Pharmaceutical composition for removing senescent cells as well as preparation method and application of pharmaceutical composition

Abstract

The invention belongs to the technical field of pharmaceutical compositions for removing senescent cells, and discloses a pharmaceutical composition for removing senescent cells and a preparation method and application thereof, and the pharmaceutical composition for removing senescent cells contains NK cells and fisetin. According to the pharmaceutical composition for removing senescent cells, the fisetin and the NK cells are combined for use, on one hand, the fisetin can enable the NK cells to easily recognize senescent cells; on the other hand, fisetin can relieve inhibition of SASP factors on NK cell activity, enough amount of cytotoxic particles such as perforin and granzyme are synthesized and released in NK cells, the killing efficiency on senescent cells is high, fisetin can directly trigger an apoptosis pathway of the senescent cells, and the effect of killing the senescent cells is achieved. The NK cells release cytotoxic particles to further accelerate death of the senescent cells, and fisetin and the NK cells have a synergistic effect, so that the senescent cell clearing efficiency is high.

Description

Technical Field

[0001] This invention relates to the field of senescent cell removal technology, and more specifically, to a method and application for removing senescent cells. Background Technology

[0002] Normal cell division replenishes the body's losses, differentiates into functional cells, and maintains tissue structure and functional stability. As cells age, their function declines, and they release inflammatory factors, matrix-degrading enzymes, and other substances that continuously damage surrounding cells, inducing tissue aging and disease. Clearing senescent cells can reduce inflammatory damage, restore tissue regeneration capacity, and delay aging.

[0003] Current technologies typically utilize NK cells (natural killer cells) to eliminate senescent cells. The principle is that activating ligands (such as MICA / B, ULBP, RAE-1, and DNAM-1) on the surface of senescent cells are upregulated. NK cells recognize these ligands and release perforin, creating pores in the cell membrane. Granulase is then injected through these pores to induce apoptosis in the senescent cells. However, the effectiveness of NK cells in eliminating senescent cells is currently limited, primarily due to the following reasons: (1) Weak ability to recognize senescent cells. On the one hand, some senescent cells do not upregulate or do not upregulate enough signaling molecules such as MICA / B, ULBP, RAE-1, and DNAM-1, making it difficult for NK cells to recognize them; on the other hand, senescent cells will actively upregulate immune checkpoint molecules such as PD-L1. PD-L1 binds to PD-1 on the surface of NK cells, transmitting inhibitory signals to block NK cells from recognizing senescent cells.

[0004] (2) Weak ability to kill senescent cells. Senescent cells secrete a large number of senescence-related SASP factors (such as IL-6, TNF-α, etc.), which inhibit the activity of NK cells. The synthesis and release of cytotoxic particles such as perforin and granzymes in NK cells will be reduced, resulting in low efficiency in killing senescent cells. Summary of the Invention

[0005] The present invention provides a pharmaceutical composition for eliminating senescent cells, which is superior to the prior art in eliminating senescent cells.

[0006] Another object of the present invention is to provide a method for preparing the pharmaceutical composition for clearing senescent cells.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows: A pharmaceutical composition for eliminating senescent cells, comprising NK cells and fisetin.

[0008] In the pharmaceutical composition for clearing senescent cells of the present invention, fisetin and NK cells are used in combination to effectively clear senescent cells, and the following technical effects are achieved: (1) Strong ability to recognize senescent cells. On the one hand, fisetin can induce senescent cells to upregulate the expression of one or more activating ligands on the surface of NK cells (such as MICA / B, ULBP, RAE-1, DNAM1 signaling molecules), and on the other hand, it can inhibit the expression of immune checkpoint molecules on the surface of senescent cells (such as PD-L1), making it easier for NK cells to recognize senescent cells.

[0009] (2) Strong killing ability against senescent cells. On the one hand, fisetin can significantly downregulate the SASP factors (such as IL-6 and TNF-α) secreted by senescent cells, thereby relieving the inhibition of NK cell activity by SASP factors. As a result, NK cells can synthesize and release sufficient amounts of perforin, granzymes and other cytotoxic particles, thus achieving high killing efficiency against senescent cells. On the other hand, fisetin can inhibit the activity of anti-apoptotic proteins (such as BCL-2 and BCL-XL) in senescent cells, directly triggering the apoptosis pathway of senescent cells. The release of cytotoxic particles by NK cells further accelerates the death of senescent cells. The synergistic effect of fisetin and NK cells results in high clearance efficiency of senescent cells.

[0010] Preferably, the content of fisetin in the pharmaceutical composition for clearing senescent cells is 10-100 μM.

[0011] The fisetin described in this invention can be fisetin or a derivative with fisetin as the main active ingredient.

[0012] Preferably, the NK cell content in the pharmaceutical composition for clearing senescent cells is 5 × 10⁻⁶. 4 ~ 4×10 6 per mL.

[0013] The NK cells in the pharmaceutical composition for clearing senescent cells described in this invention are of autologous and / or allogeneic origin.

[0014] Preferably, the NK cells in the pharmaceutical composition for eliminating senescent cells are one or more of umbilical cord blood, peripheral blood, bone marrow, and induced pluripotent stem cells.

[0015] Preferably, the senescent cells in the pharmaceutical composition for eliminating senescent cells are one or more of immune cells, nerve cells, epithelial cells, cardiomyocytes, lymphocytes, osteoblasts, fibroblasts, and germ cells.

[0016] The method for preparing senescent cells described in this invention includes induced cellular senescence under external conditions and / or natural cellular senescence.

[0017] The method for preparing cells senescence induced by external conditions described in this invention includes any one or a combination of several of the following: ionizing radiation induction, chemical drug induction, ultraviolet radiation induction, protease inhibitor induction, or overexpression of senescence genes.

[0018] Preferably, the pharmaceutical composition for clearing senescent cells further contains excipients.

[0019] More preferably, the excipients in the pharmaceutical composition for clearing senescent cells are selected from one or more of diluents, surfactants, solubilizers, antioxidants, preservatives, buffers, and pH adjusters.

[0020] Preferably, the dosage form of the pharmaceutical composition for clearing senescent cells is selected from one or more of the following: injection, suspension, solution, enema, and emulsion.

[0021] The present invention also provides a method for preparing any of the above-mentioned drug compositions for clearing senescent cells, specifically: co-culturing NK cells and fisetin to obtain a drug composition for clearing senescent cells.

[0022] Preferably, in the preparation method of the drug composition for clearing senescent cells, the ratio of fisetin to NK cells is (10~100) μM:(5×10 4 ~ 4×10 6 ) per mL.

[0023] The present invention provides a method for eliminating senescent cells, comprising the step of mixing senescent cells with a pharmaceutical composition for eliminating senescent cells as described in any one of the claims.

[0024] The method for removing senescent cells specifically includes: adjusting the density of senescent cells in the culture medium to 2.5 × 10⁻⁶. 5 ~2×10 6 Cells / mL, and co-culture with any of the above-mentioned senescent cell-clearing drug compositions at an effect-to-target ratio of 1:(0.5-5).

[0025] The culture medium described in this invention is selected from Gibco CTS NK. Xpander culture medium, OptiVitro NK cell culture medium, Yocon NK cell culture medium, AMMS NK cell culture medium, or SuperCulture N100 NK culture medium. Attached Figure Description

[0026] Figure 1 The T cell mortality rate was detected in Example 1 and Comparative Examples 4-6.

[0027] Figure 2 The results of aging biomarker detection for Example 1 and Comparative Examples 4-6 are shown, where: A is a representative flow cytometry plot and B is a statistical plot of SA-β-Gal (MFI).

[0028] Figure 3The results of senescent cell clearance detection obtained in Example 4 and Comparative Examples 1-3 are shown in Figure A, where: A is the curve of relative fluorescence intensity changing with time, and B is the relative fluorescence intensity of the last point recorded in Figure A. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments.

[0030] Example 1 A pharmaceutical composition for eliminating senescent cells, comprising NK cells and fisetin, wherein the fisetin content is 20 μM, and the ratio of fisetin to NK cells is 20 μM: 1 × 10⁻⁶. 6 per mL.

[0031] The preparation method of the pharmaceutical composition for eliminating senescent cells is as follows: co-culturing NK cells and fisetin to obtain the pharmaceutical composition for eliminating senescent cells.

[0032] The specific methods for eliminating senescent cells are as follows: The cells that need to be eliminated as senescent cells are T cells, cultured with 1 × 10⁻⁶ cells. 6 T cells were resuspended at a density of cells / ml and seeded into 48-well plates at a volume of 100 μl per well. The above-mentioned drug composition for eliminating senescent cells was added at an effector-target ratio of 1:1 and cultured overnight.

[0033] Example 2 This embodiment is the second embodiment of the present invention. The difference from Embodiment 1 is that the fisetin content is 10 μM, and the ratio of fisetin to NK cells is 10 μM: 4 × 10⁻⁶. 6 per mL.

[0034] The preparation method of the pharmaceutical composition for eliminating senescent cells is as follows: co-culturing NK cells and fisetin to obtain the pharmaceutical composition for eliminating senescent cells.

[0035] The specific methods for eliminating senescent cells are as follows: The cells that need to be eliminated as senescent cells are T cells, and they are cultured with a concentration of 2 × 10⁶ cells / mL. 6 T cells were resuspended at a density of cells / ml and seeded into 48-well plates at a volume of 100 μl per well. The above-mentioned drug composition for eliminating senescent cells was added at an effector-target ratio of 1:0.5, and the cells were cultured overnight.

[0036] Example 3 This is the third embodiment of the present invention. Unlike embodiment 1, the fisetin content is 100 μM, and the ratio of fisetin to NK cells is 100 μM: 5 × 10⁻⁶. 4 per mL.

[0037] The preparation method of the pharmaceutical composition for eliminating senescent cells is as follows: co-culturing NK cells and fisetin to obtain the pharmaceutical composition for eliminating senescent cells.

[0038] The specific methods for eliminating senescent cells are as follows: The cells that need to be eliminated as senescent cells are T cells, cultured in a medium at 2.5 × 10⁻⁶. 5 T cells were resuspended at a density of cells / ml and seeded into 48-well plates at a volume of 100 μl per well. The above-mentioned drug composition for eliminating senescent cells was added at an effector-target ratio of 1:5, and the cells were cultured overnight.

[0039] Example 4 This is the fourth embodiment of the present invention. Unlike Embodiment 1, it includes a pharmaceutical composition for eliminating senescent cells, containing NK cells and fisetin, wherein the fisetin content is 20 μM, and the ratio of fisetin to NK cells is 20 μM:7.5 × 10⁻⁶. 4 Cells / mL. Senescent cells that need to be removed are IMR-90-HRAS-GFP.

[0040] The specific methods for eliminating senescent cells are as follows: The cell type requiring senescent cell removal is IMR-90-HRAS-GFP. IMR-90 is a fibroblast isolated from human embryonic lung tissue. Overexpression of HRAS in IMR-90 cells promotes their senescence. Preparation of IMR-90-HRAS-GFP cells: IMR-90 cells were cultured at 1 × 10⁻⁶ ppm... 6 Resuspended at a density of cells / ml, and then seeded into 6-well plates at a volume of 100 μl per well. IMR-90 cells were transduced with lentivirus at an MOI of 5 IU / ml to obtain IMR-90-HRAS-GFP cells that overexpress HRAS and green fluorescent protein (GFP).

[0041] Use culture medium at 1 × 10 5 IMR-90-HRAS-GFP cells were resuspended at a density of cells / ml and seeded into 96-well plates at a volume of 100 μl per well. After being cultured overnight in an IncuCyte SX5 live cell imaging analysis system, the above-mentioned drug composition for eliminating senescent cells was added at an effector-target ratio of 0.75:1, and the cells were cultured overnight. The proliferation of IMR-90-HRAS-GFP cells was recorded in real time.

[0042] Comparative Example 1 This comparative example is the first comparative example of the present invention. Unlike Example 4, the pharmaceutical composition for clearing senescent cells does not contain NK cells or fisetin, but only a culture medium.

[0043] Comparative Example 2 This comparative example is the second comparative example of the present invention. Unlike Example 4, the pharmaceutical composition for clearing senescent cells contains only fisetin and no NK cells.

[0044] Comparative Example 3 This comparative example is the third comparative example of the present invention. Unlike Example 4, the pharmaceutical composition for clearing senescent cells contains only NK cells and no fisetin.

[0045] Comparative Example 4 This comparative example is the fourth comparative example of the present invention. Unlike Example 1, the pharmaceutical composition for clearing senescent cells contains only NK cells and no fisetin.

[0046] Comparative Example 5 This comparative example is the fifth comparative example of the present invention. Unlike Example 1, the pharmaceutical composition for clearing senescent cells contains only fisetin and no NK cells.

[0047] Comparative Example 6 This comparative example is the sixth comparative example of the present invention. Unlike Example 1, the pharmaceutical composition for clearing senescent cells does not contain NK cells or fisetin, but only a culture medium.

[0048] Performance testing: 1. Results of detection of senescent T cells by the pharmaceutical compositions for clearing senescent cells obtained in Examples 1-3 and Comparative Examples 4-6.

[0049] (1) Detection of T cell mortality rate After centrifugation at 600 ×g at room temperature for 5 min, the supernatant was discarded, and the cells were stained for 30 min with BV421-labeled anti-KLRG1 antibody, BV510-labeled anti-CD28 antibody, PE-labeled anti-CD3 antibody, APC3-labeled anti-CD56 antibody, and APC-Cy7-labeled cell viability dye. After centrifugation at 600 ×g at room temperature for 5 min, the supernatant was discarded, and the cells were washed three times with PBS. The cells were resuspended in 200 μl of PBS and analyzed by flow cytometry. See Example 1 and Comparative Examples 4-6 for the detection of T cell mortality. Figure 1 .

[0050] Figure 1Gating analysis of CD3+CD28+KLRG1- young T cells and CD3+CD28-KLRG1+ senescent T cells showed that neither fisetin nor NK cells affected the mortality rate of young cells. The senescent cell-clearing drug composition in Comparative Example 5, containing only fisetin, increased the mortality rate of senescent T cells by 6.6% when co-cultured with T cells. The senescent cell-clearing drug composition in Comparative Example 4, containing only NK cells, increased the mortality rate of senescent T cells by 24.6% when co-cultured with T cells. The combined increase in mortality rate was 31.2%. In contrast, the senescent cell-clearing drug composition in Example 1, co-cultured with both fisetin and NK cells, increased the mortality rate of senescent T cells by 35.1%. This indicates that the senescent cell-clearing drug composition of the present invention exhibits a synergistic effect between its main components, fisetin and NK cells, in the process of clearing senescent cells.

[0051] (2) Detect the expression levels of T cell senescence markers After culture, cells were centrifuged at 600 ×g for 5 min at room temperature, the supernatant was discarded, and the cells were stained with PE-labeled anti-CD3 antibody and APC-labeled anti-CD56 antibody for 30 min. After centrifugation at 600 ×g for 5 min at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. Cells were fixed with 2% paraformaldehyde for 10 min, centrifuged at 600 ×g for 5 min at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. Cells were stained with β-Gal, a senescence assessment reagent, at 37℃ for 2 h to detect changes in senescence markers. After centrifugation at 600 ×g for 5 min at room temperature, the supernatant was discarded, and the cells were washed three times with PBS. Cells were resuspended in 200 μl of PBS for flow cytometry analysis. The expression level of senescence-associated β-galactosidase (SA-β-Gal) was detected during senescence.

[0052] The results of senescent marker detection after the drug compositions for clearing senescent cells obtained in Examples 1 and Comparative Examples 4-6 are shown in the figure. Figure 2 , Figure 2 A in the diagram is a representative flow cytometry plot. Figure 2 Table B shows the SA-β-Gal (MFI) statistical chart. The decline rates of aging markers in Examples 1-3 and Comparative Examples 4-6 are shown in Table 1. Figure 2As shown in Tables A, 2B, and 1, compared to the untreated group in Comparative Example 6, the SA-β-Gal activity levels of T cells decreased after treatment with fisetin or NK cells (P<0.05). The senescence marker reduction rates of the drug compositions for clearing senescent cells obtained in Comparative Examples 5 and 4 were 14.6% and 32.6%, respectively, after clearing senescent T cells, with a combined effect of 47.2%. In Example 1, the senescence marker reduction rate of the drug composition for clearing senescent cells was 58.0% after combined use of fisetin and NK cells. This indicates that the main components of the drug composition for clearing senescent cells obtained in this invention, fisetin and NK cells, have a synergistic effect in clearing senescent T cells.

[0053] Table 1. Decline rate of aging markers in Examples 1-3 and Comparative Examples 4-6

[0054] 2. Results of the drug compositions obtained in Example 4 and Comparative Examples 1-3 for clearing senescent cells and for clearing IMR-90-HRAS-GFP.

[0055] After culture, the changes in GFP fluorescence signal of IMR-90-HRAS-GFP cells in each group were calculated using IncuCyte SX5 software. Lower signal values ​​indicated fewer cells in that group and a better effect on killing IMR-90-HRAS-GFP cells. The results of senescent cell clearance detection obtained in Example 4 and Comparative Examples 1-3 are shown below. Figure 3 , Figure 3 Figure A shows the curve of relative fluorescence intensity changing over time, and Figure B shows the relative fluorescence intensity of the last point recorded in Figure A.

[0056] Figure 3It can be seen that: the drug composition for eliminating senescent cells obtained in Comparative Example 2 showed no significant difference in fluorescence curves after eliminating IMR-90-HRAS-GFP cells (the relative fluorescence intensity at the last time point showed no statistically significant difference, P>0.05), indicating that fisetin has no killing ability against IMR-90-HRAS-GFP cells; the drug composition for eliminating senescent cells obtained in Comparative Example 3 showed a significant difference in fluorescence curves compared to Comparative Example 1 after eliminating IMR-90-HRAS-GFP cells (the relevant fluorescence intensity at the last time point of Comparative Example 3 was significantly lower than that of Comparative Example 1, P<0.05), indicating that NK... Cells exhibit cytotoxic activity against IMR-90-HRAS-GFP. The drug composition for eliminating senescent cells obtained in Example 4, after eliminating IMR-90-HRAS-GFP cells, showed a significantly different fluorescence curve compared to Comparative Examples 2 and 3 (the relevant fluorescence intensity at the last time point in Example 4 was significantly lower than that in Comparative Examples 2 and 3, P < 0.05). This indicates that the T cells in the obtained drug composition for eliminating senescent cells, after combined use with fisetin and NK cells, have a stronger cytotoxic effect against IMR-90-HRAS-GFP than the combined effect of NK cells or fisetin alone, or simply the sum of their cytotoxic effects. This demonstrates that the main components of the drug composition for eliminating senescent cells obtained in this invention, fisetin and NK cells, have a synergistic effect in eliminating IMR-90-HRAS-GFP cells.

[0057] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection. Any non-substantial changes or substitutions made by those skilled in the art based on the present invention will still fall within the scope of patent protection.

Claims

1. A pharmaceutical composition for eliminating senescent cells, characterized in that, It contains NK cells and fisetin.

2. The pharmaceutical composition for eliminating senescent cells according to claim 1, characterized in that, The fisetin content is 10~100μM.

3. The pharmaceutical composition for eliminating senescent cells according to claim 1, characterized in that, The NK cell content was 5 × 10⁶. 4 ~ 4×10 6 per mL.

4. The pharmaceutical composition for eliminating senescent cells according to claim 1, characterized in that, The NK cells are one or more of the following: umbilical cord blood, peripheral blood, bone marrow, and induced pluripotent stem cells.

5. The pharmaceutical composition for eliminating senescent cells according to claim 1, characterized in that, The senescent cells are one or more of the following: immune cells, nerve cells, epithelial cells, cardiomyocytes, lymphocytes, osteoblasts, fibroblasts, and germ cells.

6. The pharmaceutical composition for eliminating senescent cells according to claim 1, characterized in that, It also contains excipients.

7. The pharmaceutical composition for eliminating senescent cells according to claim 5, characterized in that, The excipients are selected from one or more of the following: diluents, surfactants, solubilizers, antioxidants, preservatives, buffers, and pH adjusters.

8. The pharmaceutical composition for eliminating senescent cells according to claim 1, characterized in that, The dosage form of the pharmaceutical composition is selected from one or more of the following: injection, suspension, solution, enema, and emulsion.

9. A method for preparing the pharmaceutical composition for clearing senescent cells according to any one of claims 1 to 8, characterized in that: NK cells were co-cultured with fisetin to obtain a drug composition for eliminating senescent cells.

10. The method for preparing the pharmaceutical composition for clearing senescent cells according to claim 9, characterized in that, The ratio of fisetin to NK cells was (10~100) μM:(5×10 4 ~ 4×10 6 ) per mL.

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