Pharmaceutical composition for treating gastric cancer and application

Abstract

The invention provides a pharmaceutical composition for treating gastric cancer. The pharmaceutical composition comprises an NAT10 inhibitor and a PD-1 inhibitor. The gastric cancer is treated by combining the NAT10 inhibitor Remodelin with the PD-1 inhibitor, so that the problems of tumor angiogenesis and immunosuppression in existing treatment are solved, and the clinical curative effect of immunotherapy is improved. Research finds that when the Remodelin or the PD-1 inhibitor is independently used, a certain tumor inhibition effect is achieved, but the tumor volume of a combined treatment group is remarkably reduced, and immune cell infiltration is remarkably increased.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating gastric cancer and its application. Background Art

[0002] Gastric cancer is a highly prevalent malignant tumor globally. The effectiveness of traditional treatments is often limited due to the complexity of the tumor microenvironment (TME). Abnormalization of tumor blood vessels is a typical feature of the TME, which not only affects the infiltration of immune cells but also increases tumor drug resistance. As an RNA N4-acetylcytidine (ac4C) transferase, NAT10 has been proven to play a key role in promoting the proliferation, angiogenesis, and immunosuppression of gastric cancer. In addition, immune checkpoint inhibitors (such as PD-1 inhibitors) have achieved remarkable results in cancer immunotherapy in recent years, but their effectiveness is still restricted by the immunosuppressive environment of the TME. Based on the findings, the present invention shows that tumor blood vessel normalization and remodeling of the TME can be achieved through the NAT10 inhibitor Remodelin, improving the therapeutic effect of PD-1 inhibitors. Summary of the Invention

[0003] The object of the present invention is to provide a pharmaceutical composition for treating gastric cancer, which treats gastric cancer by combining the NAT10 inhibitor Remodelin with a PD-1 inhibitor, so as to overcome the problems of abnormal tumor blood vesselization and immunosuppression in existing treatments and improve the clinical efficacy of immunotherapy.

[0004] To achieve the above object, the technical solution adopted is: a pharmaceutical composition for treating gastric cancer, comprising a NAT10 inhibitor and a PD-1 inhibitor.

[0005] Preferably, the NAT10 inhibitor is Remodelin.

[0006] Preferably, the PD-1 inhibitor is a PD-1 monoclonal antibody.

[0007] The present invention provides the application of the above-mentioned pharmaceutical composition in the preparation of a drug for treating gastric cancer.

[0008] The present invention provides the application of the combination of a NAT10 inhibitor and a PD-1 inhibitor in the preparation of a drug for treating gastric cancer.

[0009] The present invention provides the application of a NAT10 inhibitor in the preparation of a drug for treating gastric cancer.

[0010] Preferably, the NAT10 inhibitor is Remodelin.

[0011] Advantageous Effects:

[0012] The present invention has found through research that the use of Remodelin or PD-1 inhibitor alone has a certain tumor inhibitory effect, but the tumor volume in the combination treatment group is significantly reduced, and the infiltration of immune cells is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : NAT10 inhibitor Remodelin promotes the normalization of MFC tumor blood vessels; A. Analysis of the number of blood vessels in mouse tumors by CD31 fluorescence staining; B. Injection of hypoxia probe Hypoxyprobe before sampling, and observation of changes in hypoxia levels in mouse tumors by immunofluorescence staining; C. D. Immunofluorescence staining to detect the pericyte coverage (B) and basement membrane coverage rate (C) of mouse tumor blood vessels; E. F. Representative images of the perfusion capacity and leakage of tumor blood vessels by immunofluorescence staining after injection of lectin (E) and dextran (F) before sampling. Bars = means ± SEM. Scale bar: 50 μm.

[0014] Figure 2 : Inhibiting NAT10-induced immune reprogramming of the tumor microenvironment in vivo; B. Flow cytometry sorting analysis of T lymphocyte subsets in tumors, and comparison of the numbers and proportions of CD45+, CD4+, CD8+, Treg, NK, and NKT lymphocytes; C. Immunofluorescence staining of subcutaneous tumor tissues in mice after Remodelin treatment, with CD8 labeled red; D. Immunofluorescence staining of subcutaneous tumor tissues in mice after Remodelin treatment, with CD4 labeled green and CD31 labeled red; E. qCPR experiment to detect the mRNA expression levels of CXCL9, CXCL10, and CXCL11 in AGS gastric cancer cell lines infected with shNAT10 lentivirus. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0015] Figure 3 : Inhibiting NAT10-induced immune reprogramming of the tumor microenvironment in vivo; A. White light image after dissection of subcutaneous tumors in mice; B. Analysis of the subcutaneous tumor growth curve and tumor mass. DETAILED DESCRIPTION OF THE INVENTION

[0016] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0017] EXAMPLE

[0018] (1) Drug and dose design

[0019] Remodelin preparation: Oral administration at a dose of 100 mg / kg, once every two days.

[0020] PD-1 inhibitor: Intraperitoneally injected at a dose of 200 μg / mouse, once every three days.

[0021] The PD-1 monoclonal antibody was purchased from Bio X Cell (product number BPO146).

[0022] The NAT10 inhibitor Remodelin was purchased from Selleck (product number 1622921-15-6).

[0023] (2) Treatment mode

[0024] Mouse experiments showed that the use of Remodelin or PD-1 inhibitor alone had a certain tumor inhibitory effect, but the tumor volume in the combination treatment group was significantly reduced, and the infiltration of immune cells was significantly increased.

[0025] (3) Experimental data support

[0026] Vascular normalization detection: The experimental results showed that Remodelin could improve vascular density and function (such as reduced permeability and increased α-SMA coverage).

[0027] Enhanced infiltration of immune cells: The infiltration of CD8+ T cells and NK cells was significantly increased, while the infiltration ratio of Tregs was reduced.

[0028] Antitumor efficacy: The tumor volume in the combination treatment group was significantly reduced, and the survival time of animals was significantly prolonged.

[0029] I. Construction of a subcutaneous tumor-bearing model in 615 mice and treatment with the NAT10 inhibitor (Remodelin)

[0030] (1) Cell culture: Cultivate and maintain the MFC mouse gastric cancer cell line to ensure that the cells are active and healthy before the experiment. Digest the cells with trypsin containing EDTA. After digestion, terminate the digestion with complete medium. Centrifuge to remove the supernatant, resuspend the cells with 1640 medium and count, and adjust the cell density to 5*10 7 cells / mL;

[0031] (2) Mouse selection: Select 6-8-week-old 615 mice and raise them in a SPF-level environment to ensure that the mice are healthy and meet the experimental requirements;

[0032] (3) Skin preparation: Disinfect the cell inoculation area in the right groin of 615 mice with 75% ethanol;

[0033] (4) Subcutaneous injection: Inject the cell suspension (100 μl / mouse) into the skin-prepared 615 mice. After all inoculations are completed, continue to raise them in a SPF-level environment; wait for the tumor to grow to 100 mm 3After reaching the specified size, NAT10 inhibitor (Remodelin) was administered by oral gavage every 2 days, with a dosage of 100 mg / kg;

[0034] (5) Observation and recording: Observe the injection site of the 615 mice to ensure there is no bleeding or other complications. Monitor the tumor growth, and measure and record the tumor volume every 3 days using a vernier caliper;

[0035] (6) End of the experiment: After 12 days of Remodelin treatment, the nude mice were sacrificed by cervical dislocation, and fluorescence photography was performed using a live imaging instrument. The tumor tissues were collected and fixed in formalin for further analysis.

[0036] Analysis methods:

[0037] 1. Flow sorting and analysis of immune cells in tumors

[0038] (1) Preparation of single-cell suspension from tumor tissues: Weigh approximately 300 g of tumor tissues into a sterile culture dish, quickly wash twice with PBS solution containing double antibodies, and use ophthalmic scissors to remove excess fat and necrotic tissues in the tissue mass. Cut the tumor tissues into small pieces and digest with 5 ml of digestive solution for 30 minutes. After digestion, filter the tissue mass through a 70-μm filter to obtain a single-cell suspension. After centrifugation to remove the supernatant, add red blood cell lysate, and lyse on ice for 10 minutes. After the lysis is completed, add 10 ml of PBS to terminate the lysis.

[0039] (2) Put the obtained single-cell suspension into a centrifuge, centrifuge at 4°C and 500 g for 5 minutes, discard the supernatant, and resuspend the precipitate with 1 ml of flow cytometry buffer;

[0040] (3) Use a cell counter to count the cells and adjust the cell concentration to 1×10 6 cell / ml;

[0041] (4) Antibody preparation: Prepare corresponding single-stained tubes and mixed-stained tubes according to the antibody combination. Single-stained tube: Dilute the antibody at a ratio of 1:100 in a 200-μl EP tube. Mixed-stained tube: Calculate according to staining 1×106 cells per sample, and dilute the antibody with PBS at a ratio of 1:100 in a 1.5-ml EP tube;

[0042] (5) Antibody binding: Add the prepared antibodies to the corresponding single-stained tubes and mixed-stained tubes respectively, then pipette and mix well, incubate at 4°C in the dark for 30 minutes;

[0043] (6) Add 1 ml of PBS to each tube to resuspend and wash away the excess antibodies, centrifuge at 3500 rpm, discard the supernatant, and then add 1 ml of PBS to resuspend;

[0044] (7) As soon as the sample treatment is completed, sort it on the machine.

[0045] 2. Immunofluorescence immunohistochemistry detection

[0046] The immunofluorescence immunohistochemistry method was operated according to the operation instructions of the kit.

[0047] (1) Deparaffinization and hydration of paraffin sections: The sections were deparaffinized in environmental deparaffinizing agents (1), (2), and (3) for 10 min in sequence, and then in 100% ethanol, 95% ethanol, and 75% ethanol for 5 min respectively;

[0048] (2) Wash with distilled water 3 times, 3 min each time, and then soak and wash;

[0049] (3) Antigen repair: Heat the pressure cooker containing EDTA antigen repair solution (pH 9.0) on the induction cooker until it boils. Put the sections into the boiling buffer solution, start timing for 1.5 min after the pressure cooker jets steam, and cool at room temperature for 30 min after turning off the fire. Take out the sections and wash with TBST 3 times, 5 min each time;

[0050] (4) Blocking: Drop 3% H2O2 on the sections, place at room temperature for 20 min, and then wash with distilled water for 3 min;

[0051] (5) Blocking: 10% goat serum, incubate at 37°C for 30 min;

[0052] (6) Primary antibody incubation: Remove the blocking solution, mix the diluted primary antibody working solution and drop it on the sections, 50 - 100 μl per section, incubate overnight at 4°C;

[0053] (7) Secondary antibody incubation: Take out the sections and warm them at room temperature for 15 min, wash with TBST solution 3 times, 3 min each time. Drop the Goat Anti-Rabbit IgG H&L (HRP) secondary antibody working solution, incubate at room temperature for 30 min, and wash with TBST 3 times, 3 min each time;

[0054] (8) TSA staining: Drain off TBST, prepare Cy5 tyramide working solution (1:400), drop 50 - 100 μl of the prepared 488 tyramide working solution on each section, incubate at room temperature for 30 min and then soak and wash with TBST 3 times

[0055] (9) Elution: Preheat the repair box with elution solution in a 42°C water bath, then put the sections into it for elution for 20 min, take out the sections and soak and wash with TBST for 3 min;

[0056] (10) Blocking: Drop 10% donkey serum, incubate at 37°C for 30 min;

[0057] (11) Primary antibody incubation: Discard the blocking solution. Prepare the primary antibody working solution in advance according to the ratio in the instruction manual, and drop the working solution to immerse the sections, then incubate overnight at 4°C.

[0058] (12) Secondary antibody incubation: Take out the sections and warm them at room temperature for 15 min. Rinse with TBST 3 times, 3 min each time. Drop the prepared Alexa 488 donkey anti-rabbit lgG(H+L) secondary antibody working solution onto the sections, incubate at room temperature for 30 min, and then rinse with TBST 3 times, 3 min each time.

[0059] (13) Nuclear staining: Discard the TBST, and drop the prepared DAPI working solution onto the sections under light-proof conditions for nuclear staining for 5 min, then rinse with TBST.

[0060] (14) Mounting: Mount with a fluorescence mounting medium and store at 4°C in the dark.

[0061] (15) Microscopic examination: Observe the sections under a microscope and scan the pictures for analysis.

[0062] 3. HE staining of tissues

[0063] (1) Sampling, fixation, and dehydration: Trim the subcutaneous tumor tissues of mice into the size of soybeans and fix them in 10 times the volume of 4% paraformaldehyde solution for more than 24 hours. After fixation, rinse with running water overnight in an embedding frame, and then dehydrate successively with gradient ethanol (75% ethanol for 60 minutes, 85% ethanol for 45 minutes, 95% ethanol for 45 minutes, absolute ethanol for 30 minutes, absolute ethanol for 30 minutes).

[0064] (2) Paraffin embedding and sectioning of tissues: Put the tissues into a mixture of 1 / 2 volume of xylene + 1 / 2 volume of absolute ethanol for clearing for 5 minutes, and then put them into pure xylene for 10 minutes. After the tissues are well cleared, add them to the melted paraffin for embedding, and wait until they cool and solidify into blocks. After the embedded tissue blocks harden, section them.

[0065] (3) Baking the sections: Heat in an oven at 60°C for 1 hour, and note that there should be no bubbles between the sections and the glass slides.

[0066] (4) Dewaxing and hydration: Dewax the sections in xylene I and xylene II for 10 minutes respectively; then hydrate them successively with absolute ethanol for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 70% ethanol for 5 minutes.

[0067] (5) Hematoxylin staining: Determine the staining time according to the section thickness, and after staining, wash with tap water.

[0068] (6) Differentiation: Observe under a microscope. If the color is too dark, differentiate in hydrochloric acid alcohol for 1 - 3 s.

[0069] (7) Blue return: Dilute with light ammonia water for blue return to remove the residual hydrochloric acid on the sections;

[0070] (8) Eosin staining: Stain for 5 minutes, and then wash with tap water;

[0071] (9) Dehydration and mounting: Dehydrate the sections in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, 95% alcohol for 5 minutes and absolute ethanol for 5 minutes respectively, and then drop neutral resin for mounting;

[0072] (10) Observe under the microscope and take pictures for record.

[0073] Experimental results

[0074] As Figure 1 and Figure 2 shown, the NAT10 inhibitor Remodelin can significantly inhibit the angiogenesis in mouse tumors and promote the normalization of blood vessel structure and function. In addition, Remodelin significantly reduces the tumor hypoxic area ( Figure 1 ); When performing flow cytometry analysis and immunofluorescence staining on mouse tumors, it was found that compared with the control group, the density of CD45+ immune cells in the tumors treated with Remodelin was significantly increased, suggesting that the normalization of blood vessel fluid pressure promoted the infiltration of immune cells. In addition, we found that the infiltration of CD4+ T cells, CD8+ T cells, NK cells and NKT cells was significantly increased in the tumors treated with Remodelin, while the infiltration of Treg cells was reduced ( Figure 2 ).

[0075] II. In vivo animal experiments

[0076] (1) Establishment of 615 mouse subcutaneous tumor model: Digest and wash the stably cultured MFC cells with trypsin, resuspend the cells with 1640 medium, and adjust the cell concentration to 5*10 7 cells / ml after counting. Select healthy 6-8-week-old 615 mice raised in a SPF environment, and disinfect the right inguinal inoculation area of the mice with alcohol. Inject the cell suspension (100 μl / mouse) into the shaved mice.

[0077] (2) After the subcutaneous tumors of the mice grow to a certain volume, randomly divide the mice into the following three groups and treat them according to the corresponding methods: NAT10 inhibitor Remodlin group; anti-PD-1 monoclonal antibody treatment group; Remodlin + PD-1 monoclonal antibody combination treatment group. The administration method of Remodlin is oral gavage, and the administration dose is 100 mg / kg / day, once every 2 days, for 12 consecutive days. The administration method of PD-1 monoclonal antibody is intraperitoneal injection, and the administration dose is 200 μg / mouse / time, once every three days, for 12 consecutive days.

[0078] (3) Observe the tumor size daily and record it. The volume formula is V = L * W 2 / 2 (V: volume; L: long diameter; W: short diameter). Plot the obtained data as a tumor growth curve. After taking samples of the tumor, weigh it and record by taking photos.

[0079] Experimental results

[0080] As Figure 3 shown, mice were treated with Remodelin and anti-PD-1 monoclonal antibody in combination. The results showed that compared with the control group, the use of PD-1 inhibitor alone could slow down tumor growth, while the inhibitory effect of Remodelin combined with anti-PD-1 treatment on tumor growth was more significant ( Figure 3 ).

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A pharmaceutical composition for treating gastric cancer, characterized in that: Including NAT10 inhibitors and PD-1 inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that The NAT10 inhibitor is Remodelin.

3. The pharmaceutical composition according to claim 1, characterized in that PD-1 inhibitors are PD-1 monoclonal antibodies.

4. Use of the pharmaceutical composition according to claim 1 in preparing a drug for treating gastric cancer.

5. Application of the combination of NAT10 inhibitor and PD-1 inhibitor in the preparation of drugs for the treatment of gastric cancer.

6. Application of NAT10 inhibitors in the preparation of drugs for the treatment of gastric cancer.

7. The use according to claim 5 or 6, characterized in that The NAT10 inhibitor is Remodelin.

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