Application of celastrol-manganese nanoparticles as agonists of the cGAS-STING pathway

Abstract

The present disclosure generally relates to the field of pharmaceutical technologies. Specifically, it relates to the application of celastrol-manganese nanoparticles as agonists of the cGAS-STING pathway. The present disclosure has for the first time discovered that celastrol-manganese complex nanoparticles can be used as agonists of the cGAS-STNG pathway by integrating the functions of manganese complexes, and verified their pharmaceutical uses. In addition, although traditional cGAS agonists and STING agonists can enhance immune responses, they generally lack tumor specificity and controllability, and may cause systemic immune responses, thereby triggering unnecessary side effects; in some more preferred embodiments, the reactive oxygen species-responsive nCel-Mn provided by the present disclosure can achieve tumor-selective immune activation and reduce systemic side effects.

Description

Technical Field

[0001] The present disclosure generally relates to the field of pharmaceutical technologies, and more particularly, to the use of celastrol-manganese nanoparticles as agonists of the cGAS-STING pathway. Background Art

[0002] Celastrol (Cel) is a natural product extracted from Tripterygium wilfordii Hook. f., which has a wide range of pharmacological activities, especially showing significant anti-cancer activity in tumor treatment. This drug induces tumor oxidative stress and DNA damage by inhibiting the activity of peroxiredoxin 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 problems. In recent years, researchers have been committed to developing new drug delivery systems and drug derivatives to improve the therapeutic effect 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 mimic 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 ability of the immune system to recognize and eliminate tumor cells, but also act synergistically with other immunotherapeutic means such as immune checkpoint inhibitors to improve the overall therapeutic effect. Summary of the Invention

[0004] The present disclosure encompasses the following technical solutions:

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

[0006] Another aspect of the present disclosure relates to the use of celastrol-manganese nanoparticles in the preparation of a drug for treating diseases related to cGAS-STING pathway dysregulation.

[0007] Another aspect of the present disclosure relates to the celastrol-manganese nanoparticles defined in the above use.

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

[0009] a) Mixing a celastrol solution and a manganese ion solution to obtain a celastrol-manganese complex;

[0010] b) Mix the celastrol-manganese complex with an oxidation-sensitive polymer to obtain an organic phase solution;

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

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

[0013] Another aspect of the present disclosure relates to a pharmaceutical composition containing the celastrol-manganese nanoparticles as described above and a pharmaceutically acceptable carrier.

[0014] The present disclosure has for the first time discovered that the celastrol-manganese complex nanoparticles can be used as cGAS-STING pathway agonists by integrating the functions of manganese complexes, and verified their medical uses. In addition, although traditional cGAS agonists and STING agonists can enhance the immune response, they usually lack tumor specificity and controllability, and may cause systemic immune responses, thereby triggering unnecessary side effects; in some more preferred embodiments, the ROS-responsive nCel-Mn provided by 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 following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present disclosure. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

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

[0017] Figure 2 Particle size test results of nCel-Mn provided in an embodiment of the present disclosure.

[0018] Figure 3 Transmission electron microscope image of nCel-Mn provided in an embodiment of the present disclosure.

[0019] Figure 4 Stability experiment of nCel-Mn provided in an embodiment of the present disclosure.

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

[0021] Figure 6 STING activation of Cel-Mn on 4T1 tumor cells.

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

[0023] Figure 8 Cytotoxicity of Cel-Mn on 4T1 tumor cells.

[0024] Figure 9 In vitro simulated release results of nCel-Mn.

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

[0026] Figure 11 Activation of BMDC by the supernatant after treatment of 4T1 cells with nCel-Mn. Detailed implementation manners

[0027] Reference will now be provided in detail to embodiments of the present disclosure, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

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

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

[0030] As used herein, the term "treatment" and variations thereof or "amelioration" refer to therapeutic treatment, wherein the aim is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a disease (such as cancer), its associated disorders and / or symptoms. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a disease (such as 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 and refer to mammals such as human patients and non-human primates, as well as laboratory animals such as rabbits, rats and mice, and other animals. Thus, as used herein, the terms "subject" or "patient" mean any mammalian patient or subject to whom the compounds of the present disclosure can be administered.

[0033] As used herein, the alternative ranges of the terms "and / or", "or / and", "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or 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 / or", it should be understood that in the present disclosure, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, D (i.e., the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, it includes combinations of any two or any three of A, B, C, D, and also includes the combination of the four items A, B, C, D (i.e., the technical solution connected by "logical AND").

[0034] The terms "comprising", "containing" and "including" used in the present disclosure are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0035] The numerical ranges expressed by endpoints in the present disclosure include all the numerical values and fractions included in the range, as well as the recited endpoints.

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

[0037] In the present disclosure, concentration values are involved, and their meanings include fluctuations within a certain range. For example, they can fluctuate within the corresponding accuracy range. For example, for 2%, fluctuations within the range of ±0.1% are allowed. For larger values or values that do not require overly precise control, their meanings are also allowed to include larger fluctuations. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. Regarding molecular weight, fluctuations within the range of ±10% are allowed for its meaning.

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

[0039] In the present disclosure, descriptions such as "a plurality of" and "a variety of", unless otherwise specified, mean greater than or equal to 2 in number.

[0040] In the present disclosure, among the technical features described in an open-ended manner, closed technical solutions composed of the listed features are included, as well as open technical solutions that include the listed features.

[0041] In the present disclosure, "preferred", "better", "more preferable", and "preferably" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present disclosure. In the present disclosure, "optionally", "optional", and "optional" mean optional, that is, they refer to either of the two alternative options of "having" or "not having". If "optional" appears multiple times in a technical solution, unless otherwise specified and there are no contradictions or mutual restrictions, each "optional" is independent.

[0042] In the present disclosure, "Cel-Mn" represents celastrol-manganese small molecules; "nCel-Mn" represents nanoparticles after the combination of Cel-Mn and an oxidation-sensitive polymer.

[0043] All documents mentioned in the present disclosure are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the inventive purpose and / or technical solution of the present disclosure, the cited documents involved in the present disclosure are cited for 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 incorporated by reference. When the present disclosure involves cited documents, examples and preferred ways of the relevant technical features cited can also be incorporated into the present disclosure as references, but only to the extent that the present disclosure can be implemented. 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 in the present disclosure.

[0044] The first aspect of the present disclosure relates to the application of celastrol-manganese nanoparticles as agonists of the cGAS-STING pathway.

[0045] The second aspect of the present disclosure relates to the use of celastrol-manganese nanoparticles in the preparation of a medicament for treating diseases related to cGAS-STING pathway dysregulation.

[0046] Due to its crucial role in the host immune response, pharmacological modulation of cGAS-STING pathway activity has been considered a viable broad-spectrum immunotherapeutic approach for treating pathogen infections and tumors. Indeed, recent studies have shown that intratumoral administration of 2'3'-cGAMP causes deep 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 demonstrated that STING agonist therapy can induce a host immune response to control 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 colds and upper respiratory tract infections, including but not limited to paramyxoviruses, rhinoviruses, adenoviruses, human coronaviruses (including coronaviruses associated with severe acute respiratory syndrome, such as SARS-CoV-2; Middle East respiratory syndrome coronavirus and human coronavirus OC43), virus families that cause hemorrhagic fevers (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, Kyasanur Forest virus, tick-borne encephalitis virus, rabies virus, chikungunya virus). These studies have demonstrated the concept that pharmacological activation of STING is a promising immunotherapeutic approach for treating viral infections and cancer. Thus, as is known to those skilled in the art, in some embodiments, the diseases related to cGAS-STING pathway dysregulation are at least one of cancer, autoimmune diseases, and viral infections.

[0047] Due to the crucial 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 colds and upper respiratory tract infections, including but not limited to paramyxoviruses, rhinoviruses, adenoviruses, human coronaviruses (including coronaviruses associated with severe acute respiratory syndrome, such as SARS-CoV-2; Middle East respiratory syndrome coronavirus and human coronavirus OC43), virus families that cause hemorrhagic fevers (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, Kyasanur Forest virus, tick-borne encephalitis virus, rabies virus, chikungunya virus).

[0048] Various reasons cause the body to produce inflammation and stimulate the release of a large amount of cell debris containing DNA and RNA. The cGAS-STING pathway is rapidly activated and initiates the host innate immunity to quickly resist external adverse events. However, 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 persistent activation of the cGAS-STING signaling pathway is one of the initiating mechanisms of the occurrence of autoimmune diseases. It is significantly associated with the occurrence of various autoimmune diseases, and administering agonists of the cGAS-STING pathway can treat these diseases. Exemplary autoimmune diseases include psoriasis, Aicardi-Goutieres syndrome, primary Sjogren'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 includes one or more of thyroid cancer, lung cancer, pancreatic cancer, pancreatic ductal cancer, breast cancer, ovarian cancer, colorectal cancer, prostate cancer, renal cell carcinoma, head and neck tumors, lymphoma, testicular cancer, nasopharyngeal cancer, esophageal cancer, neuroblastoma, and melanoma. Exemplary tumors can be seen, for example, in Nature Reviews Cancer, 2014, 14, 173-186, the entire content of which is incorporated herein by reference.

[0051] In other embodiments, the cancer is a blood cancer. Non-limiting examples of blood cancers include leukemia, myeloma, and lymphoma.

[0052] In some embodiments, the celastrol-manganese nanoparticles are mainly obtained by binding a celastrol-manganese complex with an oxidation-sensitive polymer.

[0053] Traditional techniques load chemotherapeutic 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 a large amount of ROS in the tumor microenvironment in nanoparticles to achieve selective treatment of tumors and reduce systemic side effects.

[0054] In some embodiments, the monomers for preparing the oxidation-sensitive polymer include ROS-responsive drug carriers. The ROS-responsive groups mainly have the following functions: as a linking moiety, connecting the hydrophilic and hydrophobic parts of the nanocarrier, and the nanocarrier breaks upon encountering ROS to release the drug; the ROS-responsive groups can also be used as the part connecting the drug molecule and the nanocarrier, and release the drug under oxidation. Common ROS-responsive groups generally contain three elements: sulfur, boron, and tellurium. In addition, in order to inactivate proteins, ROS-responsive groups can also be used to block the active centers of proteins. Exemplary ROS-responsive groups applicable to the present disclosure include any one of polypropylene sulfide, borate ester, thioacetal, tellurium, selenium, ferrocene, and anthocyanin. In some embodiments, the ROS-responsive drug carrier is a carrier having a thioacetal group. Examples of carriers having a thioacetal group include 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 OH-TK-OH. In some embodiments, the carrier having a thioacetal group is OH-TK-OH [2,2-(propane-2,2-diylbisthiolane-2,2-diyl)diethanol];

[0055] In some embodiments, the monomers for preparing the oxidation-sensitive polymer further comprise a pharmaceutically acceptable crosslinking group; in some embodiments, the crosslinking group includes pyromellitic dianhydride.

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

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

[0058]

[0059] Wherein, m is the degree of polymerization, that is, the average number of water-soluble groups on the polymer macromolecular chain, corresponding to the molecular weight of the water-soluble group; 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, 14.

[0061] In some embodiments, the mass ratio of celastrol-manganese complex to the oxidation-sensitive polymer is 1:(10 - 20), such as 1:13, 1:15, 1:17.

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

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

[0064] The preparation method of the celastrol-manganese nanoparticles can be obtained through any means. According to another aspect of the present disclosure, the preparation method includes:

[0065] a) Mixing a celastrol solution and a manganese ion solution to obtain a celastrol-manganese complex;

[0066] b) Mixing the celastrol-manganese complex with an oxidation-sensitive 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 celastrol. More preferably, the molar ratio of celastrol in the celastrol solution to manganese ions in the manganese ion solution is 1:(1 - 2).

[0070] In step a), the preparation method of mixing the celastrol solution and the manganese ion solution is commonly used in the art. For example, the solvent of the celastrol solution is an organic solvent; the organic solvents used may include but are not limited to organic solvents such as dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform, etc. that can dissolve celastrol. 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 magnetic stirring, vortexing, ultrasonic waves, etc. can be used to accelerate dissolution.

[0071] An exemplary practice of step a) is: slowly dropping the manganese ion solution into the celastrol solution, fully mixing and standing for 4 - 8 hours, dropping the reaction solution into an insoluble or poorly soluble solution of the celastrol-manganese complex such as ether, and precipitating the celastrol-manganese complex.

[0072] The mixing or dispersion methods adopted in the present disclosure may include stirring, vortexing, ultrasonic treatment, etc., or combinations thereof. The solid-liquid separation methods adopted in the present disclosure may include evaporation, distillation, centrifugation, filtration, etc., or combinations thereof. Preferably, in step d), the solid-liquid separation method is centrifugation followed by filtration; the rotation speed of centrifugation may be 3000 r / min - 4000 r / min; the filtration pore size of filtration may be 0.4 μm - 0.5 μm.

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

[0074] In step d), the water used for dialysis may be selected from distilled water, deionized water, and reverse osmosis water.

[0075] In some embodiments, in step c), ultrasonic treatment and stirring are used for uniform dispersion.

[0076] According to another aspect of the present disclosure, there is also provided a pharmaceutical composition, which contains celastrol-manganese nanoparticles as described above and a pharmaceutically acceptable carrier.

[0077] As used herein, the definition of "pharmaceutically acceptable" means a compound, material, complex or dosage form that is suitable for contact with human and animal tissues within the scope of reasonable medical judgment, and which has no excessive toxicity, irritation, allergic reaction, and other problems. This term may also be replaced by "medicinally acceptable".

[0078] As used herein, the definition of "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, ingredient or vehicle, such as a liquid, solid filler, diluent, excipient, solution or coating material. Each carrier must be "acceptable" in terms of compatibility with another form of drug formulation and harmless to the patient. The following materials may be used as pharmaceutically acceptable carriers, including (1) saccharides, 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 powder; (8) adjuvants, such as viscose fiber and suppository wax; (9) oils, such as peanut oil, cottonseed oil, sunflower seed oil, sesame oil, olive oil, and castor oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol (mannitol), and 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 solution, and (21) other types of non-toxic and compatible substances used in pharmaceutical preparations.

[0079] The pharmaceutically acceptable carrier may contain physiologically acceptable agents to enhance stability, increase solubility, or increase the absorbability of the nanoparticles described in the present disclosure. The selection of the pharmaceutically acceptable carrier, including the physiologically acceptable agent, depends on the mode of administration of the agent. The preparation of the formulation or combination of agents may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The combination of agents (formulation) may also be a liposome or other polymeric matrix, and the nanoparticles or conjugates are incorporated therein. For example, liposomes or other lipid-based 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 solutions or suspensions, tablets, capsules, including pellet capsules and gel capsules, pills, powders, granule formulations, patches) and parenteral administration. As used herein, "parenteral administration" refers to other modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intratumoral, intravenous, intramuscular, intercostal, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intrasternal injection, and infusion. Pharmaceutical ingredients suitable for parenteral administration include sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions of nanoparticles pharmaceutically acceptable, sterile powders, the composition of which may be combined with sterile injectable solutions or dispersions. The pharmaceutical ingredients may contain sterilizing agents, buffers, bacteriostatic agents, solutes that make it isotonic with the blood of the intended subject, suspending agents, thickening agents, or solutes formulated into isotonic solutions.

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

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

[0083] Non-limiting examples of other therapeutic agents include: other STING agonists or partial agonists other than celastrol-manganese nanoparticles, kinase inhibitors (e.g., receptor tyrosine kinase-targeted therapeutic agents (e.g., Trk inhibitors or EGFR inhibitors) or multi-kinase inhibitors), signal transduction pathway inhibitors, checkpoint inhibitors, apoptosis pathway regulators (e.g., obataclax); cytotoxic chemotherapy, angiogenesis-targeted therapies, 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 not to limit the scope of the present disclosure. For the experimental methods without specific conditions noted in the following examples, priority should be given to the guidance provided in the present disclosure. It can also be carried out according to the experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0085] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. 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) Add 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) into a reactor and dissolve it with an organic solvent to obtain solution A;

[0089] The organic solvents used include but are not limited to organic solvents such as dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, and chloroform that can dissolve Cel.

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

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

[0092] The manganese salt used is an acetate of divalent manganese and its crystalline hydrate.

[0093] The solvents used include polar solvents such as double-distilled water, methanol, ethanol, and dimethyl sulfoxide that can dissolve the manganese salt.

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

[0095] (3) Slowly drop solution B into solution A and react for 6 hours; drop the reaction solution into diethyl ether, and a precipitate will form, that is, the Cel-Mn complex is obtained;

[0096] The molar ratio of manganese ions to Cel in Solution A and Solution B is 2:1, such that the metal ions are in excess.

[0097] The dropping and mixing process is carried out at room temperature, and the process can be accelerated by magnetic stirring, vortexing, ultrasonic treatment, etc.

[0098] (4) Synthesis method of oxidation-sensitive polymer: Weigh monomer A (2.465 g, 11 mmol) and monomer B (1.96 g, 10 mmol), dissolve them in 10 mL of ultradry DMF, react at 50 °C for 36 h, then add mPEG5000-OH (5 g, 1 mmol) to the reaction system, and react at 50 °C for 24 h. Add the reacted liquid into a dialysis bag with a molecular weight cut-off of 8000 and dialyze for 48 h, then freeze-dry to obtain the product. The 1HNMR (400 MHz, deuterated DMSO) spectrum and the synthesis route are as Figure 1 shown.

[0099] (5) Add the oxidation-sensitive polymer into the organic solvent of the Cel-Mn complex to obtain an organic phase solution; at room temperature, slowly drop the organic phase solution into 10 times the volume of deionized water solution; then transfer the liquid into a dialysis bag with MWCO3500 and dialyze in deionized water for 3 days, changing the water every 8 h; then centrifuge the liquid (3500 r / min) and filter (0.45 μm) to obtain nCel-Mn nanoparticles.

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

[0101] The mass ratio of the Cel-Mn complex to NPs is 1:(10 - 20).

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

[0103] To make the particle size meet the preparation requirements, the time for dropping the organic phase solution into the deionized water solution is controlled at one drop per second; the rotation speed is controlled at 300 - 600 rpm.

[0104] Example 2 Material Characterization of nCel-Mn

[0105] After diluting the above-prepared nCel-Mn with deionized water by 10 times, use a dynamic light scattering particle size analyzer to measure the particle size of the nanoparticles. The test results are as Figure 2 shown, and the average particle size of nCel-Mn is 72.45 nm. Use a field emission transmission electron microscope - 2200FS to observe the morphology of nCel-Mn. The test results are as Figure 3 shown, and nCel-Mn is a uniform sphere. After diluting nCel-Mn with PBS by 10 times, use a dynamic light scattering particle size analyzer to measure its stability within 5 days. The test results are asFigure 4 As shown, the particle size of nCel-Mn remained basically unchanged within 5 days, indicating its good stability under physiological conditions.

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

[0107] This example aimed to evaluate the effect of Cel-Mn on the enzymatic activity of cGAS. First, positive drugs including the cGAS inhibitor G150, Cel, Cel-Mn, and manganese acetate were diluted according to the required concentrations. Then, 150 nL of the compounds were transferred into a 96-well plate. To initiate the reaction, 2.5 μL of cGAS enzyme was added to each well. Subsequently, 2.5 μL of a mixture of ATP, GTP, and DNA was added to mimic the cGAS activation process in the in vivo environment. To monitor the change in ATP levels, 10 μL 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 emitted light signal was measured using a BMGLabtech reader, which can reflect the level of cGAMP (cyclic guanosine monophosphate-adenosine monophosphate) catalyzed by cGAS.

[0108] The results are as Figure 5 shown. Cel alone did not possess cGAS catalytic activity, while Cel-Mn and manganese acetate had the ability to effectively promote cGAS activity.

[0109] Example 4: Effect of Cel-Mn on STING Protein Phosphorylation in Breast Cancer Cells 4T1

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

[0111] The results are as Figure 6 shown. In 4T1 cells treated with 2 μM concentration of manganese acetate and Cel-Mn respectively, the expression of p-STING was observed. 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 the immune response and potential anti-cancer effects.

[0112] Example 5: Detection of DNA Damage Induced by Cel-Mn in Breast Cancer Cells 4T1

[0113] In this example, immunofluorescence technology was used to evaluate whether Cel-Mn could cause DNA damage in breast cancer cells 4T1. During this process, the evaluation was carried out by detecting the expression level of the DNA damage marker γ-H2AX in the cell nucleus, and a blank control and a manganese acetate treatment group were also set as references.

[0114] According to Figure 7 the analysis results of the immunofluorescence images shown, DAPI staining can clearly show the cell nucleus structure, and the secondary antibody labeled with red fluorescence is used to label γ-H2AX. A significant increase in γ-H2AX positive signals was observed in the 4T1 cell samples treated with Cel-Mn; in contrast, the expression levels of γ-H2AX in both the blank control and the cells treated only with manganese acetate remained at low levels. These data together confirmed 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 viability of nCel-Mn on breast cancer cells 4T1. The cells were seeded in 96-well plates at a density of 5000 cells per well and cultured for 12 h. The cells were incubated with nCel-Mn at final concentrations of 0.25, 0.15, 1, 2, 4, 8, 16 μM for 24 h. Then, 10% MTT diluted with RPMI 1640 (100 μL) was added to the wells. After culturing at 37 °C and 5% CO2 for 4 h, 100 μL of 10% SDS was added to each well, and the cells were cultured at 37 °C and 5% CO2 for 12 h. The absorbance of the wells was detected by a microplate reader at 570 nm (peak absorbance) and 650 nm (background absorbance). The cell viability was expressed as the ratio of the absorbance of the test wells to that of the control wells.

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

[0118] Example 7 Drug release of nCel-Mn in a hydrogen peroxide-mimicked oxidative environment

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

[0120] The release results are as Figure 9As shown, 14% was released in a normal environment in 48 h, while 74% was released in an environment with hydrogen peroxide present.

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

[0122] The content of manganese element in cells was detected by atomic absorption spectrometry (AAS). 2 mL of culture medium containing 3×10 6 4T1 cells was inoculated into a six-well plate and cultured in an incubator for 12 hours. Manganese acetate and nCel-Mn solutions were added respectively to make the final Mn concentration 2 μM. Three culture time periods of 1 h, 4 h, and 7 h were set, with three replicates in each group. A PBS group was set as the control. After the drug treatment was completed, the culture medium was aspirated, and the cells were gently washed three times with cold PBS to ensure that the drugs not swallowed by the cells were washed away. The six-well plate was placed in a fume hood, and 100 μL of concentrated HNO3 and 100 μL of 30% H2O2 solution were added to each well. After digestion for 3 d, the digested liquid was collected, fixed to 2 mL, and the content of manganese element was tested using AAS. The results are as Figure 10 shown, nCel-Mn can be internalized by cells more effectively.

[0123] Example 9 Effects of nCel and nCel-Mn on the activation of Bone Marrow Derived Cells (BMDC) after treatment of 4T1 cells

[0124] To evaluate the effect of the interaction between nCel-Mn and 4T1 cells on the activation of BMDCs, the following experiment can be designed: First, monocytes were isolated from the bone marrow of healthy mice and cultured in a medium containing GM-CSF until BMDCs matured; then 4T1 cells were co-incubated with different concentrations of nCel-Mn for a period of time, and then the supernatant of the treated 4T1 cells was collected and added to the BMDCs medium as a stimulant; at the same time, a control group was set, and the supernatant of untreated 4T1 cells and the supernatant containing only nCel-Mn were added respectively as controls to distinguish the effects of 4T1 cells themselves and nCel-Mn on the activation state of BMDCs; the BMDCs were continuously cultured and cell samples were collected at the designated time points, and flow cytometry was used to detect the changes in the expression of BMDC surface markers such as CD80 and CD86, so as to compare the differences in the activation states of BMDCs among the groups. The experimental results are as Figure 11 shown, and the experiment proves that nCel-Mn can effectively induce BMDC activation.

[0125] Combining the above embodiments, it can be found that although Cel alone does not have the effect of activating the cGAS-STING pathway, Cel-Mn can activate the cGAS-STING pathway more effectively 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 mainly two problems with the administration of single manganese ions. 1: Manganese ions need corresponding transport proteins to enter cells, and the expression rate of transport proteins in most cells is low, resulting in insufficient cellular internalization ability of single manganese ions. 2: Manganese ions alone activate the cGAS-STING pathway by activating cGAS activity, but do not induce DNA damage, resulting in a relatively low content of single-stranded DNA and limited activation efficiency. In addition, Cel, as a manganese carrier, internalizes Mn into cells more effectively ( Figure 10 ), and the DNA damage caused by the pharmacological action of Cel can further promote the activity of Mn-catalyzed cGAS to form an activated cGAS-STING pathway with a self-circulation.

[0126] The above embodiments only represent several implementation manners of the present disclosure. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present disclosure, several modifications and improvements can still be made, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the patent of the present disclosure should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Application of celastrol-manganese nanoparticles in the preparation of a drug for treating breast cancer; Among them, The celastrol-manganese nanoparticles are mainly obtained by combining a celastrol-manganese complex with an oxidation-sensitive polymer; The oxidation-sensitive polymer is:

2. The application according to claim 1, wherein the mass ratio of the celastrol-manganese complex to the oxidation-sensitive polymer is 1:(10-20).

3. The application according to claim 1 or 2, wherein the particle size of the celastrol-manganese nanoparticles is 50 nm to 300 nm.

4. The application according to claim 3, wherein the particle size of the celastrol-manganese nanoparticles is 50 nm to 100 nm.

5. A celastrol-manganese nanoparticle for treating breast cancer, the structure of which is defined as the celastrol-manganese nanoparticle in the application according to any one of claims 1 to 4.

6. The preparation method of the celastrol-manganese nanoparticle according to claim 5, comprising: a) Mixing a celastrol solution and a manganese ion solution to obtain a celastrol-manganese complex; b) Mixing the celastrol-manganese complex with the oxidation-sensitive 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.

7. The method according to claim 6, wherein the molar ratio of celastrol in the celastrol solution to manganese ions in the manganese ion solution is 1:(1-2).

8. A pharmaceutical composition, which contains the celastrol-manganese nanoparticle according to claim 5 and a pharmaceutically acceptable carrier.

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