A drug for effectively clearing senescent intestinal cells and its application in treating intestinal inflammatory diseases

Abstract

The present invention relates to a drug for effectively clearing senescent intestinal cells and its application in treating intestinal inflammatory diseases, belonging to the field of drugs. It is found that cobimetinib can be used as an effective drug for specifically clearing senescent intestinal cells and is a potential drug for treating inflammatory bowel disease.

Description

Technical Field

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

[0002] The accumulation of senescent cells is an important driving force for individual aging and the occurrence of 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 research on selectively clearing senescent cells is the most extensive. The drugs targeting the clearance of senescent cells are called senolytics. Compared with continuously administered drugs, 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 anti-aging field.

[0003] Inflammatory bowel disease (IBD) is a chronic intestinal disease, and the number of patients is continuously increasing globally. Its symptoms mainly include diarrhea, abdominal pain, bloody stools, etc., and it is prone to recurrent attacks, bringing a great burden to patients and seriously affecting the quality of life. Currently, the pathogenesis of IBD is not clear, and patients need to take aminosalicylate preparations, hormonal anti-inflammatory drugs, etc. for a long time. Existing studies have shown that high expressions of senescence markers p16 and p21 are observed in the colon tissues of patients with Crohn's disease and in the colon tissues of ulcerative colitis mice, proving the characteristic of an increase in senescent cells in the colon tissues during the pathogenesis of IBD. Therefore, the development of drugs that specifically clear senescent colon cells is of great significance for the treatment of IBD.

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

[0005] Senolytic drugs act by targeting survival pathways that are dependent on senescent cells. 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 such as dasatinib and quercetin: These are among the earliest discovered senolytic compounds. Dasatinib is a kinase inhibitor, while quercetin is a flavonoid. Studies have shown that their combined use can reduce the burden of senescent cells and improve healthspan in animal models. Another example is Navitoclax (ABT-263), which targets Bcl-2 family proteins and has been shown to effectively eliminate senescent cells in multiple tissues; and Fisetin: a natural flavonoid present in fruits and vegetables that has shown senolytic activity in preclinical studies.

[0006] MEK / ERK is an important signaling pathway that regulates senescence. It has been reported that the expression of phosphorylated ERK1 increases in NaBut-induced senescent cells, demonstrating that MEK1 / 2 is a target of senescent cells. Cobimetinib is an orally available MEK1 inhibitor with anti-cancer activity and is mainly used clinically to treat melanoma with BRAF V600E or V600K mutations. However, there is no clear research on the role of cobimetinib in selectively eliminating senescent intestinal cells at present.

[0007] Currently, there is no report 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 anti-cancer drug, especially in melanoma. However, the treatment research of 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 object of the present invention is to provide a drug that can effectively eliminate senescent intestinal cells and its application in the treatment of intestinal inflammatory diseases.

[0009] To achieve the above object, the technical solution of the present invention is realized as follows:

[0010] The first aspect of the present invention is to provide an application of a drug that can effectively eliminate senescent intestinal cells in the preparation of a drug for treating intestinal inflammatory diseases;

[0011] Furthermore, the active ingredient of the drug is cobimetinib, and the treatment is achieved by specifically eliminating senescent cells;

[0012] Further, the use concentration of cobimetinib is 0.78 - 12.5 μM;

[0013] Further, the dosage form of the drug includes at least one of suspension, granule, capsule, powder, tablet, dripping pill, injection, suppository, and drops;

[0014] Further, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration;

[0015] Further, the drug further includes pharmaceutically acceptable excipients, including at least one of diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, or buffer.

[0016] The second aspect of the present invention provides the use of cobimetinib in the preparation of a preparation for specifically clearing senescent intestinal cells;

[0017] Further, the use concentration of cobimetinib is 0.78 - 12.5 μM;

[0018] Further, the specific clearance is in vitro clearance.

[0019] The third aspect of the present invention is to provide a drug for effectively clearing senescent intestinal cells, which is characterized in that the drug is cobimetinib and a pharmaceutically acceptable carrier;

[0020] Further, the use concentration of cobimetinib is 0.78 - 12.5 μM;

[0021] Further, the dosage form of the drug includes at least one of suspension, granule, capsule, powder, tablet, dripping pill, injection, suppository, and drops.

[0022] The beneficial effects of the present invention different from the prior art are as follows:

[0023] 1) It is first discovered that cobimetinib can be used as an effective drug for specifically clearing senescent intestinal cells. The CCK-8 experiment proves that it has no activity of selectively clearing senescent cells in mouse cardiomyocytes (HL-1) and human non-small cell lung cancer cells (A549), but has the activity of selectively clearing senescent cells in human normal intestinal epithelial cells (NCM460) and human colorectal adenocarcinoma cells (Caco-2). This drug can be used for subsequent exploration of the treatment of IBD. Especially from the perspective of anti-aging, it will lay a foundation for analyzing the molecular mechanism of cobimetinib in the treatment of IBD. And subsequently, the combined effect of this drug with other natural compounds that can relieve IBD, such as curcumin with antibacterial and anti-inflammatory effects, can be explored to achieve better treatment effects. Brief Description of the Drawings

[0024] Figure 1 : (A) SA-β-gal staining image of untreated NCM460 cells, (B) SA-β-gal staining image of NCM460 cells treated with 15 μM cisplatin for 72 h.

[0025] 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.

[0026] Figure 3 : (A) SA-β-gal staining image of untreated A549 cells, (B) SA-β-gal staining image of A549 cells treated with 15 μM cisplatin for 72 h.

[0027] Figure 4 : (A) Viability of normal and senescent NCM460 cells treated with different concentrations of cobimetinib for 24 h, (B) Viability of normal and senescent Caco-2 cells treated with different concentrations of cobimetinib for 24 h, (C) Viability of normal and senescent HL-1 cells treated with different concentrations of cobimetinib for 24 h, (D) Viability of normal and senescent A549 cells treated with different concentrations of cobimetinib for 24 h, (E) Viability of normal and senescent NCM460 cells treated with different concentrations of dasatinib + quercetin combination and cobimetinib alone for 24 h. Detailed Embodiments

[0028] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. The methods are all conventional methods unless otherwise specified. The materials can all 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 an improper limitation of the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0029] In the experiment, cells from different tissue sources were selected for the experiment. A549 cells are non-small cell lung cancer cells, which can spread substances through alveolar diffusion and are mainly used for lung cancer research, toxicology research, etc.; HL-1 cells are a mouse cardiomyocyte cell line and are mainly used for research on cardiomyocytes in aspects such as signal transduction, cell metabolism, and transcriptional regulation; NCM460 cells are human normal colon epithelial cells and are widely used in the research of intestinal-related diseases; Caco-2 cells are human cloned colon adenocarcinoma cells, and their structure and function are similar to those of differentiated small intestinal epithelial cells and are mainly used for the study of drug intestinal permeability.

[0030] Example 1: Cobimetinib eliminates senescent cells

[0031] 1. Experimental procedures

[0032] (1) Construction of senescent cell models: NCM460 cells or A549 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.

[0033] (2) SA-β-gal staining: SA-β-gal staining was performed using a staining kit. First, cells were treated with 1 mL of cell fixation solution at room temperature for 15 min, and then washed 3 times with PBS. Subsequently, the cells were incubated with β-gal staining solution overnight at 37 °C. Finally, the cells were washed with PBS and imaged under an optical microscope for analysis.

[0034] The results are as Figure 1 shown. The experiment demonstrated that exposure to 15 μM cisplatin for 72 h could induce senescence in NCM460 cells ( Figure 1 in which A are untreated cells and B are treated cells); Figure 2 as shown, exposure to 30 g / L D-gal for 24 h could induce senescence in Caco-2 cells ( Figure 2 in which A are untreated cells and B are treated cells); similarly, Figure 3 as shown, exposure to 15 μM cisplatin for 72 h could induce senescence in A549 cells ( Figure 3 in which A are untreated cells and B are treated cells).

[0035] (3) Screening of different types of cells: Senescent and non-senescent HL-1, A549, NCM460, and Caco-2 cells (8×10 3 / well) were seeded into 96-well plates and allowed to adhere for 24 h, and then treated with different concentrations of cobimetinib for 24 h for HL-1, A549, NCM460, and Caco-2 cells respectively.

[0036] Cell viability detection: The inhibitory effect of different drugs on cell proliferation was detected using the CCK-8 method. After 24 h of drug treatment, 20 μL of CCK-8 was added to each well, incubated for 1 h, and the absorbance of each well at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell survival rate.

[0037] The results showed that: The CCK-8 experimental results indicated that cobimetinib could not selectively eliminate senescent HL-1 and A549 cells and had no senolytic activity ( Figure 4(C, D) in, but cobimetinib has the effect of selectively eliminating senescent NCM460 cells at a concentration of 0.78 - 12.5 μM ( Figure 4 (A) in, and also has the effect of selectively eliminating senescent Caco-2 cells at a concentration of 3.125 - 12.5 μM ( Figure 4 (B) in, and the effect is better than the D + Q combination ( Figure 4 (E) in, proving that cobimetinib is a drug that specifically eliminates senescent intestinal cells.

[0038] As described above, it is only a preferred embodiment of the present invention and is not used to limit the protection scope of the present invention.

Claims

1. Application of cobimetinib in preparing a preparation for specifically eliminating senescent cells in the intestine.

2. The application according to claim 1, wherein The usage concentration of cobimetinib is 0.78 - 12.5 μM.

3. The application according to claim 2, wherein The dosage form of the preparation includes at least one of suspension, granule, capsule, powder, tablet, dripping pill, injection, suppository, and drop.

4. The application according to claim 2, wherein The administration route of the preparation includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, and perfusion administration.

5. The application according to claim 2, wherein The preparation further includes pharmaceutically acceptable excipients, including at least one of diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, solubilizer, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, or buffer.

6. The application according to claim 1 or 2, characterized in that, The specific elimination is in vitro elimination.

Images