Application of neomycin in preparation of medicine for treating tumors

Abstract

The invention discloses an application of neomycin in preparation of a medicine for treating tumors, and experiments show that tumor growth can be significantly inhibited by inhibiting interference of LPS (Lipopolysaccharide) on an STING pathway. The invention further provides a pharmaceutical composition containing the neomycin and the STING agonist (such as diABZI, cGAMP or CMA), the neomycin and the STING agonist show a synergistic effect in tumor resistance, the composition can be used for preparing drugs for treating tumors such as melanoma and colon cancer, and it is indicated that the neomycin or the combination of the neomycin and the STING agonist has wide application prospects in the medical field.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of neomycin in the preparation of a drug for treating tumors. Background Art

[0002] The tumor microenvironment (TME) is a complex ecosystem composed of tumor cells, immune cells (including T cells, B cells, natural killer cells, macrophages, etc.), stromal cells (such as fibroblasts, endothelial cells), and the extracellular matrix. These components jointly regulate the occurrence, development, and metastasis processes of tumors through a complex interaction network. Recent studies have shown that microbial metabolites present in the TME, especially lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, play an important role in regulating the host immune response.

[0003] Therefore, there is an urgent need to develop a new and effective drug for treating tumors. Summary of the Invention

[0004] The object of the present invention is to provide the application of neomycin in the preparation of a drug for treating tumors. The present invention discovers that by inhibiting the interference of lipopolysaccharide (LPS) with the STING signaling pathway, tumor growth can be significantly inhibited, and a synergistic anti-tumor effect with a STING agonist is demonstrated in an animal model, indicating that neomycin or its combination with a STING agonist has broad application prospects in the medical field.

[0005] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect of the present invention, there is provided the application of neomycin in the preparation of a drug for treating tumors, wherein the tumor is a tumor sensitive to STING pathway activation.

[0006] Further, the tumor sensitive to STING pathway activation is melanoma or colon cancer.

[0007] Further, the drug for treating tumors further comprises a pharmaceutically acceptable excipient.

[0008] Further, the excipient includes at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetening agent, or a coloring agent.

[0009] Further, the dosage form of the drug includes at least one of a granule, a tablet, a pill, a capsule, an injection, or a dispersant.

[0010] Further, the dosage of neomycin in the drug is 1 - 5 mg / kg.

[0011] Furthermore, neomycin in the drug is the main active ingredient of the drug.

[0012] Furthermore, neomycin in the drug is the only active ingredient of the drug.

[0013] In the second aspect of the present invention, there is provided the use of the combination of neomycin and a STING agonist in the preparation of a drug for treating tumors.

[0014] Furthermore, in the drug, neomycin is used to inhibit the interference of lipopolysaccharide on the STING signaling pathway, and the STING agonist is used to activate the STING signaling pathway, so as to anti-tumor by restoring or enhancing the anti-tumor immune response.

[0015] Furthermore, the STING agonist is one or more of diABZI, cGAMP or CMA.

[0016] In the third aspect of the present invention, there is provided a pharmaceutical composition comprising neomycin and a STING agonist as active ingredients.

[0017] Furthermore, the tumor is a tumor sensitive to STING pathway activation.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: The present invention provides the use of neomycin in the preparation of a drug for treating tumors. Experiments show that by inhibiting the interference of LPS on the STING pathway, tumor growth can be significantly inhibited. The present invention also provides a pharmaceutical composition comprising neomycin and a STING agonist (such as diABZI, cGAMP or CMA), and the two show a synergistic effect in anti-tumor. This composition can be used to prepare drugs for treating tumors such as melanoma and colon cancer, indicating that neomycin or its combination with a STING agonist has broad application prospects in the medical field. The specific advantages are as follows: (1) Significant effect: In a melanoma mouse model and a mouse colon cancer model, monotherapy with neomycin (30 mg / kg) reduced the tumor volume by 60% (Examples 3 and 4).

[0019] (2) Wide applicability: The method of the present invention is effective for a variety of tumors (such as melanoma and colon cancer) and has wide applicability.

[0020] (3) Potential for combination therapy: The drug neomycin of the present invention can be used in combination with a STING agonist, having the potential to further enhance the therapeutic effect (Example 3). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0022] Figure 1 Are the tumor growth curves (a), tumor weights (b), and LPS immunofluorescence results of tumor tissues (c) after intraperitoneal injection of lipopolysaccharide (LPS) or Escherichia coli in a melanoma mouse model, as well as the detected cell viability and proliferation results (d) when treating tumor cells with lipopolysaccharide (LPS) in vitro.

[0023] Figure 2 Are the IFN-β mRNA levels and phosphorylation levels of TBK1 after pretreatment of mouse peritoneal macrophages (PMs) with lipopolysaccharide (LPS) and then stimulation with tumor DNA (a, b), poly(dA:dT) (c, d), cGAMP (e, f), or diABZI (g, h).

[0024] Figure 3 Are the flowcharts (a), tumor growth curves (b, d), and tumor weights (c, e), LPS immunofluorescence results of tumor tissues (f), and serum LPS levels (g) in a melanoma mouse model and a mouse colon cancer model after neomycin treatment or combined treatment of neomycin and a STING agonist. Detailed implementation manners

[0025] The following will specifically elaborate on the present invention in combination with the detailed implementation manners and embodiments, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and embodiments are used to illustrate the present invention, rather than limiting the present invention.

[0026] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0027] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

[0028] Neomycin, CAS No.: 1405-10-3, the neomycin in the embodiments of the present invention was specifically purchased from MCE, product number: HY-B0470.

[0029] STING agonist CMA, CAS number: 25178-60-3. The CMA in the examples of the present invention was specifically purchased from Sigma, catalog number: SML3185. The following will elaborate in detail on the application of a neomycin in the preparation of a drug for treating tumors in combination with examples and experimental data.

[0030] Example 1: In this example, the promoting effect of lipopolysaccharide (LPS) on tumor growth was verified in an animal model. I. Method 1. Tumor cell DNA extraction After collecting tumor cells, add 20 μl of proteinase K and 300 μl of PK buffer, and incubate in a water bath set at 56°C for 1 hour. During this period, gently stir and mix every 10 minutes. When the solution becomes clear and transparent, add 100 μl of phenol-chloroform (Polarbio, catalog number: p1013) and then centrifuge at 12000 rpm for 5 minutes at room temperature. Add an equal volume of isopropanol to 200 μl of the upper aqueous phase with a pipette. Repeat the above steps of inversion mixing and centrifugation, and add 500 μl of 75% ethanol to the precipitate for washing.

[0031] 2. Tumor model construction Inject 200 ul of B16-F10 cells (1.0×10 5 cells / mouse) suspended in sterile phosphate buffer (PBS) intraperitoneally into C57BL / 6J mice. Three days later, randomly divide the tumor-bearing mice into three groups: control group (n = 4), lipopolysaccharide (LPS) group (n = 4), and Escherichia coli group (n = 4). Starting from the 4th day, the lipopolysaccharide (LPS) (1 mg / kg) group and the Escherichia coli (1×10 7 CFU) group receive intraperitoneal injection every other day. During the entire experiment, regularly measure the tumor volume (formula: V = 1 / 2×length×width 2 ) and the body weight of the mice, and record the tumor growth curve.

[0032] 3. Observation indicators At the end of the experiment, measure the tumor weight, and detect the distribution and content of lipopolysaccharide (LPS) in the tumor tissue by immunofluorescence.

[0033] Determination of serum lipopolysaccharide (LPS) level: Place the whole blood samples of the mice at room temperature for 2 hours, then centrifuge at 1000×g for 20 minutes to extract the supernatant. Detect the lipopolysaccharide (LPS) in the mouse serum by an ELISA kit (Jianglai Biotechnology Company, catalog number: JL20691).

[0034] 4. Data analysis and processing All statistical analyses in this study were performed using GraphPad Prism (version 10.1.2). Results were presented as mean ± standard deviation. The statistical significance of differences between groups was evaluated by Student’s t-test, and a p-value < 0.05 was considered statistically significant.

[0035] II. Results As Figure 1 shown, the tumor volume of mice treated with Escherichia coli or lipopolysaccharide (LPS) increased faster over time. In addition, the tumor progression rate was the fastest in mice inoculated with lipopolysaccharide (LPS) ( Figure 1 a and 1b). Moreover, more LPS accumulation was observed in the tumor microenvironment after injection of Escherichia coli and lipopolysaccharide (LPS) ( Figure 1 c). However, when tumor cells were treated with lipopolysaccharide (LPS) in vitro, no significant changes in cell viability and proliferation were detected ( Figure 1 d).

[0036] Example 2: The production of IFN-β was significantly reduced in LPS-pretreated macrophages stimulated by tumor cell DNA I. Methods 1. Sample extraction (1) DNA was extracted from various tumor cells (including LLC, MC38, and B16).

[0037] (2) Isolation of mouse peritoneal macrophages (PMs): Four days before the experiment, each C57BL / 6J wild-type mouse was intraperitoneally injected with 1 mL of 3% sodium thioglycollate (Millipore, catalog number: 108191) to induce the aggregation of peritoneal macrophages. Subsequently, the mice were euthanized by cervical dislocation and fixed supine on an anatomical board. The abdominal skin was disinfected with 75% alcohol. The abdominal skin was lifted using sterile scissors and forceps, a small incision was made to avoid damaging the abdominal organs, and 7 mL of PBS was injected into the peritoneal cavity. The abdomen of the mouse was gently massaged for 2 - 3 minutes to fully mix the intraperitoneal fluid, and then the peritoneal lavage fluid was aspirated with a sterile syringe and collected into a centrifuge tube. The collected fluid was centrifuged at 300×g for 5 minutes, and the supernatant was discarded to obtain peritoneal macrophage pellets.

[0038] 2. Cell treatment: PMs were divided into a non-pretreated group and an LPS-pretreated group (1 μg / mL LPS for 24 h); They were stimulated with the following substances for 6 hours respectively: Tumor DNA (1 μg / mL) poly(dA:dT) (1 μg / mL) cGAMP (5 μg / mL) diABZI (1 μM) Among them, macrophages were pretreated with lipopolysaccharide (LPS) for 24 h and then stimulated with tumor cell DNA. The IFN-β level was detected by RT-qPCR.

[0039] Mouse peritoneal macrophages (PMs) were pretreated with lipopolysaccharide (LPS) for 24 hours and then washed twice with PBS. Subsequently, mouse peritoneal macrophages (PMs) were stimulated with poly(dA:dT), cGAMP, or diABZI, respectively. The mRNA level of IFN-β was detected by qRT-PCR, and the phosphorylation level of TBK1 was detected by Western Blot.

[0040] II. Results As Figure 2 shown, after LPS pretreatment, the IFN-β level produced by macrophages in response to tumor DNA stimulation was significantly reduced ( Figure 2 a and 2b), indicating that lipopolysaccharide (LPS) can inhibit the anti-tumor immune response. Further, lipopolysaccharide (LPS) showed an inhibitory effect on the production of IFN-β by macrophages induced by the double-stranded DNA analog poly(dA:dT) ( Figure 2 c and 2d). The subsequent stimulation of macrophage IFN-β production with cGAMP and the STING-specific stimulant diABZI was also inhibited, and the phosphorylation of TBK1 was significantly reduced ( Figure 2 e - h). This indicates that lipopolysaccharide (LPS) has an obvious pathway dependence on the regulation of IFN-β production in macrophages. Additionally, in a melanoma mouse model, the phosphorylation level of STING was significantly reduced in the Escherichia coli group and the lipopolysaccharide (LPS) group ( Figure 2 h).

[0041] Example 3: Inhibitory effect of neomycin on tumor growth and synergistic effect of combination of neomycin and STING agonist I. Methods 1. B16-F10 melanoma mice were selected, and a tumor model was established by intraperitoneal injection of B16-F10 cells (1×10 5 cells / mouse). Starting from the 3rd day, the tumor-bearing mice were randomly divided into a neomycin alone treatment group, a STING agonist alone treatment group, a combination treatment group, and a control group, with 4 mice in each group.

[0042] 2. Model and grouping (n = 4 / group): Control group (B16 group, intraperitoneal injection of PBS); Neomycin single treatment group (CMA group): Mice were intraperitoneally injected with neomycin (30 mg / kg) every 3 days; STING agonist single treatment group: CMA (1.5 mg / kg) was intraperitoneally injected every 3 days; Combined treatment group: After tumor inoculation, neomycin (30 mg / kg) was intraperitoneally injected every 3 days, and at the same time, STING agonist CMA (1.5 mg / kg) was intraperitoneally injected every 2 days.

[0043] 3. Detection indicators During the entire experiment, the tumor volume was regularly measured (formula: V = 1 / 2 × length × width 2 ) and the body weight of the mice was recorded, and the tumor growth curve was recorded.

[0044] At the end of the experiment, the level of lipopolysaccharide (LPS) in the serum of mice was detected by ELISA to verify the effect of neomycin on reducing the level of lipopolysaccharide (LPS).

[0045] II. Experimental results Figure 3 b shows tumor volume inhibition: The tumor inhibition rate of the combined group reached 81% (vs control group, p < 0.001); The tumor inhibition rate of neomycin alone was 48% (p < 0.01), and that of CMA alone was 52% (p < 0.01) Figure 3 c shows the tumor weight: The average weight of the combined group (0.41 ± 0.06 g) was significantly lower than that of the single-drug groups (neomycin group 0.96 ± 0.12 g, CMA group 0.92 ± 0.15 g) Figure 3 g shows the serum LPS clearance: The serum LPS in the neomycin group and the combined group decreased by 74% and 80% respectively (vs control group, p < 0.001) Example 4. Verification of the single-drug effect using the MC38 colon cancer model The MC38 intestinal cancer mouse model was selected, and a tumor model was established by intraperitoneally injecting MC38 cells (5 × 10 5 cells / mouse). Starting from the 3rd day, the tumor-bearing mice were randomly divided into a treatment group and a control group, with 4 mice in each group. The mice in the treatment group were intraperitoneally injected with neomycin (30 mg / kg) every 3 days, and the mice in the control group were injected with an equal volume of PBS. During the entire experiment, the tumor volume was regularly measured (measured using calipers, formula: V = 1 / 2 × length × width 2 ) and the body weight of the mice was recorded, and the tumor growth curve was recorded. At the end of the experiment, the distribution and content of lipopolysaccharide (LPS) in the tumor tissue were detected by immunofluorescence.

[0046] Figure 3Group d showed that the tumor volume inhibition rate of the neomycin group was 55% (on the 15th day, p < 0.01); Figure 3 Group e showed that the tumor weight of the neomycin group was significantly lower than that of the control group; Figure 3 Group f showed that the fluorescence intensity of LPS in the tumor tissue decreased by 71% (vs the control group, p < 0.001).

[0047] Based on Examples 3 and 4, it can be seen that the tumor growth rate of mice given neomycin was significantly slowed down. The increase in tumor volume was significantly lower than that of the control group, the tumor weight was relatively lighter, and the lipopolysaccharide (LPS) content in the tumor tissue and serum showed an obvious downward trend.

[0048] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0049] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Use of neomycin in the preparation of a medicament for treating tumors, characterized in that, The tumor is a tumor sensitive to STING pathway activation.

2. The application according to claim 1, characterized in that The tumor sensitive to STING pathway activation includes at least one of melanoma and colon cancer.

3. The application according to claim 1, characterized in that, The drug for treating tumors further includes a pharmaceutically acceptable excipient.

4. The application according to claim 1, wherein The dosage form of the drug for treating tumors includes at least one of granules, tablets, pills, capsules, injections, and dispersants.

5. The application according to claim 1, characterized in that, The dosage of neomycin in the drug for treating tumors is 1-5 mg / kg.

6. Use of the combination of neomycin and a STING agonist in the preparation of a medicament for treating tumors, characterized in that, The tumor is a tumor sensitive to STING pathway activation.

7. The application according to claim 6, characterized in that, In the drug, neomycin is used to inhibit the interference of lipopolysaccharide on the STING signaling pathway, and the STING agonist is used to activate the STING signaling pathway, so as to anti-tumor by restoring or enhancing the anti-tumor immune response.

8. The application according to claim 6, characterized in that, The STING agonist is one or more of diABZI, cGAMP, or CMA.

9. A pharmaceutical composition, characterized in that, It contains neomycin and a STING agonist as active ingredients.

10. Use of the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating tumors, characterized in that, The tumor is a tumor sensitive to STING pathway activation.

Images