Isopagglutinin α-Nanolyzed Micelles and Their Preparation Method

Abstract

This invention relates to the field of lyophilized formulation technology, specifically disclosing an esupagglutide α-nano-lyophilized micelle and its preparation method. The micelles of this invention utilize polylactic-co-hydroxyacetic acid copolymer and sodium phosphatidylglycerol as micelle carriers, with arginine and cysteine ​​providing branching synergistic effects. Homogenization via film formation and low-temperature lyophilization yield lyophilized nano-micelle powder. The nano-micelle powder reduces protein oxidative decomposition during lyophilization, decreases impurity fragments, and reduces various non-fucose glycosylation, basic components (succinimide, methionine oxidation products), N-terminal deletion, Asn deamidation, and Met and Trp residue oxidation impurities that affect product quality. Product quality analysis and animal experiments after reconstitution show good resistance to impurities and degradation risk. The reconstituted esupagglutide α-nano-micelle system exhibits better stability and sustained-release properties, enhancing the hypoglycemic sustained-release effect 72 hours after injection, significantly improving upon conventional non-micelle formulations.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of freeze-dried preparations, and specifically discloses a kind of esupergel peptide alpha nanometer freeze-dried micelles and a preparation method thereof. BACKGROUND

[0002] In 1977, Frank Davis and Abuchowsky first proposed the concept of pegylation, proving that the immunogenicity of proteins can be effectively reduced and their circulation periods can be prolonged by high molecular modification. Since then, pegylation has become a recognized strategy for improving the physicochemical properties and pharmacokinetic characteristics of drugs, and has developed rapidly in the research and application of protein drugs, polypeptide drugs and drug carriers. Based on this, nanodrug delivery systems have gradually become an important direction in the field of modern pharmacy. Through molecular-level design, nanocarriers can not only efficiently load therapeutic molecules, but also achieve precise positioning and controlled release by combining targeting ligand modification or environmental response mechanisms. Compared with traditional carriers, nanosystems have the advantage of subcellular level space, which can break through the limitations of biological barriers, improve the penetration and target enrichment capacity of drugs in circulation, and significantly enhance the therapeutic effect. In addition, nanoparticle delivery systems can also prolong the half-life of drugs, improve extravascular penetration efficiency, and reduce immune recognition through surface engineering modification.

[0003] As a kind of polypeptide drug, esupergel peptide alpha has the disadvantages of easy degradation and limited circulation time in vivo. The preparation of nanometer freeze-dried preparation can improve the stability of esupergel peptide alpha and prolong its action time. However, as a protein drug, esupergel peptide alpha is prone to produce special impurities due to its protein characteristics during storage or freeze-drying preparation and reconstitution process, which affects the quality of the product. These quality problems include protein aggregation and denaturation, oxidation and cleavage, etc., which introduce impurities, reduce the biological activity of the drug, and even increase the immunogenicity, affecting the efficacy. Therefore, how to improve the stability and pharmacokinetic characteristics of esupergel peptide alpha through high molecular modification and nanocarrier synergistic effect has become an important research direction of esupergel peptide alpha preparation. SUMMARY

[0004] In view of the problems in the prior art, the present application provides a preparation method of esupergel peptide alpha nanometer freeze-dried micelles, which comprises the following steps:

[0005] S1: mixing arginine, cysteine, alcohol and water for injection to obtain a protective solution;

[0006] S2: mixing the polysorbate 80 stock solution of esupergel peptide alpha with the protective solution to obtain an esupergel peptide alpha drug protective solution;

[0007] S3: The polylactic acid-glycolic acid copolymer and the sodium phosphatidylglycerol are put into alcohol to obtain a mixed solution, and the alcohol is removed from the mixed solution to form a polymer-phospholipid film;

[0008] S4: The Eptacog Alfa Drug Protection Solution is added to the polymer-phospholipid film to form a crude dispersion, and the alcohol is removed by one-time dispersion, and then water for injection is added and the second dispersion is performed to obtain an Eptacog Alfa nanomicelle solution;

[0009] S5: The Eptacog Alfa nanomicelle solution is freeze-dried to obtain an Eptacog Alfa nanofreeze-dried micelle.

[0010] In some specific embodiments of the preparation of the Eptacog Alfa nanofreeze-dried micelle, the mass of arginine added in the S1 step is 2.0-4.0 g per 1 L of water for injection, and in some specific embodiments of the preparation of the Eptacog Alfa nanofreeze-dried micelle, the mass of arginine added in the S1 step is optionally 2.3 g, 2.6 g, 2.9 g, 3.2 g, 3.5 g, or 3.8 g per 1 L of water for injection.

[0011] In some specific embodiments of the preparation of the Eptacog Alfa nanofreeze-dried micelle, the mass of cysteine added in the S1 step is 1.0-2.0 g per 1 L of water for injection, and in some specific embodiments of the preparation of the Eptacog Alfa nanofreeze-dried micelle, the mass of cysteine added in the S1 step is optionally 1.2 g, 1.4 g, 1.6 g, or 1.8 g per 1 L of water for injection.

[0012] In some specific embodiments of the preparation of the Eptacog Alfa nanofreeze-dried micelle, the volume of isopropyl alcohol added in the S1 step is 0.7-1.4 L per 1 L of water for injection, and in some specific embodiments of the preparation of the Eptacog Alfa nanofreeze-dried micelle, the volume of isopropyl alcohol added in the S1 step is optionally 0.8 L, 0.9 L, 1.0 L, 1.1 L, 1.2 L, or 1.3 L per 1 L of water for injection.

[0013] In some embodiments of the first aspect, in the S2 step, the polysorbate 80 stock solution of Eisai Pegloticase alpha is prepared by adding 8-12 g of Eisai Pegloticase alpha and 10-30 mg of polysorbate 80 into 1 L of water for injection.

[0014] In some embodiments of the first aspect, in the S2 step, 8-10 volumes of the polysorbate 80 stock solution of Eisai Pegloticase alpha is mixed with 8-9 volumes of the protective solution.

[0015] In some embodiments of the first aspect, in the S3 step, the mass ratio of the polylactic acid-glycolic acid copolymer to the sodium phosphatidylglycerol is (1.8-2.2):(0.9-1.3).

[0016] In some embodiments of the first aspect, in the S3 step, the alcohol is selected from ethanol, n-propanol, and isopropanol.

[0017] In some embodiments of the first aspect, in the S3 step, the alcohol is removed by placing the mixture in a rotary evaporator under reduced pressure at a water bath temperature of 30-50 °C.

[0018] In some embodiments of the first aspect, in the S4 step, the Eisai Pegloticase alpha drug protective solution is added to the polymer-phospholipid film by dropwise addition.

[0019] In some embodiments of the first aspect, in the S4 step, after the dropwise addition, the mixture is maintained at a temperature of 20-25 °C for 30-50 min.

[0020] In some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S4, the way of the first dispersion is ultrasonic dispersion at 15-35 kHz for 10-20 min, and in some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S4, the way of the first dispersion is optionally ultrasonic dispersion at 18 kHz, 22 kHz, 27 kHz, 31 kHz, 34 kHz for 10-20 min.

[0021] In some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S4, the way of removing alcohol is rotary evaporation at 25-35 °C under vacuum of 30-50 mbar for 40-50 min, and in some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S4, the way of removing alcohol is optionally rotary evaporation at 27 °C, 29 °C, 31 °C, 33 °C under reduced pressure for 40-50 min.

[0022] In some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S4, the way of the second dispersion is mechanical shaking at a speed of 30-40 rpm and centrifugation for 10-15 min, and in some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S4, the way of the second dispersion is optionally mechanical shaking at a speed of 32 rpm, 34 rpm, 36 rpm, 38 rpm and centrifugation for 10-15 min.

[0023] In some embodiments of the first aspect, the method for preparing the esproglifenzoe alpha nanofreezed micelles, in the step S5, the step of lyophilizing the esproglifenzoe alpha nanomicelles solution is as follows:

[0024] Step 1: The esproglifenzoe alpha nanomicelles solution is diluted with water for injection and filled into a container;

[0025] Step 2: The filled container is placed in a freeze dryer under controlled vacuum, and maintained at a first temperature for a first time, and then cooled to a second temperature for a second time;

[0026] Step 3: The temperature of the container is maintained at a third temperature for a third time, and the lyophilization is completed to obtain esproglifenzoe alpha nanofreezed micelles.

[0027] In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 1, the water for injection is added to a final concentration of 2.0-4.0 mg / mL isuplucin alpha. In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 1, the water for injection is added to a final concentration of 2.3 mg / mL, 2.6 mg / mL, 2.9 mg / mL, 3.2 mg / mL, 3.5 mg / mL, 3.8 mg / mL isuplucin alpha.

[0028] In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 1, the container is a vial.

[0029] In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the vacuum is controlled to 0.24-0.35 mbar. In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the vacuum is controlled to 0.26 mbar, 0.28 mbar, 0.30 mbar, 0.32 mbar, 0.34 mbar.

[0030] In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the first temperature is 5-8 °C. In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the first temperature is 5 °C, 6 °C, 7 °C, 8 °C.

[0031] In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the first time is 1-2 h. In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the first time is 1.2 h, 1.4 h, 1.6 h, 1.8 h.

[0032] In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the second temperature is -50--60 °C. In some embodiments of the first aspect of the application, the method for preparing the nanoscale lyophilized micelles of isuplucin alpha, in step 2, the second temperature is -50--60 °C, -50--60 °C, -50--60 °C, -50--60 °C, -50--60 °C, -50--60 °C.

[0033] In some embodiments of the first aspect, the second time is 10-14 hours in step 2, and in some embodiments of the first aspect, the second time is optionally 10 hours, 11 hours, 12 hours, 13 hours, or 14 hours in step 2.

[0034] In some embodiments of the first aspect, the third temperature is 6-10℃ in step 3, and in some embodiments of the first aspect, the third temperature is 6℃, 7℃, 8℃, 9℃, or 10℃ in step 3.

[0035] In some embodiments of the first aspect, the third time is 3-5 hours in step 3, and in some embodiments of the first aspect, the third time is optionally 3.0 hours, 3.5 hours, 4.0 hours, 4.5 hours, or 5.0 hours in step 3.

[0036] The second aspect of the present application provides a nanometer-sized lyophilized micelle of Isuplucaglucel alpha obtained by any of the preparation methods of the first aspect.

[0037] “Room temperature” refers to the indoor ambient temperature, which can be 12-37℃, 20-30℃, 25-30℃, or about 25℃.

[0038] The reagents used in the present application are not further purified and are purchased from public legal markets, for example, Shanghai Aldrin Biochemical Technology Co., Ltd., Xilong Scientific Co., Ltd., China Reagent Co., Ltd., and Shanghai McLean Biotechnology Co., Ltd.

[0039] The polylactic acid-glycolic acid copolymer used in the present application has a CAS of 34346-01-5 and a MW of 50-70 thousand, wherein LA represents a lactic acid unit, GA represents a glycolic acid unit, and LA:GA=75:25 indicates that the number ratio of lactic acid units to glycolic acid units is 75:25.

[0040] The phosphatidylglycerol sodium (L-α-phosphatidylglycerol) used in the present application has a CAS of 383907-64-0.

[0041] Advantages of the present application:

[0042] The present application micelle selects polylactic acid-glycolic acid copolymer as micelle carrier, and introduces sodium phosphatidylglycerol as surface modifier. Under the synergistic effect of arginine and cysteine providing branch, the isuplucin alpha nanomicelle is formed by film forming method homogenization, and the freeze-dried nanomicelle powder is obtained by low temperature freeze-drying. The nanomicelle powder has the advantages of freeze-drying protection, redissolution stability, in-vivo long circulation, etc. The protein oxidation decomposition in the freeze-drying process is reduced, the impurity fragments are reduced, and the generation of various influence product quality non-fucosylation, basic components (succinimidyl, methionine oxidation product), N-terminal deletion, Asn deamidation, Met and Trp residue oxidation impurities in the freeze-drying storage process is reduced. The product quality analysis and animal experiment after redissolution show good anti-impurity and degradation, aggregation risk. The stable drug carrier nanomicelle skeleton composed of polylactic acid-glycolic acid copolymer makes the isuplucin alpha nanomicelle system after redissolution exhibit better stability and sustained release in body fluid, enhances the hypoglycemic sustained release effect after 72h injection. The nanomicelle system compared with the conventional non-micellar preparation has significant improvement. DETAILED DESCRIPTION

[0043] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples. It should be pointed out that the following detailed description is exemplary and is intended to provide further description of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0044] Example 1 Isuplucin alpha nanofreeze-dried micelles

[0045] Step 1: Arginine and cysteine were weighed and dissolved in a mixed solvent (isopropyl alcohol / injection water, V i-PrOH :V H2O =50:50) to prepare a protection solution, wherein the concentration of arginine was 2.1 mg / mL and the concentration of cysteine was 1.5 mg / mL;

[0046] Step 2: 50ml isuplucin alpha polysorbate 80 stock solution (isuplucin alpha concentration 10mg / ml, polysorbate 80 concentration 0.02%, solvent injection water) was mixed with the protection solution, and the volume ratio of the stock solution to the protection solution was 100:90, to obtain isuplucin alpha drug protection solution;

[0047] Step 3: Take 4.0 g of polylactic acid-glycolic acid copolymer (LA:GA=75:25) and 2.2 g of sodium phosphatidylglycerol, dissolve them in 50 ml of ethanol, and place the obtained solution in a rotary evaporator under reduced pressure at 40°C water bath until the ethanol solvent is completely removed, and a uniform transparent polymer-phospholipid film is formed in the flask.

[0048] Step 4: Add the esproglifenzoe alpha drug protection solution dropwise to the flask containing the polymer film, stirring at a speed of 150 rpm during the dropwise addition, and after the dropwise addition is complete, stand for 30-60 min. Continue to hydrate at 23°C for 30 min to fully hydrate and peel the film to form a coarse dispersion. Then use 20 kHz ultrasonic dispersion for 10 min;

[0049] Step 5: Rotary evaporation under reduced pressure at 32°C for 45 min to completely remove isopropanol, add 20 ml of water for injection, and after 15 min of 20 kHz ultrasonic dispersion, mechanically shake at a speed of 40 rpm until clear, then centrifuge the drug solution at a speed of 9500 rpm for 10 min to remove the unencapsulated drug substance, and obtain an esproglifenzoe alpha nanomicelle solution;

[0050] Step 6: The above drug solution is diluted with water for injection to a concentration of 3.0 mg / mL (calculated as esproglifenzoe alpha), and is filled into a vial, with the thickness of the drug solution accurately controlled at 10.0 mm;

[0051] Step 7: Place the filled vial into a freeze-drying machine and cool it down, the vial containing the drug solution is put into the box at 7°C, and after the completion of the process, it is kept for 1.5 h, then the temperature is reduced from 7°C to -55°C, and the process is continued for 12 h, during which the vacuum degree of the freeze-drying box is controlled at 0.24-0.35 mbar.

[0052] Step 8: Keep the vacuum degree at 0.24-0.35 mbar, and raise the temperature of the vial to 8°C±2°C, and maintain for 4 h, then remove the vial from the freeze-drying box to obtain a freeze-dried powder.

[0053] Comparative Example 1

[0054] In Step 1 of the preparation of esproglifenzoe alpha nanomicelles in Comparative Example 1, no arginine is added, and Step 1 of Comparative Example 1 is as follows:

[0055] Step 1: Take cysteine and dissolve it in a mixed solvent (isopropanol / water for injection, V i-PrOH :V H2O =50:50) to prepare a protection solution with a concentration of 1.5 mg / mL;

[0056] Steps 2-8 of Comparative Example 1 are the same as those of Example 1, and a freeze-dried powder is obtained.

[0057] Comparative Example 2

[0058] Comparative Example 2 is different from Step 3 and Step 4 of Example 1, Comparative Example 3 does not make polymer-phospholipid film, but uses liquid mixing method to make, Step 3 and Step 4 of Comparative Example 2 are as follows:

[0059] Step 3: 4.0 g of polylactic acid-glycolic acid copolymer and 2.2 g of sodium phosphatidylglycerol are weighed and dissolved in 50 ml of ethanol to obtain a polymer-phospholipid ethanol solution.

[0060] Step 4: The esproglucar peptide alpha drug protection solution is added dropwise to the flask containing the polymer-phospholipid ethanol solution, and stirring is carried out at a speed of 150 rpm during the dropwise addition. After the dropwise addition is completed, it is left to stand for 30-60 min. Continue at 23°C for 30 min, and then use 20 kHz ultrasonic dispersion for 10 min;

[0061] Step 1, 2 and 5 to 8 of Comparative Example 2 are the same as Example 1, and a freeze-dried powder is obtained.

[0062] Comparative Example 3

[0063] Comparative Example 3 is different from Step 3 of Example 1, in Step 3 of Comparative Example 3, polylactic acid-glycolic acid copolymer (LA:GA=75:25) is replaced by equal mass of polyvinylpyrrolidone K30, and Step 3 of Comparative Example 3 is as follows:

[0064] Step 3: 4.0 g of polyvinylpyrrolidone K30 and 2.2 g of sodium phosphatidylglycerol are weighed and dissolved in 50 ml of ethanol, and the obtained solution is placed in a rotary evaporator and evaporated under reduced pressure at a water bath temperature of 40°C until the ethanol solvent is completely removed, and a uniform polymer-phospholipid film is formed in the flask.

[0065] Step 1, 2 and 4 to 8 of Comparative Example 3 are the same as Example 1, and a freeze-dried powder is obtained.

[0066] Comparative Example 4

[0067] Comparative Example 4 is different from Example 1 in that no cysteine is added in Step 1 of making esproglucar peptide alpha nanometer freeze-dried micelles, and Step 1 of Comparative Example 4 is as follows:

[0068] Step 1: Arginine is weighed and dissolved in a mixed solvent (isopropyl alcohol / injection water, V i-PrOH :V H2O =50:50) to prepare a protection solution for standby, and the concentration of arginine is 2.1 mg / mL;

[0069] Step 2 to 8 of Comparative Example 4 are the same as Example 1, and a freeze-dried powder is obtained.

[0070] Comparative Example 5

[0071] Comparative Example 5 was prepared according to the procedure of Example 1, except that cysteine was replaced by histidine in Step 1 of Comparative Example 5, which was specifically as follows:

[0072] Step 1: Arginine and histidine were weighed into a mixed solvent (isopropyl alcohol / injection water, V i-PrOH :V H2O =50:50) and configured as a protective solution for standby, wherein the concentration of arginine was 2.1 mg / mL and the concentration of histidine was 1.5 mg / mL.

[0073] Steps 2 to 8 of Comparative Example 5 were the same as Example 1, and a freeze-dried powder was obtained.

[0074] Comparative Example 6

[0075] Comparative Example 6 was prepared according to the procedure of Example 1, except that neither arginine nor cysteine was added in Step 1 of Comparative Example 6, which was specifically as follows:

[0076] Step 1: A mixed solution of isopropyl alcohol / injection water (V i-PrOH :V H2O =50:50) was used as a protective solution for standby;

[0077] Step 2: 50 ml of the polysorbate 80 stock solution of Esprosuglutide alpha (concentration of Esprosuglutide alpha 10 mg / ml, concentration of polysorbate 80 0.02%, solvent injection water) was mixed with the protective solution, and the volume ratio of the stock solution to the protective solution was 100:90, to obtain a drug protective solution of Esprosuglutide alpha;

[0078] Steps 3 to 8 of Comparative Example 6 were the same as Example 1, and a freeze-dried powder was obtained.

[0079] Comparative Example 7

[0080] Comparative Example 7 was prepared according to the procedure of Example 1, except that sodium phosphatidylglycerol was not added in Step 3 of Comparative Example 7, but an equal amount of soy lecithin was added, which was specifically as follows:

[0081] Step 3: 4.0 g of polylactic acid-glycolic acid copolymer and 2.2 g of soy lecithin were weighed, dissolved in 50 ml of ethanol, and the obtained solution was placed in a rotary evaporator and evaporated under reduced pressure at 40°C water bath until the ethanol solvent was completely removed, and a uniform polymer-phospholipid film was formed in the flask.

[0082] Steps 1, 2, and 4 to 8 of Comparative Example 7 were the same as Example 1, and a freeze-dried powder was obtained.

[0083] Comparative Example 8

[0084] Comparative Example 8 is different from Step 3 of Example 1, in Step 3 of Comparative Example 3, polylactic-glycolic acid copolymer (LA:GA=75:25) is replaced by equal mass of polylactic-glycolic acid copolymer (LA:GA=30:70), and Step 3 of Comparative Example 8 is as follows:

[0085] Step 3: 4.0 g of polylactic-glycolic acid copolymer (LA:GA=30:70) and 2.2 g of sodium phosphatidylglycerol were weighed and dissolved in 50 ml of ethanol, and the obtained solution was placed in a rotary evaporator and evaporated under reduced pressure at 40°C water bath until the ethanol solvent was completely removed, and a uniform transparent polymer-phospholipid film was formed in the flask.

[0086] Comparative Example 8 is the same as Steps 1, 2 and 4 to 8 of Example 1, and a lyophilized powder is obtained.

[0087] Example 2 Encapsulation efficiency determination, particle size determination of esprostene alpha nanometer lyophilized micelles

[0088] (1) The encapsulation efficiency was calculated according to the “Chinese Pharmacopoeia” 2020 edition Volume IV: General Principles for Injection, Guiding Principles for Microparticle Preparation 9014.

[0089] (2) The esprostene alpha nanometer lyophilized micelles were diluted with normal saline for injection, and the particle size was determined by laser particle size analyzer.

[0090] Table 1: Encapsulation efficiency and particle size determination of esprostene alpha nanometer lyophilized micelles prepared in Example 1 and Comparative Examples 1 to 3:

[0091]

[0092] Example 1 uses a combination of polylactic-glycolic acid copolymer (LA:GA=75:25) and sodium phosphatidylglycerol, and a specific film-forming micelle process to prepare a cyclophosphamide ternary nanomicelle lyophilized dosage form. The dosage form can achieve an encapsulation efficiency of 98.0±0.65% and a particle size of 80.0±4.9 nm, with high encapsulation efficiency and stable particle size.

[0093] Example 3: Detection of product variant impurities of esprostene alpha nanometer lyophilized micelles

[0094] 0.5 g of esprostene alpha lyophilized nanomicelles was taken and reconstituted with 10 mL of 0.9% sodium chloride injection, and the product variant impurities such as degradation fragments, charge variants, sugars and polypeptides were detected. The stability data of esprostene alpha lyophilized nanomicelles are shown in Table 2:

[0095] Table 2: Stability data of esprostene alpha lyophilized nanomicelles

[0096]

[0097] Table 3 Stability data table of isuplucin alpha lyophilized nanomicelles

[0098]

[0099] Isuplucin alpha lyophilized nanomicelles were reconstituted and tested for osmolality (general chapter 0632), visible particles (general chapter 0904), bacterial endotoxin (general chapter 1143), sterility (general chapter 1101) according to the Chinese Pharmacopoeia 2020 (Volume 4: General Tests for Injections); succinimidyl ratio and methionine oxidation ratio, deamidation of Asn, N-terminal deletion, Met and Trp residue oxidation were detected by LC-MS / MS; non-fucosylation was monitored by HILIC-HPLC;

[0100] The isuplucin alpha nanomicelles prepared by the embodiment 1 of the present application showed good anti-impurity and degradation, aggregation risk after reconstitution of the product quality analysis and animal experiments, reduced the production of various non-fucosylation, basic components, succinimidyl, methionine oxidation, N-terminal deletion, Asn deamidation, Met and Trp residue oxidation impurities affecting the quality of the product, for example, for N-terminal deletion, the biosynthesis of proteins starts from the N-terminal, the composition of the N-terminal sequence has a huge impact on biological function, such as the half-life of the protein, the localization of the protein in the organelle, etc., N-terminal deletion may affect the activity of the product, and non-fucosylation will enhance the binding with FcγRllla to improve the ADCC effect, the basic components are mainly derived from the oxidation of succinimidyl and methionine. Asn deamidation is one of the most common post-translational modifications in monoclonal antibodies, which usually forms 3:1 isoAsp and Asp, isoAsp and Asp will both result in negative charge, which may affect the structure and stability of the antibody, deamidation occurring in the CDR region or protein binding region of the antibody may reduce the affinity of the protein, in addition to the influence on activity, the product isoAsp of deamidation will also bring immunogenicity.

[0101] The preparation process used in embodiment 1 can reduce the generation of isuplucinol alpha impurity products during the freeze-drying production, storage and reconstitution of isuplucinol alpha nanometer freeze-dried micelles, thereby obtaining a nanometer micelle with stable use quality. The isuplucinol alpha nanometer freeze-dried micelles of embodiment 1 rely on the branched joint synergistic protection of arginine and cysteine, and simultaneously combine with the stable drug carrier micelle skeleton composed of polylactic acid-glycolic acid, sodium phosphatidylglycerol, and a film-making production method to make the isuplucinol alpha nanometer micelle system after reconstitution exhibit better stability in body fluids

[0102] Example 4

[0103] Forty male, 8-week-old CD1 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 5 groups, 8 animals in each group (n = 8), and each group was:

[0104] 1) Group a: injection of isuplucinol alpha nanometer micelle injection prepared in embodiment 1 (prepared with normal saline for injection before use);

[0105] 2) Group b: injection of isuplucinol alpha nanometer micelle injection prepared in Comparative Example 1 (prepared with normal saline for injection before use);

[0106] 3) Group c: injection of isuplucinol alpha nanometer micelle injection prepared in Comparative Example 2 (prepared with normal saline for injection before use);

[0107] 3) Group d: injection of isuplucinol alpha nanometer micelle injection prepared in Comparative Example 4 (prepared with normal saline for injection before use);

[0108] 4) Group e: injection of isuplucinol alpha polysorbate 80 stock solution (isuplucinol alpha concentration 5.0 mg / ml, polysorbate 80 concentration 0.02%, solvent normal saline for injection).

[0109] The animals were fasted overnight, and a single intraperitoneal injection of embodiment 1, Comparative Examples 1, 2 and 4, and isuplucinol alpha polysorbate 80 stock solution (all based on 300 μg / kg of isuplucinol alpha) was performed, and 30 minutes later, intraperitoneal injection of glucose 3.1 g / kg was given. Blood was collected from the tail vein at the time points after the first injection of glucose (0, 15, 30, 60, 90, 120 minutes), and 72 hours after the single intraperitoneal injection of the fusion protein, the mice were given glucose again (3.1 g / kg), and blood was collected from the tail vein at the time points after the second injection of glucose (0, 15, 30, 60, 90, 120 minutes). The blood glucose was detected by a blood glucose meter (Bayer), and the results of the intraperitoneal glucose tolerance test (IGTT) are shown in Table 4.

[0110] Table 4 Blood glucose values at each time point after 0h injection of glucose

[0111]

[0112] Table 5 Blood glucose values at each time point after 72h injection of glucose

[0113]

[0114] Example 1 prepared Isulina peptide alpha nanomicelles by film forming process, the nanomicelles have freeze-drying protection, redissolution stability, in vivo long circulation, and good sustained-release, etc. The nanomicelle system enhances the hypoglycemic effect after 72h injection, and has the advantages of freeze-drying protection, redissolution stability, in vivo long circulation and film forming enhancement, etc. Compared with conventional non-micellar preparations, it has been significantly improved.

[0115] The method of the present application has been described by preferred embodiments, and the related personnel can obviously make changes or appropriate changes and combinations to the method and application described herein within the content, spirit and scope of the present application to realize and apply the present application technology. Those skilled in the art can refer to the content herein to appropriately improve the process parameters to realize. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application.

Claims

1. A method for preparing insulin glargine alpha nanofreezed micelles, comprising the steps of: S1: mixing arginine, cysteine, alcohol and water to obtain a protective solution; S2: mixing a polysorbate 80 stock solution of insulin glargine alpha and the protective solution to obtain a drug protective solution of insulin glargine alpha; S3: putting polylactic acid-glycolic acid copolymer and sodium phosphatidylglycerol into alcohol to obtain a mixed solution, removing alcohol from the mixed solution to form a polymer-phospholipid film; S4: adding the drug protective solution of insulin glargine alpha to the polymer-phospholipid film, dispersing once, removing alcohol, adding water, dispersing twice to obtain a solution of insulin glargine alpha nanomicelles; and S5: freeze-drying the solution of insulin glargine alpha nanomicelles to obtain insulin glargine alpha nanofreezed micelles. In the step S1, the mass of arginine added per 1 L of water is 2.0-4.0 g; and / or, the mass of cysteine added per 1 L of water is 1.0-2.0 g; and / or, the alcohol is selected from any one of methanol, ethanol and isopropanol; and / or, the volume of isopropanol added per 1 L of water is 0.7-1.4 L. In the step S2, the polysorbate 80 stock solution of insulin glargine alpha per 1 L includes 8-12 g of insulin glargine alpha and 10-30 mg of polysorbate 80, and the solvent is water for injection; and / or, 8-10 volumes of the polysorbate 80 stock solution of insulin glargine alpha is mixed with 8-9 volumes of the protective solution. In the step S3, the mass ratio of polylactic acid-glycolic acid copolymer to sodium phosphatidylglycerol is (1.8-2.2):(0.9-1.3); and / or, the alcohol is selected from any one or mixture of ethanol, n-propanol and isopropanol; and / or, the mixed solution is evaporated under reduced pressure in a rotary evaporator under the condition of a water bath at 30-50℃. In the step S4, the drug protective solution of insulin glargine alpha is added to the polymer-phospholipid film by dropwise addition; and / or, after dropwise addition, the mixture is kept at 20-25℃ for 30-50 min; and / or, the first dispersion is ultrasonic dispersion at 15-35 kHz for 10-20 min; and / or, the alcohol is removed by rotary evaporation under reduced pressure at 25-35℃ for 40-50 min; and / or, the second dispersion is mechanical shaking at a speed of 30-40 rpm and centrifugation for 10-15 min. In the step S5, the freeze-drying of the solution of insulin glargine alpha nanomicelles is performed by: Step 1: diluting the solution of insulin glargine alpha nanomicelles with water for injection and filling into a container; Step 2: placing the filled container into a freeze-drying machine under a controlled vacuum, keeping at a first temperature for a first time, and then cooling to a second temperature for a second time; Step 3: keeping the temperature of the container to a third temperature for a third time, and the freeze-drying is completed to obtain insulin glargine alpha nanofreezed micelles. ​ ​ ​ ​ ​ 2. The method of claim 1, wherein the esprostene alpha nanofroze gel micelles are prepared by the process comprising: ​ 3. The method of claim 1, wherein the method is performed by a process comprising: ​ 4. The method of claim 1, wherein the method is for preparing esupergavir peptide alpha nanofroze lyophilized micelles. ​ 5. The method for preparing eosopragutin α-nano-lyophilized micelles according to claim 1, characterized in that, ​ 6. The method of claim 1, wherein the isuplumb pegol alpha nanofroze gelmicelle is prepared by, ​ ​ ​ ​ 7. The method of claim 6, wherein the method further comprises the step of lyophilizing the nanogels.

8. The method of claim 6, wherein the method further comprises the step of lyophilizing the nanogels. In the step 1, the water for injection is used to make up the volume to a concentration of 2.0-4.0 mg / mL of esprogliflopxin alpha; and / or, the container in the step 1 is a vial.

8. The method for preparing eosopragutin α-nano-lyophilized micelles according to claim 6, characterized in that, In the step 2, the vacuum degree is controlled to 0.24-0.35 mbar; and / or, in the step 2, the first temperature is 5-8℃; and / or, in the step 2, the first time is 1-2 h; and / or, in the step 2, the second temperature is -50--60℃; and / or, in the step 2, the second time is 10-14 h.

9. The method for preparing eosopragutin α-nano-lyophilized micelles according to claim 6, characterized in that, In the step 3, the third temperature is 6℃-10℃; and / or, in the step 3, the third time is 3-5 h.

10. The esprogliflopxin alpha nanofrozen lyophilized micelles prepared by the method of any one of claims 1-9.

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