Application of clofazimine in the preparation of drugs for the treatment of gastric cancer

Abstract

This invention belongs to the fields of pharmaceutical application and biomedicine, specifically relating to the application of clofazimine in the preparation of drugs for treating gastric cancer. This invention is the first to discover that clofazimine possesses significant anti-gastric cancer activity, which is confirmed through systematic experiments. This activity stems from its ability to induce mitochondrial damage and simultaneously cause lysosomal dysfunction, leading to lethal inhibition of autophagic flux. In vitro, clofazimine can inhibit gastric cancer cell viability and induce apoptosis in a concentration-dependent manner; in vivo, clofazimine effectively inhibits tumor growth and exhibits good biocompatibility. This represents the development of a novel use for a known drug, providing a potential new treatment option for gastric cancer treatment, particularly overcoming the limitations of existing treatment strategies.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical application and biomedical technology, and more specifically, to the use of clofazimine in the preparation of drugs for the treatment of gastric cancer. Background Technology

[0002] Clofazimine is a synthetic rifamycin derivative that, since its development in the 1950s, has primarily been used to treat leprosy and certain drug-resistant tuberculosis diseases. Its core pharmacological characteristic lies in its unique dual mechanism of action: potent antibacterial activity and significant anti-inflammatory / immunomodulatory activity. Despite its long history of clinical application, research reports on the effects of clofazimine on gastric cancer cells are scarce to date, and its application and mechanism of action in gastric cancer treatment remain largely unexplored. Summary of the Invention

[0003] In view of this, in order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a new use of clofazimine in the preparation of anti-gastric cancer drugs.

[0004] This invention reveals for the first time that clofazimine can exert a significant anti-gastric cancer effect by inducing mitochondrial damage and simultaneously causing lysosomal dysfunction, thereby leading to the inhibition of autophagic flux in gastric cancer cells.

[0005] In a first aspect, the present invention provides the use of clofazimine in the preparation of a medicament for the treatment of gastric cancer.

[0006] In this invention, the "treatment of gastric cancer" is demonstrated through one or more of the following aspects: In vitro killing effect: It inhibits the activity of gastric cancer cells in a concentration-dependent manner and induces apoptosis.

[0007] In vivo antitumor activity: In a tumor-bearing mouse model, it inhibited tumor growth in a dose-dependent manner.

[0008] Mechanism verification: The effect is achieved by inducing "autophagic flow arrest". This arrest is manifested as the activation of autophagy, but the degradation process of autophagosome contents is blocked.

[0009] Secondly, the present invention provides the use of clofazimine in the preparation of a drug for inducing autophagy flow inhibition in gastric cancer cells.

[0010] Thirdly, this invention provides the use of clofazimine in the preparation of a drug for inducing apoptosis in gastric cancer cells.

[0011] Fourthly, the present invention provides a pharmaceutical composition for treating gastric cancer.

[0012] The pharmaceutical composition comprises clofazimine as the active ingredient and a pharmaceutically acceptable carrier.

[0013] Preferably, the pharmaceutically acceptable carrier is suitable for injection administration; more preferably, the carrier is a solution suitable for intravenous injection, such as a physiological saline solution containing a solubilizer.

[0014] Fifthly, the present invention provides a method for treating gastric cancer, comprising administering an effective amount of clofazimine to a subject in need of such treatment.

[0015] Preferably, the method of administration is injection.

[0016] Preferably, the dosage is 2-8 mg / kg, administered once every other day.

[0017] The significant advantages of this invention compared to the prior art are as follows: This application is the first to discover that clofazimine has a significant killing effect on gastric cancer cells, and its anti-gastric cancer activity and mechanism of action have been verified through in vitro and in vivo experimental systems. This represents the secondary development and utilization of an already marketed drug, providing a potential new treatment option for gastric cancer patients.

[0018] This application experimentally demonstrates that clofazimine kills gastric cancer cells by inducing mitochondrial damage and inhibiting the maturation of lysosomal cathepsins (CTSB / CTSD), thereby blocking autophagic flux during the degradation phase. This mechanism has not been reported in the field of gastric cancer treatment both domestically and internationally, representing an important innovation in drug action mode. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 The inhibitory effect of clofazimine on the activity of gastric cancer cells and its IC50 value provided in this application. 50 calculate.

[0021] Figure 2 The flow cytometry results for clofazimine-induced apoptosis in gastric cancer cells provided in this application.

[0022] Figure 3 The antitumor effect of clofazimine provided in this application in tumor-bearing mice is shown in Figure A, which represents the actual size of tumor tissue after treatment with different drug concentrations; Figure B shows the change in tumor volume over time after treatment with different drug concentrations; and Figure C shows the tumor weight at the experimental endpoint after treatment with different drug concentrations.

[0023] Figure 4The safety evaluation results of clofazimine in experimental mice provided in this application are shown in Figure A, which shows the change in mouse body weight over time after treatment with different drug concentrations; Figure B shows histopathological sections of the major organs of mice after treatment with different drug concentrations of clofazimine.

[0024] Figure 5 RNA-seq analysis provided for this application showed that clofazimine activates the autophagy signaling pathway.

[0025] Figure 6 The Western Blot provided in this application was used to verify the effects of clofazimine on autophagy marker proteins and lysosomal structural proteins.

[0026] Figure 7 The Western Blot provided in this application was used to verify the effect of clofazimine on the expression of the lysosomal structural marker protein LAMP1.

[0027] Figure 8 The Western Blot provided in this application was used to verify the inhibitory effect of clofazimine on lysosomal functional proteins.

[0028] Figure 9 The image shows the results of transmission electron microscopy (TEM) observations provided in this application. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in multiple embodiments of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] Unless otherwise specified, specific techniques or conditions in the embodiments described herein shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available products. Example 1: The killing effect and apoptosis-inducing effect of clofazimine on gastric cancer cells in vitro. 1. Experimental Materials and Methods 1.1 Cell lines and reagents Human gastric cancer cell line MKN45 was obtained from the Cell Bank of the Chinese Academy of Sciences; NUGC4 was obtained from Cyagen Biosciences Co., Ltd., and was cultured in a conventional incubator at 37°C and 5% CO2 using a culture medium containing 10% fetal bovine serum.

[0032] Clofazimine (CLF) was dissolved in DMSO to prepare a stock solution and stored frozen. Before use, it was diluted to the required working concentration to ensure that the final concentration of DMSO was consistent across all experimental groups and did not affect cell viability.

[0033] 1.2 Cell viability assay (CCK-8 assay) MKN45 and NUGC4 cells in logarithmic growth phase were seeded at a density of 5 × 10³ cells per well in 96-well plates. After attachment, the medium was replaced with different concentrations of clofazimine (0 μM, 1.25 μM, 2.5 μM, 3 μM, 4 μM), with five replicates for each concentration. After culturing for 24 hours, 10 μL of CCK-8 solution was added to each well, and after incubation for 2 hours, the absorbance was measured at 450 nm using a microplate reader. Cell viability and half-maximal inhibitory concentration (IC50) were calculated using GraphPad Prism software. 50 ).

[0034] 1.3 Apoptosis detection (flow cytometry) MKN45 and NUGC4 cells were spaced at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. After adhesion, cells were treated with 0 μM, 1 μM, 2 μM, and 4 μM clofazimine for 24 hours, respectively. Cells were collected, analyzed using an apoptosis detection kit, and then analyzed by flow cytometry. The total apoptosis rate (total apoptosis rate = percentage of cells in quadrants Q2 + Q3) was analyzed using FlowJo software.

[0035] 2. Experimental Results 2.1 Clofazimine effectively inhibits the activity of gastric cancer cells. Please see Figure 1 CCK-8 assay results showed that clofazimine significantly inhibited the viability of MKN45 and NUGC4 gastric cancer cells in a concentration-dependent manner. The IC50 values ​​of clofazimine for MKN45 and NUGC4 cells were calculated. 50 The median lethal concentrations (LD50) were 3.267 μM and 3.962 μM, respectively. Based on these results, the drug concentrations for subsequent in vitro experiments were determined to be 0 μM, 1 μM, 2 μM, and 4 μM.

[0036] 2.2 Clofazimine-induced apoptosis in gastric cancer cells Please see Figure 2 Flow cytometry analysis showed that the apoptosis rate of gastric cancer cells significantly increased with increasing clofazimine concentration. In MKN45 cells, the total apoptosis rate increased from 3.72% in the control group to 27.2% in the 4 μM drug treatment group; in NUGC4 cells, the total apoptosis rate increased from 4.06% in the control group to 47.9% in the 4 μM drug treatment group. This example demonstrates that clofazimine has significant inhibitory effects on the proliferation of gastric cancer cells and induces apoptosis in vitro.

[0037] Example 2: Evaluation of the in vivo inhibitory effect of clofazimine on gastric cancer growth and its safety. 1. Experimental Materials and Methods 1.1 Establishment of animal models Using BALB / c nude mice, 6 weeks old, 5×10⁻⁶ mg / L was subcutaneously injected into the right back. 6 NUGC4 cells were used to establish a tumor-bearing model. When the tumor volume grew to approximately 100 mm³, the mice were randomly divided into 4 groups (n=5) and drug administration was initiated.

[0038] 1.2 Dosing regimen and observation Different groups were administered different doses of clofazimine via tail vein injection: Control group: Administered an equal volume of drug solvent.

[0039] Low-dose group: 2 mg / kg clofazimine.

[0040] Medium dose group: 4 mg / kg clofazimine.

[0041] High-dose group: 8 mg / kg clofazimine.

[0042] The medication was administered every other day until day 13. During this period, tumor volume was measured every two days using calipers, and mouse body weight was recorded daily.

[0043] Mice were sacrificed on day 13, and tumors were removed and weighed. Simultaneously, major organs such as the heart, liver, spleen, lungs, and kidneys were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) for histopathological safety assessment.

[0044] 2. Experimental Results 2.1 Clofazimine effectively inhibits tumor growth in vivo. Please see Figure 3 In vivo efficacy experiments showed that as drug concentration increased, the volume and weight of tumors in mice gradually decreased, indicating that clofazimine has good antitumor activity; details are as follows: Figure 3 A is a picture of the tumor. The results show that, compared with the 0 mg / kg control group, the tumor volume decreased significantly in a dose-dependent manner as the dosage increased from 2 mg / kg to 8 mg / kg. The tumor volume was the smallest in the 8 mg / kg group. The inhibitory effect of clofazimine on tumor growth increased with increasing dosage. Figure 3 B shows the tumor volume change over time. The results indicate that the tumor volume in the 0 mg / kg control group increased rapidly over time; the tumor growth rate in the 2 mg / kg, 4 mg / kg, and 8 mg / kg dose groups was significantly slower than that in the control group, and the higher the dose, the stronger the tumor growth inhibition effect. The differences between each dose group and the control group were statistically significant. Figure 3 C represents the experimental endpoint of gastric cancer cell tumor weight. The results showed that at the experimental endpoint, tumor weight decreased in a concentration-dependent manner with increasing drug concentration: there was no significant difference in tumor weight between the 2 mg / kg group and the control group (0 mg / kg); the tumor weight of the 4 mg / kg group was significantly lower than that of the control group; the tumor weight of the 8 mg / kg group was significantly lower than that of the control group, and the reduction was more significant, indicating the most prominent tumor-suppressing effect; all the above differences between groups were statistically significant.

[0045] In summary, as the drug concentration increased, the tumor volume and weight in mice gradually decreased, indicating that clofazimine has good anti-gastric cancer tumor activity.

[0046] 2.2 Chlorfazimine exhibits good biocompatibility. Please see Figure 4 A. Throughout the administration period, the body weight of mice in each group did not decrease significantly; Please see Figure 4 B. Pathological examination of HE-stained sections of major organs revealed no significant drug-related inflammatory cell infiltration or organic lesions.

[0047] These results indicate that clofazimine exhibits good biocompatibility at doses that effectively inhibit gastric cancer growth in vivo.

[0048] Example 3: Clofazimine exerts its anti-gastric cancer effect by inducing autophagic flux inhibition. 1. Experimental Materials and Methods 1.1 Transcriptome sequencing (RNA-seq) A control group and a treatment group were set up. The treatment group was treated with NUGC4 cells at 1 μM, 2 μM, and 4 μM chlorofazimine for 24 hours, after which total RNA was extracted from the cells. Transcriptome sequencing was performed by Shanghai Ouyi Biomedical Technology Co., Ltd. KEGG pathway analysis was performed on the sequencing data.

[0049] 1.2 Western Blot MKN45 and NUGC4 cells were treated with different concentrations (0 μM, 1 μM, 2 μM, 4 μM) of chlorozimine for 24 hours. Total protein was extracted from the cells, quantified using the BCA method, and then subjected to SDS-PAGE electrophoresis followed by membrane transfer. Cells were incubated with specific primary antibodies, followed by incubation with corresponding secondary antibodies, and then visualized by chemiluminescence. β-actin was used as an internal control to analyze the expression level of the target protein.

[0050] 2. Experimental Results 2.1 Clofazimine activates the autophagy signaling pathway and induces autophagy flow arrest. Please see Figure 5 KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in the "Mitophagy-animal" and "Autophagy-animal" pathways, suggesting that the autophagy signaling pathway (mitochondrial autophagy) was significantly activated. Please see Figure 6 Western blotting results further validated this mechanism: with increasing clofazimine concentration, the expression levels of autophagy activation markers ATG-5 and LC3-II (autophagosome membrane markers) were significantly upregulated; simultaneously, the expression level of the autophagy substrate protein p62 also significantly accumulated. This indicates that although clofazimine activates autophagy initiation, the autophagic contents are not effectively degraded by lysosomes, leading to late-stage arrest of autophagic flux.

[0051] 2.2 Chlorophosphatidylcholine does not damage lysosomal structure. As verified in section 2.1 of this embodiment, the autophagy contents were not effectively degraded by lysosomes. There are three possible reasons why lysosomes failed to effectively degrade the autophagy contents: 1. There is a structural defect in the lysosome; 2. There is a functional defect in the lysosome; 3. The autophagosome failed to fuse with the lysosome.

[0052] To investigate the reasons for the stagnation of autophagic flow, please refer to [link to relevant documentation]. Figure 7 Further investigation was conducted to examine the expression of LAMP1, a structural marker protein of lysosomes. Western blot results showed that after treatment with clofazimine, the expression level of LAMP1 not only did not decrease, but actually increased with increasing drug concentration, indicating that the lysosomal membrane structure was intact and that the autophagic flux blockade induced by clofazimine was not due to the destruction of the lysosomal structure.

[0053] Example 4: Clofazimine exerts its effects by impairing lysosomal function and inducing mitochondrial damage. Based on the results of Example 3, this invention has confirmed that clofazimine induced autophagic flux retardation without damaging the lysosomal structure; to clarify whether this retardation was due to lysosomal dysfunction or failure of autophagosome-lysosome fusion, the following experiments were conducted: 1. Experimental Materials and Methods 1.1 Western Blot MKN45 and NUGC4 cells were treated with different concentrations (0 μM, 1 μM, 2 μM, 4 μM) of clofazimine for 24 hours. Total protein was extracted from the cells and analyzed by Western blotting. The expression levels of the key lysosomal hydrolases cathepsin B (CTSB) and cathepsin D (CTSD) were detected using specific primary antibodies (Protientech; catalog numbers: CTSB (12216-1-AP); CTSD (21327-1-AP)).

[0054] 1.2 Observation by transmission electron microscopy (TEM) MKN45 and NUGC4 cells were treated with different concentrations (0 μM, 2 μM) of chlorofazimine for 24 hours. Cells were collected, fixed, dehydrated, embedded, and then ultrathin sections were prepared. After double staining with uranium acetate and lead citrate, the ultrastructure of the cells was observed under a transmission electron microscope, focusing on the morphology of autophagosomes, lysosomes, and mitochondria.

[0055] 2. Experimental Results 2.1 Late-stage autophagic flux arrest caused by lysosomal dysfunction Please see Figure 8 Western blot results showed that with increasing clofazimine concentration, the expression levels of mature CTSB and mature CTSD, representing lysosomal degradation function, significantly decreased. This preliminarily demonstrates that late-stage autophagic flux arrest is caused by lysosomal dysfunction.

[0056] 2.2 Electron microscopy confirmed autophagy flux blockade and mitochondrial damage. Please see Figure 9 Transmission electron microscopy results showed that clofazimine did not affect the fusion of lysosomes and autophagosomes, and intracellular accumulation of autophagosomes was observed due to the failure of lysosomes to perform degradation functions (arrow symbol), corroborating that the late-stage arrest of autophagic flux was caused by lysosomal dysfunction. In addition, the structural integrity of mitochondria was significantly disrupted after clofazimine treatment (triangle symbol), further indicating mitochondrial damage.

[0057] In summary, clofazimine induces mitochondrial damage in gastric cancer cells (MKN45, NUGC4), thereby initiating autophagy and mediating the maturation of CTSB and CTSD, which in turn causes lysosomal dysfunction, preventing the degradation of damaged mitochondria. These two factors, namely mitochondrial dysfunction and autophagosome accumulation in gastric cancer cells, are the main reasons for the anti-gastric cancer activity of clofazimine.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of clofazimine in the preparation of drugs for treating gastric cancer, characterized in that, The structural formula of the chloramphenicol is: 。 2. The application according to claim 1, characterized in that, The drug exerts its therapeutic effect on gastric cancer by inducing autophagy inhibition in gastric cancer cells.

3. The application according to claim 2, characterized in that, The drug achieves autophagic flux inhibition by causing lysosomal dysfunction.

4. The application according to claim 2 or 3, characterized in that, The drug is used to induce mitochondrial damage and autophagy flow arrest in gastric cancer cells.

5. The application according to claim 1, characterized in that, The drug is used to reduce the size of gastric cancer tumors.

6. The application according to claim 1, characterized in that, The drug is used to inhibit the growth of gastric cancer tumors.

7. The application according to claim 1, characterized in that, The drug is used to upregulate the expression of autophagy activation marker proteins ATG-5 and LC3-II in gastric cancer cells.

8. The application according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier suitable for injection.

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