Preparation and application of ChS-betaCD

Abstract

The invention provides ChS-betaCD as well as a preparation method and application thereof, and particularly provides application of a preparation in preparation of a medicine, the medicine is used for preventing and / or treating tumors caused by STING protein function defects, and the preparation comprises a beta cyclodextrin-chondroitin sulfate conjugate. The formulations further comprise a STING agonist, such as cGAMP. The preparation provided by the invention has organelle targeting and intracellular cholesterol distribution regulation ability, promotes STING transport and activation, induces immune response and generates lasting immune memory, and plays an anti-tumor effect. Meanwhile, the preparation provided by the invention can effectively reduce the cytotoxicity of beta-cyclodextrin, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the preparation and application of ChS-βCD, and more specifically to a pharmaceutical preparation, a drug, a single dosage form, a drug combination, and a medicine box. Background Technology

[0002] The STING pathway is a key innate immune signaling pathway, playing a crucial role in tumor immunity and antiviral defense. Although STING agonists have shown some antitumor activity in preclinical studies, their clinical efficacy has been less than ideal. Therefore, there is an urgent need to develop a safe, effective, and novel regulatory molecule that can synergistically enhance STING activation.

[0003] STING activation depends on its transport and oligomerization from the endoplasmic reticulum (ER) to the Golgi apparatus, triggering phosphorylation of downstream signaling factors such as TBK1 and IRF3, thereby initiating the production of type I interferon and other inflammatory factors. Furthermore, recent studies have found that: 1) in the ER, STING protein directly interacts with cholesterol, inhibiting STING migration; while reduced cholesterol content in the ER facilitates STING protein release and initiates transport; 2) a large amount of cholesterol and STING oligomers have been found in the STING lipid rafts on the Golgi apparatus, promoting STING pathway activation. Therefore, spatial regulation of cholesterol in subcellular organelles can effectively promote the activation of the STING signaling pathway.

[0004] Traditional cholesterol regulation tools, such as methyl-β-cyclodextrin (MβCD), can enhance STING activation by globally removing cholesterol. However, due to their lack of organelle selectivity, they often lead to problems such as high cytotoxicity, narrow in vivo therapeutic dose windows, and difficulty in translating into clinical applications. Therefore, it is still necessary to further develop an organelle-specific cholesterol regulation strategy to achieve precise regulation of STING signaling while maintaining overall cholesterol homeostasis. Summary of the Invention

[0005] This invention is based on the inventor's discoveries and understanding of the following facts and problems: Chondroitin sulfate has good cell-targeting, organelle-targeting and biocompatibility. In this invention, by coupling β-cyclodextrin and chondroitin sulfate, it was unexpectedly discovered that the compound can effectively enhance the activation effect of STING and reduce cytotoxicity, and can also synergize with cGAMP to show anti-tumor effects.

[0006] Therefore, in a first aspect, the present invention provides the use of a formulation in the preparation of a medicament. According to embodiments of the present invention, the medicament is used for the prevention and / or treatment of tumors caused by STING protein dysfunction, the formulation comprising a β-cyclodextrin-chondroitin sulfate conjugate. The β-cyclodextrin-chondroitin sulfate conjugate proposed in this invention can target the Golgi apparatus and accumulate cholesterol, significantly enhancing STING transport and activation, strengthening the immune response, inhibiting tumor growth, and generating durable immune memory, effectively preventing tumor recurrence. Therefore, the β-cyclodextrin-chondroitin sulfate conjugate differs from the mechanism by which the traditional STING agonist cGAMP induces STING activation; the two can synergistically enhance STING pathway activation.

[0007] According to embodiments of the present invention, the use of the above-described formulation in the preparation of a drug may further include at least one of the following additional technical features: According to embodiments of the present invention, the formulation further includes a STING agonist.

[0008] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0009] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0010] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20).

[0011] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0012] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0013] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0014] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0015] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0016] According to an embodiment of the present invention, the degree of modification of the coupling agent is 2 to 8.

[0017] According to an embodiment of the present invention, the degree of modification of the coupling agent is 3 to 6, more preferably 4.

[0018] In a second aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises a β-cyclodextrin-chondroitin sulfate conjugate. The medicament proposed in this invention has high organelle targeting specificity, effectively targeting the Golgi apparatus and enriching cholesterol, thereby promoting STING transport and activation, and exerting anti-tumor immunotherapy.

[0019] According to embodiments of the present invention, the above-mentioned drug may further include at least one of the following additional technical features: According to an embodiment of the present invention, the drug further comprises a STING agonist.

[0020] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0021] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0022] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20).

[0023] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0024] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0025] According to embodiments of the present invention, the drug is used to prevent and / or treat tumors caused by STING protein dysfunction.

[0026] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0027] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0028] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0029] According to an embodiment of the present invention, the degree of modification of the coupling agent is 2 to 8.

[0030] According to an embodiment of the present invention, the degree of modification of the coupling agent is 3 to 6, more preferably 4.

[0031] In a third aspect, the present invention provides a single-dose formulation. According to embodiments of the invention, the single-dose formulation comprises a β-cyclodextrin-chondroitin sulfate conjugate. According to embodiments of the invention, the single-dose formulation further comprises a STING agonist. The single-dose formulation proposed in this invention exhibits high organelle targeting, reduces cytotoxicity while achieving anti-tumor therapeutic effects, and can precisely treat tumor diseases caused by defects in the regulation of STING protein function.

[0032] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0033] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0034] According to embodiments of the present invention, the above-mentioned single dosage form may further include at least one of the following additional technical features: According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20).

[0035] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0036] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0037] According to embodiments of the present invention, the single-dose formulation is used for the prevention and / or treatment of tumors caused by STING protein dysfunction.

[0038] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0039] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0040] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0041] According to an embodiment of the present invention, the degree of modification of the coupling agent is 2 to 8.

[0042] According to an embodiment of the present invention, the degree of modification of the coupling agent is 3 to 6, more preferably 4.

[0043] According to embodiments of the present invention, the single dosage form comprises 240 mg to 25000 mg of the β-cyclodextrin-chondroitin sulfate conjugate.

[0044] According to an embodiment of the present invention, the single-dose formulation comprises 1.2 mg to 125 mg of the STING agonist.

[0045] In a fourth aspect, the present invention proposes a drug combination. According to embodiments of the invention, it includes the aforementioned β-cyclodextrin-chondroitin sulfate conjugate. According to embodiments of the invention, the drug combination further includes a STING agonist. The drug combination proposed in this invention exhibits high organelle targeting, reduces cytotoxicity while achieving anti-tumor therapeutic effects, and can precisely treat tumor diseases caused by functional defects in proteins regulating the STING pathway.

[0046] According to embodiments of the present invention, the above-described drug combination may further include at least one of the following additional technical features: According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0047] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0048] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20).

[0049] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0050] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0051] According to embodiments of the present invention, the drug is used in combination for the prevention and / or treatment of tumors caused by STING protein dysfunction.

[0052] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0053] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0054] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0055] According to an embodiment of the present invention, the degree of modification of the coupling agent is 2 to 8.

[0056] According to an embodiment of the present invention, the degree of modification of the coupling agent is 3 to 6, more preferably 4.

[0057] In a fifth aspect, the present invention provides a pillbox. According to an embodiment of the invention, the pillbox comprises a β-cyclodextrin-chondroitin sulfate conjugate. According to an embodiment of the invention, the pillbox further comprises a STING agonist. The pillbox proposed in this invention can effectively treat tumor diseases caused by STING pathway protein dysfunction, and in combination with cGAMP, can significantly activate the STING pathway, enabling precise therapeutic regulation and achieving anti-tumor therapeutic effects.

[0058] According to embodiments of the present invention, the above-mentioned medicine box may further include at least one of the following additional technical features: According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0059] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0060] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20).

[0061] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0062] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0063] According to an embodiment of the present invention, the kit is used for the prevention and / or treatment of tumors caused by STING protein dysfunction.

[0064] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0065] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0066] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0067] According to an embodiment of the present invention, the degree of modification of the coupling agent is 2 to 8.

[0068] According to an embodiment of the present invention, the degree of modification of the coupling agent is 3 to 6, more preferably 4.

[0069] The beneficial effects of this invention are at least as follows: The ChS-βCD conjugate proposed in this invention combines the ability to bind cholesterol with Golgi targeting. Compared with the use of ChS or βCD alone, the ChS-βCD conjugate significantly enhances the STING activation effect and reduces cytotoxicity in vitro and in vivo. It can synergistically exhibit anti-tumor effects with cGAMP, generate lasting immune memory, significantly reduce the dosage of cGAMP, effectively prevent tumor recurrence, and show good application prospects. Attached Figure Description

[0070] Figure 1 This is a schematic diagram of the chondroitin sulfate-β-cyclodextrin linkage site of ChS-βCD according to an embodiment of the present invention.

[0071] Figure 2 These are NMR spectra of ChS-βCD with different degrees of modification according to embodiments of the present invention.

[0072] Figure 3 The immune cell stimulating activity of ChS-βCD with different degrees of modification according to embodiments of the present invention, wherein, Figure 3 a represents the expression level of the cell surface co-stimulatory molecule CD40 as characterized by flow cytometry; Figure 3 b is the cell surface co-stimulatory molecule CD86 characterized by flow cytometry. Figure 3c represents the expression level of the cell surface co-stimulatory molecule CD80 as characterized by flow cytometry. Figure 3 d represents the expression level of cytokine IFN-β in cell supernatant as determined by ELISA; Figure 3 e represents the expression level of the cytokine IL-6 in the cell supernatant as determined by ELISA; Figure 3 f represents the expression level of cytokine CXCL10 in cell supernatant as determined by ELISA.

[0073] Figure 4 The immunostimulatory activity of different concentrations of ChS-βCD according to embodiments of the present invention, wherein, Figure 4 a represents the expression level of the cell surface co-stimulatory molecule CD40 as characterized by flow cytometry; Figure 4 b is the cell surface co-stimulatory molecule CD86 characterized by flow cytometry. Figure 4 c represents the expression level of the cell surface co-stimulatory molecule CD80 as characterized by flow cytometry.

[0074] Figure 5 This is a comparison of the immunostimulatory activities of ChS-βCD and βCD according to an embodiment of the present invention, wherein, Figure 5 a represents the expression level of the cell surface co-stimulatory molecule CD40 as characterized by flow cytometry; Figure 5 b is the cell surface co-stimulatory molecule CD86 characterized by flow cytometry. Figure 5 c represents the expression level of the cell surface co-stimulatory molecule CD80 as characterized by flow cytometry.

[0075] Figure 6 This is an embodiment of the ChS-βCD-promoted STING pathway activation detection according to the present invention.

[0076] Figure 7 The antitumor effect of ChS-βCD according to an embodiment of the present invention, wherein, Figure 7 'a' refers to the immunization process; Figure 7 b is the tumor volume growth curve; Figure 7 c represents the mouse's survival period.

[0077] Figure 8 According to an embodiment of the present invention, ChS-βCD synergistically exerts an anti-tumor effect with cGAMP, wherein, Figure 8 a is the tumor volume growth curve of the B16-OVA model; Figure 8 b represents the survival time of mice in the B16-OVA model; Figure 8 c is the tumor volume growth curve of the B16-OVA model; Figure 8 d represents the survival time of mice in the B16-OVA model. Detailed Implementation

[0078] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0079] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0080] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0081] In the process of describing this invention, the terms used herein have been explained and described. These explanations and descriptions are only for the purpose of facilitating the understanding of the solution and should not be regarded as a limitation on the protection of this invention.

[0082] In this article, the term "degree of modification" refers to the proportion of sites (or molecules) where a modifying group is successfully attached to the total number of modifiable sites (or molecules), and in this article specifically refers to the average number of β-cyclodextrin molecules successfully covalently attached to each chondroitin sulfate molecular chain.

[0083] In this article, "cGAMP" refers to cyclic guanosine monophosphate-adenosine monophosphate (Cyclic GMP-AMP), a cyclic structure formed by a guanosine monophosphate (GMP) and an adenosine monophosphate (AMP) linked by two phosphodiester bonds. It is a key second messenger in innate immunity, activating the interferon gene stimulating factor (STING) and activating the signaling cascade that leads to the production of interferon and other immune mediators.

[0084] In this article, "drug combination" refers to the simultaneous or sequential use of two or more drugs to achieve a therapeutic goal, primarily to increase the efficacy of the drugs or to reduce their toxic side effects.

[0085] In this article, "single dosage form" refers to a dosage form in which a single preparation is used up in a single dose.

[0086] The technical solution of this application is described in detail below: Pharmaceutical uses and drugs In some embodiments of the present invention, the present invention proposes the use of a formulation in the preparation of a medicament for the prevention and / or treatment of tumors caused by STING protein dysfunction, the formulation comprising a β-cyclodextrin-chondroitin sulfate conjugate. The STING protein dysfunction includes a weakened or insufficient response of STING-related proteins. β-cyclodextrin enhances STING activation by removing cholesterol, and chondroitin sulfate has Golgi apparatus targeting. The β-cyclodextrin-chondroitin sulfate conjugate proposed in this invention can significantly enhance STING transport and activation effects and organelle targeting, enhance immune stimulation, inhibit tumor growth, and generate durable immune memory, effectively preventing tumor recurrence. The β-cyclodextrin-chondroitin sulfate conjugate can also...

[0087] According to embodiments of the present invention, the formulation further includes a STING agonist.

[0088] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0089] According to some specific embodiments of the present invention, the STING agonist is cGAMP. cGAMP acts as an endogenous second messenger, activating the interferon gene-stimulating factor (STING), which in turn activates a signaling cascade leading to the production of type I interferon and other immune mediators. While cGAMP has low bioavailability when used alone, the β-cyclodextrin-chondroitin sulfate conjugate proposed in this invention synergistically enhances the immunostimulatory effect of cGAMP activation.

[0090] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20). Exemplarily, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is 50:1, 50:20, 2000:1, or 2000:20, or a range between any two of the above values. According to a preferred embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0091] According to a specific embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0092] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0093] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0094] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0095] According to embodiments of the present invention, the degree of modification of the coupling is 2 to 8. Exemplarily, the degree of modification of the coupling is 2, 3, 4, 5, 6, 6.7, 7 or 8, or a range between any two of the above values. According to some preferred embodiments of the present invention, the degree of modification of the coupling is 3 to 6, more preferably 4.

[0096] According to an embodiment of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate is obtained by reacting a chondroitin sulfate salt solution with β-cyclodextrin in a first reaction. The core principle is the dehydration condensation of the carboxyl group on the chondroitin sulfate chain with the primary hydroxyl group on the β-cyclodextrin chain to form a covalent ester bond.

[0097] According to embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (90~110):(51~70). Exemplarily, the mass ratio of chondroitin sulfate to β-cyclodextrin is 90:51, 90:70, 110:51, 100:61, or 110:70, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (95~105):(55~65). According to some specific embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is 100:61.

[0098] According to an embodiment of the present invention, the chondroitin sulfate salt solution comprises a chondroitin sulfate sodium salt solution.

[0099] According to an embodiment of the present invention, the chondroitin sulfate salt solution further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof and N-hydroxysuccinimide or a salt thereof.

[0100] According to embodiments of the present invention, the salts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide include hydrochloride, sulfate, nitrate, carbonate or phosphate, preferably hydrochloride.

[0101] According to embodiments of the present invention, the salts of the N-hydroxysuccinimide include sulfonates, hydrochlorides, sulfates, nitrates, carbonates, or phosphates, preferably sulfonates.

[0102] According to an embodiment of the present invention, the salt of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0103] According to an embodiment of the present invention, the salt of the N-hydroxysuccinimide is sodium N-hydroxysuccinimide. The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride acts as an activator to activate the carboxyl group. Sodium N-hydroxysuccinimide can significantly improve the coupling yield and is key to efficient coupling.

[0104] According to an embodiment of the present invention, the concentration of chondroitin sulfate in the chondroitin sulfate solution is 5 mg / mL.

[0105] According to an embodiment of the present invention, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt in the chondroitin sulfate salt solution is 8 mg / mL.

[0106] According to an embodiment of the present invention, the concentration of N-hydroxysuccinylsulfinic acid or its salt in the chondroitin sulfate salt solution is 9 mg / mL.

[0107] According to an embodiment of the present invention, the time of the first reaction is 18h to 30h. Exemplarily, the time of the first reaction is 18h, 20h, 22h, 24h, 26h, 28h or 30h, or a range between any two of the above values. According to some preferred embodiments of the present invention, the time of the first reaction is preferably 20h to 26h; more preferably 24h.

[0108] According to embodiments of the present invention, the chondroitin sulfate salt solution is prepared by dissolving chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt in a buffer solution at a mass ratio of (3-6):(7-9):(8-10). Exemplarily, the mass ratio of chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt is 3:7:8, 3:7:10, 3:9:8, 3:9:10, 6:7:10, 6:7:8, 6:7:10, 6:9:8, or 6:9:10, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio is preferably (4-5.8):(7.5-8.5):(8.2-9.5). According to some specific embodiments of the present invention, the mass ratio is 5:8:9.

[0109] According to embodiments of the present invention, the buffer solution comprises at least one selected from the following: PBS, MES, and HEPES. The buffer solution provides and maintains optimal reaction pH and ionic strength conditions for the preparation of the β-cyclodextrin-chondroitin sulfate conjugate.

[0110] In some embodiments of the present invention, a drug comprising a β-cyclodextrin-chondroitin sulfate conjugate is proposed. According to embodiments of the present invention, the drug further comprises a STING agonist. The drug proposed in this invention exhibits high organelle targeting, achieving anti-tumor therapeutic effects while reducing cytotoxicity. Through subcellular cholesterol spatial regulation, it can effectively promote the activation of the STING signaling pathway, enabling precise treatment of tumor diseases caused by abnormal functional defects or absence of proteins regulating the STING pathway.

[0111] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0112] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0113] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20). Exemplarily, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is 50:1, 50:20, 2000:1, or 2000:20, or a range between any two of the above values. According to a preferred embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0114] According to a specific embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0115] According to embodiments of the present invention, the drug is used to prevent and / or treat tumors caused by STING protein dysfunction.

[0116] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0117] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0118] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0119] According to embodiments of the present invention, the degree of modification of the coupling is 2 to 8. Exemplarily, the degree of modification of the coupling is 2, 3, 4, 5, 6, 6.7, 7 or 8, or a range between any two of the above values. According to some preferred embodiments of the present invention, the degree of modification of the coupling is 3 to 6, more preferably 4.

[0120] According to an embodiment of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate is obtained by reacting a chondroitin sulfate salt solution with β-cyclodextrin in a first reaction. The core principle is the dehydration condensation of the carboxyl group on the chondroitin sulfate chain with the primary hydroxyl group on the β-cyclodextrin chain to form a covalent ester bond.

[0121] According to embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (90~110):(51~70). Exemplarily, the mass ratio of chondroitin sulfate to β-cyclodextrin is 90:51, 90:70, 110:51, 100:61, or 110:70, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (95~105):(55~65). According to some specific embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is 100:61.

[0122] According to an embodiment of the present invention, the chondroitin sulfate salt solution comprises a chondroitin sulfate sodium salt solution.

[0123] According to an embodiment of the present invention, the chondroitin sulfate salt solution further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof and N-hydroxysuccinimide or a salt thereof.

[0124] According to embodiments of the present invention, the salts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide include hydrochloride and sulfate, preferably hydrochloride; and / or the salts of the N-hydroxysuccinimide include sulfonate, hydrochloride, and sulfate, preferably sulfonate.

[0125] According to embodiments of the present invention, the salt of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and / or the salt of the N-hydroxysuccinimide is sodium N-hydroxysuccinimide. The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride acts as an activator to activate the carboxyl group, while sodium N-hydroxysuccinimide can significantly improve the coupling yield and is key to efficient coupling.

[0126] According to an embodiment of the present invention, the concentration of chondroitin sulfate in the chondroitin sulfate solution is 5 mg / mL.

[0127] According to an embodiment of the present invention, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt in the chondroitin sulfate solution is 8 mg / mL, and / or the concentration of N-hydroxysuccinylsulfinic acid or its salt in the chondroitin sulfate solution is 9 mg / mL.

[0128] According to an embodiment of the present invention, the time of the first reaction is 18h to 30h. Exemplarily, the time of the first reaction is 18h, 20h, 22h, 24h, 26h, 28h or 30h, or a range between any two of the above values. According to some preferred embodiments of the present invention, the time of the first reaction is preferably 20h to 26h; more preferably 24h.

[0129] According to embodiments of the present invention, the chondroitin sulfate salt solution is prepared by dissolving chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt in a buffer solution at a mass ratio of (3-6):(7-9):(8-10). Exemplarily, the mass ratio of chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt is 3:7:8, 3:7:10, 3:9:8, 3:9:10, 6:7:10, 6:7:8, 6:7:10, 6:9:8, or 6:9:10, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio is preferably (4-5.8):(7.5-8.5):(8.2-9.5). According to some specific embodiments of the present invention, the mass ratio is 5:8:9.

[0130] According to embodiments of the present invention, the buffer solution comprises at least one selected from the following: PBS, MES, and HEPES. The buffer solution provides and maintains optimal reaction pH and ionic strength conditions for the preparation of the β-cyclodextrin-chondroitin sulfate conjugate.

[0131] According to embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier, including any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and delayed absorption agents, etc. Specific examples may be one or more of water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, the pharmaceutical composition includes isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Of course, a pharmaceutically acceptable carrier may also include trace amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, to prolong the shelf life or potency of the antibody.

[0132] According to embodiments of the present invention, the dosage form of the drug is at least one of an injectable formulation, an oral formulation, or an inhaled formulation.

[0133] According to an embodiment of the present invention, the injectable formulation is an injection solution or a lyophilized powder for injection.

[0134] According to embodiments of the present invention, the oral preparation is a tablet, capsule, granule, oral liquid / solution, or droplet.

[0135] According to embodiments of the present invention, the inhaled formulation is a solution, powder, or suspension thereof.

[0136] Single-dose formulations, combination drugs, and pillboxes In some embodiments of the present invention, the single-dose formulation includes a β-cyclodextrin-chondroitin sulfate conjugate. According to embodiments of the present invention, the single-dose formulation further includes a STING agonist. The single-dose formulation proposed in this invention exhibits high organelle targeting, achieving anti-tumor therapeutic effects while reducing cytotoxicity. The β-cyclodextrin-chondroitin sulfate conjugate combines the cholesterol-binding ability of cyclodextrin with the Golgi apparatus targeting ability of chondroitin sulfate. Through subcellular organelle cholesterol spatial regulation, it can significantly regulate STING transport and enhance STING activation in vitro and in vivo, enabling precise treatment of tumors caused by functional defects in STING pathway proteins, with enhanced safety.

[0137] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0138] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0139] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20). Exemplarily, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is 50:1, 50:20, 2000:1, or 2000:20, or a range between any two of the above values. According to a preferred embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0140] According to a specific embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0141] According to embodiments of the present invention, the single-dose formulation is used for the prevention and / or treatment of tumors caused by STING protein dysfunction.

[0142] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0143] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0144] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0145] According to embodiments of the present invention, the degree of modification of the coupling is 2 to 8. Exemplarily, the degree of modification of the coupling is 2, 3, 4, 5, 6, 6.7, 7 or 8, or a range between any two of the above values. According to some preferred embodiments of the present invention, the degree of modification of the coupling is 3 to 6, more preferably 4.

[0146] According to an embodiment of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate is obtained by reacting a chondroitin sulfate salt solution with β-cyclodextrin in a first reaction. The core principle is the dehydration condensation of the carboxyl group on the chondroitin sulfate chain with the primary hydroxyl group on the β-cyclodextrin chain to form a covalent ester bond.

[0147] According to embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (90~110):(51~70). Exemplarily, the mass ratio of chondroitin sulfate to β-cyclodextrin is 90:51, 90:70, 110:51, 100:61, or 110:70, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (95~105):(55~65). According to some specific embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is 100:61.

[0148] According to an embodiment of the present invention, the chondroitin sulfate salt solution comprises a chondroitin sulfate sodium salt solution.

[0149] According to an embodiment of the present invention, the chondroitin sulfate salt solution further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof and N-hydroxysuccinimide or a salt thereof.

[0150] According to embodiments of the present invention, the salts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide include hydrochloride and sulfate, preferably hydrochloride; and / or the salts of the N-hydroxysuccinimide include sulfonate, hydrochloride, and sulfate, preferably sulfonate.

[0151] According to embodiments of the present invention, the salt of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and / or the salt of the N-hydroxysuccinimide is sodium N-hydroxysuccinimide. The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride acts as an activator to activate the carboxyl group, while sodium N-hydroxysuccinimide can significantly improve the coupling yield and is key to efficient coupling.

[0152] According to an embodiment of the present invention, the concentration of chondroitin sulfate in the chondroitin sulfate solution is 5 mg / mL.

[0153] According to an embodiment of the present invention, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt in the chondroitin sulfate solution is 8 mg / mL, and / or the concentration of N-hydroxysuccinylsulfinic acid or its salt in the chondroitin sulfate solution is 9 mg / mL.

[0154] According to an embodiment of the present invention, the time of the first reaction is 18h to 30h. Exemplarily, the time of the first reaction is 18h, 20h, 22h, 24h, 26h, 28h or 30h, or a range between any two of the above values. According to some preferred embodiments of the present invention, the time of the first reaction is preferably 20h to 26h; more preferably 24h.

[0155] According to embodiments of the present invention, the chondroitin sulfate salt solution is prepared by dissolving chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt in a buffer solution at a mass ratio of (3-6):(7-9):(8-10). Exemplarily, the mass ratio of chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt is 3:7:8, 3:7:10, 3:9:8, 3:9:10, 6:7:10, 6:7:8, 6:7:10, 6:9:8, or 6:9:10, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio is preferably (4-5.8):(7.5-8.5):(8.2-9.5). According to some specific embodiments of the present invention, the mass ratio is 5:8:9.

[0156] According to embodiments of the present invention, the buffer solution comprises at least one selected from the following: PBS, MES, and HEPES. The buffer solution provides and maintains optimal reaction pH and ionic strength conditions for the preparation of the β-cyclodextrin-chondroitin sulfate conjugate.

[0157] According to embodiments of the present invention, the single dosage form comprises 240 mg to 25000 mg of the β-cyclodextrin-chondroitin sulfate conjugate. Exemplarily, the mass of the β-cyclodextrin-chondroitin sulfate conjugate is 240 mg, 250 mg, 300 mg, 400 mg, 500 mg, 800 mg, 1000 mg, 2000 mg, 3000 mg, 4000 mg, 5000 mg, 10000 mg, 20000 mg, or 25000 mg, or a range between any two of the above values.

[0158] According to an embodiment of the present invention, the single dosage form comprises 1.2 mg to 125 mg of the STING agonist, and exemplaryly, the mass of the STING agonist is 1.2 mg, 1.5 mg, 2 mg, 5 mg, 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg or 125 mg, or a range between any two of the above values.

[0159] The single-dose formulation can be used once, twice, or three times daily, preferably once. Through conversion between mouse and human dosages, this dosage conversion demonstrates its effectiveness in protecting humans from or alleviating tumors. Those skilled in the art will understand that the specifications of the single-dose formulation are not particularly limited. For example, the specifications can be set according to the characteristics of different target populations, such as the weight of the target population (e.g., 40kg, 60kg, 70kg, 80kg, 90kg, or 100kg), or according to the characteristics of the target population and the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist. For example, the conversion factor between mouse and human dosages is 12.3. Based on the dosage of the β-cyclodextrin-chondroitin sulfate conjugate in mice, the equivalent dose for a human weighing 40kg = mouse dose × 12.3 × 40 = 246mg.

[0160] According to embodiments of the present invention, the single dosage form may be an oral preparation, an injectable preparation, a pill, a sustained-release preparation, an implant, or an aerosol, etc., and is not particularly limited thereto.

[0161] In some embodiments of the present invention, a drug combination is proposed, comprising the aforementioned β-cyclodextrin-chondroitin sulfate conjugate. According to embodiments of the present invention, the drug combination further comprises a STING agonist. The drug combination proposed in this invention exhibits high organelle targeting, reduces cytotoxicity while achieving anti-tumor therapeutic effects, and combines anti-tumor capability with clinical safety, enabling precise treatment of tumor diseases caused by functional defects in proteins regulating the STING pathway.

[0162] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0163] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0164] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20). Exemplarily, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is 50:1, 50:20, 2000:1, or 2000:20, or a range between any two of the above values. According to a preferred embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20) or (50~1000):(1~10).

[0165] According to a specific embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0166] According to embodiments of the present invention, the drug is used in combination for the prevention and / or treatment of tumors caused by STING protein dysfunction.

[0167] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0168] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0169] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0170] According to embodiments of the present invention, the degree of modification of the coupling is 2 to 8. Exemplarily, the degree of modification of the coupling is 2, 3, 4, 5, 6, 6.7, 7 or 8, or a range between any two of the above values. According to some preferred embodiments of the present invention, the degree of modification of the coupling is 3 to 6, more preferably 4.

[0171] According to an embodiment of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate is obtained by reacting a chondroitin sulfate salt solution with β-cyclodextrin in a first reaction. The core principle is the dehydration condensation of the carboxyl group on the chondroitin sulfate chain with the primary hydroxyl group on the β-cyclodextrin chain to form a covalent ester bond.

[0172] According to embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (90~110):(51~70). Exemplarily, the mass ratio of chondroitin sulfate to β-cyclodextrin is 90:51, 90:70, 110:51, 100:61, or 110:70, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (95~105):(55~65). According to some specific embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is 100:61.

[0173] According to an embodiment of the present invention, the chondroitin sulfate salt solution comprises a chondroitin sulfate sodium salt solution.

[0174] According to an embodiment of the present invention, the chondroitin sulfate salt solution further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof and N-hydroxysuccinimide or a salt thereof.

[0175] According to embodiments of the present invention, the salts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide include hydrochloride and sulfate, preferably hydrochloride; and / or the salts of the N-hydroxysuccinimide include sulfonate, hydrochloride, and sulfate, preferably sulfonate.

[0176] According to embodiments of the present invention, the salt of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and / or the salt of the N-hydroxysuccinimide is sodium N-hydroxysuccinimide. The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride acts as an activator to activate the carboxyl group, while sodium N-hydroxysuccinimide can significantly improve the coupling yield and is key to efficient coupling.

[0177] According to an embodiment of the present invention, the concentration of chondroitin sulfate in the chondroitin sulfate solution is 5 mg / mL.

[0178] According to an embodiment of the present invention, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt in the chondroitin sulfate solution is 8 mg / mL, and / or the concentration of N-hydroxysuccinylsulfinic acid or its salt in the chondroitin sulfate solution is 9 mg / mL.

[0179] According to an embodiment of the present invention, the time of the first reaction is 18h to 30h. Exemplarily, the time of the first reaction is 18h, 20h, 22h, 24h, 26h, 28h or 30h, or a range between any two of the above values. According to some preferred embodiments of the present invention, the time of the first reaction is preferably 20h to 26h; more preferably 24h.

[0180] According to embodiments of the present invention, the chondroitin sulfate salt solution is prepared by dissolving chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt in a buffer solution at a mass ratio of (3-6):(7-9):(8-10). Exemplarily, the mass ratio of chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt is 3:7:8, 3:7:10, 3:9:8, 3:9:10, 6:7:10, 6:7:8, 6:7:10, 6:9:8, or 6:9:10, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio is preferably (4-5.8):(7.5-8.5):(8.2-9.5). According to some specific embodiments of the present invention, the mass ratio is 5:8:9.

[0181] According to embodiments of the present invention, the buffer solution comprises at least one selected from the following: PBS, MES, and HEPES. The buffer solution provides and maintains optimal reaction pH and ionic strength conditions for the preparation of the β-cyclodextrin-chondroitin sulfate conjugate.

[0182] According to embodiments of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist can be prepared together or separately. Those skilled in the art will understand that the preparation methods of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist are not particularly limited, and they can be prepared separately or together.

[0183] According to embodiments of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist may be used simultaneously or separately, including separate use in time and / or space, as long as the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist can work together to achieve the objectives of the present invention. When the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist are administered separately to the subject or test sample, the individual components may be administered simultaneously or sequentially to the subject or test sample.

[0184] It should be noted that the drug combination includes combinations that are separate in time and / or space, provided that the β-cyclodextrin-chondroitin sulfate conjugate and / or the STING agonist can work together to achieve the objectives of the present invention. For example, the components contained in the drug combination may be administered to the subject or test sample as a whole, or separately. When the components contained in the drug combination are administered separately to the subject or test sample, the individual components may be administered simultaneously or sequentially to the subject or test sample.

[0185] In some embodiments of the present invention, a pillbox is provided, the pillbox comprising the β-cyclodextrin-chondroitin sulfate conjugate. According to embodiments of the present invention, the pillbox further comprises a STING agonist. The pillbox proposed by the present invention can effectively treat tumor diseases caused by STING pathway protein dysfunction. Combined with a STING agonist, it can significantly activate the STING pathway, enabling precise therapeutic regulation and achieving anti-tumor therapeutic effects while improving clinical safety. According to embodiments of the present invention, the pillbox refers to any device or system for containing, storing, dispensing, and managing one or more drug units (such as tablets, capsules, pills, injections, powders, and liquids).

[0186] According to an embodiment of the present invention, the STING agonist includes at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281, and MK-1454.

[0187] According to an embodiment of the present invention, the STING agonist is cGAMP.

[0188] According to an embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~2000):(1~20). Exemplarily, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is 50:1, 50:20, 2000:1, or 2000:20, or a range between any two of the above values. According to a preferred embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50~1000):(1~10).

[0189] According to a specific embodiment of the present invention, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the cGAMP is 50:1.

[0190] According to embodiments of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist can be prepared together or separately. Those skilled in the art will understand that the preparation methods of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist are not particularly limited, and they can be prepared separately or together.

[0191] According to embodiments of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist may be used simultaneously or separately, including separate use in time and / or space, as long as the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist can work together to achieve the objectives of the present invention. When the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist are administered separately to the subject or test sample, the individual components may be administered simultaneously or sequentially to the subject or test sample.

[0192] According to an embodiment of the present invention, the kit is used for the prevention and / or treatment of tumors caused by STING protein dysfunction.

[0193] According to embodiments of the present invention, the tumor includes at least one of the following: melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer, and lymphoma.

[0194] According to an embodiment of the present invention, the tumor includes at least one of melanoma and colon cancer.

[0195] According to an embodiment of the present invention, the conjugate is obtained by condensing the glucuronic acid carboxyl group of chondroitin sulfate with the primary amino group at the C6 position of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond.

[0196] According to embodiments of the present invention, the degree of modification of the coupling is 2 to 8. Exemplarily, the degree of modification of the coupling is 2, 3, 4, 5, 6, 6.7, 7 or 8, or a range between any two of the above values. According to some preferred embodiments of the present invention, the degree of modification of the coupling is 3 to 6, more preferably 4.

[0197] According to an embodiment of the present invention, the β-cyclodextrin-chondroitin sulfate conjugate is obtained by reacting a chondroitin sulfate salt solution with β-cyclodextrin in a first reaction. The core principle is the dehydration condensation of the carboxyl group on the chondroitin sulfate chain with the primary hydroxyl group on the β-cyclodextrin chain to form a covalent ester bond.

[0198] According to embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (90~110):(51~70). Exemplarily, the mass ratio of chondroitin sulfate to β-cyclodextrin is 90:51, 90:70, 110:51, 100:61, or 110:70, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is (95~105):(55~65). According to some specific embodiments of the present invention, the mass ratio of chondroitin sulfate to β-cyclodextrin is 100:61.

[0199] According to an embodiment of the present invention, the chondroitin sulfate salt solution comprises a chondroitin sulfate sodium salt solution.

[0200] According to an embodiment of the present invention, the chondroitin sulfate salt solution further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or a salt thereof and N-hydroxysuccinimide or a salt thereof.

[0201] According to embodiments of the present invention, the salts of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide include hydrochloride and sulfate, preferably hydrochloride; and / or the salts of the N-hydroxysuccinimide include sulfonate, hydrochloride, and sulfate, preferably sulfonate.

[0202] According to embodiments of the present invention, the salt of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and / or the salt of the N-hydroxysuccinimide is sodium N-hydroxysuccinimide. The 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride acts as an activator to activate the carboxyl group, while sodium N-hydroxysuccinimide can significantly improve the coupling yield and is key to efficient coupling.

[0203] According to an embodiment of the present invention, the concentration of chondroitin sulfate in the chondroitin sulfate solution is 5 mg / mL.

[0204] According to an embodiment of the present invention, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt in the chondroitin sulfate solution is 8 mg / mL, and / or the concentration of N-hydroxysuccinylsulfinic acid or its salt in the chondroitin sulfate solution is 9 mg / mL.

[0205] According to an embodiment of the present invention, the time of the first reaction is 18h to 30h. Exemplarily, the time of the first reaction is 18h, 20h, 22h, 24h, 26h, 28h or 30h, or a range between any two of the above values. According to some preferred embodiments of the present invention, the time of the first reaction is preferably 20h to 26h; more preferably 24h.

[0206] According to embodiments of the present invention, the chondroitin sulfate salt solution is prepared by dissolving chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt in a buffer solution at a mass ratio of (3-6):(7-9):(8-10). Exemplarily, the mass ratio of chondroitin sulfate salt, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or its salt, and N-hydroxysuccinimide or its salt is 3:7:8, 3:7:10, 3:9:8, 3:9:10, 6:7:10, 6:7:8, 6:7:10, 6:9:8, or 6:9:10, or a range between any two of the above values. According to some preferred embodiments of the present invention, the mass ratio is preferably (4-5.8):(7.5-8.5):(8.2-9.5). According to some specific embodiments of the present invention, the mass ratio is 5:8:9.

[0207] According to embodiments of the present invention, the buffer solution comprises at least one selected from the following: PBS, MES, and HEPES. The buffer solution provides and maintains optimal reaction pH and ionic strength conditions for the preparation of the β-cyclodextrin-chondroitin sulfate conjugate.

[0208] Embodiments of the present invention will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0209] Example 1: Preparation of ChS-βCD Weigh 100 mg of chondroitin sulfate sodium salt (ChS), 160 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS No.: 7084-11-9), and 180 mg of sulfonyl-N-hydroxysuccinimide sodium salt (purchased from Bidepharm, catalog number BD30082), and dissolve them in 15 mL–20 mL of PBS buffer. Stir the mixture at room temperature for 30 minutes. Then add 61 mg of β-cyclodextrin (βCD) (CAS NO.: 7585-39-9) and continue stirring for 24 hours.

[0210] After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight of 3000 and dialyzed against deionized water for 3-5 days to remove small molecule impurities and buffer salts. After lyophilization, a white solid product, ChS-βCD, was obtained. Different degrees of modification (m=2~8) of ChS-βCD could be obtained by adjusting the amount of βCD added; subsequent reaction, dialysis, and lyophilization steps were the same as described above. The product was characterized by 1H NMR (solvent D2O, JEOL ECS-400) to determine the average number of βCD modifications. The average number of βCD modifications, m, in the product could be calculated based on the integral ratio of the characteristic peaks of βCD to those of ChS in the 1H NMR spectrum. Specific linkage site configurations are shown in the attached figure. Figure 1 The NMR spectrum results are shown in the attached figure. Figure 2 As shown.

[0211] Example 2: Cellular immune activity of ChS-βCD In this embodiment, mouse macrophage J774A.1 cells (purchased from Meisen CTCC (Hangzhou, China)) were used as the evaluation cell line to assess the immunomodulatory activity of the ChS-βCD conjugate. The specific procedures are as follows: Cells were treated for 24 hours with different levels of modification (m = 3, 4, 6.7, 8) of ChS-βCD (calculated as 0.5 mM βCD) and cGAMP (CAS No.: 849214-04-6), with a ChS-βCD:cGAMP mass ratio of 50:1. Cells were also treated for 24 hours with different concentrations of ChS-βCD (m = 4) (calculated as 0.01-2 mM βCD). Cells were further treated for 24 hours with ChS-βCD (m = 4), ChS monomer, and βCD monomer. Cells were then collected and the expression levels of surface co-stimulatory molecules CD80, CD86, and CD40 were detected by flow cytometry. The secretion of IFN-β, IL-6, and CXCL10 in the cell supernatant was detected by ELISA.

[0212] Specific experimental results are attached. Figure 3As shown in the figure, the results indicated that, compared to the control groups (PBS group and cGAMP group), this series of compounds significantly increased the expression of CD80, CD86, and CD40 on the cell surface, and significantly promoted the secretion of IFN-β, IL-6, and CXCL10. Further experiments showed, as shown in the attached figure... Figure 4 The immunomodulatory activity of ChS-βCD was dose-dependent. Furthermore, comparative experimental results are attached. Figure 5 As shown, βCD alone has a weak effect on immune activation, while ChS-βCD can significantly enhance the immunostimulatory effect of cGAMP.

[0213] Example 3: Evaluation of the activation effect of ChS-βCD on the STING pathway In vitro, mouse macrophages J774A.1 and mouse embryonic fibroblasts (MEF) (purchased from Xiehe CellBank, China Union University Center for Type Culture Collection) were used as evaluation cell lines. First, cells were stimulated with either cGAMP or ChS-βCD (m=4, with a ChS-βCD to cGAMP mass ratio of 50:1). Cells were collected at different time points, and the expression levels of STING pathway-related proteins, including STING, pSTING, TBK1, pTBK1, IRF3, and pIRF3, were detected using Western blotting.

[0214] Specific experimental results are attached. Figure 6 As shown, the results indicate that, compared with cGAMP alone, the combined treatment of ChS-βCD and cGAMP significantly upregulated the expression of STING pathway-related proteins and promoted the activation of the STING signaling pathway.

[0215] Example 4 Evaluation of the antitumor effect of ChS-βCD Female C57BL / 6J mice aged 5–6 weeks were selected and subcutaneously injected with 1.5 × 10⁻⁶ ppm of the drug into the right back. 5 A mouse melanoma (B16-OVA) model was constructed using 100 μL B16-OVA cells (suspended in PBS). Tumor length and width were measured every two days after inoculation using calipers, calculated using the formula V = 0.5 × length × (width). 2 Tumor volume was calculated, and mouse weight was recorded simultaneously. When the tumor volume reached approximately 50 mm³ to 100 mm³, the mice were randomly assigned to different groups for further treatment.

[0216] The antitumor effect of ChS-βCD was verified using the mouse melanoma (B16-OVA) model. When the mouse tumor volume was approximately 50 mm³, the mice were randomly divided into 6 groups and injected intratumorally with PBS, 0.5 mg / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, and 20.0 mg / kg ChS-βCD (m=4), respectively. The drugs were administered once every three days for a total of three administrations, and the survival time of the mice was recorded during the administration.

[0217] Specific experimental results are attached. Figure 7 As shown, the results indicate that the inhibitory effect of ChS-βCD on tumors is significantly dose-dependent. At doses ≥5 mg / kg, tumor growth can be significantly inhibited and the survival of mice can be prolonged. The median survival of the 10 mg / kg and 20 mg / kg groups reached 26 days, while that of the control group was only 19 days.

[0218] Example 5: Evaluation of the antitumor effect of the combination of ChS-βCD and cGAMP Female C57BL / 6J mice aged 5–6 weeks were selected and subcutaneously injected with 6.5 × 10⁶ mmol / L of iodine solution into the right back. 5 A mouse melanoma (B16-OVA) model was constructed using 100 μL B16-OVA cells (suspended in PBS). Tumor length and width were measured every two days after inoculation using calipers, calculated using the formula V = 0.5 × length × (width). 2 Tumor volume was calculated, and body weight was recorded simultaneously. When the tumor volume reached approximately 50 mm³ to 100 mm³, the mice were randomly assigned to different groups for further treatment.

[0219] Female C57BL / 6J mice aged 5–6 weeks were selected and subcutaneously injected with 6.5 × 10⁶ mmol / L of iodine solution into the right back. 5 A mouse colon cancer (MC38) model was constructed using 100 μL MC38 cells (100 μL PBS suspension). Tumor length and width were measured every two days after inoculation using calipers, calculated using the formula V = 0.5 × length × (width). 2 Tumor volume was calculated, and body weight was recorded simultaneously. When the tumor volume reached approximately 50 mm³ to 100 mm³, the mice were randomly assigned to different groups for further treatment.

[0220] The combined antitumor effect of ChS-βCD and the STING agonist cGAMP was verified by constructing mouse melanoma (B16-OVA) and mouse colon cancer (MC38) models. When the mouse tumor volume reached 50-100 mm³, it was randomly divided into 4 groups, including 1×PBS, 4 μg / mouse cGAMP, 10 mg / kg ChS-βCD, and ChS-βCD+cGAMP (mass ratio of 50:1). The mice were injected intratumorally every three days for a total of two times, and the survival time of the mice was recorded during the period.

[0221] Specific experimental results are attached. Figure 8 As shown, the results indicate that the combination therapy group was significantly better than the single-drug treatment group in inhibiting tumor growth and significantly prolonging the survival of mice, with 80% (8 / 10) of the tumor-bearing mice achieving a tumor-free survival state.

[0222] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0223] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a preparation in the preparation of a medicament for preventing and / or treating a tumor caused by a deficiency in the function of STING protein, wherein the preparation comprises a β-cyclodextrin-chondroitin sulfate conjugate.

2. Use according to claim 1, characterized in that, The preparation further comprises a STING agonist; Optionally, the STING agonist comprises at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281 and MK-1454; Preferably, the STING agonist is cGAMP; Optionally, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50-2000):(1-20); Preferably, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50-1000):(1-10); Optionally, the tumor comprises at least one of melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer and lymphoma; Preferably, the tumor comprises at least one of melanoma and colon cancer; Optionally, the conjugate is obtained by condensation of the carboxyl group of glucuronic acid of chondroitin sulfate with the primary amino group at C6 of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond; Optionally, the modification degree of the conjugate is 2-8; Preferably, the modification degree of the conjugate is 3-6, more preferably 4.

3. A medicament, characterized by comprising: Comprise: a β-cyclodextrin-chondroitin sulfate conjugate.

4. The medicament according to claim 3, characterized in that, The medicament further comprises a STING agonist; Optionally, the STING agonist comprises at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281 and MK-1454; Preferably, the STING agonist is cGAMP; Optionally, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50-2000):(1-20); Preferably, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate to the STING agonist is (50-1000):(1-10); Optionally, the medicament is used for preventing and / or treating a tumor caused by a deficiency in the function of STING protein; Optionally, the tumor comprises at least one of melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer and lymphoma; Preferably, the tumor comprises at least one of melanoma and colon cancer; Optionally, the conjugate is obtained by condensation of the carboxyl group of glucuronic acid of chondroitin sulfate with the primary amino group at C6 of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond; Optionally, the modification degree of the conjugate is 2-8; Preferably, the modification degree of the conjugate is 3-6, more preferably 4.

5. A single dosage form characterized in that, The single dosage form comprises a β-cyclodextrin-chondroitin sulfate conjugate.

6. The single dose of claim 5, wherein, The single dosage form further comprises a STING agonist; Optionally, the STING agonist comprises at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281 and MK-1454; Preferably, the STING agonist is cGAMP; Optionally, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist is (50~2000):(1~20); Preferably, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist is (50~1000):(1~10); Optionally, the single dosage form is used for preventing and / or treating tumors caused by STING protein function defects; Optionally, the tumors comprise at least one of melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer and lymphoma; Preferably, the tumors comprise at least one of melanoma and colon cancer; Optionally, the conjugate is obtained by condensing the carboxyl of glucuronic acid of chondroitin sulfate with the primary amino group at C6 of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond; Optionally, the modification degree of the conjugate is 2~8; Preferably, the modification degree of the conjugate is 3~6, more preferably 4; Optionally, the single dosage form comprises 240 mg ~25000 mg of the β-cyclodextrin-chondroitin sulfate conjugate; Optionally, the single dosage form comprises 1.2 mg~125 mg of the STING agonist.

7. A drug combination, characterized in that, The drug combination comprises a β-cyclodextrin-chondroitin sulfate conjugate.

8. The pharmaceutical combination according to claim 7, characterized in that, The drug combination further comprises a STING agonist; Optionally, the STING agonist comprises at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281 and MK-1454; Preferably, the STING agonist is cGAMP; Preferably, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist is (50~2000):(1~20); Optionally, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist is (50~1000):(1~10); Optionally, the drug combination is used for preventing and / or treating tumors caused by STING protein function defects; Optionally, the tumors comprise at least one of melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer and lymphoma; Preferably, the tumors comprise at least one of melanoma and colon cancer; Optionally, the conjugate is obtained by condensing the carboxyl of glucuronic acid of chondroitin sulfate with the primary amino group at C6 of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond; Optionally, the modification degree of the conjugate is 2~8; Preferably, the modification degree of the conjugate is 3~6, more preferably 4.

9. A kit characterized in that, The drug combination comprises a β-cyclodextrin-chondroitin sulfate conjugate.

10. The kit of claim 9, wherein The drug combination further comprises a STING agonist; Optionally, the STING agonist comprises at least one of cGAMP, ADU-S100, CDGSF, di-ABZi, SNX281 and MK-1454; Preferably, the STING agonist is cGAMP; Optionally, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist is (50-2000):(1-20); Optionally, the mass ratio of the β-cyclodextrin-chondroitin sulfate conjugate and the STING agonist is (50-1000):(1-10); Optionally, the kit prevents and / or treats tumors caused by defects in the function of STING protein; Optionally, the tumor comprises at least one of melanoma, colon cancer, rectal cancer, lung cancer, pancreatic cancer, breast cancer, gastric cancer, glioma, ovarian cancer and lymphoma; Preferably, the tumor comprises at least one of melanoma and colon cancer; Optionally, the conjugate is obtained by condensing the carboxyl of glucuronic acid of chondroitin sulfate with the primary amino group at C6 of 6-deoxy-6-amino-β-cyclodextrin to form an amide bond; Optionally, the modification degree of the conjugate is 2-8; Preferably, the modification degree of the conjugate is 3-6, more preferably 4.

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