WO2022166769A1 - Médicament combiné anti-âge pour la régulation négative du phénotype sécrétoire associé à la sénescence et application d'un médicament combiné anti-âge - Google Patents

Médicament combiné anti-âge pour la régulation négative du phénotype sécrétoire associé à la sénescence et application d'un médicament combiné anti-âge Download PDF

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WO2022166769A1
WO2022166769A1 PCT/CN2022/074384 CN2022074384W WO2022166769A1 WO 2022166769 A1 WO2022166769 A1 WO 2022166769A1 CN 2022074384 W CN2022074384 W CN 2022074384W WO 2022166769 A1 WO2022166769 A1 WO 2022166769A1
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cells
pharmaceutically acceptable
cancer
prodrug
hydrate
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孙宇
韩柳
张旭光
贺瑞坤
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汤臣倍健股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/385—Heterocyclic compounds having sulfur as a ring hetero atom having two or more sulfur atoms in the same ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system

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.
  • navitoclax was able to target HUVECs but was ineffective against senescent human adipose progenitor cells.
  • Evidence suggests that the efficacy of senolytics may vary even within one specific type of cell.
  • navitoclax targets and kills senescent cells in the culture-adapted IMR90 lung fibroblast-like cell line, but is less effective on senescent primary human lung fibroblasts.
  • a first aspect of the present invention provides a composition
  • a composition comprising (a) a compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof, (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or Prodrugs, and optional pharmaceutically acceptable excipients
  • R1 and R2 are each independently H or C1-C4 alkoxy.
  • R1 and R2 are C1-C4 alkoxy, preferably methoxy.
  • R1 is C1-C4 alkoxy, preferably methoxy, and R2 is H.
  • R1 and R2 are H.
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:1 190, 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • the final concentration of (a) in the composition is at least 1 ⁇ M, such as 1 ⁇ M, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 1 mM.
  • the composition further comprises an agent capable of inducing the subject to generate senescent cells.
  • 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 (a) a compound of formula I described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) lipoic acid, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, in the preparation
  • a compound of formula I described herein or a pharmaceutically acceptable salt, hydrate or prodrug thereof
  • lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof, in the preparation
  • SASP down-regulating senescence-associated secretory phenotype
  • SASP reducing the expression or activity of SASP factors, reducing the expression or activity of cell senescence marker factors, inducing apoptosis in non-proliferative cells death, reduction or elimination of non-proliferating cells, delaying aging, prolonging lifespan of subjects, reducing age-related disease burden in subjects, preventing, alleviating and treating diseases that benefit from reduction or elimination of non-proliferating cells, reducing resistance to cancer therapy , enhance the efficacy of an agent
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:1 190, 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • 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 (a) a compound of formula I described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) lipoic acid, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and ( c) Use of an agent capable of inducing a subject to produce senescent cells in the preparation of a medicament or a preparation for: promoting tumor regression, reducing tumor volume, preventing or treating cancer, and prolonging cancer survival.
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:1 190, 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • 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) and (b) are 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.
  • the kit or kit comprises container 1 and container 2 containing, respectively (a) a compound of formula I described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and optional pharmaceutically acceptable excipients, and (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof and optional pharmaceutically acceptable excipients.
  • the kit or kit further comprises a container 3 containing (c) the agent capable of inducing senescent cells in a subject and optional pharmaceutically acceptable excipients.
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:1 190, 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • compositions, kit or kit in the pharmaceutical composition, kit or kit, (a) and (b) and optional (c) are the active ingredients, and the other ingredients are pharmaceutically acceptable excipients Wait.
  • 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 subject comprising using (a) a compound of formula I described herein or a pharmaceutically acceptable salt, hydrate or prodrug thereof , and (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof to treat or administer a non-proliferating cell to a subject, the change comprising one or more selected from the group consisting of down-regulating senescence-associated secretion Phenotype (SASP), reduce the expression or activity of SASP factors, reduce the expression or activity of markers of cellular senescence, induce apoptosis in non-proliferative cells, reduce or eliminate non-proliferative cells, delay aging, prolong lifespan of subjects, reduce subjects age-related disease burden, prevention, mitigation, and treatment of diseases that benefit from the reduction or elimination of non-proliferative cells, enhancing the cytotoxicity of agents that induce cellular senescence, or reducing resistance to cancer therapies.
  • SASP down-regulating senescence-
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:1 190, 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • the final concentration of (a) is at least 1 ⁇ M, such as 1 ⁇ M, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 1 mM.
  • 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 compound of formula I described herein or a pharmaceutically acceptable salt, hydrate or prodrug thereof, (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof and (c) capable of inducing senescence in a subject Agents for cells treat cells or administer them to a 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 tumor is a prostate tumor; the cancer is prostate cancer.
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, such as 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:1 190, 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • the final concentration of (a) is at least 10 ⁇ M, such as 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 1 mM.
  • 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 is found that CA can significantly reduce genes that are significantly up-regulated in senescent cells compared to proliferating cells. Compared with BLEO group, 2653 genes were significantly down-regulated and 1847 genes were significantly up-regulated in BLEO/CA 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 CA treatment. Red star logo, typical SASP exogenous factor.
  • Figure 7 shows that the results of GSEA analysis showed 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 CA treatment of senescent cells.
  • SASP molecular marker Left, SASP molecular marker; right, NF- ⁇ B molecular marker.
  • Figure 8 shows the results of protein-protein interaction (PPI) bioinformatics analysis, showing that the senescent cell molecules significantly down-regulated by CA 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 where CA causes significant downregulation in senescent cells by KEGG pathway analysis. Left Y-axis, percentage. Right Y-axis, log10 (p-value).
  • Figure 10 shows a representative pathway of the 100 molecules on the cellular component where KEGG pathway analysis CA caused 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 CA. 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 conditions of increasing CA concentration.
  • ⁇ P>0.05; **, P ⁇ 0.01; ****, P ⁇ 0.0001.
  • the P values of CA at the concentrations of 1/100 ⁇ M, 1/200 ⁇ M, 5/200 ⁇ M, 10/400 ⁇ M, 10/600 ⁇ M, 20/600 ⁇ M and 20/80000 were similar to the data at 0 ⁇ M. The ratio was statistically significant.
  • 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 CA.
  • P values at each CA concentration are significant differences between the CTRL and BLEO groups after comparison. **, P ⁇ 0.01; ***, P ⁇ 0.001; ****, P ⁇ 0.0001.
  • the CA concentrations at each point on the abscissa are 0/0 ⁇ M, 1/100 ⁇ M, 10/400 ⁇ M, 15/600 ⁇ M, 20/800 ⁇ M and 25/1000 ⁇ M. .
  • Figure 15 shows a population doubling test for PSC27.
  • Cells were treated with BLEO injury at passage 10 (p10), followed by the addition of CA to the medium on day 8.
  • the effect of CA on cell proliferation potential was determined by comparing the doubling proliferation (PD) of CTRL group, BLEO group, CA group and BLEO/CA group. ⁇ , P>0.05; ***, P ⁇ 0.001.
  • Figure 16 shows the induction of caspase 3/7 activity during CA treatment of senescent cells.
  • PSC27 cells gradually entered the senescence stage after being treated with BLEO for 12 h.
  • 10/400 ⁇ M CA 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) (800 ⁇ M of CA was used in this experiment, while 200 ⁇ M of 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 CA 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 CA 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 the mice alone or together with the anti-aging drug CA, and the tumor size of each group was compared and analyzed 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 stromal cells SASP factors in mouse lesions following vehicle, MIT and MIT/CA 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, CA and MIT/CA group animals were considered to have severe disease when the tumor volume in vivo exceeded 2000mm 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 was administered alone or together with the anti-aging drug CA 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 CA, 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 CA 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.
  • 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.
  • R1 and R2 are each independently H or C1-C4 alkoxy.
  • the compound of formula I is curcumin; when R1 is methoxy and R2 is H, the compound of formula I is monodemethoxycurcumin; when R1 and R2 are H, The compound of formula I is bisdesmethoxycurcumin.
  • the molecular formula of lipoic acid is C 8 H 14 O 2 S 2 , and the CAS accession number is 62-46-4.
  • “compounds” may be compounds in pure form, or more than 85% pure (preferably more than 90%, such as 95% pure) %, 98%, 99%) compounds.
  • 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.
  • the compound of formula (I) or lipoic acid is also a commercial drug, so its finished product is readily available to those skilled in the art.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or animals without undue adverse side effects (oral toxicity, irritation, and allergy), ie, with a reasonable benefit/risk ratio.
  • Said “pharmaceutically acceptable salt” may be the acid salt and the base salt of the compound of formula (I) or lipoic acid.
  • “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.
  • the prodrug of the compound of formula (I) or lipoic acid is also included, and the "prodrug” refers to that the prodrug of the compound is metabolized or chemically reacted in the body of the patient after being taken by an appropriate method.
  • CA lipoic acid
  • the present invention provides the use of the compound of formula I and lipoic acid in the preparation of medicaments or preparations for: down-regulating senescence-associated secretory phenotype (SASP), reducing the expression or activity of SASP factors, and reducing cellular senescence markers expression or activity of sexual factors, induction of apoptosis in non-proliferative cells, reduction or elimination of non-proliferative cells, delaying aging, prolonging the lifespan of a subject, reducing the burden of age-related disease in a subject, preventing, relieving and treating benefiting from a reduction in non-proliferative cells or eliminate disease, 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 and reducing cellular senescence markers expression or activity of sexual factors
  • induction of apoptosis in non-proliferative cells reduction or elimination of non-pro
  • a substance eg, CA herein
  • apoptosis 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.
  • the compounds of formula I and lipoic acid can also be used to prolong lifespan and reduce 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).
  • compounds of formula I and lipoic acid can also be used to reduce patient resistance to cancer therapy.
  • the cancer therapy includes chemotherapy or radiation therapy; chemotherapy such as MIT or DOX therapy, radiation therapy such as ionizing radiation, alpha, beta or gamma radiation therapy.
  • an 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 a compound of formula I and lipoic acid in enhancing the efficacy of an agent for inducing cellular senescence, and the combined use of a compound of formula I, lipoic acid and an agent for inducing cellular senescence in promoting tumor regression and reducing tumor volume , use in the prevention or treatment of cancer, and prolongation of 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-mentioned use comprising using (a) a compound of formula I described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) sulfur Caprylic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof and optionally (c) an agent capable of inducing the production of senescent cells in a subject treats or administers senescent cells 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 the compound of formula I and lipoic acid or its salts as active components.
  • the molar concentration ratio of the compound of formula I and lipoic acid is from 1:20 to 1:500, eg, 1:30, 1:40, 1:50, 1:60, 1:70, 1 : 80, 1: 90, 1: 100, 1: 100, 1: 110, 1: 120, 1: 130, 1: 140, 1: 150, 1: 160, 1: 170, 1: 180, 1: 190 , 1:200, 1:300, 1:400, 1:500, or a range between any two ratios above.
  • containing the compound of formula I and lipoic acid 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 non-proliferating cells (senescent cells) Apoptosis, reduction or elimination of non-proliferating cells (senescent cells), delaying aging, prolonging lifespan in subjects, reducing age-related disease burden in subjects, preventing, alleviating and treating diseases that benefit from reduction or elimination of non-proliferating cells, reducing the risk of cancer resistance to therapy.
  • 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 excipients are pharmaceutically or food acceptable ingredients used in the delivery of the active ingredients in the compositions of the present invention (eg, compounds of formula I and lipoic acid and optional cellular senescence-inducing agents) to animals or humans Acceptable carriers, solvents, 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 (such as the compound of formula I and lipoic acid and optionally cell senescence induction). reagents).
  • 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 compound of formula I and lipoic acid 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 dose of the active ingredients in the composition can vary with the mode of administration and the severity of the disease to be treated, which can be based on the experience of the clinician and suggestions.
  • 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:
  • the compounds of formula I and lipoic acid 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.
  • the compound of formula I and lipoic acid 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 a compound of formula I described herein and lipoic acid and an optional cellular senescence-inducing agent, wherein the ratio of compound of formula I to lipoic acid is as previously described.
  • the kit or kit contains: container 1, and a compound of formula I described herein, or a pharmaceutically acceptable salt, hydrate or prodrug thereof, placed in container 1; and container 2, and placed in container 1 Lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof in container 2.
  • 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 compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof, (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and optional (c) pharmaceutically acceptable excipients
  • R1 and R2 are each independently H or C1-C4 alkoxy
  • R1 and R2 are C1-C4 alkoxy, preferably methoxy, or
  • R1 is C1-C4 alkoxy, preferably methoxy
  • R2 is H
  • R1 and R2 are H.
  • the molar concentration ratio of (a) and (b) is 1:20-1:500
  • the pharmaceutical composition further comprises an agent capable of inducing the subject to generate senescent cells, preferably, the agent includes an agent that causes DNA damage and/or apoptosis.
  • a substance comprising (a) a compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) lipoic acid or a pharmaceutically acceptable Salts, hydrates or prodrugs for: down-regulating senescence-associated secretory phenotype (SASP), reducing the expression or activity of SASP factors, reducing the expression or activity of hallmark factors of cellular senescence, inducing non-proliferative cells Apoptosis, reducing or eliminating non-proliferating cells, delaying aging, extending lifespan of subjects, reducing age-related disease burden in subjects, preventing, alleviating and treating diseases that benefit from the reduction or elimination of non-proliferating cells, reducing resistance to cancer therapy properties, enhance the efficacy of agents that induce cellular senescence, promote tumor regression, reduce tumor size, prevent or treat cancer, or prolong cancer survival,
  • SASP senescence-associated secretory phenotype
  • R1 and R2 are each independently H or C1-C4 alkoxy.
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, 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) a compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and (b) lipoic acid or a pharmaceutically acceptable A salt, hydrate or prodrug and (c) an agent capable of inducing a subject to produce senescent cells, the medicament or preparation being used to: promote tumor regression, reduce tumor volume, prevent or treat cancer, or prolong cancer survival,
  • R1 and R2 are each independently H or C1-C4 alkoxy
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, and/or
  • the agents capable of inducing senescent cells in a subject include agents that cause 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) a compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof and optional pharmaceutically acceptable excipients, and ( b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof and optionally a pharmaceutically acceptable adjuvant, and/or
  • the molar concentration ratio of (a) and (b) is 1:20-1:500
  • the kit or kit further comprises a container 3 containing the reagent capable of inducing the subject to produce senescent cells and optional pharmaceutically acceptable excipients.
  • a method of altering non-proliferating cells comprising treating the non-proliferating cells with a substance comprising (a) a compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof , and (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof, the alteration comprising one or more selected from the group consisting of down-regulating senescence-associated secretory phenotype (SASP), decreasing SASP factor expression or activity, reducing the expression or activity of markers of cellular senescence, inducing apoptosis in non-proliferating cells, reducing or eliminating non-proliferating cells, or reducing the resistance of cells to cancer therapy treatments,
  • SASP down-regulating senescence-associated secretory phenotype
  • R1 and R2 are each independently H or C1-C4 alkoxy
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, and/or
  • the final concentration of (a) is at least 1 ⁇ M.
  • a method of enhancing the cytotoxicity of an agent capable of inducing cellular senescence comprising treating cells with a substance comprising (a) a compound of formula I or a pharmaceutically acceptable salt, hydrate or prodrug thereof , (b) lipoic acid or a pharmaceutically acceptable salt, hydrate or prodrug thereof and (c) said agent capable of inducing cellular senescence,
  • R1 and R2 are each independently H or C1-C4 alkoxy
  • the agent capable of inducing cellular senescence causes DNA damage and/or apoptosis, and/or
  • the molar concentration ratio of (a) and (b) is from 1:20 to 1:500, and/or
  • the final concentration 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% CO2, 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.
  • SASP and GSEA signatures are 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.
  • Oligo 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.
  • CA and ABT263 were added at concentrations of 10 ⁇ M/400 ⁇ 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 curcumin extract/lipoic acid (CA) after 2 weeks ) (500 ⁇ l, 10 mg/kg dose) intraperitoneally.
  • 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 CA.
  • 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.
  • senescence (SEN) or control (CTRL) transplantation treatments using a random number generator, were assigned to mice at each interval, while mice in the middle were assigned to the other treatment modality, resulting in senescence and the body weight of control transplanted mice.
  • SEN senescence
  • CRL control transplantation treatments
  • 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 (CA) treatment by humans blinded to the design of the preclinical trial. Starting at 24-27 months of age, mice were treated with vehicle or CA every 2 weeks by oral gavage for 3 consecutive days. During the course of the study, 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 disorders; 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) ⁇ 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°).
  • Example 1 CA 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.
  • SASP factors were generally reduced in senescent cells following CA treatment, and these SASP factors were generally significantly upregulated in senescent cells (Fig. 6). While the expression profiles of some SASP-unrelated genes showed similar trends to those typical of SASP factors, the data from GSEA analysis further revealed a marked suppression of molecular signatures characterizing SASP expression or NF- ⁇ B activation, the latter of which is mediated by Major transcriptional events in the development of inflammatory SASPs ( Figure 7). Bioinformatics analysis based on protein-protein interactions revealed a highly active network involving multiple factors that were significantly up-regulated during cell senescence, but down-regulated once cells were exposed to CA (Fig. 8).
  • CA a combination drug of plant natural product and mitochondrial coenzyme, can be used to control the pro-inflammatory phenotype of senescent cells, namely SASP, especially at relatively low concentrations.
  • Example 2 CA is a new type of senolytics when used at high concentrations
  • Example 3 Therapeutic targeting of senescent cells with CA 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 prior to subcutaneous implantation of recombinants 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/CA combination exhibited the longest median survival with at least 48.1% longer survival compared to the group treated with MIT alone ( Figure 30, green vs. blue).
  • treating tumor-bearing mice with CA alone did not result in significant benefit, with only marginal survival extension.
  • VIN The mechanism of action of VIN is to attach to microtubules and inhibit the polymerization process of mitotic microtubules. Therefore, the feature that CA enhances 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.
  • CA can significantly improve the efficiency of tumor treatment or accelerate tumor regression. This treatment In a manner that does not lead to a significant increase in body morbidity, in reality it can be safely used in later stages of life.

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Abstract

La présente invention concerne une composition comprenant (a) un composé de formule I ou un sel, hydrate ou promédicament pharmaceutiquement acceptable de celui-ci, (b) de l'acide lipoïque ou un sel, hydrate ou promédicament pharmaceutiquement acceptable de celui-ci et éventuellement (c) un excipient pharmaceutiquement acceptable, R1 et R2 étant chacun indépendamment un atome d'hydrogène ou un groupe alcoxy en C1-C4.
PCT/CN2022/074384 2021-02-04 2022-01-27 Médicament combiné anti-âge pour la régulation négative du phénotype sécrétoire associé à la sénescence et application d'un médicament combiné anti-âge WO2022166769A1 (fr)

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