Polypeptide drug sustained release microsphere and pharmaceutical preparation, preparation method and application thereof

Abstract

The invention provides a sustained-release microsphere of a polypeptide drug, and a pharmaceutical preparation, a preparation method and application thereof. The preparation method comprises the following steps: forming an inner water phase; forming an oil phase; forming an outer water phase; and adding the inner water phase into the oil phase for dispersion to form primary emulsion, then adding the primary emulsion into the outer water phase for re-dispersion to form pre-multiple emulsion, then emulsifying the pre-multiple emulsion to form multiple emulsion, and finally curing the multiple emulsion to obtain the sustained-release microspheres. The preparation method is simple and convenient to operate, the drug release behavior of the obtained sustained release microspheres is ideal (low burst release in the early stage and stable release in the middle and later stages), blood sugar is reduced safely and effectively, long-term stable medication can be achieved, and patient compliance is high.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical agents, and relates to a sustained-release microsphere of a polypeptide drug, its pharmaceutical preparation, preparation method and use. Background Art

[0002] Type 2 diabetes is a disease characterized by high blood glucose levels and is associated with insulin resistance and relative insulin deficiency. Although it is usually initially controlled by increasing exercise and adjusting diet, as the disease progresses, drug treatment is usually required.

[0003] GLP-1 (glucagon-like peptide-1) is an incretin naturally present in the human body. Incretins are a group of metabolic hormones that promote a decrease in blood glucose levels, are released after eating, and increase the secretion of insulin released by pancreatic beta cells through a glucose-dependent mechanism. GLP-1 exerts its corresponding hypoglycemic effect by activating widely distributed GLP-1 receptors (GLP-1R), but it is extremely easily degraded. Liraglutide and semaglutide developed by Novo Nordisk are obtained by amino acid substitution and side-chain modification based on natural GLP-1, have a high amino acid sequence homology with natural human GLP-1, and have half-lives of approximately 12 hours and 160 hours respectively, and are injected once a day / week. Blood glucose control needs to be maintained long-term, but long-term injection of drugs results in poor patient compliance and low medication accuracy.

[0004] Microsphere formulations encapsulating drugs can utilize carrier materials to extend the drug efficacy time, improve patient compliance, optimize the therapeutic effect, and enhance medication accuracy. Microsphere formulations are widely used in the development of long-acting sustained-release injections. Currently, the application scopes of the five types of microsphere formulations already on the market in China include fields such as malignant tumors, diabetes, cardiovascular diseases, and mental diseases. With the expansion of the chronic disease patient population, the demand for related drugs is vast. Therefore, developing microsphere formulations with obvious advantages and applying them in the fields related to chronic diseases can effectively improve the health and medication experience of the corresponding patient groups and greatly enhance the accuracy of relevant clinical medications.

[0005] To overcome the problem of the short in vivo half-life of natural GLP-1, various strategies have been adopted to extend the drug half-life in the synthesis of GLP-1R agonist molecules. Among them, liraglutide and semaglutide have adopted the strategy of fatty acid acylation of specific amino residues. The fatty acid side chain is used to promote molecular self-association, thereby prolonging the diffusion time of the drug from the injection site. In addition, the fatty acid molecule can reversibly and non-covalently bind to albumin, slowing down renal clearance. However, since the microsphere preparation needs to embed the drug dose for one month or even several months, a high-concentration drug solution must be used as the internal aqueous phase during the preparation process. Liraglutide and semaglutide are prone to aggregation and even form drug macromolecular fibers under high-concentration conditions. The drug fibers will not only cause loss of drug activity but also necrosis of the surrounding tissues, resulting in drug toxicity and immunogenicity problems [1-2] . Therefore, aiming at the properties of the raw material drug, how to avoid a large amount of drug aggregation and fiber formation in the long-acting sustained-release preparation is a major challenge.

[0006] In addition, due to the amphiphilic nature of liraglutide and semaglutide molecules, during the preparation of the emulsion, a large amount of drug will be dispersed at the oil-water interface. With the rapid volatilization of the solvent during the solidification process, a large number of primary emulsion droplets will coalesce, and the drug will also migrate to the outside of the microsphere shell layer during this process, resulting in too fast release and too high Cmax at the initial stage of microsphere release, which will affect safety and make it difficult to maintain the long-term stable release of the drug in the sustained-release preparation. How to achieve low burst release and long-term stable release of active drugs in the sustained-release preparation is a difficult problem faced by such amphiphilic drugs among GLP-1R agonists at present.

[0007] [1]Zapadka KL,Becher FJ,Gomes Dos Santos AL,Jackson SE,Factorsaffecting the physical stability(aggregation)of peptide therapeutics[J],Interface Focus,2017.

[0008] [2]Venanzi M,Savioli M,Cimino R,et al.,Aspectroscopic and moleculardynamics study on the aggregation process of a long-acting lipidatedtherapeutic peptide:the case of Semaglutide[J],Soft Matter,2020. Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] In view of the above problems, the present invention aims to provide a microsphere preparation of a GLP-1R agonist with low initial burst release, stable release in the middle and late stages, which can be used for long-term medication, has high compliance and is easy to operate.

[0011] Solutions for Solving the Problems

[0012] In a first aspect, the present invention provides a method for preparing a sustained-release microsphere of a polypeptide drug, which comprises the following steps:

[0013] Mix an aggregate inhibitor, an emulsion stabilizer A and water, adjust the pH value of the mixture with a pH regulator A, and then add the polypeptide drug thereto to form an inner aqueous phase;

[0014] Mix a carrier material, an emulsion stabilizer B and an organic solvent to form an oil phase;

[0015] Mix a surfactant, an osmotic pressure regulator and water, and adjust the pH value of the mixture with a pH regulator B to form an outer aqueous phase; and,

[0016] Add the inner aqueous phase to the oil phase for dispersion to form a primary emulsion, then add the primary emulsion to the outer aqueous phase for re-dispersion to form a pre-compound emulsion, then emulsify the pre-compound emulsion to form a compound emulsion, and finally solidify the compound emulsion to obtain the sustained-release microsphere.

[0017] As a further improvement of the present invention, the polypeptide drug is a polypeptide GLP-1R agonist, preferably at least one of liraglutide and semaglutide, more preferably semaglutide.

[0018] As a further improvement of the present invention, the aggregate inhibitor is at least one of sodium caprylate, sucrose, mannitol, 8-(2-hydroxybenzamido)-sodium caprylate (SNAC), phenol, benzyl alcohol, ethanol and trifluoroethanol, preferably sucrose.

[0019] As a further improvement of the present invention, the weight ratio of the aggregate inhibitor to the polypeptide drug is 1:70 to 50:70, preferably 27:70.

[0020] As a further improvement of the present invention, the emulsion stabilizer A is at least one of tween, poloxamer, sodium oleate and dextran, preferably sodium oleate.

[0021] As a further improvement of the present invention, the weight ratio of the emulsion stabilizer A to the polypeptide drug is 1:200 to 3:2, preferably 9:1400.

[0022] As a further improvement of the present invention, the water is at least one of ultrapure water, double-distilled water and water for injection, preferably ultrapure water.

[0023] As a further improvement of the present invention, the dosage ratio of the water to the polypeptide drug is 3 mL: 10 - 650 mg, preferably 3 mL: 70 mg.

[0024] As a further improvement of the present invention, the pH regulator A is at least one of hydrogen chloride, sodium hydroxide, and phosphate.

[0025] As a further improvement of the present invention, the pH regulator A adjusts the pH value of the mixture to 6.0 - 9.5, preferably 7.6.

[0026] As a further improvement of the present invention, the organic solvent is at least one of chloroform, dichloromethane, ethyl acetate, acetone, and acetonitrile, preferably dichloromethane.

[0027] As a further improvement of the present invention, the dosage ratio of the organic solvent to the polypeptide drug is 1 - 10 mL: 50 mg, preferably 1 mL: 7 mg.

[0028] As a further improvement of the present invention, the carrier material is a biodegradable carrier material.

[0029] As a further improvement of the present invention, the carrier material is at least one of poly (lactic - co - glycolic acid) (PLGA), poly (lactic - co - polyethylene glycol) (PELA), poly (lactic acid) (PLA), polyhydroxyalkanoates (PHA), and polycaprolactone (PCL), preferably poly (lactic - co - glycolic acid), and more preferably poly (lactic - co - glycolic acid) with a ratio of lactic acid to glycolic acid of 100 - 50: 0 - 50, preferably 50:50 per 100 parts of monomers.

[0030] As a further improvement of the present invention, the molecular weight of the carrier material is 1000 - 150000 Da, preferably 20000 Da.

[0031] As a further improvement of the present invention, the weight ratio of the carrier material to the polypeptide drug is 3:1 - 100:1, preferably 240:7.

[0032] As a further improvement of the present invention, the emulsion stabilizer B is at least one of lecithin, soy lecithin, oleic acid, and span, preferably at least one of span 83 (sorbitan sesquioleate) and span 85 (sorbitan trioleate), and more preferably span 85.

[0033] As a further improvement of the present invention, the weight ratio of the emulsion stabilizer B to the polypeptide drug is 1:20 - 40:20, preferably 3:14.

[0034] As a further improvement of the present invention, the surfactant is at least one of polyvinyl alcohol (PVA), Tween, sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), and polyoxyethylene octylphenol ether, preferably Tween, and more preferably Tween 20.

[0035] As a further improvement of the present invention, the weight ratio of the surfactant to the polypeptide drug is 1:20 to 40:20, preferably 3:28.

[0036] As a further improvement of the present invention, the osmotic pressure regulator is at least one of sodium chloride and potassium chloride, preferably sodium chloride.

[0037] As a further improvement of the present invention, the weight ratio of the osmotic pressure regulator to the polypeptide drug is 1:1 to 100:1, preferably 80:1.

[0038] As a further improvement of the present invention, the water is at least one of ultrapure water, double-distilled water, and water for injection, preferably ultrapure water.

[0039] As a further improvement of the present invention, the dosage ratio of the water to the polypeptide drug is 0.001 to 10 mL:1 mg, preferably 50 mL:7 mg.

[0040] As a further improvement of the present invention, the pH regulator B is at least one of hydrogen chloride, sodium hydroxide, and phosphate.

[0041] As a further improvement of the present invention, the pH regulator B adjusts the pH value of the mixture to 6.0 to 9.5, preferably 7.8.

[0042] As a further improvement of the present invention, the dispersion is completed by at least one of stirring, homogenization, and ultrasonic treatment, preferably homogenization, and more preferably high-pressure homogenization.

[0043] As a further improvement of the present invention, the time of high-pressure homogenization is 0.5 to 20 min, preferably 2 min.

[0044] As a further improvement of the present invention, the pressure of high-pressure homogenization is 0.2 to 1200 bar, preferably 1 bar.

[0045] As a further improvement of the present invention, the redispersion is completed by at least one of stirring and homogenization, preferably stirring.

[0046] As a further improvement of the present invention, the emulsification is completed by membrane emulsification.

[0047] As a further improvement of the present invention, the membrane pore size of the membrane emulsification is 1.0 to 60 μm, preferably 40 μm.

[0048] As a further improvement of the present invention, the transmembrane pressure for membrane emulsification is 1 to 200 kPa, preferably 50 kPa.

[0049] As a further improvement of the present invention, the curing is achieved by volatilization or evaporation, preferably by reduced-pressure volatilization or thin-film evaporation, and more preferably by thin-film evaporation.

[0050] As a further improvement of the present invention, the time for thin-film evaporation is 1 to 50 min, preferably 10 min.

[0051] As a further improvement of the present invention, the temperature for thin-film evaporation is 25 to 50 °C, preferably 35 °C.

[0052] As a further improvement of the present invention, the number of cycles for thin-film evaporation is 1 to 6 times, preferably 3 times.

[0053] As a further improvement of the present invention, the preparation method further includes washing and freeze-drying after curing the multiple emulsion.

[0054] In a second aspect, the present invention provides a sustained-release microsphere for a polypeptide drug, which is prepared by using the above preparation method.

[0055] In a third aspect, the present invention provides a pharmaceutical preparation for a polypeptide drug, which contains the above sustained-release microsphere.

[0056] As a further improvement of the present invention, the pharmaceutical preparation further contains at least one pharmaceutically acceptable excipient.

[0057] As a further improvement of the present invention, the pharmaceutical preparation is a sustained-release preparation.

[0058] As a further improvement of the present invention, the pharmaceutical preparation is an injection.

[0059] In a fourth aspect, the present invention provides the use of the above sustained-release microsphere or the above pharmaceutical preparation in the preparation of a drug for preventing and / or treating at least part of the diseases and / or disorders related to GLP-1R.

[0060] As a further improvement of the present invention, the at least part of the diseases and / or disorders related to GLP-1R are diabetes, weight loss or non-alcoholic fatty liver, preferably type 2 diabetes or weight loss.

[0061] Effects of the Invention

[0062] The beneficial effects of the present invention are as follows: The preparation method of the present invention is simple to operate. The release behavior of the obtained sustained-release microspheres and preparations is stable, with low initial burst release, avoiding the hypoglycemia risk caused by the relatively high initial drug concentration of GLP-1R agonist drugs. The drug release behavior is stable in the middle and late stages, and it is safe and effective in reducing blood sugar. As a long-acting sustained-release microsphere preparation, it is conducive to long-term and stable medication and improves patient compliance. Description of the Drawings

[0063] Figure 1 is the light microscope of the primary emulsion in Example 1 and Comparative Examples 1 and 2.

[0064] Figure 2 is the emulsion and solidification process of Example 1 and Comparative Examples 1 and 2.

[0065] Figure 3 is the in vitro release trend of the microspheres in Example 1 and Comparative Examples 1, 2, and 3.

[0066] Figure 4 is the in vivo PK test result of Example 1 in rats.

[0067] Figure 5 is the in vivo PK test result of Comparative Examples 1 and 2 in rats.

[0068] Figure 6 is the aggregation situation of GLP-1 and the effect of the inhibitor.

[0069] Figure 7 is the particle size investigation of three batches of repeatability in Example 1. Detailed Embodiments

[0070] The following will detail various exemplary embodiments, features, and aspects of the present invention. The special word "exemplary" here means "serving as an example, embodiment, or illustration". Any embodiment described as "exemplary" here does not necessarily have to be construed as superior to or better than other embodiments.

[0071] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that the present invention can be implemented without certain specific details. In other instances, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0072] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the systematic errors inevitable in industrial production.

[0073] In this specification, the meaning expressed by using "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0074] In this specification, "some specific / preferred embodiments", "some other specific / preferred embodiments", "embodiments", etc. refer to the specific elements (e.g., features, structures, properties, and / or characteristics) related to the embodiment, which are included in at least one of the embodiments described herein, and may or may not exist in other embodiments. Additionally, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0075] In this specification, the numerical range represented by "numerical value A to numerical value B" refers to the range that includes the endpoint numerical values A and B.

[0076] In this specification, when "normal temperature" or "room temperature" is used, the temperature can be 10 - 40 °C.

[0077] Sustained Release Microspheres of Polypeptide Drugs and Preparation Method Thereof

[0078] The present invention provides a method for preparing a sustained-release microsphere of a polypeptide drug, which comprises the following steps:

[0079] Mix an aggregate inhibitor, an emulsion stabilizer A, and water, and adjust the pH value of the mixture using a pH regulator A, then add the polypeptide drug thereto to form an inner aqueous phase;

[0080] Mix a carrier material, an emulsion stabilizer B, and an organic solvent to form an oil phase;

[0081] Mix a surfactant, an osmotic pressure regulator, and water, and adjust the pH value of the mixture using a pH regulator B to form an outer aqueous phase; and,

[0082] Add the inner aqueous phase to the oil phase for dispersion to form a primary emulsion, then add the primary emulsion to the outer aqueous phase for re-dispersion to form a pre-compound emulsion, then emulsify the pre-compound emulsion to form a compound emulsion, and finally solidify the compound emulsion to obtain the sustained-release microsphere.

[0083] The polypeptide drug (especially polypeptide GLP-1R agonists, such as liraglutide or semaglutide) in this preparation method is prone to aggregation at high concentrations, even forming high-molecular fibers, thereby losing its activity and causing problems of drug toxicity and immunogenicity. Therefore, it is particularly important to maintain the monomer form of the drug during the preparation. Additionally, due to the amphiphilic nature of the drug molecule, the drug will migrate to the outside of the spherical shell layer during the preparation, resulting in too fast release at the initial stage of microsphere release, thus causing too high drug concentration in the early stage and making it difficult to release over a long period. Therefore, avoiding a large amount of drug dispersion at the oil-water interface is of great significance for the preparation.

[0084] By adding an aggregate inhibitor and regulating the pH value in the inner aqueous phase, the present invention controls drug aggregation, inhibits drug fibroblast formation, and avoids a decrease in drug activity. At the same time, emulsion stabilizers are added to the inner aqueous phase and the oil phase respectively, thereby regulating the molecular liquid film at the oil-water interface of the double emulsion, affecting the distribution of amphiphilic drugs in the emulsion, and ensuring uniform dispersion of the primary emulsion during the curing process, effectively inhibiting the large-scale migration of drug molecules as the organic solvent volatilizes and is removed, enabling the drug to be uniformly dispersed in the carrier material, and avoiding the migration of the drug to the oil-in-water interface of the double emulsion due to the isoelectric point factor by adjusting the pH value of the outer aqueous phase, effectively reducing the burst release of the drug and maintaining the stable release behavior of the drug.

[0085] To clearly illustrate the preparation method of the sustained-release microspheres of the polypeptide drugs of the present invention, the following takes the semaglutide sustained-release microspheres as an example for detailed description, which is also applicable to polypeptide drug active ingredients (such as liraglutide) that mainly bind to albumin and GLP-1 receptors in the form of monomers after entering the blood.

[0086] Furthermore, the present invention provides a method for preparing sustained-release microspheres of semaglutide, which comprises the following steps:

[0087] Mix an aggregate inhibitor, emulsion stabilizer A and water, and use pH regulator A to adjust the pH value of the mixture, and then add semaglutide thereto to form an inner aqueous phase;

[0088] Mix a carrier material, emulsion stabilizer B and an organic solvent to form an oil phase;

[0089] Mix a surfactant, an osmotic pressure regulator and water, and use pH regulator B to adjust the pH value of the mixture to form an outer aqueous phase; and,

[0090] Add the inner aqueous phase to the oil phase for dispersion to form a primary emulsion, then add the primary emulsion to the outer aqueous phase for re-dispersion to form a pre-double emulsion, then emulsify the pre-double emulsion to form a double emulsion, and finally cure the double emulsion to obtain semaglutide sustained-release microspheres.

[0091] In one embodiment of the present invention, the aggregate inhibitor in the above preparation method may be at least one of sodium octanoate, sucrose, mannitol, 8-(2-hydroxybenzamido)-sodium octanoate, phenol, benzyl alcohol, ethanol and trifluoroethanol, such as sucrose, mannitol or a mixture obtained by mixing the two in any proportion.

[0092] In one embodiment of the present invention, the aggregate inhibitor in the above preparation method may be sucrose.

[0093] In one embodiment of the present invention, the weight ratio of the aggregate inhibitor to semaglutide in the above preparation method can vary within a certain range, for example, 1:70 to 50:70.

[0094] In one embodiment of the present invention, the weight ratio of the aggregate inhibitor to semaglutide in the above preparation method can be 27:70.

[0095] In one embodiment of the present invention, the emulsion stabilizer A in the above preparation method can be at least one of Tween, poloxamer, sodium oleate, and dextran, for example, a mixture obtained by mixing Tween, sodium oleate, or both in any proportion.

[0096] In one embodiment of the present invention, the emulsion stabilizer A in the above preparation method can be sodium oleate.

[0097] In one embodiment of the present invention, the weight ratio of the emulsion stabilizer A to semaglutide in the above preparation method can vary within a certain range, for example, 1:200 to 3:2.

[0098] In one embodiment of the present invention, the weight ratio of the emulsion stabilizer A to semaglutide in the above preparation method can be 9:1400.

[0099] In one embodiment of the present invention, the water used for the inner aqueous phase in the above preparation method can be at least one of ultrapure water, double-distilled water, and water for injection, for example, a mixture obtained by mixing ultrapure water, double-distilled water, or both in any proportion.

[0100] In one embodiment of the present invention, the water used for the inner aqueous phase in the above preparation method can be ultrapure water.

[0101] In one embodiment of the present invention, the dosage ratio of the water used for the inner aqueous phase to semaglutide in the above preparation method can vary within a certain range, for example, 3 mL:10 to 650 mg.

[0102] In one embodiment of the present invention, the dosage ratio of the water used for the inner aqueous phase to semaglutide in the above preparation method can be 3 mL:70 mg.

[0103] In one embodiment of the present invention, the pH regulator A in the above preparation method can be at least one of hydrogen chloride, sodium hydroxide, and phosphate, for example, a mixture obtained by mixing hydrogen chloride, phosphate, or both in any proportion.

[0104] In one embodiment of the present invention, the pH regulator A in the above preparation method can adjust the pH value of the mixture within a certain range, for example, 6.0 to 9.5.

[0105] In one embodiment of the present invention, pH regulator A in the above preparation method can adjust the pH value of the mixture to 7.6.

[0106] In one embodiment of the present invention, the organic solvent in the above preparation method can be at least one of chloroform, dichloromethane, ethyl acetate, acetone and acetonitrile, such as dichloromethane, ethyl acetate or a mixture obtained by mixing the two in any ratio.

[0107] In one embodiment of the present invention, the organic solvent in the above preparation method can be dichloromethane.

[0108] In one embodiment of the present invention, the dosage ratio of the organic solvent to semaglutide in the above preparation method can vary within a certain range, such as 1 - 10 mL:50 mg.

[0109] In one embodiment of the present invention, the dosage ratio of the organic solvent to semaglutide in the above preparation method can be 1 mL:7 mg.

[0110] In one embodiment of the present invention, the carrier material in the above preparation method can be a biodegradable carrier material.

[0111] In one embodiment of the present invention, the carrier material in the above preparation method can be at least one of poly(lactic - co - glycolic acid), poly(lactic - co - polyethylene glycol), polylactic acid, polyhydroxyalkanoates and polycaprolactone, such as poly(lactic - co - glycolic acid), poly(lactic - co - polyethylene glycol) or a mixture obtained by mixing the two in any ratio.

[0112] In one embodiment of the present invention, the carrier material in the above preparation method can be poly(lactic - co - glycolic acid).

[0113] In one embodiment of the present invention, the ratio of lactic acid to glycolic acid in every 100 parts of monomers in the carrier material in the above preparation method can vary within a certain range, such as 100 - 50:0 - 50, for example, 100:0, 95:5, 85:15, 75:25, 50:50 or other ratios.

[0114] In one embodiment of the present invention, the ratio of lactic acid to glycolic acid in every 100 parts of monomers in the carrier material in the above preparation method can be 50:50.

[0115] In one embodiment of the present invention, the molecular weight of the carrier material in the above preparation method can vary within a certain range, such as 1000 - 150000 Da.

[0116] In one embodiment of the present invention, the molecular weight of the carrier material in the above preparation method can be 20,000 Da.

[0117] In one embodiment of the present invention, the weight ratio of the carrier material to semaglutide in the above preparation method can vary within a certain range, for example, 3:1 to 100:1.

[0118] In one embodiment of the present invention, the weight ratio of the carrier material to semaglutide in the above preparation method can be 240:7.

[0119] In one embodiment of the present invention, the emulsion stabilizer B in the above preparation method can be at least one of lecithin, soybean phospholipid, oleic acid, and span, for example, a mixture obtained by mixing soybean phospholipid, span, or both in any proportion.

[0120] In one embodiment of the present invention, the emulsion stabilizer B in the above preparation method can be span 83, span 85, or a mixture obtained by mixing both in any proportion.

[0121] In one embodiment of the present invention, the emulsion stabilizer B in the above preparation method can be span 85.

[0122] In one embodiment of the present invention, the weight ratio of the emulsion stabilizer B to semaglutide in the above preparation method can vary within a certain range, for example, 1:20 to 40:20.

[0123] In one embodiment of the present invention, the weight ratio of the emulsion stabilizer B to semaglutide in the above preparation method can be 3:14.

[0124] In one embodiment of the present invention, the surfactant in the above preparation method can be at least one of polyvinyl alcohol, tween, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and polyoxyethylene octylphenol ether, for example, polyvinyl alcohol, tween, or a mixture obtained by mixing both in any proportion.

[0125] In one embodiment of the present invention, the surfactant in the above preparation method can be tween.

[0126] In one embodiment of the present invention, the surfactant in the above preparation method can be tween 20.

[0127] In one embodiment of the present invention, the weight ratio of the surfactant to semaglutide in the above preparation method can vary within a certain range, for example, 1:20 to 40:20.

[0128] In one embodiment of the present invention, the weight ratio of the surfactant to semaglutide in the above preparation method can be 3:28.

[0129] In one embodiment of the present invention, the osmotic pressure regulator in the above preparation method can be sodium chloride, potassium chloride, or a mixture obtained by mixing the two in any ratio.

[0130] In one embodiment of the present invention, the osmotic pressure regulator in the above preparation method can be sodium chloride.

[0131] In one embodiment of the present invention, the weight ratio of the osmotic pressure regulator to semaglutide in the above preparation method can vary within a certain range, such as 1:1 to 100:1.

[0132] In one embodiment of the present invention, the weight ratio of the osmotic pressure regulator to semaglutide in the above preparation method can be 80:1.

[0133] In one embodiment of the present invention, the water used in the outer aqueous phase in the above preparation method can be at least one of ultrapure water, double-distilled water, and water for injection, such as ultrapure water, double-distilled water, or a mixture obtained by mixing the two in any ratio.

[0134] In one embodiment of the present invention, the water used in the outer aqueous phase in the above preparation method can be ultrapure water.

[0135] In one embodiment of the present invention, the dosage ratio of the water used in the outer aqueous phase to semaglutide in the above preparation method can vary within a certain range, such as 0.001 to 10 mL:1 mg.

[0136] In one embodiment of the present invention, the dosage ratio of the water used in the outer aqueous phase to semaglutide in the above preparation method can be 50 mL:7 mg.

[0137] In one embodiment of the present invention, the pH regulator B in the above preparation method can be at least one of hydrogen chloride, sodium hydroxide, and phosphate, such as hydrogen chloride, phosphate, or a mixture obtained by mixing the two in any ratio.

[0138] In one embodiment of the present invention, the pH regulator B in the above preparation method can adjust the pH value of the mixture within a certain range, such as 6.0 to 9.5.

[0139] In one embodiment of the present invention, the pH regulator B in the above preparation method can adjust the pH value of the mixture to 7.8.

[0140] In one embodiment of the present invention, the dispersion in the above preparation method can be carried out by at least one of stirring, homogenization, and ultrasonication, such as stirring, homogenization, or a combination of the two for dispersion.

[0141] In one embodiment of the present invention, the dispersion method in the above preparation method can be homogenization.

[0142] In one embodiment of the present invention, the dispersion method in the above preparation method can be high-pressure homogenization.

[0143] In one embodiment of the present invention, the time of high-pressure homogenization in the above preparation method can vary within a certain range, for example, 0.5 to 20 min.

[0144] In one embodiment of the present invention, the time of high-pressure homogenization in the above preparation method can be 2 min.

[0145] In one embodiment of the present invention, the pressure of high-pressure homogenization in the above preparation method can vary within a certain range, for example, 0.2 to 1200 bar.

[0146] In one embodiment of the present invention, the pressure of high-pressure homogenization in the above preparation method can be 1 bar.

[0147] In one embodiment of the present invention, redispersion in the above preparation method can be completed by at least one of stirring and homogenization.

[0148] In one embodiment of the present invention, redispersion in the above preparation method can be completed by stirring.

[0149] In one embodiment of the present invention, emulsification in the above preparation method can be completed by membrane emulsification.

[0150] In one embodiment of the present invention, the membrane pore size of membrane emulsification in the above preparation method can vary within a certain range, for example, 1.0 to 60 μm.

[0151] In one embodiment of the present invention, the membrane pore size of membrane emulsification in the above preparation method can be 40 μm.

[0152] In one embodiment of the present invention, the transmembrane pressure of membrane emulsification in the above preparation method can vary within a certain range, for example, 1 to 200 kPa.

[0153] In one embodiment of the present invention, the transmembrane pressure of membrane emulsification in the above preparation method can be 50 kPa.

[0154] In one embodiment of the present invention, curing in the above preparation method can be completed by volatilization or evaporation.

[0155] In one embodiment of the present invention, curing in the above preparation method can be completed by reduced-pressure volatilization or thin-film evaporation.

[0156] In one embodiment of the present invention, the curing in the above preparation method can be completed by thin-film evaporation.

[0157] In one embodiment of the present invention, the time of thin-film evaporation in the above preparation method can vary within a certain range, for example, 1 to 50 min.

[0158] In one embodiment of the present invention, the time of thin-film evaporation in the above preparation method can be 10 min.

[0159] In one embodiment of the present invention, the temperature of thin-film evaporation in the above preparation method can vary within a certain range, for example, 25 to 50 °C.

[0160] In one embodiment of the present invention, the temperature of thin-film evaporation in the above preparation method can be 35 °C.

[0161] In one embodiment of the present invention, the number of cycles of thin-film evaporation in the above preparation method can vary within a certain range, for example, 1 to 6 times.

[0162] In one embodiment of the present invention, the number of cycles of thin-film evaporation in the above preparation method can be 3 times.

[0163] In one embodiment of the present invention, the above preparation method may further include the following steps: washing and lyophilizing the double emulsion after curing.

[0164] Correspondingly, the present invention provides a sustained-release microsphere of a polypeptide drug, which is prepared by the above preparation method.

[0165] In one embodiment of the present invention, the polypeptide drug in the above sustained-release microsphere can be a polypeptide GLP-1R agonist.

[0166] In one embodiment of the present invention, the polypeptide drug in the above sustained-release microsphere can be semaglutide.

[0167] Pharmaceutical Preparations of Polypeptide Drugs

[0168] The present invention provides a pharmaceutical preparation of a polypeptide drug, which contains the above sustained-release microsphere.

[0169] In one embodiment of the present invention, the above pharmaceutical preparation may further contain at least one pharmaceutically acceptable excipient, such as a solvent, a solubilizer, a cosolvent, etc.

[0170] In one embodiment of the present invention, the above pharmaceutical preparation can be a preparation with a specific drug release behavior, such as a sustained-release preparation.

[0171] In one embodiment of the present invention, the above pharmaceutical preparation can be a parenteral dosage form, such as an injection.

[0172] Medical Use of Sustained Release Microspheres and Their Pharmaceutical Preparations

[0173] The present invention provides the use of the above sustained-release microspheres or the above pharmaceutical preparation in the preparation of a drug for preventing and / or treating at least partially GLP-1R-related diseases and / or disorders.

[0174] In one embodiment of the present invention, the above at least partially GLP-1R-related diseases and / or disorders can be diabetes, weight loss, or non-alcoholic fatty liver.

[0175] In one embodiment of the present invention, the above at least partially GLP-1R-related diseases and / or disorders can be type 2 diabetes or weight loss.

[0176] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0177] Example 1

[0178] Weigh 90 mg of sucrose and 1.5 mg of sodium oleate and dissolve them in 10 mL of ultrapure water. Adjust the pH to 7.6. Take 70 mg of semaglutide and dissolve it in 3 mL of this solution to obtain W1. Weigh 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and 15 mg of Span85, dissolve them in 10 mL of dichloromethane to prepare O. Prepare a 500 mL solution of 1.5 g / 100 mL of Tween 20 and 0.2 g / 100 mL of sodium chloride as the external aqueous phase, and adjust its pH to 7.8 to obtain W2. Add W1 to O and disperse it into primary emulsion by high-pressure homogenization for 2 min at a pressure of 1 bar. Add the primary emulsion to W2 and stir to form a pre-compound emulsion. Pour the pre-compound emulsion into a rapid membrane emulsifier to emulsify and disperse it into a compound emulsion with a membrane tube pore size of 40 μm and a transmembrane pressure of 50 kPa. The compound emulsion is solidified by a thin-film evaporator for 10 min at 35 °C for 3 cycles. After solidification, wash and lyophilize to obtain semaglutide-loaded sustained-release microspheres.

[0179] Take an appropriate amount of the microsphere sample, add a small amount of deionized water, disperse it evenly, and add it to a laser particle size distribution analyzer to measure the particle size of the microspheres. The obtained particle size distribution diagram is as Figure 7 .

[0180] Example 2

[0181] Weigh 30 mg of SNAC and 20 mg of poloxamer 188 and dissolve them in 10 mL of ultrapure water. Adjust the pH to 7.0. Take 400 mg of semaglutide and dissolve it in 3 mL of this solution to obtain W1. Weigh 2000 mg of PLGA (LA:GA = 75:25, Mw = 40 kDa) and 65 mg of soybean phospholipid, dissolve them in 10 mL of dichloromethane to prepare O. Prepare 200 mL of a 1.5 g / 100 mL PVA and 2.0 g / 100 mL sodium chloride solution as the outer aqueous phase and adjust its pH to 7.0, which is W2. Add W1 to O and homogenize to disperse into primary emulsion for 5 min at an average rotation speed of 10000 rpm. Add the primary emulsion to W2 and stir to form a pre-complex emulsion. Pour the pre-complex emulsion into a high-speed membrane emulsifier to emulsify and disperse into a complex emulsion. The pore size of the membrane tube used is 20 μm and the transmembrane pressure is 78 kPa. The complex emulsion is solidified by volatilization for 6 hours. After solidification, wash and lyophilize to obtain semaglutide sustained-release microspheres.

[0182] Example 3

[0183] Weigh 30 mg of phenol and 20 mg of poloxamer 188 and dissolve them in 10 mL of ultrapure water. Adjust the pH to 7.0. Take 400 mg of semaglutide and dissolve it in 3 mL of this solution to obtain W1. Weigh 2000 mg of PLGA (LA:GA = 75:25, Mw = 40 kDa) and 65 mg of soybean phospholipid, dissolve them in 10 mL of dichloromethane to prepare O. Prepare 200 mL of a 1.5 g / 100 mL PVA and 2.0 g / 100 mL sodium chloride solution as the outer aqueous phase and adjust its pH to 7.0, which is W2. Add W1 to O and homogenize to disperse into primary emulsion for 5 min at an average rotation speed of 10000 rpm. Add the primary emulsion to W2 and stir to form a pre-complex emulsion. Pour the pre-complex emulsion into a high-speed membrane emulsifier to emulsify and disperse into a complex emulsion. The pore size of the membrane tube used is 20 μm and the transmembrane pressure is 78 kPa. The complex emulsion is solidified by volatilization for 6 hours. After solidification, wash and lyophilize to obtain semaglutide sustained-release microspheres.

[0184] Example 4

[0185] Weigh 50 mg of benzyl alcohol and 50 mg of Tween 20 and dissolve them in 10 mL of ultrapure water. Adjust the pH to 7.4. Take 150 mg of semaglutide and dissolve it in 3 mL of this solution to obtain W1. Weigh 2000 mg of PLGA (LA:GA = 50:50, Mw = 5 kDa) and 300 mg of lecithin, dissolve them in 10 mL of dichloromethane to prepare O. Prepare 250 mL of 1.0 g / 100 mL PVA and 2.0 g / 100 mL sodium chloride solution as the outer aqueous phase, and adjust its pH to 7.0, which is W2. Add W1 to O and homogenize and disperse to form the primary emulsion for 5 min at a mean rotation speed of 12,000 rpm. Add the primary emulsion to W2 and stir to form a pre-multiple emulsion. Pour the pre-multiple emulsion into a high-speed membrane emulsifier to emulsify and disperse it into a multiple emulsion. The pore size of the membrane tube used is 30 μm, and the transmembrane pressure is 62 kPa. The multiple emulsion is solidified by volatilization for 6 hours. After the solidification is completed, wash and lyophilize to obtain semaglutide sustained-release microspheres.

[0186] Comparative Example 1

[0187] Weigh 90 mg of sucrose and dissolve it in 10 mL of ultrapure water. Adjust the pH to 7.6. Take 70 mg of semaglutide and dissolve it in 3 mL of this solution to obtain W1. Weigh 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and dissolve it in 10 mL of dichloromethane to prepare O. Prepare 500 mL of 1.5 g / 100 mL Tween 20 solution as the outer aqueous phase, and adjust its pH to 7.8, which is W2. Add W1 to O and disperse it into the primary emulsion by high-pressure homogenization for 2 min at a pressure of 1 bar. Add the primary emulsion to W2 and stir to form a pre-multiple emulsion. Pour the pre-multiple emulsion into a high-speed membrane emulsifier to emulsify and disperse it into a multiple emulsion. The pore size of the membrane tube used is 40 μm, and the transmembrane pressure is 50 kPa. The multiple emulsion is solidified by a thin-film evaporator for 10 min at 35 °C for 3 cycles. After the solidification is completed, wash and lyophilize to obtain semaglutide sustained-release microspheres.

[0188] Two emulsion stabilizers were added in Example 1, while no emulsion stabilizer was added in Comparative Example 1. Through Figure 1 It can be seen that the primary emulsion particles of the semaglutide sustained-release microspheres in Example 1 are fine and can be densely distributed in the emulsion with small particle sizes. The primary emulsion particles of the semaglutide sustained-release microspheres in the comparative example are large and uneven. It shows that adding an emulsion stabilizer in the preparation of the present invention is necessary for forming a uniformly dispersed primary emulsion.

[0189] Comparative Example 2

[0190] Weigh 90 mg of sucrose and dissolve it in 10 mL of ultrapure water. Take 70 mg of semaglutide and dissolve it in 3 mL of this solution to obtain W1. Weigh 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and 60 mg of Span85, dissolve them in 10 mL of dichloromethane to prepare O. Prepare 500 mL of a 1.5 g / 100 mL Tween 20 solution as the external aqueous phase and adjust its pH to 7.8, which is W2. Add W1 to O and disperse it into primary emulsion by high-pressure homogenization for 2 min at a pressure of 1 bar. Add the primary emulsion to W2 and stir to form a pre-multiple emulsion. Pour the pre-multiple emulsion into a rapid membrane emulsifier to emulsify and disperse it into a multiple emulsion with a membrane tube pore size of 40 μm and a transmembrane pressure of 50 kPa. The multiple emulsion is solidified by a thin-film evaporator for 10 min at 35 °C for 3 cycles. After solidification, wash and lyophilize to obtain semaglutide sustained-release microspheres.

[0191] In Example 1, two emulsion stabilizers were added, while in Comparative Example 2, only emulsion stabilizer B was added. By Figure 1 It can be seen that compared with the fine and uniform primary emulsion of the semaglutide sustained-release microspheres in Example 1, the primary emulsion of the semaglutide sustained-release microspheres in Comparative Example 2 is unevenly distributed, rapidly coalesces after preparing the emulsion, and a large number of large chambers are formed in the emulsion. This indicates that adding a single emulsion stabilizer in the preparation of the present invention cannot make the emulsion evenly distributed.

[0192] Comparative Example 3

[0193] Dissolve 70 mg of semaglutide in 3 mL of ultrapure water to obtain W1, and adjust the pH to 7.6. Weigh 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and 15 mg of Span85, dissolve them in 10 mL of dichloromethane to prepare O. Prepare 500 mL of a 1.5 g / 100 mL Tween 20 solution as the external aqueous phase and adjust its pH to 7.8, which is W2. Add W1 to O and disperse it into primary emulsion by high-pressure homogenization for 2 min at a pressure of 1 bar. Add the primary emulsion to W2 and stir to form a pre-multiple emulsion. Pour the pre-multiple emulsion into a rapid membrane emulsifier to emulsify and disperse it into a multiple emulsion with a membrane tube pore size of 40 μm and a transmembrane pressure of 50 kPa. The multiple emulsion is solidified by a thin-film evaporator for 10 min at 35 °C for 3 cycles. After solidification, wash and lyophilize to obtain semaglutide sustained-release microspheres.

[0194] Example 5: Characterization of Emulsion Solidification and Molding

[0195] In this example, taking the semaglutide sustained-release microspheres prepared in Example 1, Comparative Example 1 and 2 above as examples respectively, the characterization experiments were carried out. The specific methods are as follows:

[0196] The FITC was mixed with the active pharmaceutical ingredient and incubated overnight for labeling. Then, the unlabeled FITC was removed by ultrafiltration washing. After lyophilization in the dark, it was used as the fluorescently labeled active pharmaceutical ingredient for the preparation of microspheres. Nile red was mixed with the oil-phase solution to label the carrier material to characterize the drug distribution during the formation of the emulsion and the curing process of the microspheres. The experimental results are shown in Figure 2 。

[0197] Through Figure 2 It can be seen that the drug in the semaglutide sustained-release microspheres of Example 1 was densely distributed in small particle sizes in the emulsion, and the emulsion could be maintained stable during rapid curing. There was no large amount of drug distribution in the microsphere shell. In Comparative Examples 1 and 2, the particle sizes of the primary emulsions were uneven, and the drug distribution was also uneven. During the curing process, a large amount of the primary emulsion coalesced and migrated to the outside of the microspheres. It shows that the emulsion stabilizer added in the preparation of the present invention can avoid burst release and has long-term sustained-release ability.

[0198] Example 6: In vitro release detection of microspheres

[0199] In this example, taking the semaglutide sustained-release microspheres prepared in Example 1, Comparative Examples 1, 2, and 3 above as examples, in vitro release detection was carried out. The specific method is as follows:

[0200] An appropriate amount of this product (equivalent to about 2 mg of semaglutide) was placed in a 50 mL centrifuge tube, 10 mL of commercially available PBS (0.01 M) was used as the release medium, and after mixing, it was placed in a water bath at 37.5 °C and shaken at 40 rpm / min. After 0.5 hour, 24 hours, 3 days, 6 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, and 30 days, it was centrifuged at a centrifugation rate of 8000 revolutions per minute for 5 minutes. 1 mL of the supernatant was precisely measured, and 1 mL of the release medium was precisely replenished to make it evenly dispersed, and shaking was continued. For the sample solutions at each time point, they were filtered through a 0.45 μm polyvinylidene fluoride membrane (PVDF), and the subsequent filtrate was taken to obtain the test solution for testing the drug content. The experimental results are shown in Figure 3 。

[0201] Through Figure 3 It can be seen that the semaglutide sustained-release microspheres prepared in Comparative Examples 1 and 2 of the present invention had a relatively high burst release. And in Comparative Example 2, there was a release lag at the initial stage, and the drug only underwent continuous release after the excipients degraded later, and reached the release end point in advance at the later stage. The semaglutide sustained-release microspheres prepared in Comparative Example 3 had a relatively low burst release, but the release rate was relatively fast in the middle stage of release, and the release end point was relatively low, and the release rate was much lower than that of the microspheres of Example 1 and Comparative Examples 1 and 2. However, the semaglutide sustained-release microspheres prepared in Example 1 had no obvious burst release, and the release behavior was stable in the middle and later stages. It shows that the semaglutide sustained-release microspheres prepared by the present invention have long-term sustained-release ability.

[0202] Example 7: In vivo animal PK detection

[0203] In this example, taking the semaglutide sustained-release microspheres prepared in Example 1, Comparative Example 1 and Comparative Example 2 above as examples, animal PK detection was carried out. The specific method is as follows:

[0204] (1) Animal screening and grouping: Twelve male SD rats were screened and evenly divided into 3 groups according to their body weights, with 4 rats in each group. Group 1 - Example 1, Group 2 - Comparative Example 1, Group 3 - Comparative Example 2.

[0205] (2) Administration frequency and method: All rats were sequentially administered the test article solution at 1 mg / kg according to the group, and all were administered subcutaneously once.

[0206] (3) Blood sampling time: The detection time was once before administration, 0.5 h, 2 h, 4 h, 8 h, 12 h, 24 h after administration, and once at 2d, 3d, 4d, 5d, 9d, 12d, 16d, 19d, 23d, 26d, 30d.

[0207] (4) Blood sampling method: Jugular vein blood sampling was used for all groups.

[0208] (5) Detection method: The detection method for the blood drug concentration of semaglutide was to add an isotope internal standard to the standard curve samples, quality control samples and unknown samples (samples to be detected), and then perform protein precipitation extraction. After the extraction was completed, LC-MS / MS detection was carried out. The PK curves of the rats are shown in Figure 4 (Example 1) and Figure 5 (Comparative Example 1 and Comparative Example 2).

[0209] Through Figure 4 and Figure 5 It can be seen that the blood drug concentration of the semaglutide sustained-release microspheres prepared in Comparative Example 1 and Comparative Example 2 of the present invention decreased rapidly after administration. Although the blood drug concentration slightly increased in the second week after administration in Comparative Example 2, the overall level was still low. However, the semaglutide sustained-release microspheres prepared in Example 1 of the present invention had no obvious burst release, the blood drug concentration reached the highest at 15 days, and remained within a relatively high concentration range within 30 days, indicating that the semaglutide sustained-release microspheres prepared by the present invention had a low burst release and had a long-term sustained-release ability.

[0210] Example 8: Simulation of the effect of polymerization inhibitor

[0211] In this example, taking liraglutide (Lira) and semaglutide (Sema) as examples, a simulation experiment on the anti-aggregation effect was carried out. The specific method is as follows:

[0212] Take polymerization inhibitors (polymerization inhibitor 1 is sucrose, polymerization inhibitor 2 is phenol), the active pharmaceutical ingredient, and deionized water, mix them in a weight ratio of 1:3:100, and then freeze-dry. Dropwise add PBS (pH 7.4) to the freeze-dried drug powder to simulate the process of the drug being gradually infiltrated in the microspheres. Then, dissolve part of the drug in PBS and use an atomic force microscope to observe whether the drug forms fibers, simulating the structural changes of the drug during the slow penetration of the drug inside the microspheres. The aggregation results are shown in Figure 6 .

[0213] It can be seen through Figure 6 that the polymerization inhibitors can significantly inhibit the aggregation of liraglutide and semaglutide into fibers, indicating that the preparation method of the present invention can effectively inhibit the aggregation of the drug into fibers, and the semaglutide sustained-release microspheres prepared by the present invention can effectively avoid the loss of drug activity.

[0214] It should be noted that although the technical solutions of the present invention are introduced with specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0215] The embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other ordinary skilled persons in the technical field to understand the disclosed embodiments.

Claims

1. A preparation method of a sustained-release microsphere of a polypeptide drug, characterized in that, the preparation method comprises the following steps: Mix an aggregate inhibitor, an emulsion stabilizer A and water, and use a pH regulator A to adjust the pH value of the mixture, and then add the polypeptide drug thereto to form an inner aqueous phase; Mix a carrier material, an emulsion stabilizer B and an organic solvent to form an oil phase; Mix a surfactant, an osmotic pressure regulator and water, and use a pH regulator B to adjust the pH value of the mixture to form an outer aqueous phase; and, Add the inner aqueous phase to the oil phase for dispersion to form a primary emulsion, then add the primary emulsion to the outer aqueous phase for re-dispersion to form a pre-compound emulsion, then emulsify the pre-compound emulsion to form a compound emulsion, and finally solidify the compound emulsion to obtain the sustained-release microsphere.

2. The preparation method according to claim 1, characterized in that, the polypeptide drug is a polypeptide GLP-1R agonist, preferably at least one of liraglutide and semaglutide, more preferably semaglutide; and / or, the aggregate inhibitor is at least one of sodium caprylate, sucrose, mannitol, 8-(2-hydroxybenzamido)-sodium caprylate, phenol, benzyl alcohol, ethanol and trifluoroethanol, preferably sucrose; and / or, the weight ratio of the aggregate inhibitor to the polypeptide drug is 1:70 to 50:70, preferably 27:70; and / or, the emulsion stabilizer A is at least one of Tween, poloxamer, sodium oleate and dextran, preferably sodium oleate; and / or, the weight ratio of the emulsion stabilizer A to the polypeptide drug is 1:200 to 3:2, preferably 9:1400; and / or, the water is at least one of ultrapure water, double-distilled water and water for injection, preferably ultrapure water; and / or, the dosage ratio of the water to the polypeptide drug is 3 mL:10 to 650 mg, preferably 3 mL:70 mg; and / or, the pH regulator A is at least one of hydrogen chloride, sodium hydroxide and phosphate; and / or, the pH regulator A adjusts the pH value of the mixture to 6.0 to 9.5, preferably 7.

6.

3. The preparation method according to claim 1 or 2, characterized in that, the organic solvent is at least one of chloroform, dichloromethane, ethyl acetate, acetone and acetonitrile, preferably dichloromethane; and / or, the dosage ratio of the organic solvent to the polypeptide drug is 1 to 10 mL:50 mg, preferably 1 mL:7 mg; and / or, The carrier material is a biodegradable carrier material; preferably, the carrier material is at least one of poly(lactic-co-glycolic acid), poly(lactic acid-co-polyethylene glycol), polylactic acid, polyhydroxyalkanoate, and polycaprolactone, preferably poly(lactic-co-glycolic acid), more preferably poly(lactic-co-glycolic acid) with a ratio of lactic acid to glycolic acid of 100-50:0-50, preferably 50:50 per 100 parts of monomers; and / or, the molecular weight of the carrier material is 1000-150000 Da, preferably 20000 Da; and / or, the weight ratio of the carrier material to the polypeptide drug is 3:1-100:1, preferably 240:7; and / or, The emulsion stabilizer B is at least one of lecithin, soybean phospholipid, oleic acid, and span, preferably at least one of span 83 and span 85, more preferably span 85; and / or, the weight ratio of the emulsion stabilizer B to the polypeptide drug is 1:20-40:20, preferably 3:

14.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The surfactant is at least one of polyvinyl alcohol, tween, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and polyoxyethylene octylphenol ether, preferably tween, more preferably tween 20; and / or, the weight ratio of the surfactant to the polypeptide drug is 1:20-40:20, preferably 3:28; and / or, The osmotic pressure regulator is at least one of sodium chloride and potassium chloride, preferably sodium chloride; and / or, the weight ratio of the osmotic pressure regulator to the polypeptide drug is 1:1-100:1, preferably 80:1; and / or, The water is at least one of ultrapure water, double-distilled water, and water for injection, preferably ultrapure water; and / or, the dosage ratio of the water to the polypeptide drug is 0.001-10 mL:1 mg, preferably 50 mL:7 mg; and / or, The pH regulator B is at least one of hydrogen chloride, sodium hydroxide, and phosphate; and / or, the pH regulator B adjusts the pH value of the mixture to 6.0-9.5, preferably 7.

8.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The dispersion is completed by at least one of stirring, homogenization, and ultrasonication, preferably homogenization, more preferably high-pressure homogenization; preferably, the time of the high-pressure homogenization is 0.5-20 min, preferably 2 min; and / or, the pressure of the high-pressure homogenization is 0.2-1200 bar, preferably 1 bar; and / or, The redispersion is completed by at least one of stirring and homogenization, preferably stirring; and / or, The emulsification is completed by membrane emulsification; preferably, the membrane pore size of the membrane emulsification is 1.0-60 μm, preferably 40 μm; and / or, the transmembrane pressure of the membrane emulsification is 1-200 kPa, preferably 50 kPa; and / or, The curing is completed by volatilization or evaporation, preferably by vacuum volatilization or thin-film evaporation, more preferably by thin-film evaporation; preferably, the time of the thin-film evaporation is 1 to 50 min, preferably 10 min; and / or, the temperature of the thin-film evaporation is 25 to 50 °C, preferably 35 °C; and / or, the number of cycles of the thin-film evaporation is 1 to 6 times, preferably 3 times.

6. The preparation method according to any one of claims 1 to 5, wherein, the preparation method further comprises the following steps: washing and freeze-drying the cured multiple emulsion.

7. The preparation method according to any one of claims 1 to 6, wherein, the particle size of the sustained-release microspheres is 1.0 to 100 μm, preferably 50 μm; the span of the particle size distribution Span is 0.2 - 2.0; and / or, the encapsulation efficiency of the sustained-release microspheres is 80% to 105%, preferably 95%; and / or, the drug loading of the sustained-release microspheres is 0.5% to 25%, preferably 13%.

8. A sustained-release microsphere of a polypeptide drug, wherein, the sustained-release microsphere is prepared by the preparation method according to any one of claims 1 to 7.

9. A pharmaceutical preparation of a polypeptide drug, wherein, the pharmaceutical preparation comprises the sustained-release microsphere according to claim 8; preferably, the pharmaceutical preparation further comprises at least one pharmaceutically acceptable excipient; and / or, preferably, the pharmaceutical preparation is a sustained-release preparation; and / or, preferably, the pharmaceutical preparation is an injection.

10. Use of the sustained-release microsphere according to claim 8 or the pharmaceutical preparation according to claim 9 in the preparation of a drug for preventing and / or treating at least partially diseases and / or disorders related to GLP-1R; preferably, the at least partially diseases and / or disorders related to GLP-1R are diabetes, weight loss or non-alcoholic fatty liver, preferably type 2 diabetes or weight loss.

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