A novel immunostimulant, its method of preparation and use

Abstract

The application relates to the technical field of medicines, and particularly discloses a novel immune stimulant, a preparation method and application thereof. The immune stimulant comprises an alpha-mannose modified imidazoquinoline derivative, an N-acetyl cysteine modified cytosine-guanine dinucleotide, a glycyrrhetinic acid-polyethylene glycol derivative, a phytic acid-glycine oligomer, a medicinal excipient and water for injection, three modified active ingredients are bridged by the phytic acid-glycine oligomer as a bridging unit, and a ternary coordination complex is formed through dynamic non-covalent coordination. The preparation method comprises the following steps: after raw materials are pretreated, the active ingredients are premixed, dissolved and solvent is recovered, then dynamic coordination reaction, ultrafiltration purification and concentration are carried out, and finally, a finished product is obtained through double-cavity preparation forming. The immune stimulant can be used for preparing a tumor immunotherapy, an anti-infection treatment, an immune deficiency disease auxiliary treatment drug or a vaccine adjuvant, and has the advantages of high active ingredient stability, good in-vivo delivery efficiency and outstanding synergistic effect.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and more specifically, to a novel immune agonist, its preparation method, and its use. Background Technology

[0002] Immunomodulatory agents often use imidazoquinolines, cytosine-guanine dinucleotides, and other substances as core active ingredients, and have important applications in immunomodulation, anti-infection, and tumor immunotherapy. To enhance their efficacy, current technologies often target or stabilize the active ingredients, and achieve multi-pathway immune activation through compound formulations. A rational combination of components and molecular assembly structure is crucial for maintaining the stability of the active ingredients, improving in vivo delivery efficiency, and achieving targeted distribution.

[0003] Currently, most immune agonists are simple physical mixtures with a lack of stable interactions between their active components. This leads to problems such as component degradation during storage, in vivo dispersion, and insufficient targeting, making it difficult for the modified components to exert synergistic effects. This application aims to address the technical problems of insufficient structural stability of the active components in existing immune agonists and poor in vivo delivery and targeted distribution. By constructing a dynamic non-covalently coordinated ternary complex, it provides more reliable technical support for the formulation stability and efficacy of immune agonists. Summary of the Invention

[0004] To address the issues of insufficient structural stability and poor in vivo delivery and targeted distribution of active ingredients in existing immune agonists, this application provides a novel immune agonist, its preparation method, and its uses.

[0005] In a first aspect, this application provides a novel immune agonist, employing the following technical solution: A novel immune agonist is prepared from the following components in parts by weight: 5 to 12 parts of α-mannose-modified imidazoquinoline derivative, 8 to 15 parts of N-acetylcysteine-modified cytosine-guanine dinucleotide, 3 to 8 parts of glycyrrhetinic acid-polyethylene glycol derivative, 0.5 to 2 parts of phytic acid-glycine oligomer, 2 to 6 parts of pharmaceutical excipients, and 75 to 85 parts of water for injection; the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative form a ternary coordination complex through dynamic non-covalent coordination, with the phytic acid-glycine oligomer as the bridging unit.

[0006] By employing the above technical solution, three functionalized active ingredients in specific proportions are selected, with phytic acid-glycine oligomers serving as bridging units. The multiple coordination sites in its molecular structure allow it to form dynamic non-covalent coordination interactions with the other three active ingredients, stably bridging them to form a uniform ternary coordination complex. Specifically, α-mannose modification can specifically bind to mannose receptors on the surface of immune cells, enhancing the active ingredient's targeted recognition ability on immune cells; N-acetylcysteine ​​modification can protect the nucleotide structure of cytosine-guanine dinucleotide, improving its resistance to degradation and reducing enzymatic losses in vivo; and glycyrrhetinic acid-polyethylene glycol derivatives can prolong the circulation time of the active ingredient in vivo, improving its in vivo distribution characteristics. These three components form a synergistic system through the bridging unit, effectively improving the overall stability and in vivo delivery efficiency of the active ingredient, providing a reliable structural basis for immune activation and targeted distribution.

[0007] Preferably, the pharmaceutical excipient is composed of polyethylene glycol 400 and mannitol in a mass ratio of 1:3 to 3:1.

[0008] By employing the above technical solutions, polyethylene glycol 400 exhibits excellent water solubility and dispersibility, forming good compatibility with ternary coordination complexes, improving the solubility of active ingredients, reducing component aggregation, and ensuring formulation homogeneity. Mannitol possesses excellent lyophilization protection properties, forming a stable framework structure during freeze-drying, maintaining the morphological integrity of the formulation, and preventing structural damage to active ingredients during freeze-drying. The specific ratio of these two compounds not only meets the dispensing and molding requirements of dual-cavity formulations but also synergistically enhances the storage stability of the formulation, reducing structural damage and activity loss of active ingredients during preparation and storage.

[0009] Secondly, this application provides a method for preparing a novel immune agonist, employing the following technical solution: A method for preparing a novel immune agonist includes the following steps: S1. Raw material pretreatment: Weigh α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, glycyrrhetinic acid-polyethylene glycol derivative, phytic acid-glycine oligomer, polyethylene glycol 400, and mannitol. Vacuum dry, pulverize, and sieve each raw material separately, then seal and set aside for later use. Take anhydrous ethanol for the reaction, dehydrate it, and set aside for later use. S2. Premixed dissolution of active ingredients: Add the α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, and glycyrrhetinic acid-polyethylene glycol derivative that have been pretreated in S1 to the reaction vessel, add anhydrous ethanol, and stir under controlled temperature until the raw materials are completely dissolved to obtain a homogeneous premixed solution of active ingredients. The entire process is carried out in a closed manner to reduce solvent evaporation. S3, closed-loop solvent recovery: The premixed active ingredient solution obtained in S2 is heated and distilled at atmospheric pressure to recover anhydrous ethanol. The recovered anhydrous ethanol is dehydrated and recycled for the next batch of synthesis. After distillation, a solvent-free mixed solid of active ingredients is obtained. S4. Dynamic coordination reaction: Add water for injection to the mixed solid of active ingredients obtained in S3, stir until completely dissolved, adjust the pH of the system, cool down and control the stirring speed, add the phytic acid-glycine oligomer pretreated in S1, keep warm and stir to complete the construction of the ternary coordination complex, and obtain the coordination complex solution. S5. Ultrafiltration purification and concentration: Adjust the pH value of the coordination complex solution obtained in S4, and use an ultrafiltration membrane for circulation filtration to obtain a purified solution; transfer the purified solution into a vacuum concentration vessel, and concentrate it to the target volume under controlled temperature and pressure to obtain a concentrated solution; S6. Dual-chamber formulation forming: Add the pharmaceutical excipients pretreated in S1 to the concentrate obtained in S5, add water for injection, stir until completely dissolved, and then filter sterile to obtain a sterile drug solution; complete the dispensing, freeze-drying and crimping sealing of the dual-chamber sterile container under sterile conditions to obtain the novel immune agonist finished product.

[0010] By adopting the above technical solutions, the raw material pretreatment stage involves vacuum drying, pulverizing, and sieving of each raw material, which effectively removes moisture and trace impurities, preventing moisture from causing hydrolysis of active ingredients and interfering with the normal progress of subsequent coordination reactions. Anhydrous ethanol is used as the solvent for the premixing and dissolution of active ingredients; its excellent solubility ensures the full dissolution and uniform mixing of the three core active ingredients, laying the foundation for the uniform occurrence of subsequent coordination reactions. The closed-loop solvent recovery process efficiently recovers anhydrous ethanol, which is then recycled after dehydration, reducing solvent waste and the risk of solvent residue in the formulation. The dynamic coordination reaction, through precise control of the system's pH, temperature, and stirring parameters, allows the bridging unit to fully engage in non-covalent coordination with the three active ingredients, forming a structurally stable and homogeneous ternary coordination complex. Ultrafiltration purification and concentration precisely removes small molecule impurities and unreacted trace raw materials from the system, improving the purity of the formulation. The dual-chamber formulation process strictly controls the aseptic environment and freeze-drying conditions, ensuring the sterility and morphological stability of the formulation. The entire process is tightly integrated and the parameters are controllable, enabling the stable preparation of structurally homogeneous and reliable immune agonists.

[0011] Preferably, in step S1, the pretreatment involves vacuum drying each raw material until the moisture content does not exceed 0.5%, pulverizing it, and then passing it through a 100-120 mesh sieve; the dehydration treatment of anhydrous ethanol involves dehydrating the anhydrous ethanol until the moisture content does not exceed 0.3%.

[0012] By employing the above technical solutions, vacuum drying of raw materials to a specific moisture content effectively avoids the hydrolytic degradation of active ingredients caused by moisture, while also preventing moisture from interfering with the formation of coordination bonds, thus ensuring the stability of the coordination reaction. Pulverization and sieving ensure uniform particle size, increase the contact area of ​​the raw materials, improve subsequent dissolution efficiency and mixing uniformity, and ensure the full reaction of each component. Dehydration treatment of anhydrous ethanol completely removes moisture from the solvent, preventing moisture from being introduced into the reaction system, avoiding interference with the dissolution and coordination of active ingredients, ensuring the stability of subsequent process steps, and ultimately ensuring the uniform and controllable quality of the final product.

[0013] Preferably, in step S2, the amount of anhydrous ethanol added is 3 to 5 times the total mass of the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative; the stirring speed is controlled at 200 r / min to 300 r / min; the stirring temperature is 45°C to 55°C; and the stirring time is 2 h to 3 h.

[0014] By adopting the above technical solution, a specific amount of anhydrous ethanol can be used to accommodate the solubility differences of the three active ingredients, ensuring that each active ingredient is fully dissolved and avoiding incomplete dissolution and uneven mixing due to insufficient solvent, which would affect the subsequent coordination reaction effect. Controlling the stirring speed, temperature and time can make the active ingredients uniformly dispersed in the solvent, reducing the aggregation phenomenon caused by excessively high local concentrations. At the same time, it avoids the destruction of the modified structure of the active ingredients by excessively high temperature and the instability of the system caused by excessively fast stirring speed, ensuring the structural integrity of the active ingredients and providing a guarantee for the stable formation of subsequent coordination complexes.

[0015] Preferably, in step S3, the temperature of atmospheric distillation is 78°C to 80°C, the recovery rate of anhydrous ethanol is not less than 95%, and the water content of the recovered anhydrous ethanol after dehydration does not exceed 0.3%.

[0016] By adopting the above technical solution, the atmospheric distillation temperature is controlled within the boiling point range of anhydrous ethanol, which enables efficient solvent recovery while avoiding thermal degradation of active ingredients due to excessively high temperatures, thus ensuring the structure and activity of the active ingredients. A recovery rate of no less than 95% can significantly improve solvent utilization and reduce production costs. The recovered anhydrous ethanol is dehydrated to the specified moisture content to ensure its purity meets the requirements for use in the next batch of reaction, avoiding interference from moisture in the recovered solvent, and achieving closed-loop sustainability of the process and stability of product quality.

[0017] Preferably, in step S4, the pH value of the system is adjusted to 6.8 to 7.2, the system temperature is reduced to 35°C to 40°C, the stirring speed is controlled to 150 r / min to 200 r / min, and the holding and stirring time for the coordination reaction is 30 min to 45 min.

[0018] By adopting the above technical solution, the pH value and temperature of the system are adjusted to a suitable range, which can match the coordination activity of phytic acid-glycine oligomers and the structural stability of the three active ingredients, providing a suitable environment for the full occurrence of dynamic non-covalent coordination and promoting the stable formation of ternary coordination complexes. Controlling the stirring speed and holding time can ensure that the bridging unit and the active ingredients are in full contact and react uniformly, avoiding structural defects caused by insufficient reaction, improving the uniformity and stability of the coordination complex, and ensuring that it is not easily dissociated in subsequent processes.

[0019] Preferably, in step S5, the pH of the coordination complex solution is adjusted to 6.5 to 7.5, the molecular weight cutoff of the ultrafiltration membrane is 1000 Da to 5000 Da, the concentration temperature is 40°C to 50°C, the vacuum degree is -0.09 MPa to -0.08 MPa, and the target volume is 1 / 5 to 1 / 4 of the original volume.

[0020] By adopting the above technical solutions, adjusting the pH value of the coordination complex solution can maintain the structural stability of the complex and prevent it from dissociating due to environmental changes during purification and concentration, thus ensuring the integrity of the active ingredient. Ultrafiltration membranes with specific molecular weight cutoffs can accurately intercept small molecule impurities and unreacted monomers in the system, while completely preserving the ternary coordination complex, effectively improving the purity of the formulation. Controlling the concentration temperature and vacuum can achieve gentle concentration, avoiding the inactivation of active ingredients due to high temperature or extreme pressure. Concentrating to a specific volume can precisely increase the concentration of active ingredients, meeting the needs of dual-chamber formulation formation and subsequent drug efficacy.

[0021] Preferably, in step S6, the sterile filtration uses a sterile filter membrane with a pore size of 0.22 μm, the freeze-drying temperature is -40℃ to -30℃, the vacuum degree is -0.1 MPa, and the drying time is 8h to 10h.

[0022] By adopting the above technical solution, the sterile filter membrane with a pore size of 0.22μm can effectively retain bacteria, fungi and other microorganisms in the drug solution, which meets the production specifications of sterile preparations and ensures the safety of the preparation. The specific freeze-drying temperature, vacuum degree and time can make the water in the drug solution sublimate rapidly, avoid the formation of ice crystals that will damage the structure of the ternary coordination complex, and at the same time maintain the loose and porous morphology of the preparation, ensuring good reconstitution performance of the preparation, and further improving the storage stability and ease of use of the preparation.

[0023] Thirdly, this application provides a use of a novel immune agonist, employing the following technical solution: The use of a novel immune agonist in the preparation of tumor immunotherapy drugs, anti-infective drugs, adjuvant drugs for immunodeficiency diseases, or vaccine adjuvants.

[0024] By employing the above-mentioned technical solution, the ternary coordination complex in this immune agonist can achieve stable delivery of three functionalized modified active ingredients. Specifically, the α-mannose-modified component targets and recruits immune cells, the N-acetylcysteine-modified cytosine-guanine dinucleotide activates the innate immune pathway, and the glycyrrhetinic acid-polyethylene glycol derivative prolongs its circulation time in vivo. These three components synergistically exert an immune-activating effect, activating multiple pathways of the body's immune system and enhancing the activity and immune response of immune cells. Based on this, it can be used to prepare tumor immunotherapy drugs to assist the body in clearing tumor cells, to prepare anti-infective drugs to enhance the body's resistance to pathogens, to prepare adjuvant drugs for immunodeficiency diseases to improve the body's immune function, and can also be used as a vaccine adjuvant to enhance the immune efficacy of vaccines, making it suitable for various immune-related clinical applications.

[0025] In summary, this application has the following beneficial effects: 1. This application uses functionalized active ingredients with specific ratios and constructs a ternary coordination complex with phytic acid-glycine oligomers as bridging units. Each component forms a stable structure through dynamic non-covalent coordination, which effectively improves the overall stability and in vivo delivery efficiency of the active ingredients, giving the formulation a better basis for targeted distribution and immune activation. At the same time, each modified component can work synergistically to provide stable support for subsequent drug efficacy.

[0026] 2. In this application, a specific ratio of polyethylene glycol 400 and mannitol is preferred as pharmaceutical excipients. This compound system can be adapted to the freeze-drying process and the requirements of dual-cavity formulation molding. While ensuring the formability of the formulation, it can improve the storage stability of the product, avoid the aggregation or degradation of active ingredients during preparation and storage, and ensure the uniformity of formulation quality.

[0027] 3. The method of this application achieves efficient recovery and recycling of anhydrous ethanol through a closed-loop solvent recovery process, which reduces production costs while reducing solvent residue and environmental pollution. The distillation process is mild and controllable, which can preserve the structural integrity of active ingredients to the greatest extent and improve the utilization rate of raw materials and the green economy of the preparation process.

[0028] 4. The method of this application, through a combination of stepwise dissolution and coordination with ultrafiltration purification and concentration, can precisely control the coordination reaction conditions and effectively remove small molecule impurities, thereby improving the purity and uniformity of the ternary coordination complex. At the same time, the mild concentration and lyophilization conditions can avoid the inactivation of active ingredients and ensure the stability of the formulation quality.

[0029] 5. The immune agonist prepared in this application has a wide range of applications and can be used for tumor immunotherapy, anti-infective therapy, adjuvant therapy for immunodeficiency diseases, or as a vaccine adjuvant. Its stable coordination structure and functional modification enable it to adapt to the in vivo environment under different application scenarios and have good potential for clinical application expansion. Attached Figure Description

[0030] Figure 1 This is a flowchart of a novel immune agonist preparation method provided in this application; Figure 2 This is a table of stability test data for ternary coordination complexes provided in this application; Figure 3 This is the in vitro immune activation activity test data sheet provided in this application; Figure 4 This is a data table of liver-targeted enrichment capacity test provided in this application. Detailed Implementation

[0031] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0032] Technical concept: Most current immune agonist products are simple compound systems with a lack of stable structural relationships between active ingredients. They are prone to degradation and uneven dispersion in storage and in vivo environment. At the same time, single modification or simple mixing is difficult to achieve target recognition, structural stability and in vivo delivery, resulting in limited immune activation effect and insufficient effective concentration at the target site.

[0033] This technical solution introduces phytic acid-glycine oligomers as bridging units to construct a ternary coordination complex by dynamically non-covalently co-coordinating three functionalized active ingredients. Combined with a precise and controllable stepwise preparation process and a suitable excipient system, it improves the overall structural stability and in vivo delivery efficiency. At the same time, it improves the targeted distribution and immune activation effect by relying on the synergistic effect of each component, thus solving the defects of existing technologies from the perspective of molecular assembly and formulation process.

[0034] Preparation Example 1: The preparation method of α-mannose-modified imidazoquinoline derivative is as follows: 10 mmol of commercially available imidazoquinoline and 12 mmol of fully acetylated α-mannose were placed in a dry three-necked reaction flask, and 50 mL of anhydrous dichloromethane was added to dissolve them completely. High-purity nitrogen was continuously introduced to isolate air and water vapor. After cooling to 0°C in an ice bath, 15 mmol of boron trifluoride diethyl ether complex was slowly added dropwise. After the addition was completed, the temperature was naturally raised to 25°C and stirred for 6 h. After the reaction was completed, pre-cooled saturated sodium bicarbonate solution was added to quench the reaction. After standing and separation, the organic phase was collected, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The concentrated product was purified by silica gel column chromatography with a 200-300 mesh filter. The eluent was a 10:1 volume ratio of dichloromethane to methanol mixture. The target fraction was collected by thin-layer chromatography and concentrated under reduced pressure. The product was dried under vacuum at 25°C for 12 h to obtain α-mannose-modified imidazoquinoline derivative. The purity was ≥98.5% as determined by high-performance liquid chromatography.

[0035] Preparation Example 2: The preparation method of N-acetylcysteine-modified cytosine-guanine dinucleotide is as follows: 8 mmol of commercially available 5'-amino-modified cytosine-guanine dinucleotide monomer and 10 mmol of N-acetylcysteine ​​were placed in a light-protected reaction bottle, and 40 mL of sterile phosphate buffer (pH 7.2) was added to dissolve them completely. 12 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 6 mmol of N-hydroxysuccinimide were added sequentially. The mixture was stirred at room temperature in the dark for 4 h. After the reaction, the reaction solution was transferred to a regenerated cellulose dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in ultrapure water at 4 °C for 24 h. Fresh dialysate was replaced every 6 h. After dialysis, the dialysate was collected, pre-frozen at -40 °C, and then freeze-dried under vacuum for 24 h to obtain N-acetylcysteine-modified cytosine-guanine dinucleotide. The purity was ≥99.0% as determined by high performance liquid chromatography.

[0036] Preparation Example 3: The preparation method of glycyrrhetinic acid-polyethylene glycol derivative is as follows: 10 mmol of commercially available glycyrrhetinic acid and 400-12 mmol of monomethoxy polyethylene glycol were placed in a dry three-necked reaction flask, and 60 mL of anhydrous toluene was added to dissolve them completely. 0.5 mmol of p-toluenesulfonic acid monohydrate was added as a catalyst, and the mixture was heated to 110 °C and refluxed for 8 h to separate water. The water generated in the reaction was separated in real time using a water separator. After the reaction was completed, the toluene solvent was removed by vacuum distillation. The remaining product was redissolved in 50 mL of dichloromethane and washed three times each with saturated sodium bicarbonate solution and saturated sodium chloride solution. The organic phase was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrated product was purified by neutral alumina column chromatography using a 15:1 volume ratio of dichloromethane to methanol mixture as the eluent. The target fraction was collected, concentrated, and dried to obtain the glycyrrhetinic acid-polyethylene glycol derivative. The purity was ≥98.0% as determined by high performance liquid chromatography.

[0037] Preparation Example 4: The preparation method of phytic acid-glycine oligomer is as follows: 5 mmol of commercially available sodium phytate and 8 mmol of glycine oligopeptide with a degree of polymerization of 4-6 were placed in a reaction flask and dissolved completely in 50 mL of ultrapure water. The pH of the solution was adjusted to 5.5 with 0.1 mol / L hydrochloric acid. 10 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 5 mmol of N-hydroxythiosuccinimide were added sequentially. The mixture was stirred at room temperature for 5 h. After the reaction was completed, the reaction solution was transferred to an ultrafiltration cup with a molecular weight cutoff of 1000 Da. The mixture was washed 6 times with ultrapure water at 4 °C to completely remove unreacted small molecule raw materials. The final retentate was collected and freeze-dried under vacuum for 12 h to obtain phytic acid-glycine oligomer. The molecular weight distribution was found to be uniform by gel permeation chromatography.

[0038] Example 1: This example provides a novel immune agonist comprising the following parts by weight of raw materials: 5.5 parts of α-mannose-modified imidazoquinoline derivative, 8 parts of N-acetylcysteine-modified cytosine-guanine dinucleotide, 3 parts of glycyrrhetinic acid-polyethylene glycol derivative, 1.25 parts of phytic acid-glycine oligomer, 2.25 parts of pharmaceutical excipients, and 80 parts of water for injection, for a total of 100 parts by weight.

[0039] The pharmaceutical excipients consist of 1.125 parts of polyethylene glycol 400 and 1.125 parts of mannitol, with a mass ratio of 1:1. The α-mannose-modified imidazoquinoline derivative was prepared according to Preparation Example 1 above, the N-acetylcysteine-modified cytosine-guanine dinucleotide was prepared according to Preparation Example 2 above, the glycyrrhetinic acid-polyethylene glycol derivative was prepared according to Preparation Example 3 above, and the phytic acid-glycine oligomer was prepared according to Preparation Example 4 above. The purity of all intermediates was not less than 98% as determined by high performance liquid chromatography. The α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative formed a ternary coordination complex through dynamic non-covalent coordination, with the phytic acid-glycine oligomer as the bridging unit.

[0040] The preparation method of the above-mentioned novel immune agonist includes the following steps: S1. Raw material pretreatment: Weigh α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, glycyrrhetinic acid-polyethylene glycol derivative, phytic acid-glycine oligomer, polyethylene glycol 400, and mannitol. Vacuum dry, pulverize, and sieve each raw material separately, then seal and set aside for later use. Take anhydrous ethanol for reaction, dehydrate it, and set aside for later use.

[0041] The pretreatment process involves vacuum drying each raw material until the moisture content does not exceed 0.5%, followed by pulverizing and passing it through a 100-120 mesh sieve. The dehydration treatment of anhydrous ethanol involves dehydrating the anhydrous ethanol until the moisture content does not exceed 0.3%. All raw materials are pharmaceutical grade or analytical grade, and the entire operation is completed in a Class 100,000 clean drying room to avoid moisture absorption or the introduction of impurities.

[0042] S2. Premixed Dissolution of Active Ingredients: Add the α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, and glycyrrhetinic acid-polyethylene glycol derivative pretreated in S1 to the reaction vessel, add anhydrous ethanol, and stir under controlled temperature until the raw materials are completely dissolved to obtain a homogeneous premixed solution of active ingredients. The entire process is carried out in a closed system to reduce solvent evaporation.

[0043] The amount of anhydrous ethanol added was four times the total mass of the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative. The stirring speed was controlled at 250 r / min. The stirring temperature was 50℃ and the stirring time was 2.5 h. High-purity nitrogen gas was introduced throughout the reaction to isolate the active ingredients from air and prevent oxidation and degradation.

[0044] S3. Closed-loop solvent recovery: The premixed active ingredient solution obtained in S2 is heated and distilled at atmospheric pressure to recover anhydrous ethanol. The recovered anhydrous ethanol is dehydrated and recycled for the next batch of synthesis. After distillation, a solvent-free mixed solid of active ingredients is obtained.

[0045] The atmospheric distillation temperature is 79℃, the recovery rate of anhydrous ethanol is not less than 95%, and the water content of the recovered anhydrous ethanol after dehydration does not exceed 0.3%. The recovered anhydrous ethanol is sealed and stored after passing inspection and is directly used for subsequent batch production.

[0046] S4. Dynamic coordination reaction: Add water for injection to the mixed solid of active ingredients obtained in S3, stir until completely dissolved, adjust the pH of the system, cool down and control the stirring speed, add the phytic acid-glycine oligomer pretreated in S1, keep warm and stir to complete the construction of the ternary coordination complex, and obtain the coordination complex solution.

[0047] The system pH was adjusted to 7.0, the system temperature was reduced to 37℃, the stirring speed was controlled at 175 r / min, and the incubation and stirring time for the coordination reaction was 37.5 min. The entire process was carried out in the dark to avoid photodegradation of nucleotide components, and the pH was adjusted using 0.1 mol / L sterile phosphate buffer.

[0048] S5. Ultrafiltration purification and concentration: Adjust the pH value of the coordination complex solution obtained in S4, and use an ultrafiltration membrane for circulation filtration to obtain a purified solution; transfer the purified solution into a vacuum concentration vessel, and concentrate it to the target volume under controlled temperature and pressure to obtain a concentrated solution.

[0049] The pH of the coordination complex solution was adjusted to 7.0, the molecular weight cutoff of the ultrafiltration membrane was 3000 Da, the concentration temperature was 45℃, the vacuum degree was -0.085 MPa, and the target volume was 1 / 4.5 of the original volume. During the cyclic filtration process, an equal volume of sterile water for injection was added after each round of filtration for backwashing, and a total of 6 backwashes were performed to thoroughly remove small molecule impurities.

[0050] S6. Dual-chamber formulation forming: Add the pharmaceutical excipients pretreated in S1 to the concentrate obtained in S5, add water for injection, stir until completely dissolved, and then filter sterile to obtain a sterile drug solution; complete the dispensing, freeze-drying and crimping sealing of the dual-chamber sterile container under sterile conditions to obtain the novel immune agonist finished product.

[0051] The sterile filtration process uses a sterile filter membrane with a pore size of 0.22μm, the freeze-drying temperature is -35℃, the vacuum degree is -0.1MPa, and the drying time is 9h. The sterile environment is a Class A sterile environment, and the double-chamber sterile container uses a pharmaceutical-grade pre-filled double-chamber cartridge bottle. The entire dispensing process complies with GMP sterile preparation production standards.

[0052] Example 2: This example provides a novel immune agonist comprising the following parts by weight of raw materials: 5 parts of α-mannose-modified imidazoquinoline derivative, 8 parts of N-acetylcysteine-modified cytosine-guanine dinucleotide, 3 parts of glycyrrhetinic acid-polyethylene glycol derivative, 0.5 parts of phytic acid-glycine oligomer, 2 parts of pharmaceutical excipients, and 81.5 parts of water for injection, for a total of 100 parts by weight.

[0053] The pharmaceutical excipients consist of 4000.5 parts polyethylene glycol and 1.5 parts mannitol, with a mass ratio of 1:3. The α-mannose-modified imidazoquinoline derivative was prepared according to Preparation Example 1 above, the N-acetylcysteine-modified cytosine-guanine dinucleotide was prepared according to Preparation Example 2 above, the glycyrrhetinic acid-polyethylene glycol derivative was prepared according to Preparation Example 3 above, and the phytic acid-glycine oligomer was prepared according to Preparation Example 4 above. The purity of all intermediates was not less than 98% as determined by high performance liquid chromatography. The α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative formed a ternary coordination complex through dynamic non-covalent coordination, with the phytic acid-glycine oligomer as the bridging unit.

[0054] The preparation method of the above-mentioned novel immune agonist includes the following steps: S1. Raw material pretreatment: Weigh α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, glycyrrhetinic acid-polyethylene glycol derivative, phytic acid-glycine oligomer, polyethylene glycol 400, and mannitol. Vacuum dry, pulverize, and sieve each raw material separately, then seal and set aside for later use. Take anhydrous ethanol for reaction, dehydrate it, and set aside for later use.

[0055] The pretreatment process involves vacuum drying each raw material until the moisture content does not exceed 0.5%, followed by pulverizing and passing it through a 100-120 mesh sieve. The dehydration treatment of anhydrous ethanol involves dehydrating the anhydrous ethanol until the moisture content does not exceed 0.3%. All raw materials are pharmaceutical grade or analytical grade, and the entire operation is completed in a Class 100,000 clean drying room to avoid moisture absorption or the introduction of impurities.

[0056] S2. Premixed Dissolution of Active Ingredients: Add the α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, and glycyrrhetinic acid-polyethylene glycol derivative pretreated in S1 to the reaction vessel, add anhydrous ethanol, and stir under controlled temperature until the raw materials are completely dissolved to obtain a homogeneous premixed solution of active ingredients. The entire process is carried out in a closed system to reduce solvent evaporation.

[0057] The amount of anhydrous ethanol added was three times the total mass of the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative. The stirring speed was controlled at 200 r / min. The stirring temperature was 45℃ and the stirring time was 2 h. High-purity nitrogen gas was introduced throughout the reaction to isolate the active ingredients from air and prevent oxidation and degradation.

[0058] S3. Closed-loop solvent recovery: The premixed active ingredient solution obtained in S2 is heated and distilled at atmospheric pressure to recover anhydrous ethanol. The recovered anhydrous ethanol is dehydrated and recycled for the next batch of synthesis. After distillation, a solvent-free mixed solid of active ingredients is obtained.

[0059] The atmospheric distillation temperature is 78℃, the recovery rate of anhydrous ethanol is not less than 95%, and the water content of the recovered anhydrous ethanol after dehydration does not exceed 0.3%. The recovered anhydrous ethanol is sealed and stored after passing inspection and is directly used for subsequent batch production.

[0060] S4. Dynamic coordination reaction: Add water for injection to the mixed solid of active ingredients obtained in S3, stir until completely dissolved, adjust the pH of the system, cool down and control the stirring speed, add the phytic acid-glycine oligomer pretreated in S1, keep warm and stir to complete the construction of the ternary coordination complex, and obtain the coordination complex solution.

[0061] The system pH was adjusted to 6.8, the system temperature was reduced to 35℃, the stirring speed was controlled at 150 r / min, and the incubation and stirring time for the coordination reaction was 30 min. The entire process was carried out in the dark to avoid photodegradation of nucleotide components, and the pH was adjusted using 0.1 mol / L sterile phosphate buffer.

[0062] S5. Ultrafiltration purification and concentration: Adjust the pH value of the coordination complex solution obtained in S4, and use an ultrafiltration membrane for circulation filtration to obtain a purified solution; transfer the purified solution into a vacuum concentration vessel, and concentrate it to the target volume under controlled temperature and pressure to obtain a concentrated solution.

[0063] The pH of the coordination complex solution was adjusted to 6.5, the molecular weight cutoff of the ultrafiltration membrane was 1000 Da, the concentration temperature was 40℃, the vacuum degree was -0.09 MPa, and the target volume was 1 / 5 of the original volume. During the cyclic filtration process, an equal volume of sterile water for injection was added after each round of filtration for backwashing, and a total of 6 backwashes were performed to thoroughly remove small molecule impurities.

[0064] S6. Dual-chamber formulation forming: Add the pharmaceutical excipients pretreated in S1 to the concentrate obtained in S5, add water for injection, stir until completely dissolved, and then filter sterile to obtain a sterile drug solution; complete the dispensing, freeze-drying and crimping sealing of the dual-chamber sterile container under sterile conditions to obtain the novel immune agonist finished product.

[0065] The sterile filtration process uses a sterile filter membrane with a pore size of 0.22μm, the freeze-drying temperature is -40℃, the vacuum degree is -0.1MPa, and the drying time is 8h; the sterile environment is a Class A sterile environment, and the double-chamber sterile container uses a pharmaceutical-grade pre-filled double-chamber cartridge bottle, and the entire dispensing process complies with GMP sterile preparation production standards.

[0066] Example 3: This example provides a novel immune agonist comprising the following parts by weight of raw materials: 7 parts of α-mannose-modified imidazoquinoline derivative, 9 parts of N-acetylcysteine-modified cytosine-guanine dinucleotide, 4 parts of glycyrrhetinic acid-polyethylene glycol derivative, 2 parts of phytic acid-glycine oligomer, 3 parts of pharmaceutical excipients, and 75 parts of water for injection, for a total of 100 parts by weight.

[0067] The pharmaceutical excipients consist of 4002.25 parts polyethylene glycol and 0.75 parts mannitol, with a mass ratio of 3:1. The α-mannose-modified imidazoquinoline derivative was prepared according to Preparation Example 1 above, the N-acetylcysteine-modified cytosine-guanine dinucleotide was prepared according to Preparation Example 2 above, the glycyrrhetinic acid-polyethylene glycol derivative was prepared according to Preparation Example 3 above, and the phytic acid-glycine oligomer was prepared according to Preparation Example 4 above. The purity of all intermediates was not less than 98% as determined by high performance liquid chromatography. The α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative formed a ternary coordination complex through dynamic non-covalent coordination, with the phytic acid-glycine oligomer as the bridging unit.

[0068] The preparation method of the above-mentioned novel immune agonist includes the following steps: S1. Raw material pretreatment: Weigh α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, glycyrrhetinic acid-polyethylene glycol derivative, phytic acid-glycine oligomer, polyethylene glycol 400, and mannitol. Vacuum dry, pulverize, and sieve each raw material separately, then seal and set aside for later use. Take anhydrous ethanol for reaction, dehydrate it, and set aside for later use.

[0069] The pretreatment process involves vacuum drying each raw material until the moisture content does not exceed 0.5%, followed by pulverizing and passing it through a 100-120 mesh sieve. The dehydration treatment of anhydrous ethanol involves dehydrating the anhydrous ethanol until the moisture content does not exceed 0.3%. All raw materials are pharmaceutical grade or analytical grade, and the entire operation is completed in a Class 100,000 clean drying room to avoid moisture absorption or the introduction of impurities.

[0070] S2. Premixed Dissolution of Active Ingredients: Add the α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, and glycyrrhetinic acid-polyethylene glycol derivative pretreated in S1 to the reaction vessel, add anhydrous ethanol, and stir under controlled temperature until the raw materials are completely dissolved to obtain a homogeneous premixed solution of active ingredients. The entire process is carried out in a closed system to reduce solvent evaporation.

[0071] The amount of anhydrous ethanol added was 5 times the total mass of the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative. The stirring speed was controlled at 300 r / min. The stirring temperature was 55℃ and the stirring time was 3 h. High-purity nitrogen gas was introduced throughout the reaction to isolate air and avoid oxidation and degradation of the active ingredients.

[0072] S3. Closed-loop solvent recovery: The premixed active ingredient solution obtained in S2 is heated and distilled at atmospheric pressure to recover anhydrous ethanol. The recovered anhydrous ethanol is dehydrated and recycled for the next batch of synthesis. After distillation, a solvent-free mixed solid of active ingredients is obtained.

[0073] The atmospheric distillation temperature is 80℃, the recovery rate of anhydrous ethanol is not less than 95%, and the water content of the recovered anhydrous ethanol after dehydration does not exceed 0.3%. The recovered anhydrous ethanol is sealed and stored after passing inspection and is directly used for subsequent batch production.

[0074] S4. Dynamic coordination reaction: Add water for injection to the mixed solid of active ingredients obtained in S3, stir until completely dissolved, adjust the pH of the system, cool down and control the stirring speed, add the phytic acid-glycine oligomer pretreated in S1, keep warm and stir to complete the construction of the ternary coordination complex, and obtain the coordination complex solution.

[0075] The system pH was adjusted to 7.2, the system temperature was reduced to 40℃, the stirring speed was controlled at 200 r / min, and the incubation and stirring time for the coordination reaction was 45 min. The entire process was carried out in the dark to avoid photodegradation of nucleotide components, and the pH was adjusted using 0.1 mol / L sterile phosphate buffer.

[0076] S5. Ultrafiltration purification and concentration: Adjust the pH value of the coordination complex solution obtained in S4, and use an ultrafiltration membrane for circulation filtration to obtain a purified solution; transfer the purified solution into a vacuum concentration vessel, and concentrate it to the target volume under controlled temperature and pressure to obtain a concentrated solution.

[0077] The pH of the coordination complex solution was adjusted to 7.5, the molecular weight cutoff of the ultrafiltration membrane was 5000 Da, the concentration temperature was 50℃, the vacuum degree was -0.08 MPa, and the target volume was 1 / 5 of the original volume. During the cyclic filtration process, an equal volume of sterile water for injection was added after each round of filtration for backwashing, and a total of 6 backwashes were performed to thoroughly remove small molecule impurities.

[0078] S6. Dual-chamber formulation forming: Add the pharmaceutical excipients pretreated in S1 to the concentrate obtained in S5, add water for injection, stir until completely dissolved, and then filter sterile to obtain a sterile drug solution; complete the dispensing, freeze-drying and crimping sealing of the dual-chamber sterile container under sterile conditions to obtain the novel immune agonist finished product.

[0079] The sterile filtration process uses a sterile filter membrane with a pore size of 0.22μm, the freeze-drying temperature is -30℃, the vacuum degree is -0.1MPa, and the drying time is 10h. The sterile environment is a Class A sterile environment, and the double-chamber sterile container uses a pharmaceutical-grade pre-filled double-chamber cartridge bottle. The entire dispensing process complies with GMP sterile preparation production standards.

[0080] Comparative Example 1: The only difference between this comparative example and Example 1 is that: no phytic acid-glycine oligomer was added to the composition, the missing mass fraction was made up with water for injection, and the α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, and glycyrrhetinic acid-polyethylene glycol derivative were simply mixed physically and did not form a ternary coordination complex. All other components, ratios, preparation processes, and parameters were completely consistent with those of Example 1.

[0081] Comparative Example 2: The only difference between this comparative example and Example 1 is that the α-mannose-modified imidazoquinoline derivative is replaced with an equal mass of unmodified imidazoquinoline. All other components, ratios, preparation processes, and parameters are completely consistent with Example 1.

[0082] Comparative Example 3: The only difference between this comparative example and Example 1 is that the N-acetylcysteine-modified cytosine-guanine dinucleotide is replaced with an equal mass of unmodified cytosine-guanine dinucleotide. All other components, ratios, preparation processes, and parameters are completely consistent with Example 1.

[0083] Comparative Example 4: The only difference between this comparative example and Example 1 is that the glycyrrhetinic acid-polyethylene glycol derivative is replaced with an equal mass of unmodified glycyrrhetinic acid raw material. All other components, ratios, preparation processes, and parameters are completely consistent with Example 1.

[0084] Comparative Example 5: The only difference between this comparative example and Example 1 is that the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative are replaced with equal masses of unmodified imidazoquinoline, unmodified cytosine-guanine dinucleotide, and unmodified glycyrrhetinic acid raw materials, respectively. All other components, ratios, preparation processes, and parameters are completely consistent with those of Example 1.

[0085] Comparative Example 6: The only difference between this comparative example and Example 1 is that commercially available 5% imiquimod cream is used as the control drug. This product is a topical formulation of a TLR7 / 8 single agonist. The rest of the experimental system, dosage, and detection methods are completely consistent with Example 1.

[0086] Test Item 1: Stability Test of Ternary Coordination Complex. This test follows the Guidelines for Stability Testing of Drug Preparations in General Chapter 9001 of the 2020 Edition of the Pharmacopoeia of the People's Republic of China, Part IV. Products from Examples 1-3 and Comparative Examples 1-6 were used. Comparative Example 6, a commercially available 5% imiquimod cream, required reconstitution with sterile saline to the same concentration of active ingredient as in the examples. All samples were sealed and stored in a constant temperature and humidity environment at 25°C and 60% relative humidity. Samples were taken for testing at 0, 30, 60, and 90 days. High-performance liquid chromatography (HPLC) was used for testing, following the HPLC method in General Chapter 0512 of the 2020 Edition of the Pharmacopoeia of the People's Republic of China, Part IV. A C18 column was used, with a mobile phase of methanol-0.1 mol / L phosphate buffer (40:60 v / v), a detection wavelength of 260 nm, a flow rate of 1.0 mL / min, a column temperature of 30°C, and an injection volume of 20 μL. The retention rates of each active component were calculated by measuring the peak areas of α-mannose-modified imidazoquinoline derivatives, N-acetylcysteine-modified cytosine-guanine dinucleotides, and glycyrrhetinic acid-polyethylene glycol derivatives at various time points. The retention rate was calculated as (peak area at a certain time point / peak area on day 0) × 100%. Simultaneously, the presence of turbidity, precipitation, or stratification was observed in the samples. Changes in the retention rate and appearance of the active components in each sample were recorded to evaluate the coordination stability of each product. The test results are shown below. Figure 2 As shown.

[0087] Test Item 2: In vitro immune activation activity test. This test refers to the immune cell activity detection method in "Experimental Guide to Cell Biology" (Science Press, 3rd Edition) and the "Standard Operating Procedures for Isolation and Culture of Human Peripheral Blood Mononuclear Cells". Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells were isolated using density gradient centrifugation. The cell concentration was adjusted to 1×10⁶ cells / mL with RPMI 1640 medium containing 10% fetal bovine serum, and 100 μL was seeded into 96-well cell culture plates. Products from Examples 1-3 and Comparative Examples 1-6 were taken respectively. For Comparative Example 6, commercially available 5% imiquimod cream was reconstituted and diluted with other samples with sterile physiological saline to the same concentration of active ingredient. 10 μL of sample solution was added to each well. A blank control group containing only cells and culture medium was set up, with 3 parallel replicates for each group. After incubating the culture plates at 37°C and 5% CO2 for 24 hours, the supernatant from each well was collected. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were detected using enzyme-linked immunosorbent assay (ELISA). The procedure was followed according to the "ELISA Operation Guide" and the instructions for the IL-6 and TNF-α ELISA kits. The detection wavelength of the ELISA reader was set to 450 nm. The cytokine secretion levels of each sample were calculated based on the standard curve. The in vitro immune activation activity of the product was evaluated by comparing the cytokine secretion levels of each example with those of the comparative example. The test results are shown below. Figure 3 As shown.

[0088] Test Item 3: Liver-targeted enrichment capacity test. This test was conducted in accordance with the "Guiding Principles for In vivo tissue distribution assays of drugs" (National Medical Products Administration, 2021). Sixty SPF-grade Kunming mice, weighing 20±2g, were randomly divided into 12 groups of 5 mice each, corresponding to Examples 1-3 and Comparative Examples 1-6, respectively. Comparative Example 6 involved reconstituted commercially available 5% imiquimod cream and administered via tail vein injection along with other samples at the same dose of 10mg / kg based on the active ingredient. Two hours after administration, the mice were euthanized by cervical dislocation. The liver, heart, spleen, kidneys, lungs, and other major tissues were rapidly dissected and separated. The tissues were rinsed thoroughly with physiological saline, dried with filter paper, and accurately weighed. A suitable amount of physiological saline was added to prepare a tissue homogenate, which was then centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The content of α-mannose-modified imidazoquinoline derivatives in the supernatant of each tissue was determined by high-performance liquid chromatography (HPLC). The procedure was performed according to the HPLC method in General Chapter 0512 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China, with chromatographic conditions identical to those in Test Item 1. The drug concentration in each tissue was calculated in μg / g, and the liver targeting index was calculated as follows: Liver targeting index = (Drug concentration in liver / Average drug concentration in each tissue) × 100%. The liver targeting enrichment ability of the product was evaluated by comparing the liver targeting indices of each example with those of the comparative example. The test results are shown below. Figure 4As shown.

[0089] As can be seen from Examples 1-3 and Comparative Example 1, and Tables 1-3, the presence of phytic acid-glycine oligomers enables various active ingredients to form stable ternary coordination complexes. Without this bridging component, the active substances are merely in a physical mixed state, and the overall structural integrity is significantly reduced. This directly affects the delivery efficiency and targeted distribution of the formulation in vivo, resulting in significant changes in the overall immune activation effect and tissue enrichment capacity. This fully demonstrates the supporting role of the coordination bridging structure in the overall performance of the product.

[0090] As can be seen from Examples 1-3 and Comparative Example 2, and Tables 1-3, α-mannose modification of imidazoquinoline derivatives can significantly improve the targeted recognition and cellular uptake efficiency of active ingredients. After replacing them with unmodified raw materials, the effects of the relevant components are significantly limited, and the immune activation level and tissue distribution characteristics of the formulation are changed accordingly. This indicates that the targeted modification structure has an important impact on the effect of active ingredients.

[0091] As can be seen from Examples 1-3 and Comparative Example 3, and Tables 1-3, the CpG modified with N-acetylcysteine ​​can effectively improve the stability of nucleotide components and prevent them from degrading too quickly during storage and in vivo. When replaced with unmodified CpG, the stability of the components decreases, thereby affecting the overall immune activation effect, demonstrating the important significance of stabilization modification in maintaining the function of active ingredients.

[0092] Based on Examples 1-3 and Comparative Example 4, and in conjunction with Tables 1-3, it can be seen that the glycyrrhetinic acid-polyethylene glycol derivative can provide the formulation with good liver-targeted enrichment ability and in vivo circulation stability. After replacing it with unmodified glycyrrhetinic acid, the tissue targeting of the formulation is significantly reduced, the aggregation ability of the active ingredient at the target site is weakened, and thus the overall effect is affected.

[0093] As can be seen from Examples 1-3 and Comparative Example 5, and Tables 1-3, when the functional modifications of the three active ingredients are removed simultaneously, the formulation loses both its targeted delivery capability and its stabilizing and protective effect. The components cannot form an orderly synergistic system, and the overall structural stability, immune activation capability, and tissue distribution characteristics all show significant changes, which fully demonstrates that there is a significant synergistic effect among the multiple modified components.

[0094] As can be seen from Examples 1-3 and Comparative Example 6, and Tables 1-3, the ternary coordination complex immune agonist of the present invention has significant differences in composition, mechanism of action and in vivo behavior compared with commercially available single-target topical formulations. The construction of a multi-target synergistic activation and targeted delivery system enables the product to exhibit more comprehensive performance characteristics in overall effect, demonstrating the technical advantages of the present invention compared with existing commercially available products.

[0095] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A novel immune agonist, characterized in that: The product is prepared from the following components in parts by weight: 5 to 12 parts of α-mannose-modified imidazoquinoline derivative, 8 to 15 parts of N-acetylcysteine-modified cytosine-guanine dinucleotide, 3 to 8 parts of glycyrrhetinic acid-polyethylene glycol derivative, 0.5 to 2 parts of phytic acid-glycine oligomer, 2 to 6 parts of pharmaceutical excipients, and 75 to 85 parts of water for injection; wherein the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative form a ternary coordination complex through dynamic non-covalent coordination, with the phytic acid-glycine oligomer as the bridging unit.

2. The novel immune agonist according to claim 1, characterized in that: The pharmaceutical excipient is composed of polyethylene glycol 400 and mannitol in a mass ratio of 1:3 to 3:

1.

3. A method for preparing a novel immune agonist, characterized in that, A novel immune agonist according to any one of claims 1 or 2 comprises the following steps: S1. Raw material pretreatment: Weigh α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, glycyrrhetinic acid-polyethylene glycol derivative, phytic acid-glycine oligomer, polyethylene glycol 400, and mannitol. Vacuum dry, pulverize, and sieve each raw material separately, then seal and set aside for later use. Take anhydrous ethanol for the reaction, dehydrate it, and set aside for later use. S2. Premixed dissolution of active ingredients: Add the α-mannose-modified imidazoquinoline derivative, N-acetylcysteine-modified cytosine-guanine dinucleotide, and glycyrrhetinic acid-polyethylene glycol derivative that have been pretreated in S1 to the reaction vessel, add anhydrous ethanol, and stir under controlled temperature until the raw materials are completely dissolved to obtain a homogeneous premixed solution of active ingredients. The entire process is carried out in a closed manner to reduce solvent evaporation. S3, closed-loop solvent recovery: The premixed active ingredient solution obtained in S2 is heated and distilled at atmospheric pressure to recover anhydrous ethanol. The recovered anhydrous ethanol is dehydrated and recycled for the next batch of synthesis. After distillation, a solvent-free mixed solid of active ingredients is obtained. S4. Dynamic coordination reaction: Add water for injection to the mixed solid of active ingredients obtained in S3, stir until completely dissolved, adjust the pH of the system, cool down and control the stirring speed, add the phytic acid-glycine oligomer pretreated in S1, keep warm and stir to complete the construction of the ternary coordination complex, and obtain the coordination complex solution. S5. Ultrafiltration purification and concentration: Adjust the pH value of the coordination complex solution obtained in S4, and use an ultrafiltration membrane for circulation filtration to obtain a purified solution; transfer the purified solution into a vacuum concentration vessel, and concentrate it to the target volume under controlled temperature and pressure to obtain a concentrated solution; S6. Dual-chamber formulation forming: Add the pharmaceutical excipients pretreated in S1 to the concentrate obtained in S5, add water for injection, stir until completely dissolved, and then filter sterile to obtain a sterile drug solution; complete the dispensing, freeze-drying and crimping sealing of the dual-chamber sterile container under sterile conditions to obtain the novel immune agonist finished product.

4. The method for preparing a novel immune agonist according to claim 3, characterized in that: In step S1, the pretreatment involves vacuum drying each raw material until the moisture content does not exceed 0.5%, then pulverizing it and passing it through a 100-120 mesh sieve; the dehydration treatment of the anhydrous ethanol involves dehydrating the anhydrous ethanol until the moisture content does not exceed 0.3%.

5. The method for preparing a novel immune agonist according to claim 3, characterized in that: In step S2, the amount of anhydrous ethanol added is 3 to 5 times the total mass of the α-mannose-modified imidazoquinoline derivative, the N-acetylcysteine-modified cytosine-guanine dinucleotide, and the glycyrrhetinic acid-polyethylene glycol derivative. The stirring speed is controlled at 200 r / min to 300 r / min. The stirring temperature is 45°C to 55°C, and the stirring time is 2 h to 3 h.

6. The method for preparing a novel immune agonist according to claim 3, characterized in that: In step S3, the temperature of the atmospheric distillation is 78°C to 80°C, the recovery rate of the anhydrous ethanol is not less than 95%, and the water content of the recovered anhydrous ethanol after dehydration does not exceed 0.3%.

7. The method for preparing a novel immune agonist according to claim 3, characterized in that: In step S4, the pH value of the system is adjusted to 6.8 to 7.2, the system temperature is reduced to 35°C to 40°C, the stirring speed is controlled to 150 r / min to 200 r / min, and the holding and stirring time for the coordination reaction is 30 min to 45 min.

8. The method for preparing a novel immune agonist according to claim 3, characterized in that: In step S5, the pH of the coordination complex solution is adjusted to 6.5 to 7.5, the molecular weight cutoff of the ultrafiltration membrane is 1000 Da to 5000 Da, the concentration temperature is 40°C to 50°C, the vacuum degree is -0.09 MPa to -0.08 MPa, and the target volume is 1 / 5 to 1 / 4 of the original volume.

9. The method for preparing a novel immune agonist according to claim 3, characterized in that: In step S6, the sterilization filtration uses a sterile filter membrane with a pore size of 0.22 μm, the freeze-drying temperature is -40℃ to -30℃, the vacuum degree is -0.1 MPa, and the drying time is 8h to 10h.

10. The use of a novel immune agonist, characterized in that, Used as a novel immune agonist according to any one of claims 1 or 2, for the preparation of tumor immunotherapy drugs, anti-infective drugs, adjuvant drugs for immunodeficiency diseases, or vaccine adjuvants.

Images