Application of alpha-glucosidase inhibitor in preparation of medicine for treating gastric cancer

Abstract

The invention provides a new medicine for treating gastric cancer, opens up a brand new application field of an alpha-glucosidase inhibitor, and provides a potential effective, safe and economical new treatment scheme for patients with gastric cancer; the invention also discloses a brand new anti-gastric cancer action mechanism of the alpha-glucosidase inhibitor, phosphorylation of PPP1CA at a T320 site is inhibited by down-regulating expression of a tumor inhibition factor RASAL2, and finally inactivation of a downstream p38 MAPK signal channel and a downstream Smad signal channel (a key medium of a TGF-beta channel) is caused. Expression of key genes related to tumor stem cell characteristics (LGR5, CD44 and SOX9), transcription factors (BACH1) and immune escape (PD-L1) is reduced through inhibition of the pathways, so that the effects of inhibiting proliferation of gastric cancer cells and reducing the malignancy degree of the gastric cancer cells are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a new use of an alpha-glucosidase inhibitor in treating gastric cancer. BACKGROUND

[0002] Gastric cancer is a malignant tumor with high morbidity and mortality worldwide, and its treatment methods mainly include surgical resection, chemotherapy, radiotherapy and targeted therapy. For patients with advanced or metastatic gastric cancer, systemic chemotherapy is the main treatment method, but conventional cytotoxic chemotherapy drugs have problems such as poor selectivity, serious side effects, easy drug resistance and poor overall efficacy, and the prognosis of patients is poor.

[0003] In recent years, targeted drugs based on specific molecular targets (such as trastuzumab for HER2 and ramucirumab for VEGFR) have provided new treatment options for some gastric cancer patients. However, these targeted drugs have limited applicability, usually require specific biomarker expression, have high treatment costs, and have serious side effects such as myelosuppression, neurotoxicity, and gastrointestinal reactions, which seriously affect the quality of life and treatment tolerance of patients, and also face the challenge of drug resistance. Therefore, there is an urgent need for gastric cancer treatment drugs with novel mechanisms of action, good safety and wide application.

[0004] In the field of diabetes treatment, alpha-glucosidase inhibitors (such as acarbose, voglibose, and miglitol) are a mature class of oral hypoglycemic drugs that inhibit the activity of alpha-glucosidase in the intestinal tract, delay the decomposition and absorption of carbohydrates, and thus achieve the purpose of reducing postprandial hyperglycemia. Among them, voglibose has been clinically widely used for a long time due to its high potency and good safety.

[0005] Although voglibose is very mature in the treatment of diabetes, the prior art does not disclose or suggest its use in treating gastric cancer. The standard treatment regimen for gastric cancer currently does not include voglibose or other alpha-glucosidase inhibitors. SUMMARY

[0006] The present application provides a new drug for treating gastric cancer, and provides a new, effective and safer drug or method for preventing or treating gastric cancer to overcome or alleviate the defects in existing gastric cancer treatment drugs, such as high side effects, easy drug resistance or high cost. The present application provides a new treatment option for gastric cancer patients: the present application opens up a new application field for alpha-glucosidase inhibitors, and provides a potential effective, safe and economical new treatment option for gastric cancer patients, especially for patients who are intolerant or resistant to traditional chemotherapy.

[0007] The application provides use of an alpha-glucosidase inhibitor in the preparation of a drug for treating or preventing gastric cancer.

[0008] Further, the alpha-glucosidase inhibitor includes acarbose, voglibose and miglitol.

[0009] Further, the drug achieves the effect of treating or preventing gastric cancer by inhibiting p38 MAPK and Smad signaling pathways.

[0010] Further, the drug can significantly down-regulate the gene expression levels of LGR5, CD44, SOX9, BACH1 and PD-L1.

[0011] The application applies a hypoglycemic drug that has been clinically used for decades to a completely different tumor treatment field, and the core difficulty is not in the synthesis or production of the drug, but in the overturning mechanism discovery and cross-field cognitive breakthrough. Specifically, the difficulty is reflected in:

[0012] Theoretical barrier: the traditional pharmacological mechanism of alpha-glucosidase inhibitors (inhibiting intestinal alpha-glucosidase) and the regulation mechanism of tumor growth seem to belong to two completely different physiological and pathological systems, and there is no direct theoretical correlation. Crossing this cognitive gap requires strong innovative thinking and accidental scientific discovery.

[0013] Complexity of mechanism exploration: even if the anti-tumor activity of voglibose is observed in in vitro and in vivo models, further elucidation of its precise target and molecular mechanism (for example, whether it regulates glucose metabolism, affects the tumor microenvironment, activates immune response or other new pathways) is a complex and arduous scientific work.

[0014] In the examples of the application, voglibose is used as a representative of alpha-glucosidase inhibitors to treat gastric cancer and its mechanism, and acarbose and miglitol, which are also members of alpha-glucosidase inhibitors, have the same therapeutic effect on gastric cancer and the same mechanism of action.

[0015] Beneficial effects:

[0016] The application opens up a new application field of alpha-glucosidase inhibitors, and provides a potential effective, safe and economical new treatment plan for gastric cancer patients, especially for patients who are intolerant or resistant to traditional chemotherapy.

[0017] The application has significant development advantages: thanks to the good safety data and mature production process of alpha-glucosidase inhibitors, the application can greatly shorten the period from drug research and development to clinical application, significantly reduce the development risk and cost, and has the outstanding advantages of "low investment and quick effect".

[0018] The present application has important scientific value: the present application discloses a novel connection between sugar metabolism regulation and tumor growth, provides a new action target and research direction for tumor treatment, and has scientific value for inspiring other subsequent innovations.

[0019] The present application has great social and economic benefits: through "new use of old drugs", existing drug resources can be effectively revitalized, and the use value of drugs can be improved, which is expected to greatly reduce medical expenses while improving efficacy, and produce significant social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 To verify the molecular mechanism of Voglibose in the anti-tumor effect of AGS and MKN45 gastric cancer cell lines, Western blot (A) and real-time fluorescent quantitative PCR (B) were used;

[0021] Figure 2 To verify the molecular mechanism of Voglibose in the anti-tumor effect of AGS and MKN45 gastric cancer cell lines, Western blot (A) and real-time fluorescent quantitative PCR (B) were used;

[0022] Figure 3 To verify the molecular mechanism of Voglibose in the anti-tumor effect of AGS and MKN45 gastric cancer cell lines, Western blot (A) and real-time fluorescent quantitative PCR (B) were used;

[0023] Figure 4 To verify the molecular mechanism of Voglibose in the anti-tumor effect of AGS and MKN45 gastric cancer cell lines, Western blot (A) and real-time fluorescent quantitative PCR (B) were used;

[0024] Figure 5 To verify the molecular mechanism of Voglibose in the anti-tumor effect of AGS and MKN45 gastric cancer cell lines, Western blot (A) and real-time fluorescent quantitative PCR (B) were used;

[0025] Figure 6 To verify the molecular mechanism of Voglibose in the anti-tumor effect of AGS and MKN45 gastric cancer cell lines, Western blot (A) and real-time fluorescent quantitative PCR (B) were used;

[0026] Figure 7Figure 1 shows the plot of the diameter of the human gastric cancer organoids in the experiment for determining the effect of Voglibose on the growth of the human gastric cancer organoids (Human GC Organoids).

[0027] Figure 8 Figure 2 shows the plot of the diameter (A) and the number (B) of the human gastric cancer organoids after administration. DETAILED DESCRIPTION

[0028] In order to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with cases. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0029] In the following examples, the experimental methods are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0030] The drugs selected in the present embodiment are as follows:

[0031] Active ingredient: The core active ingredient in the present embodiment is Voglibose or a pharmaceutically acceptable salt thereof. The Voglibose is a known compound, and its chemical name is 5-[(1S,2S,3R,4S)-1,2,3,4-tetrabutyl-2,3,4-trihydroxy-1-cyclohexyl]amino-1,5-deoxy-1,5-imino-D-glucitol.

[0032] Pharmaceutical composition: A therapeutically effective amount of Voglibose is mixed with one or more pharmaceutically acceptable carriers, diluents or excipients to prepare a dosage form suitable for pharmaceutical administration. The dosage form includes but is not limited to oral preparations such as tablets, capsules, granules, powders, oral liquids, etc.; or injections.

[0033] The dosage of the drug for mice in the present experiment: when used for treating gastric cancer, the daily dosage of Voglibose ranges from 3 mg / kg body weight to 10 mg / kg body weight; the dosage can be taken once a day or in multiple times.

[0034] Example 1 in vitro experiment

[0035] Methods: Two human gastric cancer cell lines, AGS and MKN45 (purchased from Cellcook Biotechnology Co., Ltd., Guangzhou, China), were used for validation. AGS and MKN45 cells were cultured separately in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Subsequent experiments were performed when the cells reached the logarithmic growth phase. AGS and MKN45 cells in the logarithmic growth phase were divided into a control group (CTRL) and a voglibose treatment group (administered 2 μM voglibose). Further treatment of AGS and MKN45 cells was conducted, including a control group (CTRL) and three voglibose treatment groups with different concentrations (1 μM, 2 μM, and 4 μM) to explore the relationship between voglibose concentration and its effect on gastric cancer cells. The expression and phosphorylation levels of key signaling pathway proteins were detected by Western blotting. The characteristics of cancer stem cells and the mRNA expression levels of related genes were detected by real-time quantitative PCR (qPCR).

[0036] Experimental results:

[0037] from Figure 1 (in Figure 1 The dosage concentration administered to mice was 2 μM. Figure 2 The study showed that, compared with the untreated control group, the voglibose treatment group significantly downregulated the protein expression of the tumor-promoting factor RASAL2.

[0038] from Figure 1 (A) and Figure 2 As shown in (A), the voglibose treatment group significantly downregulated the protein levels of phosphorylated p-p38 MAPK (T180 / Y182), p-Smad2 (S465 / 467) and p-Smad3 (S423 / 425), and the magnitude of the downregulation increased with increasing drug concentration, indicating that it inhibited the p38 MAPK and TGF-β / Smad signaling pathways.

[0039] from Figure 1 and Figure 2 As can be seen, the voglibose treatment group significantly downregulated the gene expression levels of cancer stem cell markers (LGR5, CD44, SOX9), transcription factor (BACH1), and immune checkpoint protein (PD-L1), with mRNA levels decreasing by more than 60%, and the degree of downregulation increased with increasing drug concentration.

[0040] Example 2: In vivo animal experiments

[0041] Methods: Two mouse gastric cancer models were established: YTN3 cell subcutaneous transplantation tumor model and syngeneic transplantation model (MDA). The experiment was divided into control group and voglibose administration group (for example, 3 mg / kg or 10 mg / kg per day oral administration). The tumor volume was measured regularly, and the tumor weight was measured after the experiment.

[0042] Specifically, two mouse gastric cancer models were established: YTN3 cell subcutaneous transplantation tumor model and syngeneic transplantation tumor model (MDA). For the YTN3 cell subcutaneous transplantation tumor model, YTN3 RASAL2 knockdown cells or control cells were suspended in DMEM medium / Matrigel mixture (50% medium and 50% Matrigel). 3x10 6 YTN3 cells were injected subcutaneously into the flank of mice. One week after inoculation, the mice were randomly divided into 2 groups: (a) control group (CTRL); (b) voglibose treatment group (dose of 10.0 mg / kg, once a day by gavage), 5 mice in each group. The tumor volume (TV) was calculated according to the formula "TV = (length x width 2) / 2", and the growth curve was drawn according to the group average tumor volume at each time point.

[0043] For the syngeneic transplantation tumor model (MDA), APTc model-derived cancer cells were used to construct subcutaneous syngeneic transplantation tumors in C57BL / 6 mice (named MDA). The tumor was cut into 3x3x3mm 3 pieces and mixed with Matrigel (Corning, New York, USA); 4-6 week old C57BL / 6 mice were anesthetized by intraperitoneal injection of ketamine at a dose of 100mg / kg. The skin was cut open, and the tumor pieces were transplanted subcutaneously under the back of the mouse, and the cut parts of the gastric serosa and skin were covered with tissue adhesive. All operations were performed in a biological safety cabinet. When the subcutaneous tumor grew to 150mm 3, the mice were given voglibose (dose of 10.0mg / kg, once a day by gavage) for 3 weeks. The tumor volume was calculated as above.

[0044] Results of the experiment:

[0045] From Figure 3 With Figure 4 For the YTN3 cell subcutaneous transplantation tumor model, Figure 5 With Figure 6In the allogeneic tumor model (MDA), it was observed that the voglibose administration group significantly inhibited tumor growth in mice compared to the control group. Throughout the entire administration period, the mean tumor volume in the administration group was significantly smaller than that in the control group (e.g., the inhibition rate reached over 50% on day 21, *p* < 0.05).

[0046] Example 3: Human organoid experiment

[0047] Methods: Human GC Organoids were cultured using a human gastric cancer organoid model and treated with voglibose.

[0048] Specifically, a three-dimensional organoid culture system was established from human gastric tissue. Gastric antrum tissue samples were taken, washed with pre-chilled phosphate-buffered saline (PBS), and cut into 2-3 mm pieces. The tissue was digested with 2.5 mg / mL collagenase A at 37°C for 30 minutes. 5 mL of dissociation buffer was added to the tissue, and the mixture was shaken for 2 minutes. The cell suspension was filtered twice through a 70 μm FALCON cell filter, followed by centrifugation at 150 × g for 5 minutes. The cells were resuspended in pre-chilled Cultrex low-growth factor basement membrane extract (BME, type 2, Pathclear grade), a medium supplemented with various growth factors and supplements. 50 μL of BME cell suspension was added to the center of each well of a 24-well plate, followed by 0.5 mL of IntestiCult organoid growth medium. The organoids were cultured in a humidified incubator at 37°C with 5% CO2, and the medium was changed twice weekly. For second-generation organoids, they were infected overnight in 15 ml centrifuge tubes with 2 μM Voglibose. The culture medium containing Voglibose was changed every two days. After 7 days of infection, organoids from three random 100x fields of view were observed, measured, and counted using an optical microscope.

[0049] Experimental results:

[0050] like Figures 7-8 As shown, compared with the control group, the voglibose treatment group significantly inhibited the diameter growth of human gastric cancer organoids and significantly reduced the number of human gastric cancer organoids formed. This indicates that voglibose can directly inhibit the growth of human gastric cancer tissues with high physiological relevance.

[0051] Summary: Based on the above experimental results, the present application discloses a novel mechanism of action of voglibose against gastric cancer: voglibose down-regulates the expression of tumor suppressor RASAL2, and then inhibits the phosphorylation of PPP1CA at T320 site (i.e. activates the phosphatase activity of PPP1CA), ultimately leading to the inactivation of the downstream p38 MAPK signaling pathway and Smad signaling pathway (key medium of TGF-β pathway). The inhibition of these pathways further down-regulates the expression of key genes related to tumor stem cell characteristics (LGR5, CD44, SOX9), transcription factors (BACH1) and immune escape (PD-L1), thereby achieving the effect of inhibiting the proliferation of gastric cancer cells and reducing their malignancy.

Claims

1. The use of α-glucosidase inhibitors in the preparation of drugs for the treatment or prevention of gastric cancer.

2. The use as described in claim 1, characterized in that: The α-glucosidase inhibitors include acarbose, voglibose, and miglitol.

3. The use as described in claim 1, characterized in that: The drug achieves its therapeutic or preventative effects against gastric cancer by inhibiting the p38 MAPK and Smad signaling pathways.

4. The use as described in claim 1, characterized in that: The drug can downregulate the gene expression levels of LGR5, CD44, SOX9, BACH1, and PD-L1.

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