A pharmaceutical composition containing a sting agonist and a wip1 inhibitor and a liposome thereof and use thereof

Abstract

The present application relates to a kind of drug composition containing STING agonist and WIP1 inhibitor and its liposome and application.The composition can be co-encapsulated in liposome with STING agonist and WIP1 inhibitor, form stable drug delivery system.The liposome can promote efficient endocytosis of cell, and release two active ingredients in cytoplasm synchronously, block the negative feedback mechanism of STING signal path by WIP1 inhibitor, enhance and prolong the activation level of STING path, to synergistically inhibit tumor growth.Based on the mechanism, the drug composition of the present application can be used for preparing the drug for treating diseases related to STING path (such as tumor).

Description

Technical Field

[0001] This invention relates to the fields of chemistry and biomedicine, specifically to a synergistic pharmaceutical composition containing a STING agonist and a WIP1 inhibitor, and further to liposomes comprising the composition and their uses. Background Technology

[0002] The STING (Strained Interferon Gene Stimulator) signaling pathway is a core mechanism by which the body senses abnormal DNA in the cytoplasm and initiates innate immune defense. The development of its agonists is a cutting-edge focus in the field of tumor immunotherapy. However, the clinical translation of these drugs has been far slower than expected. The fundamental reason is that existing technologies have failed to systematically address the two core contradictions that limit their efficacy.

[0003] Firstly, there is a contradiction between the pharmacochemical properties of existing STING agonists and their ideal pharmacokinetic behavior. First-generation STING agonists (such as cyclic dinucleotide CDNs) are highly hydrophilic, have poor membrane permeability, and are highly sensitive to phosphodiesterases, resulting in extremely low bioavailability after systemic administration, forcing them to be administered via intratumoral injection, which significantly limits their clinical application. While subsequent developments of non-nucleotide small molecule agonists (such as MSA-2) have made progress in oral bioavailability, the risk of excessive immune activation due to their systemic distribution poses a serious challenge to clinical safety, revealing that the lack of targeted drug delivery capability is a major bottleneck in current technology.

[0004] Secondly, and more profoundly, lies the conflict between the intensity of STING pathway activation and endogenous negative feedback regulation within the cell. The initiation of STING signaling is not a unidirectional enhancement but rather subject to precise homeostatic regulation. Research in this field has found that wild-type p53-induced phosphatase 1 (WIP1 / PPM1D), as a key negative regulatory node in this pathway, directly dephosphorylates TBK1, thereby interrupting IRF3 activation and type I interferon production. Crucially, STING pathway activation feedback-induced upregulation of WIP1 expression, forming a strong self-attenuation loop. This inherent "braking" mechanism, in principle, determines that the single application of STING agonists is insufficient to achieve sustained signal amplification, constituting a fundamental mechanistic obstacle to its poor efficacy.

[0005] Faced with the aforementioned challenges, existing technological solutions mostly follow a "linear optimization" approach, focusing either on modifying the agonist molecule structure or attempting to combine it with downstream immune checkpoint inhibitors. However, these strategies fail to directly intervene in the core negative feedback mechanism of STING signaling, and cannot fundamentally solve the problems of insufficient signal intensity and persistence. Therefore, there is an urgent need in this field for a novel strategy that can remove signal self-inhibition at its mechanistic source.

[0006] Based on this, this invention proposes a groundbreaking "shackle-breaking" combination therapy: combining a STING agonist with a WIP1 inhibitor. The aim is to directly block the key negative feedback loop of the STING-TBK1-IRF3 signaling axis by inhibiting the phosphatase activity of WIP1, thereby achieving synergistic amplification and sustained activation of the signal at a mechanistic level. Furthermore, to overcome the in vivo delivery barriers of the two drugs and achieve synergistic effects, this invention designs a liposome delivery system co-loaded with the STING agonist and the WIP1 inhibitor, providing crucial technical support for the efficient and safe translation of this strategy. Summary of the Invention

[0007] Objective of the Invention: The objective of this invention is to provide a pharmaceutical composition capable of synergistically activating the STING pathway, a dual-drug liposome containing the composition, and its use in tumor therapy. Although STING agonists (such as MSA-2) have shown great potential in activating innate immunity against tumors, their monotherapy efficacy is limited, and systemic administration faces problems such as poor targeting and easy overactivation of normal tissues. Furthermore, the activation effect of the STING signaling pathway is often constrained by intracellular negative feedback mechanisms. To overcome these bottlenecks, this invention unexpectedly discovered that WIP1 inhibitors can produce a strong synergistic effect with STING agonists, significantly enhancing the latter's ability to activate the STING pathway and its anti-tumor immune response. Based on this core discovery, this invention provides a pharmaceutical composition combining a WIP1 inhibitor and a STING agonist, its preparation method, and its application. Specifically, this invention maximizes the synergistic effect through the following strategies: Combination therapy: The direct combination of WIP1 inhibitors (including the small molecule inhibitor GSK2830371 or siRNA targeting WIP1) with the STING agonist MSA-2 has been shown to significantly enhance the production of key cytokines such as IFN-β in both cell and animal models.

[0008] Innovative Delivery System: To further enhance drug targeting and stability while reducing systemic toxicity, this invention also modifies the MSA-2 molecule as a prodrug and innovatively constructs a dual-drug liposome capable of co-loading a WIP1 inhibitor and a STING agonist prodrug. This nanodelivery system can efficiently co-deliver the two drugs to the tumor lesion, enhancing STING pathway activation and anti-tumor effects while effectively improving the pharmacokinetic behavior of the drugs.

[0009] The objective of this invention is achieved through the following technical solution: This invention provides a pharmaceutical composition capable of synergistically activating the STING pathway, the pharmaceutical composition comprising a STING agonist and a WIP1 inhibitor.

[0010] Preferably, the STING agonist is a non-nucleotide small molecule agonist MSA-2 or its prodrug compound; the WIP1 inhibitor is a small molecule inhibitor GSK2830371 or a small interfering RNA (siWIP1) targeting the WIP1 gene (mouse gene number 53892, human gene number 8493).

[0011] More preferably, the pharmaceutical composition comprises MSA-2 prodrug and GSK2830371; or comprises MSA-2 prodrug and siWIP1.

[0012] Furthermore, the nucleotide sequence of siWIP1, which targets the mouse WIP1 gene, is as follows: Forward sequence: 5′-GCGGCAGUGUGAUGAACAATT-3′, Reverse sequence: 5′-UUGUUCAUCACACUGCCGCTT-3′.

[0013] The present invention also provides a dual-drug liposome that simultaneously encapsulates the STING agonist and WIP1 inhibitor from the above-mentioned pharmaceutical composition.

[0014] This invention discovers that WIP1 inhibitors can significantly enhance the activation of the STING pathway by STING agonists. A synergistic delivery system was constructed by co-encapsulating both drugs in liposomes. This dual-drug liposome effectively protects the drug components, enhances accumulation at the tumor site, and achieves synergistic drug release in the cytoplasm, thereby strongly inducing the production of cytokines such as type I interferon and reshaping the tumor immune microenvironment.

[0015] Preferably, the dual-drug liposomes are composed of lecithin, cholesterol, DSPE-PEG2000, and cationic lipid DOTAP in a mass ratio of (70~75):(10~15):(15~20):(0.5~5). This composition forms nanoliposomes with uniform particle size, high encapsulation efficiency, and good stability, enabling efficient co-delivery of drugs.

[0016] More preferably, when siWIP1 is loaded, the lipid component contains the cationic lipid DOTAP, and the nitrogen-to-phosphorus ratio (N / P) of the liposomes to siRNA is 0-24.

[0017] Furthermore, the average particle size of the dual-drug liposomes is 80~150 nm.

[0018] The present invention also provides a method for preparing the dual-drug liposomes, comprising the following steps: using an ethanol injection method, dissolving the lipid material in an organic phase, dissolving the drug in a co-solvent and mixing it with the lipid solution, then rapidly injecting this mixed solution into an aqueous phase, and subsequently removing the organic solvent by dialysis to obtain the dual-drug liposomes.

[0019] This invention utilizes liposome self-assembly technology to achieve efficient co-encapsulation of STING agonists and WIP1 inhibitors under mild conditions. The method is simple, easy to scale up, and the prepared liposomes exhibit excellent formulation properties.

[0020] The present invention also provides the use of the dual-drug liposomes in the preparation of tumor immunotherapy drugs.

[0021] The dual-drug liposomes described in this invention serve as a nanomedicine delivery system, exhibiting excellent water solubility, stability, and biocompatibility. This system can target tumor tissue through enhanced penetration and retention effects, and is effectively internalized by tumor cells and immune cells. Intracellularly, the liposomes release their carried STING agonist and WIP1 inhibitor, which synergistically activate the STING signaling pathway and inhibit its negative feedback regulation, thereby significantly enhancing the anti-tumor immune response and laying the foundation for highly effective combined immunotherapy.

[0022] This nano-drug delivery system achieves a dual synergistic effect of STING pathway activation and WIP1 phosphatase inhibition through the co-delivery of STING agonists and WIP1 inhibitors, thereby more effectively inhibiting tumor growth.

[0023] The present invention also provides the use of the aforementioned dual-drug liposomes in the preparation of medicaments for the treatment of pancreatic cancer or liver cancer.

[0024] Beneficial effects: (1) This invention is the first to discover and combine WIP1 inhibitors with STING agonists. The two exhibit a significant synergistic effect, which can strongly activate the STING pathway, induce higher levels of type I interferon and pro-inflammatory cytokines, thereby reshaping the tumor immune microenvironment.

[0025] (2) The dual-drug liposomes of the present invention can efficiently co-deliver two drugs with complementary mechanisms of action to the tumor site, solving the problems of poor targeting of free drugs, high systemic toxicity and poor in vivo stability.

[0026] (3) The dual-drug liposomes described in this invention significantly improve the pharmacokinetic behavior of the drugs, prolong the retention time of the drugs in the tumor, and generate a strong distal anti-tumor effect by activating immune cells.

[0027] (4) The preparation process of the dual-drug liposomes described in this invention is simple, with high drug loading capacity, good stability, and easy to realize industrial production, providing a brand-new formulation solution for the combined clinical application of STING agonist and WIP1 inhibitor.

[0028] [Uses and Treatments] The present invention provides the use of the above-described pharmaceutical composition or its pharmaceutically acceptable prodrug or the above-described liposome preparation in the preparation of a medicament for treating diseases related to STING.

[0029] The present invention provides the use of the above-described pharmaceutical composition or its pharmaceutically acceptable prodrug or the above-described liposome preparation in the preparation of immune adjuvants.

[0030] The present invention provides a method for treating a disease related to STING, the method comprising administering to a subject in need a therapeutically effective amount of the above-described pharmaceutical composition or a pharmaceutically acceptable prodrug or liposomal preparation thereof.

[0031] In some implementations, the disease associated with STING is selected from inflammatory diseases, autoimmune diseases, infectious diseases, cancer, or precancerous syndromes.

[0032] In some implementations, the diseases associated with STING are selected from melanoma, cervical cancer, breast cancer, oral cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial carcinoma, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, renal cancer, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasmacytoma, Wilms' tumor, hepatocellular carcinoma, and gout. Rhinitis, appendicitis, atherosclerosis, asthma, rheumatoid arthritis, allergic dermatitis, cholecystitis, cirrhosis, degenerative arthritis, dermatitis, enteritis, encephalitis, gastritis, nephritis, hepatitis, pituitary inflammation, irritable bowel syndrome, meningitis, multiple sclerosis, myocarditis, myasthenia gravis, mycosis fungoides, osteomyelitis, pancreatitis, pericarditis, pernicious anemia, pneumonia, primary biliary sclerosing cholangitis, polyarteritis nodosa, lupus erythematosus, thyroiditis, urethritis, uveitis, or vasculitis.

[0033] In some embodiments, the above-described pharmaceutical composition, or its pharmaceutically acceptable prodrug, or the above-described liposomal formulation, is administered via one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the above-described pharmaceutical composition, or its pharmaceutically acceptable prodrug, or the above-described liposomal formulation, is administered, for example, via an enteral or parenteral route. In some embodiments, the above-described pharmaceutical composition, or its pharmaceutically acceptable prodrug, or the above-described liposomal formulation, at a dose of about 0.001 mg / kg to about 50 mg / kg, is administered to the subject.

[0034] The dosing regimen can be adjusted to provide the optimal required response. For example, a single bolus injection can be administered, several fractions can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. It should be noted that dosage values ​​can vary depending on the type and severity of the disease to be alleviated and can include single or multiple doses. To further understand, for any given individual, the specific dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering the composition or supervising its administration. Attached Figure Description

[0035] Figure 1 To demonstrate the expression level of IFN-β after different concentrations of WIP1 inhibitor were combined with STING agonist in bone marrow-derived macrophages in Example 1.

[0036] Figure 2 The expression levels of IFN-β after different concentrations of WIP1 inhibitors were combined with STING agonists in Example 1 and applied to bone marrow-derived dendritic cells.

[0037] Figure 3 The expression level of IFN-β in bone marrow-derived macrophages after different treatment times with a combination of WIP1 inhibitor and STING agonist in Example 2.

[0038] Figure 4 The expression level of IFN-β in bone marrow-derived dendritic cells after different treatment times with a combination of WIP1 inhibitor and STING agonist in Example 2.

[0039] Figure 5 This describes the antitumor effect of the combination of WIP1 inhibitor and STING agonist in the KPC pancreatic cancer model in Example 3.

[0040] Figure 6 The image shows a cryo-transmission electron microscope image and particle size distribution of the dual-drug liposomes loaded with GSK2830371 in Example 5.

[0041] Figure 7The results show the stability evaluation of the dual-drug liposomes loaded with GSK2830371 in Example 5.

[0042] Figure 8 This illustrates the synergistic effect of the combined use of WIP1 inhibitor and STING agonist on dendritic cell activation in Example 6.

[0043] Figure 9 The changes in TBK1 phosphorylation levels after treatment of different cells with dual-drug liposomes loaded with GSK2830371 in Example 7 are shown.

[0044] Figure 10 The expression level of IFN-β in different cells after treatment with the dual-drug liposome loaded with GSK2830371 in Example 8.

[0045] Figure 11 The effect of dual-drug liposome therapy loaded with GSK2830371 on tumor growth in Example 9.

[0046] Figure 12 This study describes the effect of dual-drug liposome therapy loaded with GSK2830371 in Example 9 on the levels of multiple anti-tumor cytokines within the tumor.

[0047] Figure 13 The particle size distribution of the siWIP1-loaded dual-drug liposomes in Example 10 is shown.

[0048] Figure 14 The RNA encapsulation efficiency of the siWIP1-loaded dual-drug liposomes in Example 10 is shown.

[0049] Figure 15 This is a cryo-transmission electron microscopy image of the siWIP1-loaded dual-drug liposomes in Example 10.

[0050] Figure 16 This refers to the antitumor effect of the siWIP1-loaded dual-drug liposomes in the KPC orthotopic pancreatic cancer model in Example 11.

[0051] Figure 17 This refers to the antitumor effect of the siWIP1-loaded dual-drug liposomes in the Hepa1-6 orthotopic liver cancer model in Example 12. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0054] The WIP1-siRNA (siWIP1) was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., and its nucleotide sequence is as follows: Forward sequence: 5′-GCGGCAGUGUGAUGAACAATT-3′, Reverse sequence: 5′-UUGUUCAUCACACUGCCGCTT-3′.

[0055] Example 1: Evaluation of the synergistic effect of different concentrations of WIP1 inhibitors and STING agonists in promoting IFN-β secretion by bone marrow-derived macrophages and dendritic cells Dendritic cells extracted and isolated in vitro were processed at a density of 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 12-well plates and co-incubated with different concentrations of MSA-2 (20, 40 μM) and GSK2830371 (0, 10, 20, 40, 80, 160 μM), with fresh culture medium as a blank control. After 12 hours of treatment, the cell culture medium was rehydrated and collected in sterile centrifuge tubes. After centrifugation at 850 g for 5 minutes, the supernatant was collected, and the secretion level of IFN-β was detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0056] The results are as follows Figure 1 and Figure 2 As shown, under the synergistic effect of WIP1 inhibitors, the STING agonist MSA-2 can significantly increase the level of IFN-β in the supernatant of macrophages and dendritic cells. For example, different concentrations of WIP1 inhibitors can increase the IFN-β secretion level induced by 20 μM MSA-2 by approximately 20-fold, indicating that WIP1 inhibitors can effectively enhance the activation ability of MSA-2 on the STING pathway.

[0057] Example 2: Effects of combined treatment with WIP1 inhibitor and STING agonist at different time points on cellular IFN-β expression levels. Dendritic cells extracted and isolated in vitro were processed at a density of 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 12-well plates and co-incubated with MSA-2 (30 μM) and GSK2830371 (20 μM), respectively, with fresh culture medium as a blank control. After 0, 1, 3, 6, 12, and 24 hours of treatment, the cell culture medium was collected and centrifuged at 850 g for 5 minutes. The supernatant was collected and the IFN-β secretion level was detected using an ELISA kit.

[0058] The results are as follows Figure 3 and Figure 4 As shown, the STING agonist can significantly increase the level of IFN-β in the supernatants of macrophages and dendritic cells at different time points under the synergistic effect of the WIP1 inhibitor, further confirming that the WIP1 inhibitor can effectively enhance the activation effect of MSA-2 on the STING pathway.

[0059] Example 3 Anti-tumor effect of the combination of WIP1 inhibitor and STING agonist in a KPC pancreatic cancer model Construct a murine KPC pancreatic cancer subcutaneous xenograft model: Digest KPC cells with trypsin, prepare a cell suspension after washing with PBS, and inoculate 5×10 6 cells per mouse subcutaneously into the right abdominal wall of healthy male C57BL / 6 mice, with an injection volume of 100 μL per mouse. When the tumor volume grows to about 100 mm 3 , randomly divide the mice into 4 groups (n = 6). To evaluate the synergistic anti-tumor activity of the WIP1 inhibitor and the STING agonist, intraperitoneally inject free MSA-2 (25 mg / kg), GSK2830371 (25 mg / kg), or an equimolar mixture of both on days 0, 3, and 6, with normal saline as the control. Measure and record the tumor volume of the mice every three days, measure the longest diameter (L, mm) and the shortest diameter (W, mm) of the tumor using a vernier caliper, and calculate the tumor volume according to the formula V=(L×W 2 ) / 2 (where W < L).

[0060] The anti-tumor efficacy results are as Figure 5 shown. Compared with any single drug treatment group, the combination of the WIP1 inhibitor and the STING agonist can significantly inhibit the growth rate of the tumor volume in mice.

[0061] Example 4 Synthesis of a STING agonist prodrug compound

[0062] Weigh MSA-2 (200 mg, 0.68 mmol, 1.0 eq) and 4-(4-morpholino)-1-butanol (140 mg, 0.88 mmol, 1.3 eq) into 3 mL of anhydrous DCM. Then add TBTU (284 mg, 0.88 mmol, 1.3 eq) and DIEA (114 mg, 0.88 mmol, 1.3 eq) to it. After that, stir the reaction solution at 43 °C for 24 h. After the reaction is completed, extract the reaction solution with DCM. The organic layer obtained by liquid separation is washed sequentially with 5% citric acid, saturated sodium bicarbonate, and saturated sodium chloride, and then dehydrated with anhydrous sodium sulfate. Using dichloromethane and ethyl acetate as eluents, separate and purify by silica gel column chromatography to obtain a pink solid with a yield of 276 mg and a yield of 91.1%.

[0063] Example 5 Preparation of GSK2830371-loaded dual-drug liposomes STING agonist prodrug liposomes were prepared using the ethanol injection method: 70.0 mg of lecithin, 10.0 mg of cholesterol, and 15.0 mg of DSPE-PEG2000 were weighed at a mass ratio of 14:2:3 and dissolved in 0.9 mL of ethanol. Then, STING agonist prodrug compound (MSA-2 concentration of 100 mg / mL) and GSK2830371 (concentration of 100 mg / mL) dissolved in 0.1 mL of dimethyl sulfoxide (DMSO) were added. 1 mL of the above mixture was rapidly injected into 9 mL of phosphate-buffered saline (PBS) to obtain a STING agonist prodrug liposome solution co-loaded with GSK2830371 (MSA-2 and GSK2830371 concentrations were both 0.5 mg / mL). The liposome solution was dialyzed for 24 hours in a dialysis bag with a molecular cutoff of 3 kDa to remove organic solvents, and the drug concentration was subsequently determined by reversed-phase high-performance liquid chromatography (RP-HPLC).

[0064] The morphology of the obtained dual-drug liposomes was observed by transmission electron microscopy as follows: Figure 6 As shown, it exhibits a vesicular, single-chamber structure. The particle size distribution is as follows... Figure 7 As shown, the nanoparticles are monodisperse and uniformly distributed, with an average particle size of approximately 100 nm. Stability experiments indicate that the liposomes can be stably stored in PBS for over 30 days, demonstrating good physiological stability.

[0065] Example 6: Synergistic effect of combined use of WIP1 inhibitor and STING agonist on dendritic cell activation The maturation status of bone marrow-derived dendritic cells (BMDCs) was detected by flow cytometry: cells were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded in 12-well plates, and free MSA-2 (30 μM), GSK2830371 (20 μM), or single-drug or dual-drug liposomes at equal concentrations were added, with fresh culture medium as a blank control. After 12 hours of treatment, BMDCs were collected, washed once with PBS, and incubated on ice for 10 minutes with anti-mouse CD16 / 32 antibody. Subsequently, staining was performed using the following antibodies: anti-mouse CD45-APC, anti-mouse CD11c-PE / Cy7, anti-mouse CD80-FITC, and anti-mouse CD86-PerCP / Cy5.5. Cells were incubated on ice in the dark for 20 minutes, washed with PBS, resuspended, and analyzed by flow cytometry.

[0066] The results are as follows Figure 8As shown, compared with other groups, the expression level of co-stimulatory molecules CD80 / CD86 on the surface of dendritic cells stimulated by dual-drug liposomes was significantly increased, and significantly higher than that of the single STING agonist liposome stimulation group, indicating that GSK2830371 can synergistically enhance the activation ability of STING agonists on dendritic cells.

[0067] Example 7: Changes in TBK1 phosphorylation levels after treatment of different cells with GSK2830371-loaded dual-drug liposomes Dendritic cells or KPC pancreatic cancer cells extracted in vitro were used at a concentration of 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 12-well plates, and single-drug liposomes (30 μM), GSK2830371 (20 μM), or dual-drug liposomes loaded with GSK2830371 at equivalent drug concentrations were added, with fresh culture medium as a blank control. After 12 hours of treatment, cells were collected, fixed, and permeabilized with permeabilization solution, followed by incubation with anti-p-TBK1 flow cytometry antibody at room temperature in the dark. After washing with PBS, fluorescently labeled secondary antibody was added and incubation was continued in the dark. After washing again, cells were resuspended in PBS, and TBK1 phosphorylation levels were detected by flow cytometry.

[0068] The results are as follows Figure 9 As shown, dual-drug liposomes loaded with GSK2830371 can significantly increase the level of p-TBK1 in dendritic cells and KPC pancreatic cancer cells, indicating that WIP1 inhibitors can effectively enhance STING pathway activation, and the liposome co-delivery strategy helps to improve the drug's activation effect on the STING signaling pathway.

[0069] Example 8: Expression level of IFN-β in different cells after treatment with GSK2830371-loaded dual-drug liposomes Dendritic cells or KPC pancreatic cancer cells were injected at a rate of 1 × 10⁻⁶ cells per well. 5 Each well was seeded into a 12-well plate, and liposomes containing a single STING agonist (30 μM), GSK2830371 (20 μM), or a dual-drug liposome loaded with GSK2830371 at the same concentration were added. Fresh culture medium was used as a blank control. After 12 hours of treatment, the supernatant was collected, centrifuged at 850 g for 5 minutes, and the IFN-β secretion level was detected using an ELISA kit.

[0070] The results are as follows Figure 10 As shown, the dual-drug liposomes significantly increased the IFN-β levels in both cell supernatants, further validating the enhancing effect of WIP1 inhibitors on STING pathway activation and the effectiveness of liposome co-delivery.

[0071] Example 9: Antitumor efficacy of GSK2830371-loaded dual-drug liposomes in a large-volume pancreatic cancer model. Construction of KPC subcutaneous xenograft model: The method is the same as in Example 3, until the tumor grows to about 300 mm. 3 Mice were randomly divided into 5 groups (n=9). On days 12, 14, and 16, mice were intraperitoneally injected with free GSK2830371 (15 mg / kg) or a mixture of free MSA-2 (15 mg / kg) / GSK2830371 (15 mg / kg), or via tail vein injection with a single STING agonist liposome (15 mg / kg) or a dual-drug liposome loaded with GSK2830371. Saline was used as a control. Tumor volume was measured every two days and calculated as before. After treatment, mouse tumor tissue was collected, homogenized with an appropriate amount of PBS, and centrifuged. The supernatant was collected after centrifugation, and the levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), and IFN-β in the supernatant were detected using enzyme-linked immunosorbent assay (ELISA).

[0072] The results are as follows Figure 11 As shown, compared with the free drug mixture group, the dual-drug liposomes loaded with GSK2830371 can significantly inhibit tumor growth, indicating that the liposome delivery system can effectively enhance the anti-tumor effect of the drug.

[0073] In addition, such as Figure 12 As shown, the results of cytokine detection in tumor tissue homogenate showed that the levels of TNF-α, IFN-γ, IL-6 and IFN-β in the local tumor microenvironment of mice treated with dual-drug liposomes were significantly higher than those in other control groups, further demonstrating that the dual-drug liposomes can effectively activate the immune response in the tumor.

[0074] Example 10 Preparation of siWIP1-loaded dual-drug liposomes The liposomes were prepared using the ethanol injection method: 70.0 mg of lecithin, 10.0 mg of cholesterol, 15.0 mg of DSPE-PEG2000, and 1.0 mg of DOTAP were weighed in a mass ratio of 14:2:3:0.2 and dissolved in 0.9 mL of ethanol. Then, a STING agonist prodrug compound (MSA-2 concentration of 100 mg / mL) dissolved in 0.1 mL of DMSO was added. 1 mL of the mixed solution was rapidly injected into 9 mL of PBS containing 89.75 μg siWIP1 to obtain siWIP1-co-loaded dual-drug liposomes (MSA-2 concentration of 0.5 mg / mL) with a nitrogen-to-phosphorus ratio (N / P) of 1. Liposomes with different N / P ratios could be prepared by adjusting the amount of DOTAP. The liposome solution was dialyzed in a 3 kDa dialysis bag for 24 hours to remove organic solvents. The MSA-2 concentration was determined by reversed-phase high-performance liquid chromatography (RP-HPLC), and the siWIP1 encapsulation efficiency was determined using an RNA detection kit.

[0075] like Figure 13 As shown, the addition of DOTAP significantly reduced the liposome particle size; particles exhibited monodisperse distribution at different N / P ratios, with an average particle size of approximately 100 nm. With increasing N / P, the encapsulation efficiency of siWIP1 gradually increased, approaching 100% at N / P=6. Figure 14 Transmission electron microscopy images ( ) Figure 15 The results show that the liposomes have a single-chamber vesicle structure.

[0076] Example 11: Antitumor effect of siWIP1-loaded dual-drug liposomes in a KPC orthotopic pancreatic cancer model. KPC-luc cells were mixed with matrix gel at a 1:1 volume ratio. Six-week-old male C57BL / 6 mice were anesthetized, fixed, and their abdomens were shaved and disinfected. A 1 cm incision was made along the linea alba, and the pancreas was gently extracted. KPC-luc cell mixture (5 × 10⁻⁶ cells / mL) was injected using an insulin needle. 6 / each). After pressing the injection site for 1 minute, the pancreas was repositioned, and the incision was sutured layer by layer. Tumor growth was monitored using an in vivo imaging system. Dual-drug liposomes loaded with siWIP1 (MSA-2 equivalent dose 15 mg / kg) were injected via tail vein on days 0, 3, and 6. Control groups included single-drug liposomes loaded with siWIP1, single-STING agonist liposomes, and saline. Bioluminescence signals were monitored periodically.

[0077] The results are as follows Figure 16 As shown, the tumor fluorescence signal in the dual-drug liposome group increased slowly. On day 25 of treatment, the control group and the single-drug group showed significant signals, while the dual-drug group showed almost no signal, indicating that it significantly inhibited the growth of pancreatic cancer in situ. Survival analysis showed that the median survival (MST) of siWIP1 liposome alone and STING agonist liposome alone was 14 days and 18 days, respectively, while the MST of the dual-drug liposome group was prolonged to more than 60 days.

[0078] Example 12: Antitumor effect of siWIP1-loaded dual-drug liposomes in the Hepa1-6 orthotopic liver cancer model.

[0079] Hepa1-6-luc cells were mixed with matrix gel at a 1:1 volume ratio. Six-week-old male C57BL / 6 mice were anesthetized, fixed, and their abdomens were shaved and disinfected. A 1 cm incision was made along the linea alba to expose the left lobe of the liver, and the Hepa1-6-luc cell mixture (5 × 10⁻⁶ cells / mL) was injected. 6 / each). After pressing the puncture site for 1 minute, the liver was repositioned and sutured layer by layer. Tumor growth was monitored using a live imaging system. The treatment method and grouping were the same as in Example 11.

[0080] The results are as follows Figure 17As shown, the tumor fluorescence signal in the dual-drug liposome group increased slowly. On day 15 of treatment, the control group and the single-drug group showed significant signals, while the dual-drug group showed almost no signal, indicating that it significantly inhibited the growth of in situ hepatocellular carcinoma. In addition, the dual-drug liposome effectively reduced the levels of liver biochemical indicators AST and ALT, alleviating the damage to liver function caused by tumors.

Claims

1. A pharmaceutical composition, characterized in that, It contains STING agonists and WIP1 inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that, The STING agonist is a cyclic dinucleotide STING agonist, a non-cyclic dinucleotide STING agonist, or a pharmaceutically acceptable prodrug thereof; The WIP1 inhibitor is selected from small molecule inhibitors, their pharmaceutically acceptable salts or solvates, or small interfering RNA, messenger RNA, or antisense oligonucleotides that target the WIP1 gene.

3. The pharmaceutical composition according to claim 2, characterized in that, The STING agonist is the noncyclic dinucleotide small molecule agonist MSA-2 or its pharmaceutically acceptable prodrug; The WIP1 inhibitor is GSK2830371 or a pharmaceutically acceptable salt or solvate thereof, or a small interfering RNA targeting the WIP1 gene, the sequence of which comprises a forward sequence: 5′-GCGGCAGUGUGAUGAACAATT-3′ and a reverse sequence: 5′-UUGUUCAUCACACUGCCGCTT-3′.

4. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of the STING agonist to the WIP1 inhibitor is 1:0.02~50.

5. A liposome, characterized in that, It comprises a lipid layer and a pharmaceutical composition encapsulated therein, wherein the pharmaceutical composition is any one of claims 1-4.

6. The liposomes according to claim 5, characterized in that, The lipid layer contains lecithin, cationic lipids, cholesterol, and DSPE-PEG.

7. Use of the pharmaceutical composition of any one of claims 1-4 or the liposome of claim 5 or 6 in the preparation of a medicament for treating diseases related to STING.

8. The use according to claim 7, characterized in that, The diseases associated with STING are selected from at least one of inflammatory diseases, autoimmune diseases, infectious diseases, cancer, and precancerous syndromes.

9. The use according to claim 7, characterized in that, The diseases associated with STING include pancreatic cancer, hepatocellular carcinoma, melanoma, cervical cancer, breast cancer, oral cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial carcinoma, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, kidney cancer, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasmacytoma, Wilms' tumor, gout, rhinitis, and appendicitis. Tail inflammation, atherosclerosis, asthma, rheumatoid arthritis, allergic dermatitis, cholecystitis, cirrhosis, degenerative arthritis, dermatitis, enteritis, encephalitis, gastritis, nephritis, hepatitis, pituitary inflammation, irritable bowel syndrome, meningitis, multiple sclerosis, myocarditis, myasthenia gravis, mycosis fungoides, osteomyelitis, pancreatitis, pericarditis, pernicious anemia, pneumonia, primary biliary sclerosing cholangitis, polyarteritis nodosa, lupus erythematosus, thyroiditis, urethritis, uveitis, or vasculitis.

10. The use of the pharmaceutical composition of any one of claims 1-4 or the liposome of claim 5 or 6 in the preparation of an immune adjuvant.

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