A glucan-lysine-azepine conjugate, its preparation method and application as a sting pathway agonist

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a dextran-lysine-azepine conjugate, a preparation method thereof and application as a STING pathway agonist. The present application discloses a preparation method and application of a conjugate for activating a STING signal pathway, which is constructed by using dextran (Dex) as a polysaccharide main chain, lysine as a connecting arm and azepine. The conjugate can significantly improve the STING activation effect and promote the reprogramming of macrophages and dendritic cells through the lysine connecting arm. Compared with the prior art, the present application does not need to use a complex high-molecular polymerization method, and uses a biocompatible and targeted polysaccharide as a high-molecular carrier, thereby greatly enhancing the STING activation efficiency through the lysine connecting arm.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials technology, specifically relating to an immune-activating conjugate based on a polysaccharide backbone, particularly a conjugate in which aziridine heptane is covalently linked to the dextran backbone via a lysine linker, and its application in activating the STING pathway. Background Technology

[0002] Activation of the interferon gene-stimulating factor (STING) signaling pathway can reprogram antigen-presenting cells, reverse the tumor suppressor microenvironment, and improve the efficacy of tumor treatment. Nature Cancer (2022, 3, 1452-1463). Natural or synthetic small molecule STING agonists have been widely reported; however, the development of macromolecular agonists with STING activation activity remains a significant challenge with very few reports.

[0003] Publicly available prior art ( Nat. Nanotechnol. 2017, 12, 648-654; Nat. Biomed. Eng. (2021, 5, 455-466) reveals that a synthetic polymer containing a nitrogen-containing heterocyclic heptane structure, polyethylene glycol-poly(heptane ethyl methacrylate) (PEG-PC7A), can activate the STING signaling pathway. However, the polymer backbone is a non-degradable synthetic polymer (polymethacrylic acid), the synthesis steps are complex, and it needs to be assembled into nanoparticles for drug delivery, posing a biocompatibility risk. Based on the above-disclosed technology, an upgraded technology ( Nat. Commun. A study (2020, 11, 5828) demonstrated that a synthetic polymer with a degradable polycarbonate backbone and a nitrogen-containing heterocyclic heptane structure (PEG-PSC7A) can also activate the STING signaling pathway. Although this upgraded technology solves the problem of the degradability of the synthetic polymer backbone, it still requires stepwise preparation through specific polymerization and photoreaction reactions, followed by assembly into nanoparticles. The preparation process is complex and lacks controllability.

[0004] The technical problem of this invention: Those skilled in the art have traditionally thought of using azircycloheptane monomers to synthesize polymers containing azircycloheptane through polymerization reactions. No one has yet attempted to directly attach azircycloheptane to biocompatible macromolecules to overcome the difficulties of polymerization and biocompatibility issues, such as the natural polysaccharide dextran. However, unexpectedly, the directly prepared "dextran-azircycloheptane" conjugate has almost no STING activation activity (see Experimental Results section). Therefore, how to effectively attach azircycloheptane to the polysaccharide backbone while maintaining STING activation activity remains an unsolved technical problem. Summary of the Invention

[0005] The purpose of this invention is to provide a dextran-lysine-aziridine heptane conjugate, its preparation method, and its application as a STING pathway agonist. The dextran-lysine-aziridine heptane conjugate solves the technical problem of the prior art requiring the preparation of polyaziridine heptane polymers through polymer polymerization.

[0006] Breakthrough of the invention: The inventors surprisingly discovered that when a simple lysine molecule is introduced as a linker between dextran and aziridine heptane, the resulting dextran-lysine-aziridine heptane conjugate regains a strong and specific STING pathway activation ability. This "linker switch effect" is completely unexpected from the prior art. The dextran-lysine-aziridine heptane conjugate is described in detail below.

[0007] The first objective of this invention is to provide a dextran-lysine-aziridine heptane conjugate having a dextran backbone and an aziridine heptane group attached to the dextran backbone via a lysine molecule.

[0008] Furthermore, the molecular weight range of the dextran is 5 kDa to 70 kDa.

[0009] Furthermore, the lysine molecule forms an amide bond with the dextran backbone through its α-amino group, and its α-carboxyl group and / or ε-amino group forms an ester bond or a carbamate bond with the heptyl group of the azaheptanyl group.

[0010] Furthermore, the grafting rate of the conjugate ranges from 10 to 80 lysine-azine-heptanyl groups per 100 dextran repeating units.

[0011] A second objective of this invention is to provide a method for preparing the dextran-lysine-azacycloheptane conjugate, comprising the following steps: Step a: After dissolving the dextran and Boc-protected lysine together, a condensation agent is added to carry out a condensation reaction, introducing the lysine linker; Step b: Obtain the dextran-lysine intermediate through a deprotection reaction; Step c: Add an activating agent to couple the azircycloheptane with the dextran-lysine intermediate described in step b to obtain the conjugate.

[0012] Furthermore, the condensing agent is any one of DCC (dicyclohexylcarbodiimide), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), CDI (N,N'-carbonyldiimidazole), or their hydrochloride salts.

[0013] Furthermore, the activating agent is either CDI (N,N'-carbonyldiimidazole) or 4-nitrophenylchloroformate.

[0014] A third objective of this invention is to provide the use of the aforementioned dextran-lysine-azacycloheptane conjugate in the preparation of a drug for activating the STING pathway.

[0015] Furthermore, the drug is one or more of the following: vaccine adjuvant, antitumor drug, and antiviral drug.

[0016] A fourth objective of this invention is to provide a pharmaceutical composition for activating the STING pathway, comprising the aforementioned dextran-lysine-azacycloheptane conjugate and a pharmaceutically acceptable carrier and / or excipients.

[0017] The present invention discloses a dextran-lysine-aziridine heptane conjugate, which is mainly constructed by chemical coupling between a dextran backbone, lysine linkers, and aziridine. This conjugate, using a biocompatible polysaccharide as a carrier and lysine as a linker, significantly enhances its STING activation activity and biocompatibility.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The dextran-lysine-azacycloheptane conjugate of the present invention uses natural polysaccharides as a carrier, which avoids the traditional strategy of constructing aazacycloheptane polymers by polymer polymerization. The preparation process is simpler, the cost is lower, it can be mass-produced, and it is easy to commercialize. It also improves biocompatibility and biodegradability.

[0019] 2. The dextran-lysine-azacycloheptane conjugate of the present invention greatly enhances the STING activation activity of the conjugate through the lysine linker arm. This linker arm effect is completely unpredictable in the prior art. Attached Figure Description

[0020] Figure 1 The 1H NMR spectrum of dextran-azacycloheptane (low grafting rate) (DC7A-L) from Example 1; Figure 2 The 1H NMR spectrum of dextran-azacycloheptane (high grafting rate) (DC7A-H) in Example 2; Figure 3 The 1H NMR spectrum of dextran-lysine-azacycloheptane (low grafting rate) (DLC7A-L) in Example 3; Figure 4 The 1H NMR spectrum of dextran-lysine-azacycloheptane (high grafting rate) (DLC7A-H) in Example 4; Figure 5 Western blot characterization of STING pathway activation in macrophages from different drug administration groups in Example 5; Figure 6The results of flow cytometry analysis of the expression level of CD86, a macrophage surface marker, in different drug administration groups in Example 6; This means P < 0.05. This means P < 0.01. This means P < 0.001. This represents P < 0.0001; Figure 7 Example 7 shows the expression levels of STING downstream genes CXCL10 and IFN-β in macrophages from different drug administration groups; This means P < 0.05. This means P < 0.01. This means P < 0.001. This means P < 0.0001. Detailed Implementation

[0021] The present invention will be described in detail below with reference to the embodiments and accompanying drawings. All specific embodiments are implemented based on the technical solutions of the present invention, including detailed implementation methods and specific operating procedures, but the scope of protection of the present invention is not limited to the following embodiments.

[0022] Example 1: Preparation of a low-grafting-rate dextran-azacycloheptane conjugate (DC7A-L) This example is a control conjugate without lysine modification, used for subsequent comparative verification of the "connector arm effect".

[0023] 2-(azacycloheptan-1-yl)ethanol (89.1 μL, 600 μmol) and 4-dimethylaminopyridine (DAMP) (73.4 mg, 600 μmol) were dissolved in 10–15 mL of anhydrous dichloromethane, and then 4-nitrophenyl chloroformate (121.2 mg, 600 μmol) was slowly added. The reaction was carried out at room temperature for 8 h. Aminoglucan (100 mg, 10 kDa, low amino number) was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and added to the above reaction mixture. After stirring for about 5 min, TEA (33 μL) was added, and the reaction was continued at room temperature overnight. After the reaction was complete, the product, DC7A-L, was obtained by precipitation with anhydrous ethanol / methyl tert-butyl ether.

[0024] By nuclear magnetic resonance hydrogen spectrum (1 The chemical structure of DC7A-L was determined by ¹H NMR. Figure 1 As shown, the signal peaks at 7.43 ppm–7.22 ppm are characteristic of the carbamate bond; the signal peaks at 5.12 ppm–4.44 ppm are characteristic of Dex; the signal peaks at 4.27 ppm–4.05 ppm are characteristic of the methylene group adjacent to the carbamate bond; the signal peaks at 3.90 ppm–2.97 ppm are characteristic of Dex; and the signal peaks at 1.84 ppm–1.44 ppm are characteristic of the azircycloheptane and methylene groups. These experimental results demonstrate the successful synthesis of DC7A-L.

[0025] Example 2: Preparation of a high-grafting-rate dextran-azacycloheptane conjugate (DC7A-H) Together with Example 1, they form a grafting rate gradient control and serve as a control group for the "lysine-free linker".

[0026] 2-(azacycloheptan-1-yl)ethanol (142.5 μL, 960 μmol) and 4-dimethylaminopyridine (DAMP) (117.3 mg, 960 μmol) were dissolved in 10–15 mL of anhydrous dichloromethane, and then 4-nitrophenyl chloroformate (193.9 mg, 960 μmol) was slowly added. The reaction was carried out at room temperature for 8 h. Aminoglucan (100 mg, 10 kDa, high amino number) was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO). After complete dissolution, it was added to the above reaction mixture. After stirring for about 5 min, TEA (33 μL) was added, and the reaction was continued at room temperature overnight. After the reaction was completed, the product, DC7A-H, was obtained by precipitation with anhydrous ethanol / methyl tert-butyl ether.

[0027] By nuclear magnetic resonance hydrogen spectrum ( 1 (H NMR) was used to determine the chemical structure of DC7A-H. For example... Figure 2 As shown, the signal peaks at 7.42 ppm–7.22 ppm are characteristic of the carbamate bond; the signal peaks at 5.14 ppm–4.44 ppm are characteristic of Dex; the signal peaks at 4.30 ppm–4.06 ppm are characteristic of the methylene group adjacent to the carbamate bond; the signal peaks at 3.90 ppm–2.94 ppm are characteristic of Dex; and the signal peaks at 1.85 ppm–1.43 ppm are characteristic of the azircycloheptane and methylene groups. These experimental results demonstrate the successful synthesis of DC7A-H.

[0028] Example 3: Preparation of a low-grafting-rate dextran-lysine-azacycloheptane conjugate (DLC7A-L) This embodiment introduces a lysine linker arm and uses a low grafting rate to form a parallel control with or without a linker arm compared to Example 1.

[0029] 2,6-Di-tert-butoxycarbonylaminohexanoic acid (138.6 mg, 400 μmol) and N-hydroxysuccinimide (NHS) (46.1 mg, 400 μmol) were dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO), and then dicyclohexylcarbodiimide (DCC) (96.1 mg, 500 μmol) was added. The reaction was carried out at room temperature for 1.5 h. Aminoglucan (100 mg, 10 kDa, 11.5% low amino group) was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and added to the above reaction mixture. After stirring for about 5 min, TEA (33 μL) was added, and the reaction was continued at room temperature overnight. Preliminary purification was performed using a dialysis bag (1.0 kDa) and dialyzed for 14 h. The dialyzed liquid was added to a 100 mL round-bottom flask, and 1 / 4 to 1 / 5 of the volume of the dialyzed liquid was added to trifluoroacetic acid. The reaction was carried out at room temperature with stirring for 48 h. After the reaction was complete, the solution was adjusted to neutral with 1 M sodium hydroxide aqueous solution, and then purified using a dialysis bag (1.0 kDa) for 8 h. The product was then freeze-dried to obtain the dextran-lysine product. 2-(azacycloheptan-1-yl)ethanol (89.1 μL, 600 μmol) and triethylamine (TEA) (60.7 mg, 600 μmol) were dissolved in 10–15 mL of anhydrous dichloromethane, and 4-nitrophenyl chloroformate (121.2 mg, 600 μmol) was slowly added. The reaction was carried out at room temperature for 8 h. The dextran-lysine product was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and added to the above reaction mixture. After stirring for approximately 5 min, TEA (33 μL) was added, and the reaction was continued at room temperature overnight. After the reaction was complete, the product was obtained by precipitation with anhydrous ethanol / methyl tert-butyl ether, DLC7A-L, with a grafting rate of 11.5%.

[0030] By nuclear magnetic resonance hydrogen spectrum ( 1 (H NMR) was used to determine the chemical structure of DC7A-H. For example... Figure 3As shown, the signal peaks at 8.35 ppm–7.01 ppm are characteristic of carbamate and amide bonds; the signal peaks at 5.10 ppm–4.45 ppm are characteristic of Dex; the signal peaks at 4.22 ppm–3.95 ppm are characteristic of the methylene group adjacent to carbamate; the signal peaks at 3.90 ppm–2.93 ppm are characteristic of Dex; and the signal peaks at 1.82 ppm–1.14 ppm are characteristic of azircycloheptane and methylene groups. These experimental results demonstrate the successful synthesis of DLC7A-L.

[0031] Example 4: Preparation of a high-grafting-rate dextran-lysine-azacycloheptane conjugate (DLC7A-H) This embodiment introduces a lysine linker and uses a high grafting rate to form a parallel control with and without a linker in Example 2, and to form a grafting rate gradient with Example 3 to examine the effect of grafting rate on activity.

[0032] 2,6-Di-tert-butoxycarbonylaminohexanoic acid (226.2 mg, 640 μmol) and N-hydroxysuccinimide (NHS) (77.5 mg, 640 μmol) were dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (161.7 mg, 800 μmol) was added. The reaction was allowed to proceed at room temperature for 1.5 h. Aminoglucan (100 mg, 10 kDa, with a high amino group grafting rate of 18.1%) was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and added to the above reaction mixture. After stirring for approximately 5 min, TEA (44 μL) was added, and the reaction was allowed to continue overnight at room temperature. Preliminary purification was performed using a dialysis bag (1.0 kDa), and dialyzing was performed for 18 h. The dialyzed liquid was added to a 100 mL round-bottom flask, and 1 / 4 to 1 / 5 of the volume of the dialyzed liquid in trifluoroacetic acid was added. The mixture was stirred at room temperature for 48 h. After the reaction was completed, the solution was adjusted to neutral with 1 M sodium hydroxide aqueous solution, and then purified using a dialysis bag (1.0 kDa) for 8 h. The product was then freeze-dried to obtain the dextran-lysine product. 2-(azacycloheptan-1-yl)ethanol (142.6 μL, 960 μmol) and 4-dimethylaminopyridine (DAMP) (117.5 mg, 960 μmol) were dissolved in 10–15 mL of anhydrous dichloromethane, and then 4-nitrophenyl chloroformate (194.0 mg, 960 μmol) was slowly added. The mixture was reacted at room temperature for 8 h. The dextran-lysine product was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and added to the above reaction mixture. After stirring for about 5 min, TEA (44 μL) was added, and the reaction was continued overnight at room temperature. After the reaction was completed, the product DLC7A-H was obtained by precipitation with anhydrous ethanol / methyl tert-butyl ether, with a grafting rate of 18.1%.

[0033] By nuclear magnetic resonance hydrogen spectrum ( 1 (H NMR) was used to determine the chemical structure of DC7A-H. For example... Figure 4 As shown, the signal peaks at 8.39 ppm–7.07 ppm are characteristic of carbamate and amide bonds; the signal peaks at 5.11 ppm–4.45 ppm are characteristic of Dex; the signal peaks at 4.38 ppm–4.00 ppm are characteristic of the methylene group adjacent to carbamate; the signal peaks at 3.97 ppm–2.86 ppm are characteristic of Dex; and the signal peaks at 1.95 ppm–1.12 ppm are characteristic of azircycloheptane and methylene groups. These experimental results demonstrate the successful synthesis of DLC7A-H.

[0034] Bioactivity verification: Example 5: Western Blot detection of phosphorylation activation levels of the STING pathway in macrophages by different conjugates. The core indicator is the expression level of p-STING, which directly verifies whether the STING pathway is activated and is key evidence of the "connector switch effect".

[0035] M2 macrophages were used at a rate of 5 × 10 5 Cells were seeded in 6-well plates and cultured at 37 °C in 5% CO2 for 24 h. Cell supernatant was aspirated and drug-containing medium (DMEM) with 10% FBS (drug concentration 100 μg / mL) was added, followed by incubation for another 48 h. After cell lysis, cells were centrifuged (12,000 × g, 15 min, 4 °C), and the supernatant was collected. An equal volume of protein was transferred to a PVDF membrane (220 mA, 2 h), then incubated overnight with primary antibody at 4 °C, and co-incubated with HRP-labeled secondary antibody for 2 h. Signal was detected and imaged using enhanced chemiluminescence immunoassay (ChemiDoc MP system, Bio-Rad). The list of antibodies used is as follows: GAPDH (Proteintech, 81640–5-RR, 1:10000), STING (Cell Signaling, #50494S, 1:1000), p-STING (Cell Signaling, #72971S, 1:800).

[0036] like Figure 5 As shown, conjugates with lysine linkers (DLC7A-L and DLC7A-H) exhibited significantly enhanced STING activation compared to conjugates without lysine linkers (DC7A-L and DC7A-H), with significantly increased p-STING expression. This indicates that lysine linkers can significantly enhance the STING pathway activation of polysaccharide conjugates.

[0037] Example 6: Flow cytometry detection of the effects of different conjugates on the expression of CD86, a macrophage M1 polarization marker. M2 macrophages were used at a rate of 1×10 5Cells were seeded into 24-well plates and cultured at 37 °C in 5% CO2 for 24 h. Cell supernatant was aspirated and added to DMEM medium containing 10% FBS at a drug concentration of 100 μg / mL, and incubated for another 24 h. Cells were then digested from the 24-well plates with EDTA-free trypsin, collected into flow cytometry tubes, washed, and stained with PE-labeled anti-mouse CD86 antibody (BD Biosciences, catalog number #553692) at 4 °C for 30 min. After staining, cells were washed twice with PBS containing 0.1% BSA, centrifuged (300 g, 5 min, 4 °C), and resuspended in 400 μL PBS for flow cytometry analysis.

[0038] like Figure 6 As shown, conjugates with lysine linkers (DLC7A-L and DLC7A-H) exhibited significantly enhanced CD86 expression levels compared to conjugates without lysine linkers (DC7A-L and DC7A-H) (P<0.0001). This indicates that lysine linkers can significantly enhance the effect of polysaccharide conjugates in inducing pro-inflammatory (M1) polarization in M2 macrophages.

[0039] Example 7: Real-time quantitative PCR detection of the expression levels of macrophage STING downstream pro-inflammatory gene CXCL10 and IFN-β by different conjugates. M2 macrophages were used at a rate of 5 × 10 5 Cells were seeded in 6-well plates and cultured at 37 °C in 5% CO2 for 24 h. The cell supernatant was aspirated, and DMEM containing 10% FBS (drug concentration 100 μg / mL) was added, followed by incubation for another 48 h. Total RNA was extracted and reverse transcribed into cDNA using the BeyoRT II cDNA synthesis kit. 1 μg of cDNA was then used as a template for real-time PCR amplification using gene-specific primers. The amplification reaction was performed on a QuantStudio™ 6 Flex real-time PCR system (Thermo Fisher Scientific, USA) under the following conditions: 50 °C pre-denaturation for 2 min, 95 °C denaturation for 2 min, followed by 40 cycles (95 °C for 15 s, 60 °C for 30 s). Relative mRNA expression levels were calculated using the comparison ΔΔCt method (RQ = 2^(-ΔΔCt)) and normalized to the corresponding reference gene.

[0040] like Figure 7As shown, the polysaccharide conjugates with lysine linkers (DLC7A-L and DLC7A-H) exhibited significantly enhanced CXCL10 and IFN-β mRNA expression levels compared to those without lysine linkers (DC7A-L and DC7A-H). This further demonstrates that lysine linkers can significantly enhance STING pathway activation and induce enhanced expression of its downstream genes.

[0041] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A dextran-lysine-azacycloheptane conjugate, characterized in that, The conjugate has a dextran backbone and a nitrogen-containing heptyl group attached to the dextran backbone via a lysine molecule.

2. The dextran-lysine-azacycloheptane conjugate as described in claim 1, characterized in that, The molecular weight range of dextran is 5 to 70 kDa.

3. The dextran-lysine-azacycloheptane conjugate as described in claim 1, characterized in that, The lysine molecule forms an amide bond with the dextran backbone through its α-carboxyl group, and its α-amino and / or ε-amino groups form ester bonds or carbamate bonds with the heptyl group of the azaheptanyl group.

4. The dextran-lysine-azacycloheptane conjugate as described in claim 1, characterized in that, The grafting rate of the conjugate is in the range of 10 to 80 lysine-nitroheptanyl groups per 100 dextran repeating units.

5. The method for preparing a dextran-lysine-azacycloheptane conjugate according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step a: After dissolving the dextran and Boc-protected lysine together, a condensation agent is added to carry out a condensation reaction, introducing the lysine linker; Step b: Obtain the dextran-lysine intermediate through a deprotection reaction; Step c: Add an activating agent to couple the azircycloheptane with the dextran-lysine intermediate described in step b to obtain the conjugate.

6. The method for preparing a dextran-lysine-azacycloheptane conjugate as described in claim 5, characterized in that, The condensing agent is any one of DCC (dicyclohexylcarbodiimide), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), CDI (N,N'-carbonyldiimidazole), or their hydrochloride salts.

7. The method for preparing a dextran-lysine-azacycloheptane conjugate as described in claim 5, characterized in that, The activating agent is either CDI (N,N'-carbonyldiimidazole) or 4-nitrophenylchloroformate.

8. The use of the dextran-lysine-azacycloheptane conjugate as described in any one of claims 1 to 4 in the preparation of a drug for activating the STING pathway.

9. The application as described in claim 8, characterized in that, The drug is one or more of the following: vaccine adjuvant, antitumor drug, and antiviral drug.

10. A pharmaceutical composition for activating the STING pathway, characterized in that, It comprises the dextran-lysine-azacycloheptane conjugate according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier and / or excipient.

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