细胞)，达到细胞死亡并被清除的效果。在示例性实施方案中，物质(例如PCC1)可通过诱导细胞凋亡来消除或清除非增殖态细胞。
本文所述“SASP因子”包括胞外基质蛋白、炎症性细胞因子及癌细胞生长因子。所述SASP因子可包括图6所示的因子或选自：IL6、CXCL8、MCP2、CXCL1、GM-CSF、MMP3、AREG、SFRP2、ANGPTL4、IL1a中的一种或多种。
本文所述“受益于非增殖态细胞减少或消除的疾病”通常是年龄相关疾病，包括但不限于癌症、心脑血管疾病、骨质疏松、年龄相关的退行性关节疾病(如关节炎)、代谢性疾病、神经退行性疾病。优选地，所述癌症是前列腺癌。
本文中，原花青素(例如PCC1)还可用于延长对象寿命、减少对象的年龄相关疾病负担。在一些具体实施方案中，所述对象是年长对象，例如对应于至少20月龄小鼠或至少60岁人类的对象。优选地，所述年长对象是对应于至少24月龄小鼠或至少75岁人类的对象。更优选地，所述年长对象是对应于24-27月龄小鼠或75-90岁人类的对象。虽然具体实施例中采用年长对象作为研究对象，但这仅为便于进行结果分析的示例(例如年长对象的年龄相关疾病较多)。基于本发明发现的原花青素消除衰老细胞的功效，本领域技术人员应知晓它们可用于任何年龄的对象来消除衰老细胞、延长寿命、减少年龄相关疾病负担。
本文中，原花青素(例如PCC1)还可用于降低患者对癌症疗法的耐药性。所述癌症疗法包括化疗或辐射疗法；化疗例如MIT或DOX等细胞毒疗法，辐射治疗例如 电离辐射，主要包括α、β、γ和X射线以及质子、中子流治疗等。
此外，发明人发现，当与某些试剂联用时，原花青素(例如PCC1)可以增强诱导细胞衰老的试剂的细胞毒性。诱导细胞衰老的试剂可以是通过导致DNA损伤和/或细胞凋亡而诱导产生衰老细胞的试剂，例如化疗剂或放射线。
因此，本发明还提供原花青素在增强诱导细胞衰老的试剂的功效中的用途，以及原花青素和诱导细胞衰老的试剂的联用在促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、延长癌症存活期中的用途。示例性的，所述细胞是肿瘤细胞；所述肿瘤是前列腺肿瘤；所述癌症是前列腺癌。
在本发明的另一方面提供一种实现上述用途的方法，所述方法包括用(a)本文所述原花青素或其药学上可接受的盐、水合物或前药，和任选的(b)能诱导对象产生衰老细胞的试剂处理衰老细胞或将其给予有需要的对象。本文所用术语“给予”或“给药”是指向患有待治疗或预防的疾病或病症或具有其风险的对象提供本发明的化合物或药物组合物。
组合物
本发明的组合物以原花青素或其盐等物质作为活性组分。如上所述，含有原花青素(例如PCC1)可以下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞(衰老细胞)凋亡、减少或消除非增殖态细胞(衰老细胞)、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、降低对癌症疗法的耐药性。
当所述组合物还包含细胞衰老诱导试剂(例如化疗剂或辐射)作为活性组分时，所述组合物可促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、延长癌症存活期。
本文所述组合物作为药物使用时，还包含药学上可接受的辅料。“药学上可接受的辅料”是用于将本发明组合物中的活性组分(例如原花青素和可选的细胞衰老诱导试剂)传送给动物或人的药学上或食品上可接受的载体、溶剂、悬浮剂或赋形剂。示例性的辅料可以是液体或固体，包括但不限于：pH调节剂，表面活性剂，碳水化合物，佐剂，抗氧化剂，螯合剂，离子强度增强剂、防腐剂、载剂、助流剂、甜味剂、染料/着色剂、增味剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂、乳化剂、喷雾剂、压缩空气或其它适宜的气体，或其它适宜的与药效化合物合用的非活性成分。更具体而言，合适的辅料可以是本领域常用于小分子化合物给药的辅料。辅料的示例包括各种乳糖、甘露醇，油类如玉米油，缓冲剂如PBS、盐水、聚乙二醇、甘油、聚丙二醇、二甲亚砜，酰胺如二甲基乙酰胺，蛋白质如白蛋白，和去污剂如吐温80， 单糖和低聚多糖如葡萄糖、乳糖、环糊精和淀粉。
通常，组合物中含有治疗有效量的本文所述活性组分。治疗有效量是指可在受试者中实现治疗、预防、减轻和/或缓解疾病或病症的剂量。可根据患者年龄、性别、所患病症及其严重程度、患者的其它身体状况等因素确定治疗有效量。治疗有效量可作为单一剂量施用，或者可依据有效的治疗方案在多个剂量中给药。本文中，受试者或患者通常指哺乳动物，尤其指人。示例性地，所述组合物含有按照重量比例为例如0.001-50％，优选0.01-30％，更优选0.05-10％的活性组分(例如原花青素和可选的细胞衰老诱导试剂)。
本发明的药物组合物或混合物可以通过常规方法制成任何常规的制剂形式。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，输液剂，片剂，胶囊剂，丸剂。其中活性组分(例如原花青素和可选的细胞衰老诱导试剂)可以存在于适宜的固体或液体的载体或稀释液中。本发明的活性组分的混合物或药物组合物也可储存在适宜于注射或滴注的消毒器具中。
组合物中活性组分(例如原花青素和可选的细胞衰老诱导试剂)的有效剂量可随给药的模式和待治疗的疾病的严重程度而变化，这可以根据临床医师的经验和建议。
本发明的具体实施例中，提出了原花青素和可选的细胞衰老诱导试剂按照不同摩尔比或质量比的一系列给药方案。本发明中，也运用了小鼠作为实验动物，从小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：
A＝k×(W 2/3)/10000。
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，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。
原花青素和可选的细胞衰老诱导试剂或药物组合物可通过口服以及静脉、肌肉或皮下等途径给药。优选地可以是口服给药。适应于口服的药物形式包括但不限于片剂、粉剂、胶囊剂、缓释剂等。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。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。
必要的时候，原花青素和可选的细胞衰老诱导试剂还可与其它活性成分或药物联合给药。
本发明还提供了一种用于下调或清除衰老细胞，或延长机体生存期的药盒或试剂盒，所述药盒或试剂盒含有本文任一实施方案所述的药物组合物。或者，所述的药盒或试剂盒含有本文所述的原花青素和可选的细胞衰老诱导试剂的混合物。或者，所 述药盒或试剂盒中含有：容器1，以及置于容器1中的本文所述原花青素或其药学上可接受的盐、水合物或前药；和容器2，以及置于容器2中的细胞衰老诱导试剂。
所述药盒或试剂盒中还可以含有一些辅助用药的材料，例如使用或施用各种剂型的组合物所需的量具、容器例如注射器等。所述药盒或试剂盒中还可含有使用说明书，说明治疗下调或清除衰老细胞或延长机体生存期的方法。
示例性实施方案
1、一种药物组合物，包含(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导对象产生衰老细胞的试剂，和任选的药学上可接受的辅料，
优选地，所述原花青素是低聚原花青素。
2、如项目1所述的药物组合物，其特征在于，
所述原花青素或其药学上可接受的盐、水合物或前药在药物组合物中的终浓度为至少1μM，和/或
所述原花青素是原花青素C1，和/或
所述试剂包括导致DNA损伤和/或细胞凋亡的试剂。
3、原花青素或其药学上可接受的盐、水合物或前药在制备药物或制剂中的用途，所述药物或制剂用于：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、降低对癌症疗法的耐药性、增强能诱导细胞衰老的试剂的功效、促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期，
优选地，所述原花青素是低聚原花青素。
4、如项目3所述的用途，其特征在于，
所述原花青素是原花青素C1，和/或
所述SASP因子包括胞外基质蛋白、炎症性细胞因子及癌细胞生长因子，和/或
非增殖态细胞是衰老细胞，优选为自然衰老细胞或受损细胞，和/或
所述受益于非增殖态细胞减少或消除的疾病是年龄相关疾病，优选为癌症、心脑血管疾病、骨质疏松、年龄相关的退行性关节疾病、代谢性疾病、神经退行性疾病，和/或
所述能诱导细胞衰老的试剂包括导致DNA损伤和/或细胞凋亡的试剂，和/或
所述对象是年长对象，和/或
所述癌症疗法包括化疗或辐射治疗。
5、如项目3或4所述的用途，其特征在于，所述肿瘤是前列腺肿瘤，和/或所述癌症是前列腺癌。
6、物质在制备药物或制剂中的用途，所述物质包括(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导对象产生衰老细胞的试剂，所述药物或制剂用于：促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期，
优选地，所述原花青素是低聚原花青素，更优选为原花青素C1，
优选地，所述能诱导对象产生衰老细胞的试剂包括导致DNA损伤和/或细胞凋亡的试剂。
7、如项目6所述的用途，其特征在于，所述肿瘤是前列腺肿瘤，和/或所述癌症是前列腺癌。
8、一种药盒或试剂盒，其包括项目1或2所述的药物组合物，
优选地，所述药盒或试剂盒包括容器1及容器2，分别装有(a)原花青素或其药学上可接受的盐、水合物或前药和任选的药学上可接受的辅料，和(b)能诱导对象产生衰老细胞的试剂和任选的药学上可接受的辅料，
优选地，所述原花青素是低聚原花青素，更优选为原花青素C1。
9、一种使非增殖态细胞发生变化的方法，所述方法包括用原花青素或其药学上可接受的盐、水合物或前药处理非增殖态细胞，所述变化包括选自以下的一种或多种：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、或降低细胞对癌症疗法处理的耐药性，
优选地，
所述原花青素是低聚原花青素，更优选为原花青素C1，和/或
原花青素或其药学上可接受的盐、水合物或前药的终浓度为至少1μM。
10、一种增强能诱导细胞衰老的试剂的细胞毒性的方法，所述方法包括用(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导细胞衰老的试剂处理细胞，
优选地，
所述原花青素是低聚原花青素，更优选为原花青素C1，和/或
所述能诱导细胞衰老的试剂能导致DNA损伤和/或细胞凋亡，和/或
原花青素或其药学上可接受的盐、水合物或前药的终浓度为至少10μM。
除非另外定义，否则，本文中所使用的所有技术和科学术语都具有本发明所属领域普通技术人员通常所理解的同样含义。虽然可采用与本文所述类似或等同的任何方法和材料实施或测试本发明，但现在描述优选的方法和材料。本文具体提及的所有出版物和专利都通过引用全文纳入本文用于所有目的，包括描述和公开所述出版物报道的可与本发明关联使用的化学物质、设备、统计分析和方法。本说明书引用的所有参考文献都应看作对本领域技术水平的指示。本文中所有内容均不应解释为承认本发明不能凭借在先发明而先于这些公开内容。
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。
实施例
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。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，我们使用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盖片(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。小鼠单克隆抗体抗-phospho-Histone H2A.X(Ser139)(克隆JBW301，Millipore)和小鼠单克隆抗体抗-BrdU(Cat#347580，BD Biosciences)，及二级抗体Alexa
Figure PCTCN2021077495-appb-000003
488(or 594)-欧联的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/m的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。
双端转录组读数(Paired-end transcriptomic reads)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。参考接头连接(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错误发现率(FDR)-校正的P值＜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/)修剪读数(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Majorbio 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)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(PPI)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)。DESeq2独立筛选(independent 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的GSEA标记(signature)如我们过往发表文献所述(Zhang等人，2018a)。
7.定量PCR(RT-PCR)测定基因表达
(1)细胞总RNA的提取
以Trizol试剂抽提处于生长期或停滞期细胞的总RNA，每T25培养瓶细胞加入1ml Trizol，用细胞刮刀刮下细胞层后将其转移至离心管中，充分混匀至不粘稠。每1ml Trizol加0.2ml氯仿，剧烈震荡15sec，室温孵育5-10min；4℃，11,000g离心15min；将无色上清液移入一新的离心管中，按每1ml Trizol加0.5ml异丙醇，室 温孵育10分钟，11,000g，4℃离心10min；倒掉上清，用75％乙醇(每1ml Trizol至少用1ml 75％乙醇)洗涤，4℃，7,500g离心5min；室温干燥RNA沉淀5-10分钟(RNA不能干燥)，用DEPC-H 2O溶解沉淀。分光光度计定量RNA之后，取少量总RNA进行1％琼脂糖电泳，检查RNA状态和质量。
(2)逆转录反应
寡聚dT 23V N(50uM)，1ul；总RNA，1-2ug；无RNase的ddH 2O加至8ul。65℃加热5min，迅速置于冰上骤冷，并静置2min。
配置第一链cDNA合成液：2x RT Mix，10ul；HiScript II Enzyme Mix，2ul。按照以下条件进行第一链cDNA合成：25℃5min，50℃45min，85℃5min。
(3)实时定量PCR反应
将逆转录反应产物cDNA稀释50倍作为模板。如下配置PCR反应液：AceQ SYBR Green Master Mix，10ul；引物1(10uM)，0.4ul；引物2(10uM)，0.4ul；Rox Reference Dye，0.4ul；模板，2ul；ddH 2O加至20ul。
按照以上标准加样，反应条件为：95℃预变性15sec，然后95℃5sec，60℃31sec，40个循环；融解曲线条件为95℃15sec，60℃30sec，95℃15sec。样品于ABI ViiA7(ABI)仪上进行反应。以β-肌动蛋白的表达作内参。反应完成后，经软件分析查看每个基因的扩增情况，导出相应的域值循环数，采用2-ΔΔCt方法，计算每个基因的相对表达量。对融解曲线(melting curve)的波峰和波形进行分析以确定得到的扩增产物是否为特异性单一目的片段。
其中，所用检测引物序列如下所示，F表示正向引物，R表示反向引物：
IL6(F：SEQ ID NO：1，R：SEQ ID NO：2)；CXCL8(F：SEQ ID NO：3，R：SEQ ID NO：4)；SPINK1(F：SEQ ID NO：5，R：SEQ ID NO：6)；WNT16B(F：SEQ ID NO：7，R：SEQ ID NO：8)；GM-CSF(F：SEQ ID NO：9，R：SEQ ID NO：10)；MMP3(F：SEQ ID NO：11，R：SEQ ID NO：12)；IL-1α(F：SEQ ID NO：13，R：SEQ ID NO：14)；p16INK4a(F：SEQ ID NO：15，R：SEQ ID NO：16)；IL-1β(F：SEQ ID NO：17，R：SEQ ID NO：18)；AREG(F：SEQ ID NO：19，R：SEQ ID NO：20)；CXCL1(F：SEQ ID NO：21，R：SEQ ID NO：22)；CXCL3(F：SEQ ID NO：23，R：SEQ ID NO：24)；p21CIP1(F：SEQ ID NO：25，R：SEQ ID NO：26)；BMP6(F：SEQ ID NO：27，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，操作执行以往报道程序(Debacq-Chainiaux等人，2009年)。简单地说，细胞培养皿中经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，按过往文献所述进行(Duan等人，2015年；Wu等人，2018年)。简单地说，细胞被铺板于明胶涂层的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50μM和1.0μM的浓度加入PCC1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)。选取代表性视野进行拍照。
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小鼠，ICR)(体重约25g)用于本专利相关动物实验。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((l+w)/2) 3(V，体积；l，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/或原花青素C1(PCC1)(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，PCC1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。
12.小鼠寿命研究
在细胞移植研究中，我们在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。我们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(载剂)或药物组(PCC1)组处理。从24-27个月龄开始，小鼠每2周用载剂或PCC1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。滚筒跑步(RotaRod)和悬挂(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，我们对小鼠进行安乐死；如果它们表现出以下几种症状之一，我们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，我们采用Cox比例风险模型(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。保存的尸体被运到尸体解剖(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。通过综合实验动物监测系统(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开路量热计系统(Oxymax Open Circuit Calorimeter System，Columbus Instruments公司)。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
本专利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，通过单因素方差分析(one-way analysis of variance，ANOVA)或双尾斯氏t测试(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皮尔逊积差相关系数(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比例风险模进行生存分析。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，我们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，增强了对我们结果的自信度。生存分析使用统计软件R(version 3.4.1；库‘coxme’)。在大多数实验和结果评估中，研究者对分配采取盲选。我们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。我们根据过往的实验确定样本量，因此没有使用统计功效分析(statistical power analysis)。本研究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，PCC1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，我们利用一个由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，我们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。我们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-图3)。我们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)。
我们对这些细胞进行了RNA-seq测序。而随后获得的高通量数据表明，一种植物原料，原花青素C1(procyanidin C1，PCC1)显著改变了衰老细胞的表达谱。其中4406个基因出现显著下调，同时2766个基因发生上调，这里热图(heatmap)中每个基因的倍数变化为2.0(P＜0.01)(图5)。重要的是，PCC1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-κB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PCC1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PCC1在体外条件下对SASP表达的影响，我们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10μM时的PCC1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，PCC1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更彰显其效果。
实施例2，当在高浓度使用时PCC1是一种新型的senolytics
鉴于PCC1在控制SASP表达方面的显著疗效，我们接下来探究了这种天然产物在较高浓度下杀死衰老细胞的潜力。为此，我们测量了随着PCC1浓度的增加，体外条件下所处理的衰老细胞的生存百分比。SA-β-Gal染色数据表明，在PCC1浓度达到50μM之前，衰老细胞不会被消除(图12)。随着浓度的增加，PCC1对衰老细胞(80％染色阳性)的杀伤效果进一步增强，而当PCC1在150μM时达到阈值(衰老细胞此时剩余20％)；当其浓度升高到200μM时，PCC1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，我们做了验证性实验。细胞活力测定表明，与其增殖态对照细胞相比较，PCC1从50μM浓度开始诱导衰老细胞显著死亡(图14)。当PCC1浓度增加到200μM时，存活衰老细胞的百分比下降到约10％。然而，即使在PCC1的200μM时，增殖细胞也并未明显减少。这些结果，证实了PCC1对衰老细胞高度的选择性和突出的特异性，而这种特征实际是目前国际上对senolytics作为一类独特的抗衰老药的基本技术要求。
我们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PCC1的联合处理组表现出显著增高的PD能力(图15)。然而有趣的是，PCC1本身似乎不影响增殖细胞的PD，这一数据进一步表明PCC1在衰老细胞与正常细胞之间的选择性。
为了探究PCC1是否通过诱导凋亡的方式造成衰老细胞丧失存活能力，我们使用PCC1在培养条件下分别处理增殖组细胞和衰老组细胞。随后观察到的胱冬酶(caspase)-3/7活性变化结果，表明PCC1引起衰老细胞发生凋亡；从PCC1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，泛胱冬酶抑制剂QVD可防止PCC1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，PCC1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PCC1对衰老细胞产生的明显影响，我们随后分析了PCC1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，我们的数据一致性支持PCC1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PCC1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PCC1在体外较高浓度条件下清除衰老细胞中的突出选择性，我们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，我们通过将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，我们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，我们在第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44.0％，P＜0.0001)(图23)。值得注意的是，虽然PCC1本身并不会引起肿瘤收缩，但对治疗MIT后的小鼠，PCC1给药却可显著减小肿瘤(与MIT相比，肿瘤体积减少55.9％，P＜0.001；与安慰剂治疗相比，肿瘤体积减少74.9％，P＜0.0001)(图23)。
接下来，我们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，PCC1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、IL1A，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肿瘤微环境(TME)中产生了获得性耐药。然而，在使用PCC1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的那样(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，我们在第一次PCC1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PCC1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PCC1一起使用时，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PCC1在治疗过程中应用时，胱冬酶3切割活性升高，这是细胞凋亡的一个典型标志(图29)。
接下来我们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，我们对动物进行了前列腺肿瘤生长监测，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/PCC1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，绿色与蓝色相比)。然而，仅用PCC1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。我们没有观察到尿素、肌酐、碱性磷酸酶、谷丙转氨酶或体重的显著波动(图31；图32)。更重要的是，在本研究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PCC1在提高化疗治疗效果方面是否具有药物依赖性或特异性，我们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PCC1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PCC1联用可以大致重复MIT与PCC1联合治疗所造成的显著效果(图35)。而DOC与VIN尽管在单独使用时可以降低肿瘤体积，但当PCC1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)。MIT和DOX均为基因毒药物，可以造成造成典型的DNA双链断裂，继而引起细胞衰老。VIN作用机理是附着在微管之上，抑制有丝分裂过程。因此，PCC1在体内条件下提高化疗治疗效果这一特征，可以与诱导机体产生衰老细胞的药物联用，具有药物类型 依赖性。
实施例4，PCC1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PCC1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，我们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。值得我们注意、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PCC1组，其治疗后中位生存期比载剂组延长了64.2％，同时具有较低的死亡危险(HR＝0.35，PCC1组/载剂组；P＜0.0001)(图38，图39)。这一发现，表明PCC1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，我们评估了这些小鼠的身体功能。尽管PCC1组小鼠的剩余寿命较长，但经过PCC1每两周一次给药处理的小鼠，在生命的最后2个月的身体功能跟载剂处理组的小鼠在雄、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PCC1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。即，PCC1给药的对象并未出现显著变化的疾病发生率；而一旦机体出现某些衰老相关疾病(如肿瘤)，PCC1则可以显著提高肿瘤治疗效率或加快肿瘤退行。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。

Claims (10)

  1. 一种药物组合物，包含(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导对象产生衰老细胞的试剂，和任选的药学上可接受的辅料，
    优选地，所述原花青素是低聚原花青素。
  2. 如权利要求1所述的药物组合物，其特征在于，
    所述原花青素或其药学上可接受的盐、水合物或前药在药物组合物中的终浓度为至少1μM，和/或
    所述原花青素是原花青素C1，和/或
    所述试剂包括导致DNA损伤和/或细胞凋亡的试剂。
  3. 原花青素或其药学上可接受的盐、水合物或前药在制备药物或制剂中的用途，所述药物或制剂用于：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、延缓衰老、延长对象寿命、减少对象的年龄相关疾病负担、预防、缓解和治疗受益于非增殖态细胞减少或消除的疾病、降低对癌症疗法的耐药性、增强能诱导细胞衰老的试剂的功效、促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期，
    优选地，所述原花青素是低聚原花青素。
  4. 如权利要求3所述的用途，其特征在于，
    所述原花青素是原花青素C1，和/或
    所述SASP因子包括胞外基质蛋白、炎症性细胞因子及癌细胞生长因子，和/或
    非增殖态细胞是衰老细胞，优选为自然衰老细胞或受损细胞，和/或
    所述受益于非增殖态细胞减少或消除的疾病是年龄相关疾病，优选为癌症、心脑血管疾病、骨质疏松、年龄相关的退行性关节疾病、代谢性疾病、神经退行性疾病，和/或
    所述能诱导细胞衰老的试剂包括导致DNA损伤和/或细胞凋亡的试剂，和/或
    所述对象是年长对象，和/或
    所述癌症疗法包括化疗或辐射治疗。
  5. 如权利要求3或4所述的用途，其特征在于，所述肿瘤是前列腺肿瘤，和/ 或所述癌症是前列腺癌。
  6. 物质在制备药物或制剂中的用途，所述物质包括(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导对象产生衰老细胞的试剂，所述药物或制剂用于：促进肿瘤消退、减小肿瘤体积、预防或治疗癌症、或延长癌症存活期，
    优选地，所述原花青素是低聚原花青素，更优选为原花青素C1，
    优选地，所述能诱导对象产生衰老细胞的试剂包括导致DNA损伤和/或细胞凋亡的试剂。
  7. 如权利要求6所述的用途，其特征在于，所述肿瘤是前列腺肿瘤，和/或所述癌症是前列腺癌。
  8. 一种药盒或试剂盒，其包括权利要求1或2所述的药物组合物，
    优选地，所述药盒或试剂盒包括容器1及容器2，分别装有(a)原花青素或其药学上可接受的盐、水合物或前药和任选的药学上可接受的辅料，和(b)能诱导对象产生衰老细胞的试剂和任选的药学上可接受的辅料，
    优选地，所述原花青素是低聚原花青素，更优选为原花青素C1。
  9. 一种使非增殖态细胞发生变化的方法，所述方法包括用原花青素或其药学上可接受的盐、水合物或前药处理非增殖态细胞，所述变化包括选自以下的一种或多种：下调衰老相关分泌表型(SASP)、降低SASP因子的表达或活性、降低细胞衰老标志性因子的表达或活性、诱导非增殖态细胞凋亡、减少或消除非增殖态细胞、或降低细胞对癌症疗法处理的耐药性，
    优选地，
    所述原花青素是低聚原花青素，更优选为原花青素C1，和/或
    原花青素或其药学上可接受的盐、水合物或前药的终浓度为至少1μM。
  10. 一种增强能诱导细胞衰老的试剂的细胞毒性的方法，所述方法包括用(a)原花青素或其药学上可接受的盐、水合物或前药，和(b)能诱导细胞衰老的试剂处理细胞，
    优选地，
    所述原花青素是低聚原花青素，更优选为原花青素C1，和/或
    所述能诱导细胞衰老的试剂能导致DNA损伤和/或细胞凋亡，和/或
    原花青素或其药学上可接受的盐、水合物或前药的终浓度为至少10μM。
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