Application of ecripara in preparation of cGAS-STING agonist

Abstract

The invention provides an application of ecripara in the preparation of a cGAS-STING agonist (cGAS-STING agonist). Studies prove that the ekripara can obviously activate an STING signal channel in tumor cells, induce a downstream I-type interferon reaction and increase the infiltration level of T lymphocytes in tumor tissues. The invention provides a small molecule compound capable of activating an STING signal channel, provides a new disease application for ecrista, and provides a new possibility for development of drugs for enhancing anti-tumor immunity.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the application of ectelida in the preparation of cGAS-STING agonists. Background Technology

[0002] The development of immune checkpoint inhibitors (ICIs) has significantly changed the landscape of clinical oncology treatment. In particular, a series of monoclonal antibody drugs developed based on the PD-1 / PD-L1 axis have become standard therapy for many types of solid tumors, improving patient survival and prognosis. However, except for melanoma and Hodgkin's lymphoma, the response rate of PD-1 / PD-L1 monoclonal antibodies in the treatment of most solid tumors is generally below 30%, making it difficult for most cancer patients to benefit from immunotherapy. Improving patient responsiveness to immunotherapy has become a critical issue that urgently needs to be addressed in clinical oncology treatment. Current research indicates that low tumor immunogenicity and the lack of immune cell infiltration in the tumor microenvironment (TME) are among the main reasons for the poor clinical response to immune checkpoint inhibitors. Therefore, finding effective intervention strategies to promote antigen presentation and increase T lymphocyte infiltration holds promise for improving the low clinical response rate of immunotherapy.

[0003] The Stimulator of Interferon Genes (STING) pathway is a crucial step in the host's perception of pathogen invasion and the initiation of an immune response. Recent studies have shown that STING pathway activation can induce a Type I interferon (IFN-I) response, mediating the recruitment and activation of natural killer (NK) cells and increasing T lymphocyte infiltration in the tumor microenvironment (TME). Furthermore, the STING signaling pathway can also induce the release of tumor antigens and promote the expression of Major Histocompatibility Complex Class I (MHC-I), which helps to enhance T lymphocyte recognition and killing of tumor cells. Therefore, finding effective STING agonists can reshape the tumor immune microenvironment, thereby sensitizing immunotherapy.

[0004] Given the crucial role of the STING pathway in anti-tumor immune responses, the development of STING agonists has become a new hot topic in anti-tumor drug research both domestically and internationally. Although some CDNs analogs, such as MK-1454, and non-CDNs compounds like TAK-676 and SNX-281, have gradually entered clinical trials, no STING agonist has yet been successfully approved for clinical cancer treatment. Therefore, the development of STING agonists has not yet achieved a breakthrough and requires continued in-depth research.

[0005] Elacridar is an oral P-glycoprotein inhibitor. Its pharmacological mechanism involves selectively inhibiting the efflux transport function of P-glycoprotein against chemotherapeutic drugs, thereby increasing the concentration of chemotherapeutic drugs in tumor cells and effectively sensitizing chemotherapy. Elacridar has demonstrated good safety and favorable pharmacokinetic properties in clinical studies, but it has not yet been approved for clinical treatment. Recent studies have found that Elacridar downregulates PD-L1 protein expression in tumors, but its other functions in tumor immunity remain unclear. Summary of the Invention

[0006] The purpose of this invention is to provide the application of Eclazamide in the preparation of cGAS-STING agonists, particularly in combination with immunotherapy for the treatment of solid tumors. Eclazamide's chemical name is N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridin-4-carboxamide, and its molecular formula is C2. 34 H 33 N3O5 has a molecular weight of 563.64.

[0007] Experiments have confirmed that Eclatida (10 μM) activates the STING signaling pathway in vitro, as observed by Western blotting and immunofluorescence, and promotes downstream type I interferon responses, as observed by qRT-PCR. Furthermore, in the CT26 animal model, Eclatida significantly enhances the infiltration of cytotoxic T lymphocytes in tumor tissue.

[0008] Therefore, the present invention provides the following: Application of Ectodextrin in the preparation of cGAS-STING agonists.

[0009] Ekrita can achieve at least one of the following functions: 1) Enhances the phosphorylation level of TBK1 and STING proteins in tumor cells.

[0010] 2) Increases the level of dsDNA in the cytoplasm of tumor cells; 3) Induces type I interferon response in tumor cells; 4) Enhances the infiltration of cytotoxic T lymphocytes in tumor tissue.

[0011] A STING agonist, the active ingredient of which is ecrigrade, can activate the cGAS-STING signaling pathway.

[0012] Application of Ekritarash in the preparation of therapeutic drugs that enhance STING-mediated immune responses.

[0013] Application of Eclata in the preparation of drugs that enhance anti-tumor immunotherapy.

[0014] A drug that enhances anti-tumor immunotherapy, whose active ingredient contains ecrifa.

[0015] The tumors are non-small cell lung cancer and colorectal cancer.

[0016] The drug is made from eclipta and pharmaceutically acceptable excipients and is formulated as an oral preparation.

[0017] Eclareta, a P-glycoprotein inhibitor, has undergone clinical trials to evaluate its role in overcoming chemotherapy resistance, but it has not yet been approved for clinical treatment. Furthermore, studies have reported the downregulation of PD-L1 protein expression in tumor cells by clareta, but its other functions in tumor immunity remain unclear. This invention demonstrates through in vitro experiments and animal model studies that clareta can activate the STING signaling pathway in tumor cells and induce a type I interferon response, possessing the potential to sensitize immunotherapy, thereby expanding the clinical application of the chemotherapeutic drug clareta in tumor immunotherapy. The significance of this invention lies not only in clarifying the possibility of using clareta for clinical tumor immunotherapy, but also in providing new ideas and potential targets for the development of STING agonists.

[0018] The inventors discovered in their research that eclipta significantly activates the cGAS-STING signaling pathway in tumor cells and induces a downstream type I interferon response. Currently, no STING agonists are approved for clinical use in tumor immunotherapy, and no research or inventions related to eclipta's activation of the STING signaling pathway have been found. This invention provides a small molecule compound for clinical tumor immunotherapy, offering a new pharmaceutical application for eclipta. Attached Figure Description

[0019] Figure 1 The images show the concentration-dependent enhancement of phosphorylation levels of TBK1 and STING proteins in tumor cells by ectelidae, and the immunoblot and statistical graphs of proteins from ectelidae (2.5 μM, 5 μM and 10 μM) acting on H1299 non-small cell lung cancer cells.

[0020] Figure 2 This image shows that Eclatide significantly increases the level of dsDNA in the cytoplasm of tumor cells. It is an immunofluorescence image of Eclatide (10 μM) acting on H1299 non-small cell lung cancer cells.

[0021] Figure 3 The graph shows that ectelida significantly induces type I interferon response. It is a statistical graph of qRT-PCR results of ectelida (2.5 μM, 5 μM and 10 μM) acting on non-small cell lung cancer cells H1299 and mouse colorectal cancer cells CT26.

[0022] Figure 4 This is a statistical graph showing that ectelida significantly enhances the infiltration of cytotoxic T lymphocytes in tumor tissue, and that ectelida (200 mg / kg) increased the infiltration of T lymphocytes and cytotoxic T lymphocytes in tumor tissue in the CT26 xenograft model. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1: Eclatrin significantly upregulated the phosphorylation levels of key proteins TBK1 and STING in the STING signaling pathway after acting on H1299 non-small cell lung cancer cells. The specific steps are as follows: Non-small cell lung cancer cells H1299 were selected and cultured at a concentration of 1.0 × 10⁻⁶. 6 Cells were seeded at a density of 6-well plates and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with ectodiclofenac (2.5 μM, 5 μM, and 10 μM) for 24 h. Cells were then collected and lysed, and proteins were extracted. Phosphorylation levels of TBK1 and STING proteins in the cells were detected by Western blotting. The enhancing effect of ectodiclofenac on the phosphorylation levels of TBK1 and STING proteins in H1299 non-small cell lung cancer cells is shown in [the figure below]. Figure 1 Experimental results showed that ectidone can increase the phosphorylation levels of TBK1 and STING proteins in tumor cells in a concentration-dependent manner.

[0025] Example 2: Eclipta prostrata significantly increased the dsDNA level in the cytoplasm of non-small cell lung cancer H1299 cells after treatment. The specific steps are as follows: Non-small cell lung cancer cells H1299 were selected and cultured at a concentration of 1.5 × 10⁻⁶. 4Cells were seeded at a density of 1 / well in confocal microscopy chambers and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with ectodiclofenac (10 μM) for 24 h. Cellular dsDNA was detected using dsDNA fluorescence detection of antibody-labeled cytoplasmic dsDNA, and DAPI was used as the nuclear dye. Images were acquired using a laser confocal microscope. The effect of ectodiclofenac on dsDNA levels in non-small cell lung cancer cells H1299 is shown in [the table below]. Figure 2 Experimental results showed that Eclatide significantly increased the level of dsDNA in the cytoplasm of tumor cells.

[0026] Example 3: Eclipta significantly induced a type I interferon response in non-small cell lung cancer H1299 cells. The specific steps are as follows: Non-small cell lung cancer cells H1299 and mouse colorectal cancer cells CT26 were selected and subjected to a 1.0×10⁻⁶ m²⁻¹. 6 Cells were seeded at a density of 6-well plates and cultured overnight at 37°C with 5% CO2. The following day, cells were treated with ectodiclofenac (2.5 μM, 5 μM, and 10 μM) for 24 h. Cell RNA was collected and extracted, and the RNA was detected by qRT-PCR. CCL5 , CXCL10 and IFN-β Changes in mRNA levels. The effect of ecridia on the type I interferon response of tumor cells is shown in [reference needed]. Figure 3 Experimental results show that Eclipta can significantly increase CCL5 , CXCL10 and IFN-β The mRNA levels suggest that ecrifa can significantly induce type I interferon response in tumor cells.

[0027] Example 4: Eclatide significantly enhanced the infiltration of cytotoxic T lymphocytes in tumor tissue. The specific steps are as follows: CT26 colorectal cancer cells from mice were seeded into the left axilla of 6-week-old male BALB / c mice (5×10⁻⁶ cells / year). 5 Cells / animal) were used to establish a xenograft tumor model. The tumor was allowed to grow to 50-100 mm. 3 Mice were randomly divided into two groups: a control group and an ectelidae treatment group (200 mg / kg), with six mice in each group. Ecelidae was administered once daily by gavage, or an equal volume of the solvent. On day 10 after ectelidae administration, the mice were sacrificed, and tumor tissue was isolated. Flow cytometry was used to detect the presence of CD45+ T lymphocytes in the tumor tissue. + CD3 + ) and cytotoxic T lymphocytes (CD45) + CD8 + The level of invasion of ectodiclofenac in tumor tissue. The effect of ectodiclofenac on the level of immune cell infiltration in tumor tissue is shown in [reference needed]. Figure 4Experimental results showed that Eclatide significantly increased the infiltration level of cytotoxic T lymphocytes in tumor tissue.

[0028] The embodiments described above are merely some preferred embodiments of the present invention, and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Application of Eclipta in the preparation of cGAS-STING agonists.

2. The application according to claim 1, characterized in that, Eclata enhances the phosphorylation levels of TBK1 and STING proteins in tumor cells.

3. The application according to claim 1, characterized in that, Eclipta increases the level of dsDNA in the cytoplasm of tumor cells.

4. The application according to claim 1, characterized in that, Eclipta-methyl induces type I interferon response in tumor cells.

5. The application according to claim 1, characterized in that, Eclipta enhances the infiltration of cytotoxic T lymphocytes in tumor tissue.

6. A STING agonist, characterized in that, Its active ingredient is ecrigrade, which can activate the cGAS-STING signaling pathway.

7. Application of Ekritarash in the preparation of therapeutic drugs that enhance STING-mediated immune responses.

8. Application of Ekritarash in the preparation of drugs that enhance anti-tumor immunotherapy.

9. A drug for enhancing anti-tumor immunotherapy, characterized in that, Its active ingredient contains ecrifa.

10. The medicament according to claim 9, characterized in that, The tumors are non-small cell lung cancer and colorectal cancer.

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