Application of tripterine-manganese nanoparticles as cGAS-STING pathway agonist

Abstract

The invention generally relates to the technical field of medicines, in particular to application of a tripterine-manganese nanoparticle as a cGAS-STING pathway agonist. According to the invention, the tripterine manganese complex nanoparticle can be used as a cGAS-STNG pathway agonist by integrating the functions of a manganese complex for the first time, and the medical application of the tripterine manganese complex nanoparticle is verified. Besides, although the traditional cGAS agonist and STING agonist can enhance the immune response, the traditional cGAS agonist and STING agonist generally lack tumor specificity and controllability, may cause systemic immune response and cause unnecessary side effects; in some preferable schemes, the active oxygen response type nCel-Mn provided by the invention can realize selective immune activation of tumors and reduce side effects of the whole body.

Description

Technical Field

[0001] The present disclosure relates generally to the field of medical technology, and more particularly to the use of tripterygium wilfordii-manganese nanoparticles as a cGAS-STING pathway agonist. Background Art

[0002] Celastrol (Cel) is a natural product extracted from Tripterygium wilfordii with a wide range of pharmacological activities, especially showing significant anticancer activity in tumor therapy. The drug induces tumor oxidative stress and induces DNA damage by inhibiting the activity of peroxidase 1 (PRDX1), and inhibits the survival and proliferation of cancer cells through this mechanism. However, Cel has some problems that limit its clinical application, such as low water solubility, insufficient bioavailability, and potential toxicity. In recent years, researchers have been committed to developing new drug delivery systems and drug derivatives to improve therapeutic effects and reduce side effects.

[0003] The cGAS (cyclic GMP-AMP synthase) and STING (stimulator of interferon genes) pathways have shown great potential in the field of tumor immunity. This pathway triggers an immune response by recognizing intracellular DNA, thereby activating the innate immune system. cGAS agonists and STING agonists are designed to simulate the presence of intracellular DNA or directly activate STING, thereby promoting the production of type I interferon (IFN-I) and the expression of other antiviral genes. These agonists can not only enhance the immune system's ability to recognize and eliminate tumor cells, but also work in conjunction with other immunotherapy methods such as immune checkpoint inhibitors to improve the overall therapeutic effect. Summary of the invention

[0004] This disclosure covers the following technical solutions:

[0005] The present disclosure relates to the use of tripterygium wilfordii-manganese nanoparticles as cGAS-STING pathway agonists.

[0006] Yet another aspect of the present disclosure relates to the use of tripterygium wilfordii-manganese nanoparticles in the preparation of drugs for treating diseases related to cGAS-STING pathway disorders.

[0007] Yet another aspect of the present disclosure relates to the tripterygium wilfordii-manganese nanoparticles defined in the use.

[0008] Another aspect of the present disclosure relates to a method for preparing the tripterygium wilfordii-manganese nanoparticles, comprising:

[0009] a) mixing a tripterygium wilfordii solution and a manganese ion solution to obtain a tripterygium wilfordii-manganese complex;

[0010] b) mixing the tripterygium wilfordii-manganese complex with an oxidation-sensitive high molecular polymer to obtain an organic phase solution;

[0011] c) uniformly dispersing the organic phase solution in water to obtain a mixed solution;

[0012] d) dialyzing the mixed solution and then performing solid-liquid separation to obtain a solid component.

[0013] Yet another aspect of the present disclosure relates to a pharmaceutical composition, which contains the tripterygium wilfordii-manganese nanoparticles as described above and a pharmaceutically acceptable carrier.

[0014] The present disclosure first discovered that tripterygium wilfordii manganese complex nanoparticles can be used as cGAS-STNG pathway agonists by integrating the function of manganese complexes, and verified its medical use. In addition, although traditional cGAS agonists and STING agonists can enhance immune responses, they usually lack tumor specificity and controllability, and may cause systemic immune responses, thereby causing unnecessary side effects; in some preferred schemes, the active oxygen-responsive nCel-Mn provided in the present disclosure can achieve tumor-selective immune activation and reduce systemic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 A schematic diagram and NMR image of the synthesis of an oxidation-sensitive polymer provided in one embodiment of the present disclosure;

[0017] Figure 2 This is a test result of nCel-Mn particle size provided by an embodiment of the present disclosure.

[0018] Figure 3 A transmission electron microscope image of nCel-Mn provided for one embodiment of the present disclosure.

[0019] Figure 4 This is a stability experiment of nCel-Mn provided by one embodiment of the present disclosure.

[0020] Figure 5 Effects of different molecules on cGAS activity; G150, 1: Cel, 2: Cel-Mn, 3: Manganese acetate.

[0021] Figure 6 Cel-Mn agonistic effect on STING in 4T1 tumor cells.

[0022] Figure 7 Cel-Mn can induce DNA damage in 4T1 tumor cells. Scale bar is 50 μm.

[0023] Figure 8 The killing effect of Cel-Mn on 4T1 tumor cells.

[0024] Fig. 9 Simulated release results of nCel-Mn in vitro.

[0025] Fig.10 Uptake of manganese acetate and nCel-Mn by 4T1 tumor cells.

[0026] Fig.11 Activation effect of the supernatant on BMDCs after 4T1 cells were treated with nCel-Mn. DETAILED DESCRIPTION

[0027] References to embodiments of the present disclosure will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0028] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present disclosure is the same as that commonly understood by those of ordinary skill in the art to which the present disclosure belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0029] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0030] As used herein, the term "treat" and variations thereof or "ameliorate" refer to therapeutic treatment, wherein the objective is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of a disease (e.g., cancer), its associated conditions and / or symptoms. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a disease (e.g., cancer).

[0031] As used herein, "therapeutically effective" and "effective dose" refer to a substance or amount that elicits a desired biological activity or effect.

[0032] Unless otherwise indicated, the terms "subject" or "patient" are used interchangeably to refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats and mice, and other animals. Therefore, as used herein, the term "subject" or "patient" means any mammalian patient or subject to which the compounds of the present disclosure can be administered.

[0033] The terms "and / or", "or / and", and "and / or" used herein have a selection range that includes any one of two or more related listed items, and also includes any and all combinations of related listed items, wherein the any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present disclosure, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0034] As used in this disclosure, the terms "comprising," "including," and "including" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0035]

[0013] Numerical ranges recited in this disclosure as endpoints include all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0036] As used herein, reference to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0037] The present disclosure relates to concentration values, and the meaning includes fluctuations within a certain range. For example, fluctuations can be allowed within a corresponding precision range. For example, 2%, a fluctuation within the range of ±0.1% can be allowed. For values ​​that are large or do not require too fine control, the meaning is also allowed to include greater fluctuations. For example, 100mM, fluctuations within the range of ±1%, ±2%, ±5%, etc. can be allowed. Involving molecular weight, the meaning is allowed to include fluctuations of ±10%.

[0038] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0039] In the present disclosure, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0040] In the present disclosure, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0041] In the present disclosure, "preferred", "better", "more preferred", and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0042] In the present disclosure, "Cel-Mn" represents Celastrol-manganese small molecules; and "nCel-Mn" represents nanoparticles formed by combining Cel-Mn with an oxidation-sensitive high molecular polymer.

[0043] All documents mentioned in the present disclosure are cited as references in the present disclosure, just as each document is cited as a reference individually. Unless they conflict with the invention objectives and / or technical solutions of the present disclosure, the cited documents involved in the present disclosure are cited with all their contents and all their purposes. When the present disclosure involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present disclosure involves cited documents, the examples and preferred methods of the cited relevant technical features may also be incorporated into the present disclosure as references, but are limited to the implementation of the present disclosure. It should be understood that when the cited content conflicts with the description in the present disclosure, the present disclosure shall prevail or be modified adaptively according to the description of the present disclosure.

[0044] A first aspect of the present disclosure relates to the use of tripterygium wilfordii-manganese nanoparticles as cGAS-STING pathway agonists.

[0045] A second aspect of the present disclosure relates to the use of tripterygium wilfordii-manganese nanoparticles in the preparation of drugs for treating diseases related to cGAS-STING pathway disorders.

[0046] Due to its critical role in host immune responses, pharmacological modulation of cGAS-STING pathway activity has been proposed as a viable broad-spectrum immunotherapeutic approach for the treatment of pathogen infections and tumors. Indeed, recent studies have shown that intratumoral administration of 2'3'-cGAMP induces profound regression of established tumors in mice and generates a substantial systemic immune response capable of rejecting distant metastases and providing long-term immune memory (Corrales et al., 2015; Iurescia et al., 2018). STING agonists have also been shown to enhance the efficacy of immune checkpoint blockade therapy (Ghaffari et al., 2018; Wang et al., 2017) and enhance the immunogenicity of vaccines (Fu et al., 2015; Hanson et al., 2015). In addition, studies have also shown that STING agonist therapy can induce host immune responses to control influenza A virus (Shirey et al., 2011), hepatitis B virus (HBV) (Guo et al., 2015; Hou et al., 2015; Guo et al., 2017), herpes simplex virus (HSV) (Skouboe et al., 2018) and human immunodeficiency virus (HIV) (Aroh et al., 2017). These studies have demonstrated the concept that pharmacological activation of STING is an attractive immunotherapy method for treating viral infections and cancers. Therefore, according to what is known to those skilled in the art, in some embodiments, the disease associated with the disorder of the cGAS-STING pathway is at least one of cancer, autoimmune disease and viral infection.

[0047] Due to the critical role of the cGAS-STING pathway in the host immune response, viral infections that can be treated by activating it include hepatitis C virus (HCV), as well as DNA and RNA viruses that cause acute infections, such as influenza virus and other virus families that cause common cold and upper respiratory tract infections, including but not limited to paramyxoviruses, rhinoviruses, adenoviruses, human coronaviruses (including coronaviruses associated with severe respiratory syndrome, such as new coronavirus; Middle East respiratory syndrome coronavirus and human coronavirus OC43), virus families that cause hemorrhagic fever (including but not limited to viruses belonging to the Flaviviridae, Filoviridae, Arenaviridae and Bunyaviridae families), and viruses that cause encephalitis (including but not limited to West Nile virus, La Crosse virus, California encephalitis virus, Venezuelan equine encephalitis virus, Western equine encephalitis, Japanese encephalitis virus, Kwasana Forest virus, tick-borne encephalitis virus, rabies virus, chikungunya virus).

[0048] Various reasons cause the body to produce inflammation and stimulate it to release a large amount of cell fragments containing DNA and RNA. The cGAS-STING pathway is quickly activated and starts the host's innate immunity to quickly resist adverse events from the outside world, but chronic and persistent activation of the pathway can lead to chronic inflammation and even induce autoimmune diseases. In recent years, more evidence suggests that the continuous activation of the cGAS-STING signaling pathway is one of the initiation mechanisms of autoimmune diseases. It is significantly associated with the occurrence of a variety of autoimmune diseases, and the administration of cGAS-STING pathway agonists can treat these diseases. Exemplary autoimmune diseases include psoriasis, Aicardi-Goutieres syndrome, primary Sjögren's syndrome, systemic lupus erythematosus, inflammatory bowel disease (IBD), etc.

[0049] In some embodiments, the cancer is a solid tumor or a blood cancer.

[0050] In some embodiments, the solid tumor comprises one or more of thyroid cancer, lung cancer, pancreatic cancer, pancreatic ductal carcinoma, breast cancer, ovarian cancer, colorectal cancer, prostate cancer, renal cell carcinoma, head and neck tumors, lymphoma, testicular cancer, nasopharyngeal carcinoma, esophageal cancer, neuroblastoma and melanoma. Exemplary tumors can be found in, for example, Nature Reviews Cancer, 2014, 14, 173-186, the entire contents of which are incorporated herein by reference.

[0051] In other embodiments, the cancer is a blood cancer. Non-limiting examples of blood cancers include leukemias, myelomas, and lymphomas.

[0052] In some embodiments, the tripterygium wilfordii-manganese nanoparticles are mainly obtained by combining a tripterygium wilfordii-manganese complex with an oxidation-sensitive high molecular polymer.

[0053] Traditional technology is to load chemotherapy drugs through carriers such as liposomes and albumin to improve their water solubility and biocompatibility. However, these carriers lack responsiveness and cannot achieve selective delivery and treatment of tumors. The present disclosure utilizes the characteristics of nanoparticles to resist a large amount of ROS in the tumor microenvironment, which can achieve selective treatment of tumors and reduce systemic side effects.

[0054] In some embodiments, the monomers for preparing the oxidation-sensitive high molecular polymer include ROS-responsive drug carriers. The ROS-responsive group mainly has the following functions: as a connecting part, connecting the hydrophilic part and the hydrophobic part of the nanocarrier, the nanocarrier breaks and releases the drug when encountering ROS: the ROS-responsive group can also be used as a part connecting the drug molecule and the nanocarrier, releasing the drug under oxidation. Common ROS-responsive groups generally contain three elements: sulfur, boron and tellurium. In addition, in order to inactivate the protein, the ROS-responsive group can also be used to block the active center of the protein. Exemplary ROS-responsive genes that can be used in the present disclosure include polypropylene sulfide, borate, thioketal, tellurium, selenium, ferrocene, and anthocyanin. In some embodiments, the ROS-responsive drug carrier has a carrier with a thioketal group. As the carrier having a thioketal group, one or more of TK-COOH, TK-NH2, Вос-TK-NH2, Fmoc-TK-NH2, Biotin-TK-NH2, FA-TK-NH2, FITC-TK-NH2, NH2-TK-NH2, СООН-ТK-СOOН and ОН-ТK-ОН can be cited. In some embodiments, the carrier having a thioketal group is OH-TK-OH [2,2-(propane-2,2-diylbisulphanedyl)diethanol];

[0055] In some embodiments, the monomers used to prepare the oxidation-sensitive high molecular polymer further comprise a pharmaceutically acceptable cross-linking group; in some embodiments, the cross-linking group comprises pyromellitic anhydride.

[0056] In some embodiments, the monomer for preparing the oxidation-sensitive high molecular polymer further comprises a water-soluble group; in some embodiments, the water-soluble group comprises a methoxy PEG hydroxyl group, and its molecular weight is preferably 4000-6000, for example 4000, 4500, 5000, 5500, 6000; specifically preferably 5000, for example mPEG5000-OH.

[0057] In some embodiments, the oxidation-sensitive polymer is:

[0058]

[0059] Among them, m is the degree of polymerization, that is, the average number of water-soluble groups contained in the polymer macromolecular chain, corresponding to the molecular weight of the water-soluble groups; for example, when the water-soluble group is methoxy PEG hydroxyl and the molecular weight is 5000, m is specifically 113.

[0060] In some embodiments, the value of n is selected from 10 to 15, such as 11, 12, 13, or 14.

[0061] In some embodiments, the mass ratio of the tripterygium wilfordii-manganese complex to the oxidation-sensitive high molecular polymer is 1:(10-20), for example, 1:13, 1:15, or 1:17.

[0062] In some embodiments, the particle size of the tripterygium wilfordii-manganese nanoparticles is 50 nm to 300 nm, preferably 50 nm to 100 nm.

[0063] According to yet another aspect of the present disclosure, it also relates to the tripterygium wilfordii-manganese nanoparticles defined in the above application.

[0064] The preparation method of the tripterygium wilfordii-manganese nanoparticles can be obtained by any route. According to another aspect of the present disclosure, the preparation method comprises:

[0065] a) mixing a tripterygium wilfordii solution and a manganese ion solution to obtain a tripterygium wilfordii-manganese complex;

[0066] b) mixing the tripterygium wilfordii-manganese complex with an oxidation-sensitive high molecular polymer to obtain an organic phase solution;

[0067] c) uniformly dispersing the organic phase solution in water to obtain a mixed solution;

[0068] d) dialyzing the mixed solution and then performing solid-liquid separation to obtain a solid component.

[0069] In step a), the manganese ions are in excess relative to tripterygium wilfordii. Preferably, the molar ratio of tripterygium wilfordii in the tripterygium wilfordii solution to the manganese ions in the manganese ion solution is 1:(1-2).

[0070] In step a), the preparation method of mixing the tripterygium wilfordii solution and the manganese ion solution is commonly used in the art. For example, the solvent used for the tripterygium wilfordii solution is an organic solvent; the organic solvent used may include but is not limited to dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform and other organic solvents that can dissolve tripterygium wilfordii. For example, the manganese ion solution includes polar solvents such as water, methanol, ethanol, dimethyl sulfoxide, etc. that can dissolve manganese salts. The dissolution process can be carried out at room temperature, and the dissolution can be accelerated by magnetic stirring, vortexing, ultrasound, etc.

[0071] An exemplary method of step a) is: slowly adding the manganese ion solution dropwise to the tripterygium wilfordii solution, thoroughly mixing and standing for 4 to 8 hours, adding the reaction solution dropwise to an insoluble or poorly soluble solution of the tripterygium wilfordii-manganese complex, such as ether, to precipitate the tripterygium wilfordii-manganese complex.

[0072] The mixing or dispersing method adopted in the present disclosure may be selected from stirring, vortexing, ultrasound and the like or a combination thereof. The solid-liquid separation method adopted in the present disclosure may be selected from evaporation, distillation, centrifugation, filtration and the like or a combination thereof. Preferably, the solid-liquid separation method in step d) is first centrifugation and then filtration; the centrifugal speed may be 3000r / min-4000r / min; and the filtration pore size may be 0.4μm-0.5μm.

[0073] In step c), the water may be distilled water, deionized water or reverse osmosis water.

[0074] In step d), the water used for dialysis can be distilled water, deionized water, or reverse osmosis water.

[0075] In some embodiments, in step c), ultrasound and stirring are used to achieve uniform dispersion.

[0076] According to yet another aspect of the present disclosure, there is also provided a pharmaceutical composition comprising the above-mentioned tripterygium wilfordii-manganese nanoparticles and a pharmaceutically acceptable carrier.

[0077] The definition of "pharmaceutically acceptable" as described herein refers to compounds, materials, complexes or dosage forms that are suitable for contact with human and animal tissues within the scope of reasonable medical judgment, and do not have excessive toxicity, irritation, allergic reactions and other problems. The term can also be replaced by "pharmaceutically acceptable".

[0078] The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, ingredient or vehicle, such as a liquid or solid filler, diluent, excipient, solution or coating material. Each carrier must be "acceptable" in terms of compatibility with another form of pharmaceutical formulation and not harmful to the patient. The following materials can be used as pharmaceutically acceptable carriers, including (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as viscose fiber and suppository wax; (9) oils, such as peanut oil, cotton oil, sunflower oil, sesame oil, olive oil, carob oil; (10 ) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol (mannitol), mannan; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer and (21) other types of non-toxic compatible substances used in pharmaceutical preparations.

[0079] The pharmaceutically acceptable carrier may include a physiologically acceptable agent to improve stability, increase solubility or increase the absorptive capacity of the nanoparticles described in the present disclosure. The pharmaceutically acceptable carrier, including the selection of a physiologically acceptable agent, depends on the mode of administration of the agent. The preparation of the preparation or the pharmaceutical combination may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical combination (preparation) may also be a liposome or other polymer matrix, and the nanoparticle or conjugate is incorporated therein. For example, liposomes or other lipid-composition liposomes are non-toxic, biocompatible, metabolizable carriers, and are easy to prepare and use.

[0080] The pharmaceutical composition can be administered by any of a variety of routes of administration, including oral administration (e.g., dissolved in water or non-aqueous solution or suspension, tablets, capsules, including micro-pellet capsules and gel capsules, pills, powders, granular preparations, adhesives) and parenteral administration. "Parenteral administration" as described herein refers to other modes of administration other than enteral and topical administration, usually by injection, including but not limited to intratumoral, intravenous, intramuscular, intercostal, interthecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intravertebral, intrasternal injection and infusion. Pharmaceutical ingredients suitable for parenteral administration include nanoparticles that are pharmaceutically usable aseptic isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, sterile powders, and their composition may be the same as sterile injections or dispersions. The pharmaceutical ingredients may contain sterilizing agents, buffers, antibacterial agents, solutes that can make them compatible with the blood of the intended subject, suspending agents, thickeners, or are formulated into isotonic solutions.

[0081] According to yet another aspect of the present disclosure, there is also provided a method for treating diseases related to cGAS-STING pathway disorder, comprising administering an effective dose of celastrol-manganese nanoparticles to a patient.

[0082] In some embodiments, the method of the disease associated with cGAS-STING pathway dysregulation is cancer. In some embodiments of any of the methods described herein, the tripterygium wilfordii-manganese nanoparticles are administered in combination with an effective amount of at least one other therapeutic agent selected from one or more other therapeutic or treatment (e.g., chemotherapeutic) agents.

[0083] Non-limiting examples of other therapeutic agents include: other STING agonists or partial agonists other than tripterygium wilfordii-manganese nanoparticles, kinase inhibitors (e.g., receptor tyrosine kinase targeted therapeutics (e.g., Trk inhibitors or EGFR inhibitors) or multi-kinase inhibitors), signal transduction pathway inhibitors, checkpoint inhibitors, apoptosis pathway modulators (e.g., obataclax); cytotoxic chemotherapy, angiogenesis targeted therapy, immune targeted agents, including immunotherapy and radiotherapy.

[0084] The embodiments of the present disclosure will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present disclosure, and can also be based on the experimental manuals or conventional conditions in the art, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0085] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0086] Example 1 Preparation of nCel-Mn

[0087] (1) Celastrol (Cel)

[0088] ((2R,4aS,6aS,12bR,14aS,14bR)-10-hydroxy-2,4a,6a,9,12b,14a-hexamethyl-11-oxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-tetradecahydropicene-2-carboxylic acid) was added into a reactor and dissolved with an organic solvent to obtain a solution A;

[0089] The organic solvent used includes, but is not limited to, dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform and other organic solvents that can dissolve Cel.

[0090] The dissolution process is carried out at room temperature and can be accelerated by magnetic stirring, vortexing, ultrasound, etc.

[0091] (2) dissolving a manganese salt in a solvent to obtain a solution B;

[0092] The manganese-containing salt used is divalent manganese acetate and its crystalline hydrate.

[0093] The solvents used include double distilled water, methanol, ethanol, dimethyl sulfoxide and other polar solvents that can dissolve manganese salts.

[0094] The dissolution process is carried out at room temperature and can be accelerated by magnetic stirring, vortexing, ultrasound, etc.

[0095] (3) Slowly add solution B to solution A and react for 6 hours; add the reaction solution to ether to obtain a precipitate, thus obtaining a Cel-Mn complex;

[0096] The molar ratio of manganese ions to Cel in solution A and solution B was 2:1, so that the metal ions were in excess.

[0097] The dropwise mixing process is carried out at room temperature and can be accelerated by magnetic stirring, vortexing, ultrasound, etc.

[0098] (4) Synthesis method of oxidation-sensitive polymer: Monomer A (2.465 g, 11 mmol) and monomer B (1.96 g, 10 mmol) were weighed and dissolved in 10 mL of ultra-dry DMF, and reacted at 50 °C for 36 h. mPEG5000-OH (5 g, 1 mmol) was then added to the reaction system and reacted at 50 °C for 24 h. The liquid after the reaction was added to a dialysis bag with a molecular weight cutoff of 8000 and dialyzed for 48 h, and the product was lyophilized. 1HNMR (400 MHZ, deuterated DMSO) spectrum and synthesis route are shown in Figure 1 shown.

[0099] (5) Adding an oxidation-sensitive polymer to an organic solvent of a Cel-Mn complex to obtain an organic phase solution; slowly adding the organic phase solution dropwise to a 10-fold volume of a deionized water solution at room temperature; then transferring the liquid to a dialysis bag of MWCO3500 and dialyzing it in deionized water for 3 days, changing the water every 8 hours; then centrifuging the liquid (3500 r / min) and filtering (0.45 μm) to obtain nCel-Mn nanoparticles.

[0100] The oxidation-sensitive polymer used is NPs.

[0101] The mass ratio of Cel-Mn complex and NPs was 1:(10-20).

[0102] The volume dosage of the organic solvent is 2%-10% of the deionized water solution, preferably 4%-6%.

[0103] In order to make the particle size meet the preparation requirements, the time for adding the organic phase solution to the deionized water solution is controlled to be one drop per second; and the rotation speed is controlled to be 300-600 rpm.

[0104] Example 2 Material Characterization of nCel-Mn

[0105] The prepared nCel-Mn was diluted 10 times with deionized water, and the particle size of the nanoparticles was measured using a dynamic laser particle size analyzer. The test results are as follows: Figure 2 As shown in Figure 2, the average particle size of nCel-Mn is 72.45nm. The morphology of nCel-Mn was observed using a field emission transmission electron microscope-2200FS. The test results are shown in Figure 2. Figure 3 As shown in Figure 2, nCel-Mn is a uniform sphere. After diluting nCel-Mn 10 times with PBS, a dynamic laser particle size analyzer was used to measure its stability within 5 days. The test results are shown in Figure 2. Figure 4 As shown, the particle size of nCel-Mn remained basically unchanged within 5 days, indicating that it has good stability under physiological conditions.

[0106] Example 3 Effect of Cel-Mn on cGAS activity

[0107] This example aims to evaluate the effect of Cel-Mn on cGAS enzyme activity. First, positive drugs including cGAS inhibitor G150, Cel, Cel-Mn and manganese acetate were diluted according to the required concentration. Next, 150nL of the compound was transferred to a 96-well plate. To start the reaction, 2.5μL of cGAS enzyme was added to each well. Subsequently, 2.5μL of ATP, GTP and DNA mixture was added to simulate the cGAS activation process in vivo. To monitor changes in ATP levels, 10μL of Kinase Glo Max working solution was added, which is an ATP detection method based on the luciferase reporter system. After incubation at 25°C for 10 minutes, the light signal emitted was measured using a BMGLabtech reader, which reflects the level of cGAMP (cyclic guanosine monophosphate adenylate) catalyzed by cGAS.

[0108] The results are as follows Figure 5 As shown in Figure 2, Cel alone does not have cGAS catalytic activity, while Cel-Mn and manganese acetate have the ability to effectively promote cGAS activity.

[0109] Example 4 Effect of Cel-Mn on STING protein phosphorylation in breast cancer cell line 4T1

[0110] This example aims to evaluate the effect of Cel-Mn on STING protein phosphorylation (p-STING) at the cellular level by Western Blot (WB) method, in order to further explore the potential of Cel-Mn as a cGAS-STING pathway activator. A blank control group (Control) and a manganese acetate treatment group were set up for comparison.

[0111] The results are as follows Figure 6 As shown in the figure, p-STING expression was observed in 4T1 cells treated with 2 μM concentration of manganese acetate and Cel-Mn, respectively. This indicates that Cel-Mn can effectively promote the phosphorylation process of STING protein, thereby triggering the downstream signal transduction pathway. This finding supports the application value of Cel-Mn in enhancing immune response and potential anti-cancer effects.

[0112] Example 5 Detection of DNA damage in breast cancer cell line 4T1 induced by Cel-Mn

[0113] This example uses immunofluorescence technology to evaluate whether Cel-Mn can cause DNA damage in breast cancer cells 4T1. In this process, the expression level of the DNA damage marker γ-H2AX in the cell nucleus is detected for evaluation, and a blank control and manganese acetate treatment group are also set as references.

[0114] according to Figure 7 The results of immunofluorescence image analysis shown in the figure show that DAPI staining can clearly show the nuclear structure, while a red fluorescent labeled secondary antibody is used to mark γ-H2AX. A significantly increased γ-H2AX positive signal was observed in 4T1 cell samples treated with Cel-Mn; in contrast, the expression of γ-H2AX remained at a low level in both the blank control and cells treated with manganese acetate alone. These data together confirm that Cel-Mn can not only activate the cGAS-STING signaling pathway, but also promote tumor cell apoptosis by inducing DNA damage.

[0115] Example 6 Effect of nCel-Mn on the Viability of Breast Cancer Cells 4T1

[0116] The MTT method was used to detect the activity of nCel-Mn on breast cancer 4T1 cells. The cells were seeded in a 96-well plate at a density of 5000 cells per well and cultured for 12h. The cells were incubated with nCel-Mn at a final concentration of 0.25, 0.15, 1, 2, 4, 8, and 16μM for 24h. Then, 10% MTT diluted with RPMI 1640 (100μL) was added to the wells. After culturing for 4h at 37℃, 5% CO2, 100μL 10% SDS was added to each well and cultured for 12h at 37℃, 5% CO2. The absorbance of the wells was detected by a microplate reader at 570nm (peak absorbance) and 650nm (background absorbance). Cell viability was expressed as the ratio of the absorbance of the test wells to the control wells.

[0117] MTT results are as follows Figure 8 As shown, nCel-Mn has a concentration-dependent killing activity against 4T1 tumor cells.

[0118] Example 7 Drug release of nCel-Mn in an oxidative environment simulated by hydrogen peroxide

[0119] The dialysis method was used to test the release data of nCel-Mn in a simulated normal environment and an oxidative stress environment simulated by 10mM hydrogen peroxide. The molecular weight of the dialysis bag was 10kDa, the liquid volume ratio of the inner and outer liquids was 1:100, the temperature was set to 37°C, 2% tween 80 was added to the outer liquid to achieve the sink condition, and samples were taken at specific times to calculate the release ratio.

[0120] Release results such as Fig. 9As shown, 14% was released in a normal environment within 48 hours, while 74% was released in the presence of hydrogen peroxide.

[0121] Example 8 Delivery effect of nCel-Mn targeting tumor cells

[0122] Atomic absorption spectroscopy (AAS) was used to detect the intracellular manganese content. 6 2mL culture medium of 4T1 cells was inoculated into a six-well plate and cultured in an incubator for 12 hours. Manganese acetate and nCel-Mn solution were added respectively to make the final Mn concentration 2μM, and three culture time periods of 1h, 4h, and 7h were set. Three replicate wells were set for each group, and the PBS group was set as the control. After the drug treatment was completed, the culture medium was aspirated and gently washed three times with cold PBS to ensure that the drugs that were not swallowed into the cells were rinsed clean. The six-well plate was placed in a fume hood, and 100μL of concentrated HNO3 and 100μL of 30% H2O2 solution were added to each well. After 3 days of digestion, the digested liquid was collected, the volume was fixed to 2mL, and the manganese content was tested using AAS. The results are as follows Fig.10 As shown, nCel-Mn can be internalized by cells more efficiently.

[0123] Example 9 Effects of nCel and nCel-Mn on Activation of Bone Marrow Derived Cells (BMDC) after Treatment of 4T1 Cells

[0124] In order to evaluate the effect of nCel-Mn on BMDCs activation after interaction with 4T1 cells, the following experiment can be designed: first, monocytes are isolated from the bone marrow of healthy mice and cultured in a medium containing GM-CSF until BMDCs mature; then 4T1 cells are co-incubated with different concentrations of nCel-Mn for a period of time, and then the treated 4T1 cell supernatant is collected and added to the BMDCs culture medium as a stimulator; at the same time, a control group is set up, and untreated 4T1 cell supernatant and supernatant containing only nCel-Mn are added as controls, so as to distinguish the effects of 4T1 cells themselves and nCel-Mn on the activation state of BMDCs; BMDCs are continued to be cultured and cell samples are collected at designated time points, and flow cytometry is used to detect changes in the expression of BMDCs surface markers such as CD80 and CD86, so as to compare the differences in the activation state of BMDCs between the groups. The experimental results are as follows. Fig.11 As shown, the experiments proved that nCel-Mn can effectively induce BMDC activation.

[0125] In combination with the above examples, it can be found that although Cel alone does not have the effect of activating the cGAS-STING pathway, Cel-Mn can more effectively activate the cGAS-STING pathway than Mn, and can more effectively promote STING phosphorylation and induce DNA damage (such as Figure 6 , Figure 7 ). It is speculated that there are two main problems with single manganese ion administration: 1. Manganese ions need corresponding transporters to enter cells, and the transporter expression rate of most cells is low, resulting in insufficient cellular internalization capacity of single manganese ions. 2. Manganese ions activate the cGAS-STING pathway by activating cGAS activity without inducing DNA damage, resulting in a single dsDNA content of less and limited excitation efficiency. In addition, Cel, as a manganese carrier, internalizes Mn into cells more efficiently ( Fig.10 ), and the DNA damage caused by Cel pharmacological action can further promote the activity of Mn-catalyzed cGAS to form a self-circulating cGAS-STING pathway activation.

[0126] The above-described embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure shall be based on the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. Application of tripterygium wilfordii-manganese nanoparticles as cGAS-STING pathway agonists.

2. Application of tripterygium wilfordii-manganese nanoparticles in the preparation of drugs for treating diseases related to cGAS-STING pathway disorders.

3. The use according to claim 2, wherein the disease associated with cGAS-STING pathway disorder is at least one of cancer, autoimmune disease and viral infection.

4. The use according to claim 3, wherein the cancer is a solid tumor or a blood cancer.

5. The use according to claim 4, wherein the solid tumor comprises one or more of thyroid cancer, lung cancer, pancreatic cancer, pancreatic ductal carcinoma, breast cancer, ovarian cancer, colorectal cancer, prostate cancer, renal cell carcinoma, head and neck tumors, lymphoma, testicular cancer, nasopharyngeal carcinoma, esophageal cancer, neuroblastoma and melanoma.

6. The use according to any one of claims 1 to 5, wherein the tripterygium wilfordii-manganese nanoparticles are mainly obtained by combining a tripterygium wilfordii-manganese complex with an oxidation-sensitive high molecular polymer.

7. The use according to claim 6, wherein the monomers for preparing the oxidation-sensitive polymer include ROS-responsive drug carriers; The ROS-responsive drug carrier is preferably a carrier having a thioketal group; The carrier having a thioketal group is preferably OH-TK-OH; Preferably, the monomers for preparing the oxidation-sensitive high molecular polymer further include pyromellitic anhydride; Preferably, the monomers for preparing the oxidation-sensitive high molecular polymer further comprise a water-soluble group, more preferably a methoxy PEG hydroxyl group; Preferably, the oxidation-sensitive high molecular polymer is: Preferably, the mass ratio of the tripterygium wilfordii-manganese complex to the oxidation-sensitive high molecular polymer is 1:(10-20); Preferably, the particle size of the tripterygium wilfordii-manganese nanoparticles is 50 nm to 300 nm; more preferably 50 nm to 100 nm.

8. The tripterygium wilfordii-manganese nanoparticles as defined in the use according to claim 6 or 7.

9. The method for preparing the tripterygium wilfordii-manganese nanoparticles according to claim 8, comprising: a) mixing a tripterygium wilfordii solution and a manganese ion solution to obtain a tripterygium wilfordii-manganese complex; b) mixing the tripterygium wilfordii-manganese complex with an oxidation-sensitive high molecular polymer to obtain an organic phase solution; c) uniformly dispersing the organic phase solution in water to obtain a mixed solution; d) dialyzing the mixed solution and then performing solid-liquid separation to obtain a solid component; Optionally, the molar ratio of tripterygium wilfordii in the tripterygium wilfordii solution to the manganese ions in the manganese ion solution is 1:(1-2). 10 . A pharmaceutical composition comprising the tripterygium wilfordii-manganese nanoparticles according to claim 8 and a pharmaceutically acceptable carrier.

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