WO2021032213A1 - 靶向组织微环境中衰老细胞的抗衰老药物d/s及其应用 - Google Patents

靶向组织微环境中衰老细胞的抗衰老药物d/s及其应用 Download PDF

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WO2021032213A1
WO2021032213A1 PCT/CN2020/110860 CN2020110860W WO2021032213A1 WO 2021032213 A1 WO2021032213 A1 WO 2021032213A1 CN 2020110860 W CN2020110860 W CN 2020110860W WO 2021032213 A1 WO2021032213 A1 WO 2021032213A1
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dasatinib
cells
srt2104
senescent cells
gene
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French (fr)
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孙宇
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中国科学院上海营养与健康研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents

Definitions

  • the invention belongs to the field of pharmacy. More specifically, the invention relates to an anti-aging drug D/S with the activity of targeting senescent cells in the tissue microenvironment and its application.
  • senescent cells in most organs and tissues will increase; under clinical conditions, such cells will also appear in organs related to many chronic diseases, and after radiotherapy and chemotherapy.
  • Cell senescence refers to the process in which cells lose their functions (including the ability to divide and replicate) but still resist death. It has been proven to drive a variety of age-related diseases, such as osteoporosis, osteoarthritis, atherosclerosis and idiopathic disease Pulmonary fibrosis and so on.
  • Senescent cells will continue to exist under the conditions of the body and have long-term effects on surrounding cells. This is mainly because senescent cells often develop a senescence-associated secretory phenotype (SASP), which makes these cells continue to secrete a large number of pro-inflammatory and microenvironmental remodeling molecules.
  • SASP senescence-associated secretory phenotype
  • the purpose of the present invention is to provide an anti-aging drug D/S with the activity of targeting senescent cells in the tissue microenvironment and its application.
  • the composition in the preparation of a medicine or preparation for down-regulating or eliminating senescent cells; wherein the composition includes dasatinib and SRT2104.
  • the cells include: natural senescent cells; or damaged cells; preferably damaged cells in the tissue microenvironment; more preferably damaged cells after chemotherapy or radiation treatment.
  • the radiation therapy includes ionizing radiation, alpha, beta or gamma radiation therapy.
  • the cells do not include or substantially do not include proliferating cells.
  • composition in the preparation of a medicine or preparation for prolonging the survival period (life) of the body; wherein the composition includes dasatinib and SRT2104.
  • the composition is also used to reduce the level of senescence-related secreted phenotype (SASP) cell senescence marker factors; preferably, the marker factors include (but not limited to): SASP marker factor IL6, IL8, MCP2, CXCL1, GM-CSF, MMP3, AREG, SFRP2, ANGPTL4 or IL 1a; cell senescence marker factor p16 INK4a or p21 CIP1 .
  • SASP marker factor IL6, IL8, MCP2, CXCL1, GM-CSF, MMP3, AREG, SFRP2, ANGPTL4 or IL 1a cell senescence marker factor p16 INK4a or p21 CIP1 .
  • the composition is also used to: improve body function and avoid ataxia of multiple organs.
  • Dasatinib:SRT2104 in terms of molar ratio or mass ratio, is 1:(2-100); preferably 1:(4-80); more preferably The ground is 1:(5 ⁇ 50).
  • Dasatinib:SRT2104 is 1:6; 1:8; 1:10; 1:12; 1:15; 1:20; 1:30; 1 :50; 1:60; 1:70; 1:90, etc.
  • the final concentration of dasatinib in the composition is 1 nM-10mM, such as 10nM, 100nM, 1uM, 10uM, 100uM, 1mM.
  • a pharmaceutical composition for down-regulating or eliminating senescent cells which includes dasatinib and SRT2104; preferably, it also includes a pharmaceutically acceptable carrier or excipient.
  • Dasatinib:SRT2104 is 1:(2 ⁇ 100); preferably 1:(4 ⁇ 80); more preferably 1:(5 ⁇ 50) ).
  • the use of the pharmaceutical composition is provided for preparing a kit or kit for down-regulating or eliminating senescent cells.
  • kits or kit for down-regulating or eliminating senescent cells which includes the pharmaceutical composition.
  • a kit or kit for down-regulating or eliminating senescent cells which comprises a container 1 and a container 2, respectively containing dasatinib and SRT2104; preferably, in terms of molar ratio or mass
  • the ratio, Dasatinib:SRT2104 is 1:(2 ⁇ 100); preferably 1:(4 ⁇ 80); more preferably 1:(5 ⁇ 50).
  • composition, kit or kit uses dasatinib and SRT2104 as active ingredients, and the other ingredients are pharmaceutical carriers or excipients.
  • composition only consists of dasatinib and SRT2104.
  • the dosage form of the pharmaceutical composition is: oral agent, injection, infusion, tablet, powder, capsule, pill; preferably oral agent.
  • a method for down-regulating or eliminating senescent cells including treating senescent cells with a composition; wherein the composition includes dasatinib and SRT2104.
  • SRT2104 is provided to promote the activity of dasatinib to down-regulate or eliminate the activity of senescent cells or to prolong the life of the body.
  • SRT2104 is provided for the preparation of drugs or preparations that promote the activity of dasatinib to down-regulate or eliminate the activity of senescent cells or prolong the life of the body.
  • a method for promoting the activity of dasatinib to down-regulate or eliminate senescent cells includes the combined application of SRT2104 and dasatinib.
  • the above-mentioned uses or methods are not directly aimed at the treatment of clinical diseases.
  • genes as down-regulation targets in the preparation of drugs or preparations for down-regulating or eliminating senescent cells
  • the genes are selected from ABL (ABL proto-oncogene), PTGS2 (prostaglandin-endoperoxide synthase) 2) Or BCL2A1 (BCL2 related protein A1).
  • a gene down-regulating agent for the preparation of drugs or preparations for down-regulating or eliminating senescent cells; wherein the gene is selected from ABL (Gene ID: 25), PTGS2 (Gene ID: 5743) Or BCL2A1 (Gene ID: 597).
  • the gene downregulator includes: interference molecules that specifically interfere with the expression of the gene, small molecule compounds that specifically inhibit the gene, and a gene editing reagent that specifically knocks out the gene; preferably
  • the interfering molecule is a siRNA, antisense nucleic acid, miRNA, dsRNA, shRNA, or a construction capable of expressing or forming the siRNA, antisense nucleic acid, miRNA, dsRNA, or shRNA with the gene as the target of inhibition or silencing More preferably, the interfering molecule is shRNA, which targets the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SED ID NO: 3.
  • FIG. 1 The human prostate primary stromal cell line PSC27 was treated with the chemotherapeutic drug BLEO on the 7th day, and RNA-Seq was used for whole transcriptome sequencing, and it was found that a large number of SASP factors showed an up-regulation trend. Red asterisks, BCL2A1 and SERPINB2.
  • FIG. 1 Using SA-B-Gal staining technique to analyze PSC27 cells. On the left, representative stained pictures, with proliferative (PRE) and senescent (SEN) cells on the top and bottom, with a scale of 20 ⁇ m; on the right, a statistical comparison based on 100 cells per group. ***, P ⁇ 0.001.
  • PRE proliferative
  • SEN senescent
  • Figure 4 Parallel comparison of the degree of DNA damage after immunofluorescence staining.
  • the primary antibody is ⁇ H2AX; it is graded according to the number of DNA damage foci in each nucleus (0foci; 1-3foci; 4-10foci; >10foci). ***, P ⁇ 0.001.
  • Figure 9 Use Dasatinib in a certain concentration range (0, 200, 400, 600, 800, 1000 nM) to treat PSC27 cells in PRE and SEN states, and analyze their survival. Each group of data normalizes the number of cells in this group at the beginning of the experiment. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • FIG 10. Similar to Figure 9, Dasatinib was used to treat WI38 cells in PRE and SEN states within a certain concentration range (0, 200, 400, 600, 800, 1000 nM) and analyze their survival. Each group of data normalizes the number of cells in this group at the beginning of the experiment. *, P ⁇ 0.05; **, P ⁇ 0.01.
  • Figure 11 Use SRT2104 to treat PSC27 cells in PRE and SEN states within a certain concentration range (0, 5, 10, 15, 20, 25, 30, 35, 40 ⁇ M), and analyze their survival. Each group of data normalizes the number of cells in this group at the beginning of the experiment.
  • FIG. 13 The micrograph shows that Dasatinib and SRT2104 are used alone or in combination to treat PSC27 cells in the SEN state and analyze their survival. Ruler, 20 ⁇ m. D, dasatinib; S, SRT2104.
  • Figure 14 Use certain concentrations of Dasatinib (0, 400, 1000 nM) and SRT2104 (0, 5, 10 ⁇ M) separately or in combination to treat PSC27 cells during proliferation or senescence, and analyze their survival. Green dotted line, the starting cell number is used as the baseline level of data analysis.
  • FIG. 15 Dasatinib and SRT2104 were used to treat PSC27 cells during proliferation or senescence at concentrations of 1000 nM and 10 ⁇ M, respectively, and analyze their apoptosis.
  • FIG. 16 Immunofluorescence staining analysis of the signal intensity of caspase 3 (cleaved) and p16 INK4a in PSC27 cells under proliferation and senescence conditions, respectively. Ruler, 20 ⁇ m. D, dasatinib; S, SRT2104.
  • Figure 17 Use gamma rays to treat wild-type mice at a certain dose (10 Gy) and analyze their aging progress in vivo after 3 months. The results of histochemical staining showed that the p16 INK4a positive and p21 CIP1 positive cells were statistically compared within and between groups. D, dasatinib; S, SRT2104.
  • Figure 18 Staining analysis of mouse tissues irradiated by ⁇ -rays by SA-B-Gal staining technique, the proportion of positive cells was compared in parallel within and between groups.
  • IR ionizing radiation.
  • Figure 19 Representative pictures of mouse tissue sections after SA-B-Gal staining. Ruler, 200 ⁇ m. D, dasatinib; S, SRT2104. IR, ionizing radiation.
  • Figure 20 Technical flow chart of pre-clinical anti-aging test in aging mice. 20-month-old wild-type mice were selected for combined administration of Dasatinib (5 mg/kg)/SRT2104 (50 mg/kg); oral administration was given once every two weeks, and after the end of the 4-month treatment course, a comprehensive analysis Physiological ability.
  • FIG. 23 Technical schematic diagram of anti-aging treatment for wild-type mice in the extremely aging stage in the late life. Wild-type mice aged 24-27 months were selected for combined administration of Dasatinib(5mg/kg)/SRT2104(50mg/kg); oral administration was given every two weeks until pathological symptoms appeared.
  • dasatinib and SIRT1 activator SRT2104 has an extremely excellent effect on down-regulating or eliminating senescent cells in the body, and thus can be used to eliminate tissue microenvironment. Damaged cells, such as damaged cells after chemotherapy or radiation treatment, can also be used to remove naturally senescent cells, thereby prolonging the body's survival time.
  • the present invention was formed on this basis.
  • the English name of Dasatinib is Dasatinib; its CAS number is 302962-49-8; its molecular formula is C 22 H 26 ClN 7 O 2 S.
  • the chemical structural formula is as follows (I):
  • the "dasatinib” may be a compound represented by formula (I) in a pure form, or a purity greater than 85% (preferably greater than 90%, such as 95%, 98%, 99%). %) of the compound represented by formula (I).
  • the compound of formula (I) is generally obtained by chemical synthesis. It is a commercial drug, so its finished product is easily obtained by those skilled in the art.
  • a pharmaceutically acceptable salt of the compound of formula (I) is also included, which also retains the chemical activity of dasatinib.
  • the "pharmaceutically acceptable salt” may be a salt formed by the reaction of dasatinib with an inorganic acid or an organic acid.
  • the precursor of the compound of formula (I) is also included, and the "precursor” refers to the precursor of the compound undergoing metabolism or chemical reaction in the patient's body to transform into the structural formula ( A compound of I), or a salt or solution composed of a compound of formula (I).
  • dasatinib is a polytyrosine kinase inhibitor and is used in adult patients of all stages of chronic myelogenous leukemia who have been treated, including imatinib mesylate-resistant or intolerable. ; At the same time, it is also used to treat adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia who are resistant or intolerant to other therapies.
  • dasatinib in the field of cell aging is not very satisfactory.
  • SRT2104 is a selective SIRT1 activator involved in the regulation of energy balance. Its CAS number is 1093403-33-8. The chemical structure of SRT2104 is as follows:
  • the SRT2104 can be obtained by chemical synthesis.
  • a pharmaceutically acceptable salt of SRT2104 is also included, which also retains the chemical activity of SRT2104.
  • the "pharmaceutically acceptable salt” may be a salt formed by the reaction of SRT2104 with an inorganic acid or an organic acid.
  • the precursor of SRT2104 is also included.
  • the "precursor” refers to the precursor of the compound undergoing metabolism or chemical reaction in the patient's body to convert it into the one of structural formula (II) after being taken by a proper method.
  • Dasatinib (Dasatinib) within a certain concentration range can cause a significant decrease in the survival rate of senescent cells, while proliferating cells are basically Not affected.
  • the "proliferative state (PRE) cell” refers to a cell that can maintain a state of continuous, active division and continuous proliferation.
  • the "senescent (SEN) cell” refers to a cell whose ability to proliferate and divide and whose physiological function is degraded.
  • the inventors also found that the SIRT1 activator SRT2104 does not affect the survival of senescent cells within a limited concentration range.
  • the present invention provides the use of a mixture or composition of dasatinib and SRT2104 to prepare a pharmaceutical composition for the preparation of a drug or preparation for down-regulating or eliminating senescent cells; and for preparing a pharmaceutical composition for prolonging the life span (life) of the body Drugs or preparations.
  • the cells can be naturally senescent cells or damaged cells.
  • the damaged cells may be damaged cells in the tissue microenvironment, or damaged cells after chemotherapy or radiation treatment.
  • Radiotherapy is a local treatment method that uses radiation to treat diseases, especially tumors.
  • Radiation includes alpha, beta, and gamma rays produced by radioisotopes and x-rays, electron rays, proton beams and other particle beams produced by various x-ray treatment machines or accelerators.
  • the role and status of radiotherapy in tumor treatment have become increasingly prominent, and it has become one of the main methods for the treatment of malignant tumors. However, its side effects are also very significant, and the damage to tissues and organs can have a pathological impact that cannot be ignored.
  • the mixture or composition of dasatinib and SRT2104 of the present invention is expected to be applied to alleviate this damage.
  • the mixture or composition of dasatinib and SRT2104 is also used to: reduce the level of senescence-associated secreted phenotype (SASP) marker factors; preferably, the marker factors include but are not limited to: IL6, IL8, MCP2, CXCL1, GM-CSF, MMP3, AREG, SFRP2, ANGPTL4, IL 1a, p16 INK4a , p21 CIP1 .
  • SASP senescence-associated secreted phenotype
  • the present invention provides a mixture containing: SRT2104 and dasatinib as active components.
  • the molar ratio or mass ratio of SRT2104 and Dasatinib is 1:(2 ⁇ 100); preferably 1:(4 ⁇ 80); more preferably 1:( 5 ⁇ 50).
  • the molar ratio or mass ratio it is 1:6; 1:8; 1:10; 1:12; 1:15; 1:20; 1:30; 1:50; 1:60; 1:70; 1 :90 etc.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) an effective amount of dasatinib or a pharmaceutically acceptable salt thereof; (b) an effective amount of SRT2104 or a pharmaceutically acceptable salt thereof; and (c ) A pharmaceutically acceptable carrier or excipient.
  • the term "containing” means that various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “mainly consisting of” and “consisting of” are included in the term “containing”.
  • pharmaceutically acceptable ingredients are substances that are suitable for humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances that have a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier is a pharmaceutically acceptable solvent, suspending agent or excipient used to deliver dasatinib and SRT2104 of the present invention to animals or humans.
  • the carrier can be liquid or solid.
  • the pharmaceutical composition or mixture of the present invention can be prepared into any conventional preparation form by conventional methods.
  • the dosage form can be various, as long as it can make the active ingredient reach the mammalian body effectively. For example, it can be selected from: injections, infusions, tablets, capsules, and pills.
  • Dasatinib or SRT2104 can be present in a suitable solid or liquid carrier or diluent.
  • the mixture or pharmaceutical composition of Dasatinib and SRT2104 of the present invention can also be stored in a sterile device suitable for injection or drip.
  • the effective dose of dasatinib and SRT2104 used can vary with the mode of administration and the severity of the disease to be treated, which can be based on the experience and recommendations of the clinician.
  • some mixtures of dasatinib and SRT2104 are given.
  • a series of dasatinib and SRT2104 according to different molar ratios or mass ratios are proposed Dosing regimen.
  • mice are also used as experimental animals. The conversion from the dose of mice to the dose suitable for humans is easily made by those skilled in the art. For example, it can be calculated according to the Meeh-Rubner formula:
  • the dasatinib and SRT2104 and their mixtures or pharmaceutical compositions can be administered orally, intravenously, intramuscularly, or subcutaneously. Preferably it can be administered orally.
  • Pharmaceutical forms suitable for oral administration include but are not limited to tablets, powders, capsules, sustained-release agents, and the like.
  • 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 to facilitate the ejection of fluid from the syringe.
  • Dasatinib and SRT2104 can also be administered in combination with other active ingredients or drugs.
  • the present invention also provides a kit for down-regulating or removing senescent cells, or prolonging the survival period of the body.
  • the kit contains: container 1 and a dasatinib group placed in container 1 And container 2 and the SRT2104 component placed in container 2.
  • the kit contains the mixture of dasatinib and SRT2104, wherein the ratio of SRT2104 to dasatinib is as described above.
  • the kit contains a pharmaceutical composition including a mixture of dasatinib and SRT2104 and a pharmaceutically acceptable carrier.
  • 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.
  • the present invention provides a use of a down-regulating agent of ABL, PTGS2 or BCL2A1 genes or proteins for the preparation of drugs or preparations for down-regulating or eliminating senescent cells.
  • the down-regulators of ABL, PTGS2 or BCL2A1 include inhibitors, antagonists, blockers, blockers and the like.
  • the down-regulator of ABL, PTGS2 or BCL2A1 gene or protein refers to any agent that can reduce the activity of ABL, PTGS2 or BCL2A1 protein, reduce the stability of ABL, PTGS2 or BCL2A1 gene or protein, or down-regulate the expression of ABL, PTGS2 or BCL2A1 protein , Reduce the effective time of ABL, PTGS2 or BCL2A1 protein, or inhibit the transcription and translation of ABL, PTGS2 or BCL2A1 gene, these substances can be used in the present invention, as a substance useful for down-regulation of ABL, PTGS2 or BCL2A1, which can be used Inhibit tumors.
  • the downregulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the ABL, PTGS2 or BCL2A1 gene; or an antibody or ligand that specifically binds to the protein encoded by the ABL, PTGS2 or BCL2A1 gene ,and many more.
  • the downregulator is a small molecule compound targeting ABL, PTGS2 or BCL2A1.
  • Those skilled in the art can use conventional screening methods in the art to screen such small molecule compounds.
  • the down-regulating agent is a specific interfering RNA molecule (shRNA) specific for ABL, PTGS2 or BCL2A1.
  • shRNA interfering RNA molecule
  • the present invention has no particular limitation on the preparation method of interfering RNA molecules, including but not limited to: chemical synthesis method, in vitro transcription method and the like.
  • the interfering RNA can be delivered into the cell by using an appropriate transfection reagent, or can also be delivered into the cell by using various techniques known in the art.
  • the CRISPR/Cas9 system can be used for targeted gene editing, thereby knocking out the ABL, PTGS2 or BCL2A1 genes in the targeted disease region.
  • Common methods for knocking out ABL, PTGS2 or BCL2A1 genes include: co-transferring sgRNA or the nucleic acid capable of forming the sgRNA, Cas9 mRNA or the nucleic acid capable of forming the Cas9 mRNA into a targeted region or a targeted cell. After the target site is determined, known methods can be used to introduce sgRNA and Cas9 into the cell.
  • Example 1 Chemotherapeutic drugs induce senescence of human stromal cells and present a typical senescence-related secretory phenotype
  • the inventors used the genotoxic chemotherapy drug bleomycin (bleomycin, BLEO) to treat the human prostate primary stromal cell line PSC27.
  • PSC27 cells were cultured in DMEM (10% FBS) medium, and BLEO was added to a final concentration of 50 g/ml, and the whole transcriptome was sequenced by RNA-Seq on the 7th day after treatment.
  • proliferative (PRE) and senescent (SEN) cells For cultured cells, the SA- ⁇ -Gal and BrdU staining techniques are used to define the senescent cells are SA- ⁇ -Gal positive, and the proliferative cells are BrdU positive.
  • the inventors subsequently detected the degree of DNA damage, and the immunofluorescence staining (IF staining) results for ⁇ H2AX showed that after BLEO treatment (50 ⁇ g/ml final concentration, treatment for 12 hours), PSC27 showed a higher proportion of damaged cells (Figure 4).
  • Example 2 The kinetic study of anti-aging drug candidates dasatinib and SIRT1 activator on cell survival under in vitro conditions
  • the inventors investigated the effects of selective knockout of some genes on stromal cells. Combined with the up-regulated expression of PSC27 after senescence and related anti-apoptotic genes such as PTGS2, BCL2A1, SERPINB2, the present inventors used shRNA to knock out ABL, EFNB1, EFNB3, PTGS2, BCL2A1, SERPINB2, ATPLite, respectively. The results showed that only the deletion of ABL, PTGS2, and BCL2A1 can significantly reduce the survival rate of senescent stromal cells (PSC27, human embryonic lung fibroblast WI38) (Figure 5, Figure 6).
  • shRNA targeting site sequence used to knock out ABL, PTGS2, and BCL2A1 is as follows:
  • ABL CCGCCTTCATCCCTCTCATAT (SEQ ID NO:1);
  • PTGS2 AGAGTATGCGATGTGCTTAAA (SEQ ID NO: 2);
  • BCL2A1 GTTGCGGAGTTCATAATGAAT (SEQ ID NO: 3).
  • Dasatinib within a certain concentration range (400-1000 nM) can cause a significant decrease in the survival rate of senescent PSC27 and WI38 cells, and In contrast, proliferating cells were basically unaffected (Figure 9, Figure 10).
  • SRT2104 is a selective SIRT1 activator involved in the regulation of energy balance. In in vitro experiments, SRT2104 failed to produce a significant effect of inhibiting the survival of senescent cells. Under 5 ⁇ M conditions, the survival of senescent stromal cells could not be significantly reduced ( Figure 11, Figure 12). When the concentration increased to 10 ⁇ M, the cell survival rate remained unchanged, and when the concentration was subsequently increased to 40 ⁇ M, the overall effect did not continue to rise; both PSC27 and WI38 showed a similar trend ( Figure 11, Figure 12). These results indicate that SRT2104 does not affect the survival of senescent cells within a limited concentration range.
  • dasatinib caused a significant decrease in the survival rate of stromal cells at both 400 nM and 1000 nM concentrations, although the latter was more significant (Figure 14).
  • SRT2104 did not significantly change the survival of senescent cells under 5 ⁇ M and 10 ⁇ M conditions.
  • dasatinib (1000nM) and SRT2104 (10 ⁇ M) were used at the same time, the survival rate of senescent stromal cells continued to decrease based on the results caused by dasatinib (1000nM) alone, but Proliferating cells were almost unchanged (Figure 14).
  • the inventors used gamma rays (10 Gy) to treat wild-type mice and conducted in-depth analysis after 3 months. One month after the radiation treatment, Dasatinib and SRT2104 were taken, and the dosage was Dasatinib (5mg/kg) and SRT2104 (50mg/kg).
  • the inventors performed SA-B-Gal staining analysis on tissue sections, and the evaluation results confirmed that ionizing radiation itself can significantly increase the proportion of senescent cells in the tissue, while anti-aging drugs cannot.
  • the experimental mice were treated with D/S by oral administration, the number of senescent cells in the microenvironment was significantly reduced within 15 days after one use ( Figure 18, Figure 19).
  • D/S can effectively reduce the number of senescent cells in the body, and does not require multiple treatments or administrations, and it does not cause premature aging or premature aging in the microenvironment under normal physiological conditions And so on.
  • Example 4 Under natural aging conditions, D/S can improve the physiological functions of multiple organs by inducing the elimination of senescent cells in the tissue microenvironment
  • the present inventors next selectively used a group of experimental mice aged 20 months and administered them intermittently for a period of 4 months (orally once every two weeks), mainly divided into placebo group Simultaneously with the D/S drug group ( Figure 20).
  • the inventors comprehensively evaluated the physiological indicators of the two groups of mice.
  • D/S drugs can improve multiple indicators of the body by reducing senescent cells in tissues, and avoid ataxia in multiple organs.
  • Example 5 D/S can prolong the overall lifespan without causing pathological symptoms by inducing the elimination of senescent cells in the tissue microenvironment under extreme aging conditions
  • mice can anti-aging drugs play a role in delaying aging even when the body is nearing the end of life?
  • the inventors used a batch of mice (24-27 months old) in a more advanced stage.
  • mice in the D/S group showed a general and significant decrease in the expression levels of SASP marker factors, including but not limited to IL6, IL8, MCP2, and CXCL1 (Figure 24). More importantly, this oral D/S drug that is used every other week from the age of 24-27 months is: Dasatinib (5 mg/kg), SRT2104 (50 mg/kg), which makes mice The median survival period was significantly prolonged after treatment, from 244 days to 305 days ( Figure 25, 21.5%, P ⁇ 0.001). Correspondingly, the overall survival of mice treated with oral D/S drugs also increased significantly, from 974 days to 1035 days ( Figure 26, 25%, P ⁇ 0.001).
  • SASP marker factors including but not limited to IL6, IL8, MCP2, and CXCL1
  • D/S drugs can reduce the number of senescent cells and reduce the expression of a broader spectrum of SASP exocrine factors, which ultimately significantly prolongs the body's survival time.

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Abstract

本发明提供了具有靶向组织微环境中衰老细胞作用的抗衰老药物D/S及其应用。将达沙替尼与SRT2104联合应用，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损伤细胞如化疗或辐射治疗后的受损细胞，也可应用于清除随年龄增长而自然衰老的细胞，从而延长机体的生存时间。

Description

靶向组织微环境中衰老细胞的抗衰老药物D/S及其应用 技术领域
本发明属于制药领域，更具体地，本发明涉及具有靶向组织微环境中衰老细胞活性的抗衰老药物D/S及其应用。
背景技术
随着年龄的增长，多数器官和组织中的衰老细胞会增多；临床条件下，这类细胞还会出现在与许多慢性疾病相关的器官中，以及放化疗之后。细胞衰老是指细胞失去功能(包括分裂和复制能力)但仍抵抗死亡的过程，已被证明可驱动多种与年龄有关的疾病，如骨质疏松、骨关节炎、动脉粥样硬化和特发性肺纤维化等。
体内条件下衰老细胞会持续存在，并对周围细胞产生长期的影响。这主要是因为衰老细胞往往会发展为一种名为衰老相关分泌表型(senescence-associated secretory phenotype，SASP)，使得这些细胞持续分泌大量促炎和微环境重塑分子。通过转录分析，研究人员发现，与癌细胞一样，衰老细胞增强了“促生存网络”相关因子的表达，从而帮助它们抵抗细胞凋亡或其它形式的程序性细胞死亡。
美国斯克里普斯研究所(TSRI)、梅奥诊所和其他机构的一个研究小组，2015年报道了一类新的药物，可在动物模型中显著减缓老化过程，缓解虚弱症状，改善心脏功能，延长健康寿命。科学家将这类新的药物统称为“senolytics”。进一步的细胞培养实验表明，这些化合物确实可选择性地诱导衰老细胞的死亡。有趣的是，这两种化合物有不同的特异性，即达沙替尼(dasatinib)可消除衰老的人脂肪细胞祖细胞，而槲皮素(quercetin)则能更有效地对抗衰老的人内皮细胞和小鼠骨髓干细胞；在此基础上，两者的结合方是更有效的。然而，国际范围内大量后续研究则表明，这种方法在人类正式使用前，还需要更多的测试。除了这两种药物可能在长期治疗时带来一些未知的副作用，更因其无法对组织微环境中占据数量更多的成纤维细胞等基质细胞群予以有效清除，而使其适用性、准确性和实际效果受到一定限制。此外，槲皮素是天然植物成分，激活的靶点并不唯一，涉及多个激酶，这也限制了其应用。
因此，本领域还亟需进一步寻找抗衰老的有效药物。
发明内容
本发明的目的在于提供具有靶向组织微环境中衰老细胞活性的抗衰老药物D/S及其应用。
在本发明的第一方面，提供组合物在制备下调或清除衰老细胞的药物或制剂中的用途；其中，所述的组合物包括达沙替尼和SRT2104。
在一个优选例中，所述的细胞包括：自然衰老细胞；或损伤细胞；较佳地为组织微环境中的损伤细胞；更佳地为化疗或辐射治疗后的受损细胞。
在另一优选例中，所述的辐射治疗包括：电离辐射，α、β或γ射线治疗。
在另一优选例中，所述细胞不包括或基本不包括增殖态细胞。
在本发明的另一方面，提供组合物在制备延长机体生存期(寿命)的药物或制剂中的用途；其中，所述的组合物包括达沙替尼和SRT2104。
在另一优选例中，所述的组合物还用于：降低衰老相关分泌表型(SASP)细胞衰老标志性因子的水平；较佳地，所述的标志性因子包括(但不限于)：SASP标志因子IL6、IL8、MCP2、CXCL1、GM-CSF、MMP3、AREG、SFRP2、ANGPTL4或IL 1a；细胞衰老标志因子p16 INK4a或p21 CIP1。
在另一优选例中，所述的组合物还用于：提高机体机能，避免多个器官共济失调。
在另一优选例中，所述的组合物中，按照摩尔比或质量比，达沙替尼:SRT2104为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。
在另一优选例中，按照摩尔比或质量比，达沙替尼:SRT2104为1:6；1:8；1:10；1:12；1:15；1:20；1:30；1:50；1:60；1:70；1:90等。
在另一优选例中，达沙替尼在组合物中的终浓度为1nM～10mM，如10nM、100nM、1uM、10uM、100uM、1mM。
在本发明的另一方面，提供一种用于下调或清除衰老细胞的药物组合物，其包括达沙替尼和SRT2104；较佳地，其还包括药学上可接受的载体或赋形剂。
在一个优选例中，按照摩尔比或质量比，达沙替尼:SRT2104为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。
在本发明的另一方面，提供所述的药物组合物的用途，用于制备下调或清除衰老细胞的药盒或试剂盒。
在本发明的另一方面，提供用于下调或清除衰老细胞的药盒或试剂盒，其包括所述的药物组合物。
在本发明的另一方面，提供用于下调或清除衰老细胞的药盒或试剂盒，其包括容器1及容器2，分别装有达沙替尼和SRT2104；较佳地，按照摩尔比或质量比，达沙替尼:SRT2104为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。
在另一优选例中，所述的组合物、药盒或试剂盒中，以达沙替尼和SRT2104为活性成分，其它成分为药学载体或赋形剂等。
在另一优选例中，所述的组合物仅由达沙替尼和SRT2104组成。
在另一优选例中，所述的药物组合物的剂型是：口服剂，注射剂，输液剂，片剂，粉剂，胶囊剂，丸剂；较佳地为口服剂。
在本发明的另一方面，提供一种下调或清除衰老细胞的方法，包括以组合物处理衰老细胞；其中，所述的组合物包括达沙替尼和SRT2104。
在本发明的另一方面，提供SRT2104的用途，用于促进达沙替尼下调或清除衰老细胞的活性或延长机体生存期的活性。
在本发明的另一方面，提供SRT2104的用途，用于制备促进达沙替尼下调或清除衰老细胞活性或延长机体生存期活性的药物或制剂。
在本发明的另一方面，提供一种促进达沙替尼下调或清除衰老细胞的活性的方法，包括将SRT2104与达沙替尼联合应用。
在另一优选例中，上述各用途或方法为不以临床疾病治疗为直接目的的。
在本发明的另一方面，提供基因作为下调靶点在制备下调或清除衰老细胞的药物或制剂中的用途；其中，所述基因选自ABL(ABL proto-oncogene)、PTGS2(prostaglandin-endoperoxide synthase 2)或BCL2A1(BCL2related protein A1)。
在本发明的另一方面，提供基因下调剂的用途，用于制备下调或清除衰老细胞的药物或制剂；其中，所述基因选自ABL(Gene ID:25)、PTGS2(Gene ID:5743)或BCL2A1(Gene ID:597)。
在一个优选例中，所述的基因下调剂包括：特异性干扰所述基因表达的干扰分子，特异性抑制所述基因的小分子化合物，特异性敲除所述基因的基因编辑试剂；较佳地，所述的干扰分子是以所述基因为抑制或沉默靶标的siRNA、反义核酸、miRNA、dsRNA、shRNA，或能表达或形成所述siRNA、反义核酸、miRNA、dsRNA、shRNA的构建物；更佳地，该干扰分子是shRNA，靶向于SEQ ID NO:1、SEQ ID NO:2或SED ID NO:3所示的核苷酸序列。
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、人源前列腺原代基质细胞系PSC27经过化疗药物BLEO处理之后第7天，以RNA-Seq进行全转录组测序，发现大量SASP因子呈现上调趋势。红色星号，BCL2A1和SERPINB2。
图2、使用SA-B-Gal染色技术对PSC27细胞进行分析。左，代表性染色图片， 上下分别为增殖态(PRE)和衰老(SEN)细胞，标尺20μm；右，基于每组100个细胞的统计学比较。***，P<0.001。
图3、通过BrdU染色确定细胞分裂和增殖状态。基于每组100个细胞的统计学比较。***，P<0.001。
图4、经过免疫荧光染色之后对DNA损伤程度进行平行比较。一抗为γH2AX；根据最终每个细胞核中DNA damage foci的数量进行分级(0foci；1-3foci；4-10foci；>10foci)。***，P<0.001。
图5、使用shRNA在PSC27细胞分别敲除多个生存相关基因之后，利用ATPLite检测细胞信号并进行平行对比。**，P<0.01；*，P<0.05。
图6、同图5类似，使用shRNA在WI38细胞中分别敲除多个生存相关基因之后，利用ATPLite检测细胞信号并进行平行对比。**，P<0.01。
图7、使用shRNA在PSC27细胞分别敲除多个生存相关基因之后，利用结晶紫染色技术检测细胞存活数量并进行平行对比。**，P<0.01；*，P<0.05。
图8、同图7类似，使用shRNA在WI38细胞中分别敲除多个生存相关基因之后，利用结晶紫染色技术检测细胞信号并进行平行对比。**，P<0.01。
图9、使用Dasatinib在一定浓度范围内(0、200、400、600、800、1000nM)处理PRE和SEN状态下的PSC27细胞，并分析其生存情况。每组数据对实验开始时的本组细胞数量进行规范化。*，P<0.05；**，P<0.01。
图10、同图9相似，使用Dasatinib在一定浓度范围内(0、200、400、600、800、1000nM)处理PRE和SEN状态下的WI38细胞，并分析其生存情况。每组数据对实验开始时的本组细胞数量进行规范化。*，P<0.05；**，P<0.01。
图11、使用SRT2104在一定浓度范围(0、5、10、15、20、25、30、35、40μM)内处理PRE和SEN状态下的PSC27细胞，并分析其生存情况。每组数据对实验开始时的本组细胞数量进行规范化。
图12、同图11相似，使用SRT2104在一定浓度范围内(0、5、10、15、20、25、30、35、40μM)处理PRE和SEN状态下的WI38细胞，并分析其生存情况。每组数据对实验开始时的本组细胞数量进行规范化。
图13、显微图片显示Dasatinib和SRT2104单独或联合使用处理SEN状态下的PSC27细胞，并分析其生存情况。标尺，20μm。D，dasatinib；S，SRT2104。
图14、分别或者联合使用一定浓度的Dasatinib(0、400、1000nM)和SRT2104(0、5、10μM)处理增殖或衰老时期的PSC27细胞，并分析其生存情况。绿色虚线，起始细胞数量作为数据分析的基线水平。
图15、Dasatinib和SRT2104分别在1000nM和10μM浓度下处理增殖或衰老时期的PSC27细胞，并分析其凋亡情况。D，dasatinib；S，SRT2104。
图16、免疫荧光染色分析PSC27细胞分别在增殖态和衰老条件下的caspase 3(cleaved)和p16 INK4a的信号强度。标尺，20μm。D，dasatinib；S，SRT2104。
图17、使用γ射线以一定剂量(10Gy)处理野生型小鼠并在3个月之后分析其体内衰老进展情况。组化染色结果显示的p16 INK4a阳性和p21 CIP1阳性细胞，经过统计之后进行组内和组间平行对比。D，dasatinib；S，SRT2104。
图18、通过SA-B-Gal染色技术对经过γ射线照射过的小鼠组织进行染色分析，阳性细胞比例在组内和组间平行对比。D，dasatinib；S，SRT2104。IR，ionizing radiation。
图19、小鼠组织切片经过SA-B-Gal染色之后的代表性图片。标尺，200μm。D，dasatinib；S，SRT2104。IR，ionizing radiation。
图20、在衰老小鼠中进行预临床抗衰老试验的技术流程图。选择20个月龄的野生型小鼠进行Dasatinib(5mg/kg)/SRT2104(50mg/kg)联合给药处理；每两周予以一次口服给药，4个月疗程结束之后，综合分析其多项生理能力。
图21、系统检测各组实验小鼠在多个方面包括左心室射血频率，最大行走速度，悬挂耐久力，咬合力量的平均能力。N＝10。
图22、平行比较各组实验小鼠在常规体能项目包括塌车耐力和每日活动量；同时，基本生理测验包括体重和食物摄入量也予以检测并对获取的数据进行综合比较。N＝10。
图23、针对处于生命晚期极度衰老阶段的野生型小鼠进行抗衰老治疗的技术示意图。选择24-27个月龄的野生型小鼠进行Dasatinib(5mg/kg)/SRT2104(50mg/kg)联合给药处理；每两周予以一次口服给药，直至其出现病理症状。
图24、通过qRT-PCR检测小鼠组织微环境中基质细胞表达SASP典型外泌因子的水平。选择3月龄小鼠作为分析对比的基线，安慰剂(placebo)组和Dasatinib/SRT2104(D/S)组数据进行规范化并平行比较。RPL13A表达值为内参对照。N＝3。
图25、抗衰老治疗后的小鼠无病生存曲线分析(healthspan)。Dasatinib/SRT2104(D/S)组，n＝80；中位生存期＝305天；Placebo组，n＝85。中位生存期＝244天。P＝0.0008。
图26、抗衰老治疗前后小鼠生命期间无病总生存曲线分析(lifespan)。Dasatinib/SRT2104(D/S)组，n＝80,中位生存期＝1035天；Placebo组，n＝85,中位生存期＝974天。P＝0.0008。
具体实施方式
本发明人经过长期而广泛的研究，意外地发现，将达沙替尼与SIRT1活化剂SRT2104联合应用，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如化疗或辐射治疗后的受损细胞，也可应用于清除自然衰老的细胞，从而延长机体的生存时间。在此基础上形成了本发明。
达沙替尼
达沙替尼的英文名称为Dasatinib；CAS号为302962-49-8；分子式为C 22H 26ClN 7O 2S。化学结构式如下式(I)：
Figure PCTCN2020110860-appb-000001
在本发明中，所述的“达沙替尼”可以是纯净形式存在的式(I)所示的化合物，或纯度大于85％(较佳地大于90％，例如95％，98％，99％)的式(I)所示的化合物。
现有技术中，所述的式(I)化合物一般通过化学合成的方式获得，其是一种商品化的药物，因此其成品是本领域技术人员易于获得的。
在本发明中，还包括式(I)化合物的药学上可接受的盐，其也保留有达沙替尼的化学活性。所述的“药学上可接受的盐”可以是达沙替尼与无机酸或有机酸反应生成的盐。
在本发明中，还包括式(I)化合物的前体，所述的“前体”指当用适当的方法服用后，该化合物的前体在病人体内进行代谢或化学反应而转变成结构式(I)的一种化合物，或化学结构式(I)的一个化合物所组成的盐或溶液。
现有技术中，已知达沙替尼属多酪氨酸激酶抑制剂，用于已经治疗，包括甲磺酸伊马替尼耐药或不能耐受的慢性骨髓性白血病所有病期的成人患者；同时其也被应用于治疗对其他疗法耐药或不能耐受的费城染色体阳性的急性淋巴细胞性白血病成人患者。然而，达沙替尼在细胞衰老相关领域的研究或应用效果并不十分理想。
SRT2104
SRT2104是一种选择性SIRT1活化剂，参与能量平衡的调节，其CAS号为1093403-33-8。SRT2104的化学结构式如下：
Figure PCTCN2020110860-appb-000002
所述的SRT2104可通过化学合成的方式获得。此外，现有技术中已经有商品化的SRT2104，因此其是本领域技术人员易于获得的。
在本发明中，还包括SRT2104的药学上可接受的盐，其也保留有SRT2104的化学活性。所述的“药学上可接受的盐”可以是SRT2104与无机酸或有机酸反应生成的盐。
在本发明中，还包括SRT2104的前体，所述的“前体”指当用适当的方法服用后，该化合物的前体在病人体内进行代谢或化学反应而转变成结构式(II)的一种化合物，或化学结构式(II)的一个化合物所组成的盐或溶液。
达沙替尼与SRT2104的联用用途
经过基于大量的小分子药物的体外药效动力学筛选分析，本发明人发现达沙替尼(Dasatinib)在一定浓度范围内能够造成衰老细胞存活率显著下降，而相比之下增殖态细胞基本不受影响。
如本发明所用，所述的“增殖态(PRE)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。
如本发明所用，所述的“衰老(SEN)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。
本发明人也发现，SIRT1活化剂SRT2104在有限浓度范围内不影响衰老细胞存活。
然而，令人惊讶的是，当达沙替尼和SRT2104同时使用时，衰老基质细胞的生存率在达沙替尼单独处理造成的结果基础上进一步降低，但增殖态细胞大致没有变化。因此，两者联合用药具有显著的增效效果，且基本上仅针对衰老细胞而不会影响增殖态细胞。
因此，本发明提供了达沙替尼与SRT2104的混合物或组合物的用途，用于制备下调或清除衰老细胞的药物或制剂的药物组合物；以及，用于制备延长机体生存期(寿命)的药物或制剂。所述的细胞可以是自然衰老细胞或受损细胞。所述受损细胞可以是组织微环境中的受损细胞，或为化疗或辐射治疗后的受损细胞。
放射治疗是利用放射线治疗疾病尤其肿瘤的一种局部治疗方法。放射线包括放射性同位素产生的α、β、γ射线和各类x射线治疗机或加速器产生的x射线、电子线、质子束及其他粒子束等。大约70％的癌症患者在治疗癌症的过程中需要用放射治疗，约有40％的癌症可以用放疗根治。放射治疗在肿瘤治疗中的作用和地位日益突出，已成为治疗恶性肿瘤的主要手段之一。然而，其副作用也十分显著，对于组织器官的损伤能够产生不可忽视的病理影响。而本发明的达沙替尼与SRT2104的混合物或组合物则有望被应用于缓解这种损伤。
所述的达沙替尼与SRT2104的混合物或组合物还用于：降低衰老相关分泌表型(SASP)标志性因子的水平；较佳地，所述的标志性因子包括但不限于：IL6、IL8、MCP2、CXCL1、GM-CSF、MMP3、AREG、SFRP2、ANGPTL4、IL 1a，p16 INK4a、p21 CIP1。
组合物或混合物
本发明提供了一种混合物，含有：SRT2104和达沙替尼作为活性组分。较佳地，所述的混合物中，SRT2104与达沙替尼按照摩尔比或质量比为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。例如，按照摩尔比或质量比为1:6；1:8；1:10；1:12；1:15；1:20；1:30；1:50；1:60；1:70；1:90等。
本发明提供了一种药物组合物，含有：(a)有效量的达沙替尼或其药学上可接受的盐；(b)有效量的SRT2104或其药学上可接受的盐；以及(c)药学上可接受的载体或赋形剂。
本发明中，术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
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。
本发明中，“药学上可接受的载体”是用于将本发明的达沙替尼和SRT2104传送给动物或人的药学上可接受的溶剂、悬浮剂或赋形剂。载体可以是液体或固体。
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。其中达沙替尼或SRT2104可以存在于适宜的固体或液体的载体或稀释液中。
本发明的达沙替尼和SRT2104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。
所用的达沙替尼和SRT2104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，给出了一些达沙替尼和SRT2104的混合物，例如一个实施例中，在细胞实验中，提出了达沙替尼和SRT2104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：
Meeh-Rubner公式：A＝k×(W 2/3)/10,000。
式中A为体表面积，以m 2计算；W为体重，以g计算；K为常数，随动物种类而不同，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。
所述达沙替尼和SRT2104及其混合物或药物组合物可通过口服以及静脉内、肌内或皮下等途径给药。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，达沙替尼和SRT2104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用于下调或清除衰老细胞，或延长机体生存期的药盒，所述的药盒中，含有：容器1，以及置于容器1中的达沙替尼组分；以及容器2以及置于容器2中的SRT2104组分。
或者，所述的药盒中，含有所述的达沙替尼和SRT2104的混合物，其中SRT2104与达沙替尼的比例如前述。
或者，所述的药盒中，含有包括达沙替尼和SRT2104的混合物，以及药学上可接受的载体的药物组合物。
此外，所述的药盒中还可以还有一些辅助用药的材料。
此外，所述的药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。
基因靶向抑制
本发明人还发现，ABL、PTGS2或BCL2A1的缺失可以造成衰老基质细胞的显著性下降。基于本发明人的这一新发现，本发明提供了一种ABL、PTGS2或BCL2A1基因或蛋白的下调剂的用途，用于制备下调或清除衰老细胞的药物或制剂。
如本文所用，所述的ABL、PTGS2或BCL2A1的下调剂包括了抑制剂、拮抗剂 剂、阻滞剂、阻断剂等。
所述的ABL、PTGS2或BCL2A1基因或蛋白的下调剂是指任何可降低ABL、PTGS2或BCL2A1蛋白的活性、降低ABL、PTGS2或BCL2A1基因或蛋白的稳定性、下调ABL、PTGS2或BCL2A1蛋白的表达、减少ABL、PTGS2或BCL2A1蛋白有效作用时间、或抑制ABL、PTGS2或BCL2A1基因的转录和翻译的物质，这些物质均可用于本发明，作为对于下调ABL、PTGS2或BCL2A1有用的物质，从而可用于抑制肿瘤。例如，所述的下调剂是：特异性干扰ABL、PTGS2或BCL2A1基因表达的干扰RNA分子或反义核苷酸；或是特异性与ABL、PTGS2或BCL2A1基因编码的蛋白结合的抗体或配体，等等。
作为本发明的一种选择方式，所述的下调剂是针对ABL、PTGS2或BCL2A1的小分子化合物。本领域技术人员可以采用本领域的常规筛选方法，来进行这类小分子化合物的筛选。
作为本发明的一种选择方式，所述的下调剂是一种ABL、PTGS2或BCL2A1特异性的干扰RNA分子(shRNA)，本发明人观察到，采用本发明的干扰RNA分子，可显著地下调ABL、PTGS2或BCL2A1，对于下调或清除衰老细胞具有作用。
本发明对干扰RNA分子的制备方法没有特别的限制，包括但不限于：化学合成法，体外转录法等。所述的干扰RNA可通过采用适当的转染试剂被输送到细胞内，或还可采用本领域已知的多种技术被输送到细胞内。
作为本发明的另一种选择，可采用CRISPR/Cas9系统进行靶向的基因编辑，从而在靶向疾病的区域敲除ABL、PTGS2或BCL2A1基因。常见的敲除ABL、PTGS2或BCL2A1基因的方法包括：将sgRNA或能形成所述sgRNA的核酸、Cas9mRNA或能形成所述Cas9mRNA的核酸共转到靶向区域或靶向细胞中。在确定了靶位点之后，可以采用已知的方法来使得sgRNA及Cas9被引入到细胞内。
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1、化疗药物诱导人源基质细胞衰老并呈现典型的衰老相关分泌表型
本发明人使用基因毒化疗药物博来霉素(bleomycin，BLEO)处理人源前列腺原代基质细胞系PSC27。PSC27细胞在DMEM(10％FBS)培养基中培养，添加BLEO至终 浓度50μg/ml，处理后第7天以RNA-Seq进行全转录组测序。
增殖态(PRE)和衰老(SEN)细胞的界定：对于培养的细胞，通过SA-β-Gal和BrdU染色技术界定，衰老细胞呈现SA-β-Gal阳性，增殖态细胞呈现BrdU阳性。
结果表明，衰老(SEN)细胞相比于增殖态(PRE)细胞，基因表达谱已经发生深刻改变。在上调的基因列表中，出现大量SASP典型外泌因子，如IL8、CSF2、CCL20、IL1a等(图1)。
与此同时，PSC27细胞在SA-B-Gal染色后的阳性呈现显著性升高(图2)，而BrdU阳性率则显著性降低(图3)。说明细胞衰老程度有显著增加，细胞增殖显著下降。
本发明人随后检测了DNA损伤程度，而针对γH2AX的免疫荧光染色(IF staining)结果表明，BLEO处理(50μg/ml终浓度，处理12小时)之后，PSC27呈现更高比例的受损细胞(图4)。
实施例2、体外条件下抗衰老候选药物达沙替尼和SIRT1激活剂对细胞存活的动力学研究
首先，本发明人考察了选择性敲除一些基因对于基质细胞的影响情况。结合PSC27衰老之后发生上调表达的、同抗凋亡相关的个别基因如PTGS2、BCL2A1、SERPINB2，本发明人使用shRNA分别敲除了ABL、EFNB1、EFNB3、PTGS2、BCL2A1、SERPINB2，ATPLite。结果显示仅有ABL、PTGS2、BCL2A1的缺失可以造成衰老基质细胞(PSC27，人胚肺成纤维细胞WI38)存活率显著降低(图5，图6)。同样，结晶紫染色证实PTGS2、BCL2A1在这两个细胞系中对于生存具有显著影响(图7，图8)。由于同时影响衰老(SEN)细胞和增殖态(PRE)细胞，可能影响正常生理条件下的微环境。
用于敲除ABL、PTGS2、BCL2A1的shRNA靶向位点序列如下：
ABL：CCGCCTTCATCCCTCTCATAT(SEQ ID NO:1)；
PTGS2：AGAGTATGCGATGTGCTTAAA(SEQ ID NO:2)；
BCL2A1：GTTGCGGAGTTCATAATGAAT(SEQ ID NO:3)。
经过基于大量的小分子药物的体外药效动力学筛选分析，本发明人发现达沙替尼(Dasatinib)在一定浓度范围内(400-1000nM)能够造成衰老PSC27和WI38细胞存活率显著下降，而相比之下增殖态细胞基本未受影响(图9，图10)。
SRT2104是一种参与能量平衡的调节的选择性SIRT1活化剂。在体外实验中SRT2104未能产生显著的抑制衰老细胞生存的效果。在5μM条件下，不能显著降低衰老基质细胞的生存(图11，图12)。当浓度上升为10μM时，细胞存活率不变，而随后继续提高浓度至40μM时，总体效果也并未继续上升；无论PSC27还是WI38 均表现为类似的一种趋势(图11，图12)。这些结果表明，SRT2104在有限浓度范围内不影响衰老细胞存活。
进一步的显微图像分析结果表明，达沙替尼(1000nM)在48小时内可以大幅减少存活衰老细胞的数量，而残余细胞也出现典型的凋亡特征；相比之下，SRT2104(1μM)造成衰老细胞死亡的程度不及前者，但使得细胞呈现更加典型的纤维样拉伸，而非明显的凋亡形态；有趣的是，两者一旦合用，无论PSC27还是WI38均在数量下降低的同时表现为凋亡和牵拉伴随的迹象(图13)。那么，衰老细胞的生存究竟在哪种条件下受到的影响最显著呢？
随后的药效动力学实验中，达沙替尼在400nM和1000nM浓度下均造成基质细胞存活率显著降低，尽管后者更加显著(图14)。相比之下SRT2104在5μM和10μM条件下均没有显著改变衰老细胞的生存。然而，令人惊讶的是，当达沙替尼(1000nM)和SRT2104(10μM)同时使用时，衰老基质细胞的生存率在达沙替尼(1000nM)单独处理造成的结果基础上继续降低，但增殖态细胞大致没有变化(图14)。
同时进行的凋亡检测实验结果证实，达沙替尼(1000nM)可以引起衰老细胞凋亡比例显著上升，而SRT2104(10μM)没有类似影响；二者合用，衰老细胞凋亡程度进一步显著上扬，高于达沙替尼单独使用的效果(图15)。以上任何条件下，对照组即增殖态细胞均未受明显影响。
免疫荧光染色结果清除地表明，相比于增殖态细胞，衰老细胞呈现p16 INK4a表达上调，其高表达说明细胞衰老程度的提高；达沙替尼与SRT2104(D/S)联用，可以造成Caspase 3(cleaved)信号明显上升，而相比之下的增殖态细胞基本不变(图16)。Caspase 3(cleaved)信号增强意味着衰老细胞的细胞凋亡增加。
实施例3、放疗导致的组织微环境中衰老细胞可以通过达沙替尼和SRT2104联用的方式得以大量去除
本发明人使用γ射线(10Gy)处理野生型小鼠并在3个月之后进行深入分析。射线处理之后1个月服用达沙替尼和SRT2104，服用量为Dasatinib(5mg/kg),SRT2104(50mg/kg)。
组化染色结果证实，微环境中有近60％的细胞出现p16 INK4a阳性，但在D/S联用条件下却显著降低(幅度大约60％)。同时，相比于对照(安慰剂组)，小鼠组织中p21 CIP1阳性细胞比例也显著下降(图17)。
本发明人对组织切片进行了SA-B-Gal染色分析，评估结果证实电离辐射本身可以显著增加组织中衰老细胞的比例，而抗衰老药物不能。但是，在D/S以口服给药的 方式处理实验小鼠时，一次使用之后的15天之内，微环境中衰老细胞的数量即呈显著降低(图18，图19)。
这些结果证明，D/S作为抗衰老组合药物，能够有效降低体内衰老细胞的数量，并且不需要多次治疗或者给药，而其本身对于正常生理条件下的微环境不会造成提前衰老或早衰等影响。
实施例4、自然衰老条件下D/S通过诱导组织微环境中衰老细胞的清除可以提高多器官的生理功能
上述论证后，需要思考一个关键问题是：即便能够清除因抗癌治疗等外界胁迫因素造成的组织中被动衰老细胞，是否可以在正常生理条件下尤其自然衰老已经发生的前提背景下同样达到类似目的？D/S的使用是否需要可以通过多次进行、即间歇式给药？是否会对机体的生理功能造成影响？
鉴于此，本发明人接下来选择性使用了一组年龄为20个月的实验小鼠，对其施以为期4个月的间歇给药(每两周一次口服)，主要分为安慰剂组与D/S药物组同时进行(图20)。在4个月的疗程结束时，本发明人全面评估了这两组小鼠的生理指标。
分析数据表明，D/S组动物在多个方面包括左心室射血频率，最大行走速度，悬挂耐久力，咬合力量等检测结果中，均比对照组呈现更高的能力(图21)。同时，常规体能项目如塌车耐力(即小鼠自主跑轮能力，可以用于监测小鼠的自发性活动。这个跑轮的设计：每套设备的跑轮上有一个磁力指示器，在笼子外面有个霍尔传感器，用来监测跑轮的旋转)和每日活动也发生明显增强，但一组基本生理测验如体重和食物摄入均未出现明显改变(图22)。
以上结果表明，在不影响基本生理功能的情况下，D/S药物能够通过降低组织中衰老细胞的方式提高机体的多项指标，避免多个器官发生共济失调。
实施例5、极度衰老条件下D/S通过诱导组织微环境中衰老细胞的清除可以延长总体寿命但不引起病理症状
那么，使用抗衰老药物，是否在机体已经接近生命终点时，也能起到延缓老化的作用呢？为回答这一问题，本发明人进而使用了处于更晚期阶段的一批小鼠(24-27月龄)。
通过每两周一次的口服给药，D/S组小鼠在SASP标志性因子的表达水平上出现普遍的显著下降，包括但不限于IL6、IL8、MCP2、CXCL1(图24)。更重要的则是这种从24-27月龄开始每隔一周使用的口服D/S药物，每次服用量为：Dasatinib(5 mg/kg)，SRT2104(50mg/kg)，使得小鼠的疗后阶段中位生存期显著延长，从244天显著延长到305天(图25，21.5％，P<0.001)。相应的，口服D/S药物处理下的小鼠总生存期也出现大幅增长，从974天显著延长到1035天(图26，25％，P<0.001)。
因而，对于生命晚期阶段的实验动物，D/S药物能够降低衰老细胞数量，减少较为广谱的SASP外泌因子表达，最终使得机体的生存时间显著延长。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (18)

  1. 组合物在制备下调或清除衰老细胞的药物或制剂中的用途；其中，所述的组合物包括达沙替尼和SRT2104。
  2. 如权利要求1所述的用途，其特征在于，所述的细胞包括：
    自然衰老细胞；或
    损伤细胞；较佳地为组织微环境中的损伤细胞；更佳地为化疗或辐射治疗后的受损细胞。
  3. 如权利要求1所述的用途，其特征在于，所述细胞不包括增殖态细胞。
  4. 组合物在制备延长机体生存期的药物或制剂中的用途；其中，所述的组合物包括达沙替尼和SRT2104。
  5. 如权利要求1～4任一所述的用途，其特征在于，所述的组合物还用于：降低衰老相关分泌表型细胞衰老标志性因子的水平；较佳地，所述的标志性因子包括：SASP标志因子IL6、IL8、MCP2、CXCL1、GM-CSF、MMP3、AREG、SFRP2、ANGPTL4或IL 1a；细胞衰老标志因子p16 INK4a或p21 CIP1。
  6. 如权利要求1～4任一所述的用途，其特征在于，所述的组合物还用于：提高机体机能，避免多个器官共济失调。
  7. 如权利要求1～4任一所述的用途，其特征在于，所述的组合物中，按照摩尔比或质量比，达沙替尼:SRT2104为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。
  8. 一种用于下调或清除衰老细胞的药物组合物，其包括达沙替尼和SRT2104；较佳地，其还包括药学上可接受的载体或赋形剂。
  9. 如权利要求8所述的药物组合物，其特征在于，按照摩尔比或质量比，达沙替尼:SRT2104为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。
  10. 权利要求8或9所述的药物组合物的用途，用于制备下调或清除衰老细胞的 药盒或试剂盒。
  11. 用于下调或清除衰老细胞的药盒或试剂盒，其包括权利要求8～9任一所述的药物组合物。
  12. 用于下调或清除衰老细胞的药盒或试剂盒，其包括容器1及容器2，分别装有达沙替尼和SRT2104；较佳地，按照摩尔比或质量比，达沙替尼:SRT2104为1:(2～100)；较佳地为1:(4～80)；更佳地为1:(5～50)。
  13. 一种下调或清除衰老细胞的方法，包括以组合物处理衰老细胞；其中，所述的组合物包括达沙替尼和SRT2104。
  14. SRT2104的用途，用于促进达沙替尼下调或清除衰老细胞的活性或延长机体生存期的活性；或
    用于制备促进达沙替尼下调或清除衰老细胞活性或延长机体生存期活性的药物或制剂。
  15. 一种促进达沙替尼下调或清除衰老细胞的活性的方法，包括将SRT2104与达沙替尼联合应用。
  16. 基因作为下调靶点在制备下调或清除衰老细胞的药物或制剂中的用途；其中，所述基因选自ABL、PTGS2或BCL2A1。
  17. 基因下调剂的用途，用于制备下调或清除衰老细胞的药物或制剂；其中，所述基因选自ABL、PTGS2或BCL2A1。
  18. 如权利要求16所述的用途，其特征在于，所述的基因下调剂包括：特异性干扰所述基因表达的干扰分子，特异性抑制所述基因的小分子化合物，特异性敲除所述基因的基因编辑试剂；较佳地，所述的干扰分子是以所述基因为抑制或沉默靶标的siRNA、反义核酸、miRNA、dsRNA、shRNA，或能表达或形成所述siRNA、反义核酸、miRNA、dsRNA、shRNA的构建物；更佳地，该干扰分子是shRNA，靶向于SEQ ID NO:1、SEQ ID NO:2或SED ID NO:3所示的核苷酸序列。
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