Medicine for effectively removing aged intestinal cells and application of medicine in treating intestinal inflammation

Abstract

The invention relates to a medicine for effectively removing aging intestinal cells and application of the medicine in treating intestinal inflammation diseases, belongs to the field of medicines, and finds that cobiitinib can be used as a medicine for effectively and specifically removing aging intestinal cells and is a potential medicine for treating inflammatory bowel diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a drug for effectively removing senescent intestinal cells and its application in treating intestinal inflammatory diseases, and specifically to the application of cobimetinib in treating inflammatory bowel disease by removing senescent cells. Background Art

[0002] The accumulation of senescent cells is an important driving force for individual aging and aging-related diseases, and is also recognized as one of the important targets for the treatment of aging-related diseases. Among the treatment methods targeting senescent cells, the most extensive research is on the selective elimination of senescent cells. Drugs that target the elimination of senescent cells are called senolytics. Compared with drugs that are taken continuously, senolytics can be used intermittently to reduce off-target or other side effects. Currently, the development of new senolytics has become one of the hottest topics in the field of anti-aging.

[0003] Inflammatory bowel disease (IBD) is a chronic intestinal disease, and the number of patients continues to grow worldwide. Its main symptoms include diarrhea, abdominal pain, bloody stools, etc., which are prone to recurrence, bringing great burdens to patients and seriously affecting their quality of life. At present, the pathogenesis of IBD is still unclear, and patients need to take aminosalicylic acid preparations, hormone anti-inflammatory drugs, etc. for a long time. Studies have shown that high expression of senescence markers p16 and p21 was observed in the colon tissue of Crohn's disease patients and the colon tissue of ulcerative colitis mice, proving that the colon tissue shows the characteristics of increased senescent cells during the onset of IBD. Therefore, the development of drugs that specifically eliminate senescent colon cells is of great significance for the treatment of IBD.

[0004] "Senolytic" refers to a class of agents that selectively induce the death of senescent cells. Senescent cells are old or damaged cells that have stopped dividing but remain metabolically active. These cells accumulate in tissues over time and promote aging and age-related diseases by secreting inflammatory cytokines, growth factors, and proteases (collectively known as the senescence-associated secretory phenotype, SASP).

[0005] Senolytic drugs work by targeting survival pathways that senescent cells rely on. These pathways include Bcl-2 family proteins, PI3K / AKT, and other pathways involved in cell survival and anti-apoptosis (programmed cell death). By disrupting these pathways, senolytic drugs can specifically induce apoptosis in senescent cells without affecting normal cells. Typical senolytic drugs are dasatinib and quercetin: this is one of the earliest discovered senolytic compounds. Dasatinib is a kinase inhibitor, while quercetin is a flavonoid. Studies have shown that their combination can reduce the burden of senescent cells and improve health lifespan in animal models. Another example is Navitoclax (ABT-263), which targets Bcl-2 family proteins and has been shown to effectively clear senescent cells in multiple tissues; and Fisetin: a natural flavonoid found in fruits and vegetables that has shown senolytic activity in preclinical studies.

[0006] MEK / ERK is an important signaling pathway for regulating aging. Studies have reported that the expression of phosphorylated ERK1 in NaBut-induced senescent cells increases, proving that MEK1 / 2 is a target of senescent cells. Cobimetinib is an orally available MEK1 inhibitor with anticancer activity. It is mainly used clinically to treat melanoma with BRAF V600E or V600K mutations. However, at this stage, there is no research to clarify the role of cobimetinib in selectively eliminating senescent intestinal cells.

[0007] Currently, there are no reports on the direct use of cobimetinib in the treatment of inflammatory bowel disease (IBD). Most research and clinical applications focus on its use as an anticancer drug, especially in melanoma. However, therapeutic research in IBD has been exploring different molecular targets and drugs, especially in regulating immune responses and inflammation. Summary of the invention

[0008] In view of this, the main purpose of the present invention is to provide a drug for effectively removing senescent intestinal cells and its use in treating intestinal inflammatory diseases.

[0009] To achieve the above object, the technical solution of the present invention is achieved as follows: The first aspect of the present invention is to provide a drug for effectively removing senescent intestinal cells for use in the preparation of a drug for treating intestinal inflammatory diseases; Furthermore, the active ingredient of the drug is cobimetinib, and the treatment is achieved by specifically removing senescent cells; Furthermore, the concentration of cobimetinib used is 0.78-12.5 μM; Furthermore, the dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops; Furthermore, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration; Furthermore, the drug also includes pharmaceutically acceptable excipients, including: at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a solubilizing agent, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

[0010] The second aspect of the present invention provides the use of cobimetinib in the preparation of a preparation for specifically eliminating intestinal senescent cells; Furthermore, the concentration of cobimetinib used is 0.78-12.5 μM; Furthermore, the specific clearance is in vitro clearance.

[0011] The third aspect of the present invention is to provide a drug that effectively removes senescent intestinal cells, characterized in that the drug is cobimetinib and a pharmaceutically acceptable carrier; Furthermore, the concentration of cobimetinib used is 0.78-12.5 μM; Furthermore, the dosage form of the drug includes at least one of a suspension, a granule, a capsule, a powder, a tablet, a pill, an injection, a suppository, and a drop.

[0012] The beneficial effects of the present invention compared with the prior art are: 1) For the first time, it was discovered that cobimetinib can be used as an effective drug for the specific removal of senescent intestinal cells. CCK-8 experiments have shown that it has no selective removal of senescent cells in mouse cardiomyocytes (HL-1) and human non-small cell lung cancer cells (A549), but has selective removal of senescent cells in human normal intestinal epithelial cells (NCM460) and human colorectal adenocarcinoma cells (Caco-2). This drug can be used for subsequent research on the treatment of IBD, especially from the perspective of anti-aging, which will lay the foundation for analyzing the molecular mechanism of cobimetinib in the treatment of IBD. In addition, the combined effects of this drug and other natural compounds that can alleviate IBD, such as curcumin with antibacterial and anti-inflammatory effects, can be explored in the future to achieve better therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : (A) SA-β-gal staining images of untreated NCM460 cells, (B) SA-β-gal staining images of NCM460 cells treated with 15 μM cisplatin for 72 h.

[0014] Figure 2 : (A) SA-β-gal staining image of untreated Caco-2 cells, (B) SA-β-gal staining image of Caco-2 cells treated with 30 g / L D-gal for 24 h.

[0015] Figure 3 : (A) SA-β-gal staining images of untreated A549 cells, (B) SA-β-gal staining images of A549 cells treated with 15 μM cisplatin for 72 h.

[0016] Figure 4 :(A) The viability of normal and senescent NCM460 cells treated with different concentrations of cobimetinib for 24 h,(B) The viability of normal and senescent Caco-2 cells treated with different concentrations of cobimetinib for 24 h,(C) The viability of normal and senescent HL-1 cells treated with different concentrations of cobimetinib for 24 h,(D) The viability of normal and senescent A549 cells treated with different concentrations of cobimetinib for 24 h,(E) The viability of normal and senescent NCM460 cells treated with different concentrations of dasatinib + quercetin and cobimetinib alone for 24 h. DETAILED DESCRIPTION

[0017] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0018] The experiment selected cells from different tissues. A549 cells are non-small cell lung cancer cells, which can diffuse substances through the alveoli and are mainly used in lung cancer research, toxicology research, etc.; HL-1 cells are mouse cardiomyocyte cell lines, which are mainly used for the study of cardiomyocytes in signal transduction, cell metabolism and transcriptional regulation; NCM460 cells are normal human colon epithelial cells, which are widely used in the study of intestinal related diseases; Caco-2 cells are human cloned colon adenocarcinoma cells, which have structures and functions similar to differentiated small intestinal epithelial cells and are mainly used in the study of drug small intestinal permeability.

[0019] Example 1 Cobimetinib eliminates senescent cells 1. Experimental Procedure (1) Construction of senescent cell model: NCM460 cells or A549 cells were exposed to 15 μM cisplatin for 72 h; Caco-2 cells were exposed to 30 g / L D-gal for 24 h; HL-1 cells were exposed to 500 nm doxorubicin for 24 h.

[0020] (2) SA-β-gal staining: SA-β-gal staining was performed using a staining kit. First, cells were treated with 1 mL of cell fixative at room temperature for 15 min and then washed three times with PBS. Subsequently, cells were incubated with β-gal staining solution at 37°C overnight. Finally, cells were washed with PBS and imaged under an optical microscope for analysis.

[0021] The results are as follows Figure 1 As shown, the experiment proved that 15 μM cisplatin induction for 72 hours can induce NCM460 cell senescence ( Figure 1 A represents untreated cells, and B represents treated cells); Figure 2 As shown, 30 g / L D-gal induction for 24 h can induce Caco-2 cell senescence ( Figure 2 A in the figure represents untreated cells and B represents treated cells); similarly, Figure 3 As shown, 15 μM cisplatin induced A549 cell senescence for 72 h ( Figure 3 A represents untreated cells and B represents treated cells).

[0022] (3) Screening of different cell types: Senescent and non-senescent HL-1, A549, NCM460, and Caco-2 cells (8×10 3 / well) were inoculated into 96-well plates and adhered to the wall for 24h, and HL-1, A549, NCM460, and Caco-2 cells were treated with different concentrations of cobimetinib for 24h.

[0023] Cell viability detection: CCK-8 method was used to detect the inhibitory effect of different drugs on cell proliferation. 20 μL CCK-8 was added to each well 24 hours after drug treatment, incubated for 1 hour, and the absorbance of each well at 450 nm was measured with an enzyme-labeled instrument to calculate the cell survival rate.

[0024] The results showed that the CCK-8 experimental results showed that cobimetinib could not selectively eliminate senescent HL-1 and A549 cells and had no senolytic activity ( Figure 4 C, D), but cobimetinib has the effect of selectively eliminating senescent NCM460 cells at a concentration of 0.78-12.5 μM ( Figure 4A in the figure), 3.125-12.5 μM concentration also has the effect of selectively eliminating senescent Caco-2 cells ( Figure 4 B in the above figure), and the effect is better than the combination of D+Q ( Figure 4 E), demonstrating that cobimetinib is a drug that specifically eliminates senescent intestinal cells.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention.

Claims

1. Use of a drug for effectively removing senescent intestinal cells in the preparation of a drug for treating intestinal inflammatory diseases, characterized in that: The active ingredient of the drug is cobimetinib, and the treatment is achieved by specifically eliminating senescent cells.

2. The use according to claim 1, characterized in that: The concentration of cobimetinib used is 0.78-12.5 μM.

3. The use according to claim 2, characterized in that: The dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops.

4. The use according to claim 1, characterized in that: The administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

5. The use according to any one of claims 1 to 4, characterized in that: The drug also includes pharmaceutically acceptable excipients, including at least one of a diluent, a binder, a wetting agent, a lubricant, a disintegrant, a solvent, an emulsifier, a cosolvent, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant or a buffer.

6. The application of cobimetinib in the preparation of preparations for specific elimination of intestinal senescent cells.

7. The use according to claim 6, characterized in that: The concentration of cobimetinib used is 0.78-12.5 μM.

8. The use according to claim 6 or 7, characterized in that: The specific clearance is in vitro clearance.

9. A drug for effectively removing senescent intestinal cells, characterized in that: The drug is cobimetinib and a pharmaceutically acceptable carrier; characterized in that the concentration of cobimetinib is 0.78-12.5 μM.

10. The drug according to claim 9, characterized in that The dosage form of the drug includes at least one of suspension, granules, capsules, powders, tablets, pills, injections, suppositories, and drops.

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