Sustained-release microsphere for polypeptide drugs, pharmaceutical formulation thereof, method for preparing same, and use thereof

Abstract

Provided are a sustained-release microsphere for polypeptide drugs, a pharmaceutical formulation thereof, a method for preparing same, and use thereof. The method comprises the following steps: forming an inner aqueous phase; forming an oil phase; forming an outer aqueous phase; and dispersing the inner aqueous phase into the oil phase to give a primary emulsion, then re-dispersing the primary emulsion into the outer aqueous phase to give a pre-secondary emulsion, emulsifying the pre- secondary emulsion to form a secondary emulsion, and finally curing the secondary emulsion to give the sustained-release microsphere. The method features ease to operate, and the obtained sustained-release microsphere possesses ideal drug release behaviors (low early burst release, and stable mid- and later-stage releases) and safety and potency in reducing blood glucose, and is thus suitable for stable, long-term use with good patient compliance.

Description

A sustained-release microsphere of a polypeptide drug and its pharmaceutical preparation, preparation method and use

[0001] Citation of Related Applications

[0002] This disclosure claims priority to an invention patent application filed with the Patent Office of China on November 24, 2023, with application number 202311585750.5 and titled “Sustained-release microspheres of a polypeptide drug and its pharmaceutical preparation, preparation method and use,” and all of its contents are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of pharmaceutical preparations, and relates to sustained-release microspheres of polypeptide drugs, pharmaceutical preparations thereof, a preparation method and uses thereof. Background Art

[0004] Type 2 diabetes is a disease characterized by high blood sugar levels associated with insulin resistance and relative insulin deficiency. While often initially managed with increased exercise and dietary adjustments, medication is often required as the disease progresses.

[0005] GLP-1 (glucagon-like peptide-1) is a naturally occurring incretin insulin in the human body. Incretins are a group of metabolic hormones that promote the reduction of blood sugar levels. They are released after eating and increase the secretion of insulin released by pancreatic beta cells through a blood sugar-dependent mechanism. GLP-1 exerts its corresponding hypoglycemic effect by stimulating the widely distributed GLP-1 receptor (GLP-1R), but it is itself extremely susceptible to degradation. Liraglutide and semaglutide developed by Novo Nordisk are based on natural GLP-1 through amino acid substitution and side chain modification. They have high homology with the amino acid sequence of natural human GLP-1, with half-lives of approximately 12 hours and 160 hours, respectively, and are injected once a day / week. Sugar control needs to be maintained for a long time, but long-term injections of drugs lead to poor patient compliance and low medication accuracy.

[0006] Drug-encapsulated microsphere formulations (microsphere formulation) can utilize carrier materials to prolong the duration of drug efficacy, improve patient compliance, optimize treatment effects, and enhance medication accuracy. Microsphere formulations are widely used in the development of long-acting sustained-release injections. Currently, the five types of microsphere formulations currently on the market in China are used in areas such as malignant tumors, diabetes, cardiovascular disease, and mental illness. As the size of the chronic disease patient population expands, the demand for related drugs is vast. Therefore, the development of microsphere formulations with obvious advantages and their application in chronic disease-related fields can effectively improve the health and medication experience of the corresponding patient population and greatly improve the accuracy of related clinical medication.

[0007] In order to overcome the problem of short half-life of natural GLP-1 in vivo, synthetic GLP-1R agonist molecules have adopted a variety of strategies to extend drug half-life. Among them, liraglutide and semaglutide have adopted the strategy of acylation of specific amino residues with fatty acids. Fatty acid side chains are used to promote molecular self-association, thereby prolonging the diffusion time of the drug from the injection site. In addition, fatty acid molecules can undergo reversible non-covalent binding with albumin, slowing down renal clearance. However, since the microsphere preparation needs to embed a month or even several months of doses of drugs, 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 polymer fibers under high concentration conditions. Drug fibers will not only lead to loss of drug activity, but may even cause necrosis of surrounding tissues, and there are problems of drug toxicity and immunogenicity. [1-2] Therefore, considering the properties of the API, how to avoid large-scale drug aggregation and fiber formation in long-acting sustained-release preparations is a major challenge.

[0008] Furthermore, due to the amphiphilic nature of liraglutide and semaglutide, a large amount of drug is dispersed at the oil-water interface during emulsion preparation. Rapid solvent evaporation during solidification causes a large number of primary emulsion droplets to coalesce, and the drug migrates to the exterior of the microsphere shell during this process. This results in excessively rapid initial release and a high Cmax, compromising safety and making it difficult to maintain stable drug release over a long period in sustained-release formulations. Achieving a low burst release and long-term, stable release of active drugs in sustained-release formulations is a current challenge facing amphiphilic GLP-1R agonists.

[0009] [1]Zapadka KL,Becher FJ,Gomes Dos Santos AL,Jackson SE,Factors affecting the physical stability(aggregation) of peptide therapeutics[J],Interface Focus,2017.

[0010] [2] Venanzi M, Savioli M, Cimino R, et al., A spectroscopic and molecular dynamics study on the aggregation process of a long-acting lipidated therapeutic peptide: the case of Semaglutide[J], Soft Matter, 2020.

[0011] Summary of the Invention

[0012] Problems to be solved by the invention

[0013] In view of the above problems, the present invention aims to provide a microsphere preparation of a GLP-1R agonist with low early burst release, stable release in the middle and late stages, long-term medication, high compliance and simple operation.

[0014] Solutions for solving problems

[0015] In a first aspect, the present invention provides a method for preparing sustained-release microspheres of polypeptide drugs, comprising the following steps:

[0016] Mixing an aggregation inhibitor, an emulsion stabilizer A, and water, adjusting the pH value of the mixture using a pH adjuster A, and then adding the polypeptide drug thereto to form an inner aqueous phase;

[0017] Mixing the carrier material, the emulsion stabilizer B and the organic solvent to form an oil phase;

[0018] Mixing a surfactant, an osmotic pressure regulator, and water, and adjusting the pH value of the mixture using a pH regulator B to form an external aqueous phase; and,

[0019] The inner aqueous phase is added to the oil phase for dispersion to form colostrum, and the colostrum is then added to the outer aqueous phase for redispersion to form a pre-emulsion. The pre-emulsion is then emulsified to form a composite emulsion, and finally the composite emulsion is solidified to obtain the sustained-release microspheres.

[0020] 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.

[0021] As a further improvement of the present invention, the aggregation inhibitor is at least one of sodium octanoate, sucrose, mannitol, sodium 8-(2-hydroxybenzamido)-octanoate (SNAC), phenol, benzyl alcohol, ethanol and trifluoroethanol, preferably sucrose.

[0022] As a further improvement of the present invention, the weight ratio of the aggregation inhibitor to the polypeptide drug is 1:70 to 50:70, preferably 27:70.

[0023] 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.

[0024] 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.

[0025] As a further improvement of the present invention, the water is at least one of ultrapure water, redistilled water and water for injection, preferably ultrapure water.

[0026] As a further improvement of the present invention, the usage ratio of the water to the polypeptide drug is 3 mL:10-650 mg, preferably 3 mL:70 mg.

[0027] As a further improvement of the present invention, the pH regulator A is at least one of hydrogen chloride, sodium hydroxide and phosphate.

[0028] As a further improvement of the present invention, the pH adjuster A adjusts the pH value of the mixture to 6.0-9.5, preferably 7.6.

[0029] 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.

[0030] As a further improvement of the present invention, the usage ratio of the organic solvent to the polypeptide drug is 1-10 mL:50 mg, preferably 1 mL:7 mg.

[0031] As a further improvement of the present invention, the carrier material is a biodegradable carrier material.

[0032] As a further improvement of the present invention, the carrier material is at least one of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid-polyethylene glycol copolymer (PELA), polylactic acid (PLA), polyhydroxyalkanoate (PHA) and polycaprolactone (PCL), preferably polylactic acid-glycolic acid copolymer, more preferably polylactic acid-glycolic acid copolymer in which the ratio of lactic acid to glycolic acid per 100 parts of monomers is 100-50:0-50, preferably 50:50.

[0033] As a further improvement of the present invention, the molecular weight of the carrier material is 1000 to 150000 Da, preferably 20000 Da.

[0034] As a further improvement of the present invention, the weight ratio of the carrier material to the polypeptide drug is 3:1 to 100:1, preferably 240:7.

[0035] As a further improvement of the present invention, the emulsion stabilizer B is at least one of lecithin, soybean lecithin, oleic acid and Span, preferably at least one of Span 83 (sorbitan sesquioleate) and Span 85 (sorbitan trioleate), more preferably Span 85.

[0036] As a further improvement of the present invention, the weight ratio of the emulsion stabilizer B to the polypeptide drug is 1:20 to 40:20, preferably 3:14.

[0037] As a further improvement of the present invention, the surfactant is at least one of polyvinyl alcohol (PVA), Tween, sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS) and polyoxyethylene octylphenol ether, preferably Tween, more preferably Tween 20.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] As a further improvement of the present invention, the water is at least one of ultrapure water, redistilled water and water for injection, preferably ultrapure water.

[0042] As a further improvement of the present invention, the usage ratio of the water to the polypeptide drug is 0.001-10 mL:1 mg, preferably 50 mL:7 mg.

[0043] As a further improvement of the present invention, the pH regulator B is at least one of hydrogen chloride, sodium hydroxide and phosphate.

[0044] As a further improvement of the present invention, the pH adjuster B adjusts the pH value of the mixture to 6.0-9.5, preferably 7.8.

[0045] As a further improvement of the present invention, the dispersion is accomplished by at least one of stirring, homogenizing and ultrasound, preferably homogenizing, more preferably high-pressure homogenizing.

[0046] As a further improvement of the present invention, the high-pressure homogenization time is 0.5 to 20 minutes, preferably 2 minutes.

[0047] As a further improvement of the present invention, the pressure of the high-pressure homogenization is 0.2 to 1200 bar, preferably 1 bar.

[0048] As a further improvement of the present invention, the redispersion is accomplished by at least one of stirring and homogenizing, preferably stirring.

[0049] As a further improvement of the present invention, the emulsification is accomplished by membrane emulsification.

[0050] As a further improvement of the present invention, the pore size of the membrane emulsification is 1.0 to 60 μm, preferably 40 μm.

[0051] As a further improvement of the present invention, the transmembrane pressure of the membrane emulsification is 1 to 200 kPa, preferably 50 kPa.

[0052] As a further improvement of the present invention, the solidification is accomplished by volatilization or evaporation, preferably by reduced pressure volatilization or thin film evaporation, more preferably by thin film evaporation.

[0053] As a further improvement of the present invention, the thin film evaporation time is 1 to 50 minutes, preferably 10 minutes.

[0054] As a further improvement of the present invention, the temperature of the thin film evaporation is 25-50°C, preferably 35°C.

[0055] As a further improvement of the present invention, the number of cycles of the thin film evaporation is 1 to 6 times, preferably 3 times.

[0056] As a further improvement of the present invention, the preparation method further comprises washing and freeze-drying the emulsion after solidification.

[0057] In a second aspect, the present invention provides a sustained-release microsphere of a polypeptide drug, which is prepared using the above-mentioned preparation method.

[0058] In a third aspect, the present invention provides a pharmaceutical preparation of a polypeptide drug, which comprises the above-mentioned sustained-release microspheres.

[0059] As a further improvement of the present invention, the pharmaceutical preparation further comprises at least one pharmaceutically acceptable excipient.

[0060] As a further improvement of the present invention, the pharmaceutical preparation is a sustained-release preparation.

[0061] As a further improvement of the present invention, the pharmaceutical preparation is an injection.

[0062] In a fourth aspect, the present invention provides use of the sustained-release microspheres or the pharmaceutical preparations described above in the preparation of drugs for preventing and / or treating at least some of the diseases and / or conditions associated with GLP-1R.

[0063] As a further improvement of the present invention, the disease and / or condition at least partially related to GLP-1R is diabetes, weight loss or non-alcoholic fatty liver disease, preferably type 2 diabetes or weight loss.

[0064] Effects of the Invention

[0065] 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, the early burst release is low, avoiding the risk of hypoglycemia caused by the high initial concentration of GLP-1R agonist drugs, the drug release behavior is stable in the middle and late stages, and blood sugar is safely and effectively lowered. As a long-acting sustained-release microsphere preparation, it is conducive to long-term stable medication and improves patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 is a primary milk light microscope of Example 1 and Comparative Examples 1 and 2.

[0067] FIG2 is the emulsion and curing process of Example 1 and Comparative Examples 1 and 2.

[0068] FIG3 shows the in vitro release trends of the microspheres of Example 1 and Comparative Examples 1, 2, and 3.

[0069] FIG4 is the in vivo PK test result of rats in Example 1.

[0070] FIG5 is the in vivo PK test results of rats of Comparative Examples 1 and 2.

[0071] FIG6 shows the aggregation of GLP-1 and the effect of polymerization inhibitors.

[0072] FIG7 is a particle size study of three batches of repeatability in Example 1. DETAILED DESCRIPTION

[0073] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0074] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced 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 in order to highlight the main points of the present invention.

[0075] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0076] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0077] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0078] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0079] In this specification, when "normal temperature" or "room temperature" is used, the temperature may be 10-40°C.

[0080] Sustained-release microspheres of polypeptide drugs and preparation method thereof

[0081] The present invention provides a method for preparing sustained-release microspheres of polypeptide drugs, which comprises the following steps:

[0082] Mixing an aggregation inhibitor, an emulsion stabilizer A, and water, adjusting the pH value of the mixture using a pH adjuster A, and then adding the polypeptide drug thereto to form an inner aqueous phase;

[0083] Mixing the carrier material, the emulsion stabilizer B and the organic solvent to form an oil phase;

[0084] Mixing a surfactant, an osmotic pressure regulator, and water, and adjusting the pH value of the mixture using a pH regulator B to form an external aqueous phase; and,

[0085] The inner aqueous phase is added to the oil phase for dispersion to form colostrum, and the colostrum is then added to the outer aqueous phase for redispersion to form a pre-emulsion. The pre-emulsion is then emulsified to form a composite emulsion, and finally the composite emulsion is solidified to obtain the sustained-release microspheres.

[0086] The peptide drugs used in this preparation method (especially peptide GLP-1R agonists, such as liraglutide or semaglutide) tend to aggregate at high concentrations, even forming high-molecular-weight fibers, which can lead to loss of activity and toxicity and immunogenicity. Therefore, maintaining the monomeric form of the drug is particularly important during preparation. Furthermore, due to the amphiphilic nature of the drug molecules, the drug can migrate to the outside of the spherical shell during preparation, resulting in an overly rapid initial release from the microspheres. This results in excessively high early drug concentrations and difficulty in long-term release. Therefore, avoiding large-scale dispersion of the drug at the oil-water interface is crucial for preparation.

[0087] The present invention controls drug aggregation and inhibits drug fiberization by adding an aggregation inhibitor to the inner aqueous phase and regulating the pH value, thereby avoiding a reduction in drug activity. At the same time, an emulsion stabilizer is 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 the amphiphilic drug in the emulsion, ensuring uniform dispersion of the primary emulsion during the curing process, effectively inhibiting the large-scale migration of drug molecules as the organic solvent is volatilized and removed, allowing the drug to be uniformly dispersed in the carrier material, and preventing the drug from migrating to the water-in-oil interface of the double emulsion due to isoelectric point factors by regulating the pH value of the outer aqueous phase, effectively reducing drug burst release, and maintaining stable drug release behavior.

[0088] In order to clearly illustrate the preparation method of the sustained-release microspheres of the polypeptide drug of the present invention, the following detailed description is given using semaglutide sustained-release microspheres as an example. The same is also applicable to the active ingredients of polypeptide drugs (such as liraglutide) that bind to albumin and GLP-1 receptors mainly in the form of monomers after entering the blood.

[0089] Furthermore, the present invention provides a method for preparing semaglutide sustained-release microspheres, which comprises the following steps:

[0090] An aggregation inhibitor, an emulsion stabilizer A, and water are mixed, and the pH value of the mixture is adjusted using a pH adjuster A, and semaglutide is added thereto to form an inner aqueous phase;

[0091] Mixing the carrier material, the emulsion stabilizer B and the organic solvent to form an oil phase;

[0092] Mixing a surfactant, an osmotic pressure regulator, and water, and adjusting the pH value of the mixture using a pH regulator B to form an external aqueous phase; and,

[0093] The inner aqueous phase is added to the oil phase for dispersion to form colostrum, and the colostrum is then added to the outer aqueous phase for redispersion to form a pre-emulsion. The pre-emulsion is then emulsified to form a double emulsion, and finally the double emulsion is solidified to obtain semaglutide sustained-release microspheres.

[0094] In one embodiment of the present invention, the aggregation inhibitor in the above preparation method can 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 of the two in any proportion.

[0095] In one embodiment of the present invention, the aggregation inhibitor in the above preparation method may be sucrose.

[0096] In one embodiment of the present invention, the weight ratio of the aggregation inhibitor to semaglutide in the above preparation method can vary within a certain range, for example, 1:70 to 50:70.

[0097] In one embodiment of the present invention, the weight ratio of the aggregation inhibitor to semaglutide in the above preparation method can be 27:70.

[0098] 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, such as Tween, sodium oleate or a mixture thereof in any proportion.

[0099] In one embodiment of the present invention, the emulsion stabilizer A in the above preparation method may be sodium oleate.

[0100] 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.

[0101] 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.

[0102] In one embodiment of the present invention, the water used in the internal aqueous phase in the above preparation method can be at least one of ultrapure water, redistilled water and water for injection, such as ultrapure water, redistilled water or a mixture of the two in any proportion.

[0103] In one embodiment of the present invention, the water used in the internal aqueous phase in the above preparation method may be ultrapure water.

[0104] In one embodiment of the present invention, the ratio of water to semaglutide used in the internal aqueous phase in the above preparation method can vary within a certain range, for example, 3 mL: 10-650 mg.

[0105] In one embodiment of the present invention, the ratio of water to semaglutide used in the internal aqueous phase in the above preparation method can be 3 mL:70 mg.

[0106] In one embodiment of the present invention, the pH adjuster A 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 thereof in any proportion.

[0107] In one embodiment of the present invention, the pH adjuster 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.

[0108] In one embodiment of the present invention, the pH adjuster A in the above preparation method can adjust the pH value of the mixture to 7.6.

[0109] 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 thereof in any proportion.

[0110] In one embodiment of the present invention, the organic solvent in the above preparation method may be dichloromethane.

[0111] In one embodiment of the present invention, the ratio of the organic solvent to semaglutide in the above preparation method can vary within a certain range, for example, 1-10 mL:50 mg.

[0112] In one embodiment of the present invention, the ratio of the organic solvent to semaglutide in the above preparation method can be 1 mL:7 mg.

[0113] In one embodiment of the present invention, the carrier material in the above preparation method may be a biodegradable carrier material.

[0114] In one embodiment of the present invention, the carrier material in the above preparation method can be at least one of polylactic acid-co-glycolic acid, polylactic acid-polyethylene glycol copolymer, polylactic acid, polyhydroxyalkanoate and polycaprolactone, for example, polylactic acid-co-glycolic acid, polylactic acid-polyethylene glycol copolymer or a mixture thereof in any proportion.

[0115] In one embodiment of the present invention, the carrier material in the above preparation method may be poly(lactic acid-glycolic acid) copolymer.

[0116] In one embodiment of the present invention, the ratio of lactic acid to glycolic acid per 100 parts of monomers in the carrier material in the above preparation method can vary within a certain range, for example, 100-50:0-50, for example, 100:0, 95:5, 85:15, 75:25, 50:50 or other ratios.

[0117] 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 may be 50:50.

[0118] 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, for example, 1000 to 150000 Da.

[0119] In one embodiment of the present invention, the molecular weight of the carrier material in the above preparation method may be 20,000 Da.

[0120] 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.

[0121] In one embodiment of the present invention, the weight ratio of the carrier material to semaglutide in the above preparation method may be 240:7.

[0122] In one embodiment of the present invention, the emulsion stabilizer B in the above preparation method can be at least one of lecithin, soybean lecithin, oleic acid and Span, such as soybean lecithin, Span or a mixture thereof in any proportion.

[0123] 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 thereof in any proportion.

[0124] In one embodiment of the present invention, the emulsion stabilizer B in the above preparation method may be Span 85.

[0125] 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.

[0126] 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.

[0127] 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 dodecylbenzene sulfonate and polyoxyethylene octylphenol ether, for example, polyvinyl alcohol, Tween or a mixture of the two in any proportion.

[0128] In one embodiment of the present invention, the surfactant in the above preparation method may be Tween.

[0129] In one embodiment of the present invention, the surfactant in the above preparation method may be Tween 20.

[0130] 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.

[0131] In one embodiment of the present invention, the weight ratio of the surfactant to semaglutide in the above preparation method may be 3:28.

[0132] 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 of the two in any proportion.

[0133] In one embodiment of the present invention, the osmotic pressure regulator in the above preparation method may be sodium chloride.

[0134] 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, for example, 1:1 to 100:1.

[0135] 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.

[0136] In one embodiment of the present invention, the water used in the external aqueous phase in the above preparation method can be at least one of ultrapure water, redistilled water and water for injection, such as ultrapure water, redistilled water or a mixture of the two in any proportion.

[0137] In one embodiment of the present invention, the water used in the external aqueous phase in the above preparation method may be ultrapure water.

[0138] In one embodiment of the present invention, the ratio of water to semaglutide used in the external aqueous phase in the above preparation method can vary within a certain range, for example, 0.001-10 mL:1 mg.

[0139] In one embodiment of the present invention, the ratio of water to semaglutide used in the external aqueous phase in the above preparation method can be 50 mL:7 mg.

[0140] In one embodiment of the present invention, the pH adjuster 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 thereof in any proportion.

[0141] In one embodiment of the present invention, the pH adjuster B in the above preparation method can adjust the pH value of the mixture within a certain range, for example, 6.0 to 9.5.

[0142] In one embodiment of the present invention, the pH adjuster B in the above preparation method can adjust the pH value of the mixture to 7.8.

[0143] In one embodiment of the present invention, the dispersion in the above preparation method can be performed by at least one of stirring, homogenizing and ultrasonication, for example, stirring, homogenizing or a combination of the two.

[0144] In one embodiment of the present invention, the dispersion method in the above preparation method can be homogenization.

[0145] In one embodiment of the present invention, the dispersion method in the above preparation method may be high-pressure homogenization.

[0146] 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 minutes.

[0147] In one embodiment of the present invention, the high-pressure homogenization time in the above preparation method can be 2 minutes.

[0148] In one embodiment of the present invention, the pressure of the high-pressure homogenization in the above preparation method can vary within a certain range, for example, 0.2 to 1200 bar.

[0149] In one embodiment of the present invention, the pressure of the high-pressure homogenization in the above preparation method can be 1 bar.

[0150] In one embodiment of the present invention, the redispersion in the above preparation method can be accomplished by at least one of stirring and homogenizing.

[0151] In one embodiment of the present invention, the redispersion in the above preparation method can be accomplished by stirring.

[0152] In one embodiment of the present invention, the emulsification in the above preparation method can be accomplished by membrane emulsification.

[0153] In one embodiment of the present invention, the pore size of the membrane emulsification in the above preparation method can vary within a certain range, for example, 1.0 to 60 μm.

[0154] In one embodiment of the present invention, the pore size of the membrane emulsification in the above preparation method may be 40 μm.

[0155] In one embodiment of the present invention, the transmembrane pressure of the membrane emulsification in the above preparation method can vary within a certain range, for example, 1 to 200 kPa.

[0156] In one embodiment of the present invention, the transmembrane pressure of the membrane emulsification in the above preparation method may be 50 kPa.

[0157] In one embodiment of the present invention, the solidification in the above preparation method can be accomplished by volatilization or evaporation.

[0158] In one embodiment of the present invention, the solidification in the above preparation method can be accomplished by reduced pressure volatilization or thin film evaporation.

[0159] In one embodiment of the present invention, the solidification in the above preparation method can be accomplished by thin film evaporation.

[0160] In one embodiment of the present invention, the time for thin film evaporation in the above preparation method can vary within a certain range, for example, 1 to 50 minutes.

[0161] In one embodiment of the present invention, the thin film evaporation time in the above preparation method can be 10 minutes.

[0162] In one embodiment of the present invention, the temperature of thin film evaporation in the above preparation method can be varied within a certain range, for example, 25-50°C.

[0163] In one embodiment of the present invention, the temperature of thin film evaporation in the above preparation method can be 35°C.

[0164] 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.

[0165] In one embodiment of the present invention, the number of cycles of thin film evaporation in the above preparation method can be 3 times.

[0166] In one embodiment of the present invention, the preparation method may further comprise the following steps: solidifying the multiple emulsion, followed by washing and freeze-drying.

[0167] Accordingly, the present invention provides a sustained-release microsphere of a polypeptide drug, which is prepared by the above-mentioned preparation method.

[0168] In one embodiment of the present invention, the polypeptide drug in the sustained-release microspheres may be a polypeptide GLP-1R agonist.

[0169] In one embodiment of the present invention, the polypeptide drug in the sustained-release microspheres may be semaglutide.

[0170] Pharmaceutical preparations of polypeptide drugs

[0171] The present invention provides a pharmaceutical preparation of a polypeptide drug, which comprises the above-mentioned sustained-release microspheres.

[0172] In one embodiment of the present invention, the pharmaceutical preparation may further comprise at least one pharmaceutically acceptable excipient, such as a solvent, a solubilizer, a cosolvent, and the like.

[0173] In one embodiment of the present invention, the pharmaceutical preparation can be a preparation with a specific drug release behavior, such as a sustained-release preparation.

[0174] In one embodiment of the present invention, the pharmaceutical preparation can be a parenteral dosage form, such as an injection.

[0175] Medical uses of sustained-release microspheres and their pharmaceutical preparations

[0176] The present invention provides use of the sustained-release microspheres or the pharmaceutical preparation in preparing a drug for preventing and / or treating at least some of the diseases and / or conditions related to GLP-1R.

[0177] In one embodiment of the present invention, the aforementioned disease and / or disorder at least partially related to GLP-1R may be diabetes, weight loss or non-alcoholic fatty liver disease.

[0178] In one embodiment of the present invention, the aforementioned disease and / or disorder at least partially related to GLP-1R may be type 2 diabetes or weight loss.

[0179] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0180] Example 1

[0181] 90 mg of sucrose and 1.5 mg of sodium oleate were weighed and dissolved in 10 mL of ultrapure water, the pH of which was adjusted to 7.6. 70 mg of semaglutide was dissolved in 3 mL of this solution to obtain W1. 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and 15 mg of Span85 were weighed and dissolved in 10 mL of dichloromethane to obtain O. 500 mL of a 1.5 g / 100 mL Tween 20 and 0.2 g / 100 mL sodium chloride solution was prepared as the external aqueous phase and the pH was adjusted to 7.8 to obtain W2. W1 was added to O and dispersed into colostrum by high-pressure homogenization for 2 minutes at a pressure of 1 bar. The colostrum was added to W2 and stirred to form a pre-emulsion. The pre-emulsion was then poured into a rapid membrane emulsifier for emulsification to obtain a complex emulsion. The membrane tube used had a pore size of 40 μm and a transmembrane pressure of 50 kPa. The emulsion was solidified by thin film evaporation for 10 min at 35°C for 3 cycles; after solidification, it was washed and freeze-dried to obtain semaglutide-containing sustained-release microspheres.

[0182] Take an appropriate amount of microsphere sample, add a small amount of deionized water, disperse evenly, and add it to the laser particle size distribution analyzer to test the microsphere particle size. The obtained particle size distribution diagram is shown in Figure 7.

[0183] Example 2

[0184] 30 mg of SNAC and 20 mg of poloxamer 188 were weighed and dissolved in 10 mL of ultrapure water, the pH of which was adjusted to 7.0. 400 mg of semaglutide was dissolved in 3 mL of this solution to obtain W1. 2000 mg of PLGA (LA:GA = 75:25, Mw = 40 kDa) and 65 mg of soybean lecithin were weighed and dissolved in 10 mL of dichloromethane to obtain O. 200 mL of 1.5 g / 100 mL of PVA and 2.0 g / 100 mL of sodium chloride solution were prepared as the external aqueous phase and the pH was adjusted to 7.0 to obtain W2. W1 was added to O and homogenized to form colostrum for 5 minutes at an average speed of 10,000 rpm. The colostrum was added to W2 and stirred to form a pre-emulsion. The pre-emulsion was then poured into a rapid membrane emulsifier and emulsified to form a complex emulsion. The membrane tube used had a pore size of 20 μm and a transmembrane pressure of 78 kPa. The emulsion is solidified by volatilization for 6 hours; after the solidification is completed, it is washed and freeze-dried to obtain semaglutide-containing sustained-release microspheres.

[0185] Example 3

[0186] 30 mg of phenol and 20 mg of poloxamer 188 were weighed and dissolved in 10 mL of ultrapure water, the pH of which was adjusted to 7.0. 400 mg of semaglutide was dissolved in 3 mL of this solution to obtain W1. 2000 mg of PLGA (LA:GA = 75:25, Mw = 40 kDa) and 65 mg of soybean lecithin were weighed and dissolved in 10 mL of dichloromethane to obtain O. 200 mL of 1.5 g / 100 mL PVA and 2.0 g / 100 mL sodium chloride solution were prepared as the external aqueous phase and adjusted to pH 7.0 to obtain W2. W1 was added to O and homogenized to form colostrum for 5 minutes at an average speed of 10,000 rpm. The colostrum was added to W2 and stirred to form a pre-emulsion. The pre-emulsion was then poured into a rapid membrane emulsifier for emulsification and dispersion to form a complex emulsion. The membrane tube used had a pore size of 20 μm and a transmembrane pressure of 78 kPa. The emulsion is solidified by volatilization for 6 hours; after the solidification is completed, it is washed and freeze-dried to obtain semaglutide-containing sustained-release microspheres.

[0187] Example 4

[0188] 50 mg of benzyl alcohol and 50 mg of Tween 20 were weighed and dissolved in 10 mL of ultrapure water, the pH of which was adjusted to 7.4. 150 mg of semaglutide was dissolved in 3 mL of this solution to obtain W1. 2000 mg of PLGA (LA:GA = 50:50, Mw = 5 kDa) and 300 mg of lecithin were weighed and dissolved in 10 mL of dichloromethane to obtain O. 250 mL of 1.0 g / 100 mL of PVA and 2.0 g / 100 mL of sodium chloride solution were prepared as the external aqueous phase and the pH was adjusted to 7.0 to obtain W2. W1 was added to O and homogenized to form colostrum for 5 minutes at an average speed of 12,000 rpm. The colostrum was added to W2 and stirred to form a pre-emulsion. The pre-emulsion was poured into a rapid membrane emulsifier and emulsified to form a complex emulsion. The membrane tube used had a pore size of 30 μm and a transmembrane pressure of 62 kPa. The emulsion is solidified by volatilization for 6 hours; after the solidification is completed, it is washed and freeze-dried to obtain semaglutide-containing sustained-release microspheres.

[0189] Comparative Example 1

[0190] 90 mg of sucrose was weighed and dissolved in 10 mL of ultrapure water, the pH of which was adjusted to 7.6. 70 mg of semaglutide was dissolved in 3 mL of this solution to obtain W1. 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) was weighed and dissolved in 10 mL of dichloromethane to obtain O. 500 mL of a 1.5 g / 100 mL Tween 20 solution was prepared as the external aqueous phase and its pH was adjusted to 7.8 to obtain W2. W1 was added to O and dispersed into colostrum by high-pressure homogenization for 2 minutes at a pressure of 1 bar. The colostrum was added to W2 and stirred to form a pre-emulsion. The pre-emulsion was poured into a rapid membrane emulsifier and emulsified to form a complex emulsion. The membrane tube used had a pore size of 40 μm and a transmembrane pressure of 50 kPa. The emulsion was solidified by thin film evaporation for 10 min at 35°C for 3 cycles; after solidification, it was washed and freeze-dried to obtain semaglutide-containing sustained-release microspheres.

[0191] Example 1 incorporates two emulsion stabilizers, while Comparative Example 1 does not. As shown in Figure 1, the colostrum particles of the semaglutide sustained-release microspheres of Example 1 are fine and densely distributed in the emulsion as small particles, while the colostrum particles of the semaglutide sustained-release microspheres of the Comparative Example are large and uneven. This indicates that the addition of an emulsion stabilizer in the preparation of the present invention is necessary to form a uniformly dispersed colostrum.

[0192] Comparative Example 2

[0193] 90 mg of sucrose was dissolved in 10 mL of ultrapure water, and 70 mg of semaglutide was dissolved in 3 mL of this solution to obtain W1. 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and 60 mg of Span85 were dissolved in 10 mL of dichloromethane to obtain O. 500 mL of a 1.5 g / 100 mL Tween 20 solution was prepared as the external aqueous phase and adjusted to a pH of 7.8 to obtain W2. W1 was added to O and dispersed into colostrum via high-pressure homogenization for 2 minutes at a pressure of 1 bar. The colostrum was added to W2 and stirred to form a pre-emulsion. The pre-emulsion was then poured into a rapid membrane emulsifier for emulsification and dispersion into a complex emulsion using a 40 μm pore size membrane and a transmembrane pressure of 50 kPa. The emulsion was solidified by thin film evaporation for 10 min at 35°C for 3 cycles; after solidification, it was washed and freeze-dried to obtain semaglutide-containing sustained-release microspheres.

[0194] Example 1 incorporates two emulsion stabilizers, while Comparative Example 2 incorporates only Emulsion Stabilizer B. As shown in Figure 1, compared to the fine and uniform colostrum of the semaglutide sustained-release microspheres of Example 1, the colostrum of the semaglutide sustained-release microspheres of Comparative Example 2 is unevenly distributed. After the emulsion is prepared, it rapidly aggregates, forming numerous large cavities within the emulsion. This indicates that the addition of a single emulsion stabilizer in the preparation of the present invention does not result in a uniform emulsion distribution.

[0195] Comparative Example 3

[0196] 70 mg of semaglutide was dissolved in 3 mL of ultrapure water to obtain W1, and the pH was adjusted to 7.6. 2400 mg of PLGA (LA:GA = 50:50, Mw = 20 kDa) and 15 mg of Span85 were weighed and dissolved in 10 mL of dichloromethane to obtain O. 500 mL of a 1.5 g / 100 mL Tween 20 solution was prepared as the external aqueous phase and its pH was adjusted to 7.8 to obtain W2. W1 was added to O and dispersed into colostrum via high-pressure homogenization for 2 minutes at a pressure of 1 bar. The colostrum was then added to W2 and stirred to form a pre-emulsion. The pre-emulsion was then emulsified and dispersed into a complex emulsion using a rapid membrane emulsifier with a 40 μm pore size and a transmembrane pressure of 50 kPa. The complex emulsion was solidified by thin-film evaporation for 10 minutes at 35°C, with three cycles. After solidification, the microspheres were washed and freeze-dried to obtain sustained-release microspheres containing semaglutide.

[0197] Example 5: Emulsion Curing Molding Characterization

[0198] In this example, the semaglutide sustained-release microspheres prepared in Example 1, Comparative Examples 1 and 2 were used as examples to conduct characterization experiments. The specific methods are as follows:

[0199] FITC was mixed with the API and incubated overnight for labeling. Unlabeled FITC was then removed by ultrafiltration and washed. After freeze-drying in the dark, the API was used to prepare microspheres as a fluorescently labeled API. Nile red was mixed with the oil phase solution to label the carrier material to characterize the drug distribution after emulsion formation and during microsphere solidification. The experimental results are shown in Figure 2.

[0200] As shown in Figure 2, the drug in the semaglutide sustained-release microspheres of Example 1 is densely distributed in the emulsion as small particles, maintaining emulsion stability during rapid solidification, with no significant distribution of drug on the outer shell of the microspheres. In contrast, in Comparative Examples 1 and 2, the colostrum particles are unevenly sized and the drug distribution is also uneven. During solidification, a large amount of colostrum coalesces and migrates to the outside of the microspheres. This indicates that the emulsion stabilizer added during the preparation of the present invention can prevent burst release and provide long-term sustained-release capability.

[0201] Example 6: In vitro release assay of microspheres

[0202] In this example, the semaglutide sustained-release microspheres prepared in Example 1, Comparative Examples 1, 2 and 3 were used as examples to perform in vitro release tests. The specific methods are as follows:

[0203] An appropriate amount of the product (approximately equivalent to 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. After mixing, the tube was placed in a 37.5°C water bath and shaken at 40 rpm / min. After 0.5 hours, 24 hours, 3 days, 6 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, and 30 days, the tube was centrifuged at 8000 rpm for 5 minutes. 1 mL of the supernatant was accurately measured and added with 1 mL of the release medium to ensure uniform dispersion. The tube was then shaken again. The sample solution at each time point was filtered through a 0.45 μm polyvinylidene fluoride membrane (PVDF). The filtrate was collected to obtain the test solution for drug content. The experimental results are shown in Figure 3.

[0204] As can be seen from Figure 3, the semaglutide sustained-release microspheres prepared in Comparative Examples 1 and 2 of the present invention have a high burst release, and there is a release hysteresis in the initial stage of Comparative Example 2. Subsequently, the drug is continuously released after the degradation of the excipients, and the release endpoint is reached early in the later stage. The semaglutide sustained-release microspheres prepared in Comparative Example 3 have a low burst release, but a faster release rate in the middle release period, a lower release endpoint, and a release rate much lower than that of the microspheres in Example 1 and Comparative Examples 1 and 2. The semaglutide sustained-release microspheres prepared in Example 1 have no obvious burst release, and the release behavior is stable in the middle and late stages. This shows that the semaglutide sustained-release microspheres prepared in the present invention have long-term sustained release ability.

[0205] Example 7: In vivo animal PK assay

[0206] In this example, the semaglutide sustained-release microspheres prepared in Example 1, Comparative Examples 1 and 2 were used as examples to conduct animal PK tests. The specific methods are as follows:

[0207] (1) Animal screening and grouping: 12 male SD rats were screened and divided into 3 groups according to their body weight, with 4 rats in each group: Group 1: Example 1, Group 2: Comparative Example 1, and Group 3: Comparative Example 2.

[0208] (2) Dosing frequency and method: All rats were given 1 mg / kg test solution in sequence according to the group, and all were given a single subcutaneous dose.

[0209] (3) Blood collection time: The detection time is once before administration, and once at 0.5h, 2h, 4h, 8h, 12h, 24h, and 2d, 3d, 4d, 5d, 9d, 12d, 16d, 19d, 23d, 26d, and 30d after administration.

[0210] (4) Blood collection method: All groups received blood from the jugular vein.

[0211] (5) Detection method: The method for detecting the blood concentration of semaglutide is to add an isotope internal standard to the standard curve sample, quality control sample and unknown sample (sample to be tested), and then perform protein precipitation extraction. After the extraction is completed, LC-MS / MS detection is performed. The PK curves of rats are shown in Figure 4 (Example 1) and Figure 5 (Comparative Examples 1 and 2).

[0212] As can be seen from Figures 4 and 5, the blood concentration of the semaglutide sustained-release microspheres prepared in Comparative Examples 1 and 2 of the present invention decreased rapidly after administration. Although the blood concentration of Comparative Example 2 increased slightly in the second week after administration, it was still low overall. The semaglutide sustained-release microspheres prepared in Example 1 of the present invention had no obvious burst release, and the blood concentration reached the highest at 15 days and remained in a relatively high concentration range within 30 days, indicating that the semaglutide sustained-release microspheres prepared in the present invention had a low burst release and had long-term sustained release ability.

[0213] Example 8: Simulation of the effect of polymerization inhibitor

[0214] In this embodiment, liraglutide (Lira) and semaglutide (Sema) are used as examples to perform a simulation experiment on the anti-aggregation effect. The specific method is as follows:

[0215] Inhibitors (inhibitor 1: sucrose, inhibitor 2: phenol), the API, and deionized water were mixed in a weight ratio of 1:3:100 and then lyophilized. PBS (pH 7.4) was added dropwise to the lyophilized drug powder to simulate the gradual infiltration of the drug into the microspheres. A portion of the drug was then dissolved in PBS and atomic force microscopy was used to observe whether the drug formed fibers, simulating the structural changes of the drug during slow penetration into the microspheres. The aggregation results are shown in Figure 6.

[0216] As can be seen from Figure 6, the inhibitor 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 drugs into fibers, and the semaglutide sustained-release microspheres prepared by the present invention can effectively avoid the loss of drug activity.

[0217] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0218] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing sustained-release microspheres of polypeptide drugs, characterized in that: The preparation method comprises the following steps: The aggregation inhibitor, the emulsion stabilizer A and water are mixed, and the pH value of the mixture is adjusted using the pH adjuster A, and then the polypeptide drug is added thereto to form an inner water phase; Mixing a carrier material, an emulsion stabilizer B and an organic solvent to form an oil phase; Mixing a surfactant, an osmotic pressure regulator, and water, and adjusting the pH value of the mixture using a pH regulator B to form an external aqueous phase; and, The inner water phase is added to the oil phase for dispersion to form colostrum, and the colostrum is then added to the outer water phase for redispersion to form a pre-emulsion, and then the pre-emulsion is emulsified to form a composite emulsion, and finally the composite emulsion is solidified to obtain the sustained-release microspheres.

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 aggregation inhibitor is at least one of sodium octanoate, sucrose, mannitol, 8-(2-hydroxybenzamido)-sodium octanoate, phenol, benzyl alcohol, ethanol and trifluoroethanol, preferably sucrose; and / or, the weight ratio of the aggregation 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, redistilled water and water for injection, preferably ultrapure water; and / or, the amount ratio of the water to the polypeptide drug is 3 mL:10-650 mg, preferably 3 mL:70 mg; and / or, The pH adjuster A is at least one of hydrogen chloride, sodium hydroxide and phosphate; and / or, the pH adjuster A adjusts the pH value of the mixture to 6.0-9.5, 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 ratio of the organic solvent to the polypeptide drug is 1-10 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 polylactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polylactic acid, polyhydroxyalkanoate and polycaprolactone, preferably polylactic acid-glycolic acid copolymer, more preferably polylactic acid-glycolic acid copolymer in which the ratio of lactic acid to glycolic acid per 100 parts of monomer is 100-50:0-50, preferably 50:50; and / or, the molecular weight of the carrier material is 1000-150000Da, preferably 20000Da; 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 lecithin, 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 to 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 dodecylbenzene sulfonate 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 to 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 to 100:1, preferably 80:1; and / or, The water is at least one of ultrapure water, redistilled water and water for injection, preferably ultrapure water; and / or, the amount ratio of the water to the polypeptide drug is 0.001-10 mL:1 mg, preferably 50 mL:7 mg; and / or, The pH adjuster B is at least one of hydrogen chloride, sodium hydroxide and phosphate; and / or, the pH adjuster 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 accomplished by at least one of stirring, homogenization and ultrasound, preferably homogenization, more preferably high-pressure homogenization; preferably, the high-pressure homogenization time is 0.5 to 20 minutes, preferably 2 minutes; and / or, the high-pressure homogenization pressure is 0.2 to 1200 bar, preferably 1 bar; and / or, The redispersion is accomplished by at least one of stirring and homogenizing, preferably stirring; and / or, The emulsification is accomplished by membrane emulsification; preferably, the membrane pore size of the membrane emulsification is 1.0 to 60 μm, preferably 40 μm; and / or, the transmembrane pressure of the membrane emulsification is 1 to 200 kPa, preferably 50 kPa; and / or, The curing is accomplished by volatilization or evaporation, preferably reduced pressure volatilization or thin film evaporation, more preferably thin film evaporation; preferably, the time of the thin film evaporation is 1 to 50 minutes, preferably 10 minutes; 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, characterized in that The preparation method further comprises the following steps: After the multiple emulsion is solidified, it is washed and freeze-dried.

7. The preparation method according to any one of claims 1 to 6, characterized in that The particle size of the sustained-release microspheres is 1.0-100 μm, preferably 50 μm; the particle size distribution span is 0.2-2.0; and / or, The encapsulation rate 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, characterized in that: The sustained-release microspheres are prepared by the preparation method according to any one of claims 1 to 7.

9. A pharmaceutical preparation of a polypeptide drug, characterized in that: The pharmaceutical preparation comprises the sustained-release microspheres 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 microspheres according to claim 8 or the pharmaceutical preparation according to claim 9 in the preparation of a medicament for preventing and / or treating at least part of a disease and / or condition related to GLP-1R; Preferably, the disease and / or disorder at least partially related to GLP-1R is diabetes, weight loss or non-alcoholic fatty liver disease, preferably type 2 diabetes or weight loss.

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