KR20070042598A - Method for preparing sustained release polymeric microspheres comprising a protein drug - Google Patents

Abstract

The present invention relates to a method for producing a protein drug-containing sustained-release polymer microspheres, comprising: (1) preparing a protein drug mixture by mixing and drying a protein drug, a hydrophilic polymer and a cyclodextrin derivative in an aqueous solution; (2) preparing a primary emulsion by adding a viscous water-soluble aqueous polymer solution to an organic solvent containing a biodegradable polymer; (3) dispersing the protein drug mixture in the primary emulsion; And (4) adding the emulsion obtained in step (3) to an external aqueous continuous phase to prepare a secondary emulsion, and then separating the solids produced in the secondary emulsion, the protein drug comprising: By stably and uniformly containing, it is possible to produce sustained-release polymer microspheres capable of minimizing initial release of the drug and continually releasing the drug.

Description

METHODS FOR PREPARING SUSTAINED RELEASE POLYMERIC MICROSPHERES COMPRISING A PROTEIN DRUG}

1 is a scanning electron microscope (SEM) photograph of the polymer microspheres prepared in Example 1,

Figure 2 is a confocal micrograph of the polymer microspheres prepared in Example 1 showing the distribution of lysozyme in the microspheres,

3 to 13 is a graph showing the change in drug release rate with time of the polymer microspheres prepared in Examples 1 to 32 and Comparative Example 7,

14 shows the results of SDS-PAGE analysis of recombinant human growth hormone extracted from the polymer microspheres prepared in Examples 23, 26, 29 and 32.

The present invention relates to a method for producing sustained-release polymer microspheres which can induce a uniform and stable encapsulation of a protein drug, thereby minimizing the initial release of the drug and continuously releasing the drug for a long time.

Stable encapsulation of protein drugs has been the basis of formulation in the study of the formulation of polymer microspheres of protein drugs, and the technology of suppressing the initial over-release of drugs and controlling the controlled release of drugs is also a long-term release (sustained release) polymer. There is a continuing study of microsphere formulations.

For example, US Pat. No. 6,113,947 discloses a complex of protein drug and zinc by adding zinc carbonate or zinc acetate as a protein drug stabilizer, and then dispersing the composite particles in an organic solvent in which the biodegradable polymer is dissolved (solid -in-oil) is disclosed a method for producing a polymer microspheres by spray drying. However, according to this method of coating the protein-zinc complex particles with the biodegradable polymer, the shape of the microspheres can be changed according to the size of the protein-zinc complex, it is difficult to control the sustained release of the drug, and the organic solvent and the protein Since the direct contact, the drug may be denatured.

U.S. Patent No. 4,652,441 discloses a technique for improving the encapsulation efficiency of water-soluble peptides by using the viscosity of gelatin, but gelatin is obtained by acid or base treatment on animal collagen protein, and the viscosity varies depending on temperature, and thus the variation in the preparation of microspheres There is a risk of occurrence.

In Korean Patent No. 329336, sodium hyaluronate having an average molecular weight of 2 million daltons and human growth hormone was dissolved in phosphate buffer solution and spray dried to prepare microparticles, and the drug-coated microparticles were dispersed in ethanol containing lecithin. After spray drying, the sustained release microspheres were manufactured. At this time, sodium hyaluronate coated with the drug contributes to the stabilization of the protein drug but has the disadvantage of releasing the drug in 1-2 weeks.

US Pat. No. 6,120,787 discloses a process for preparing polymeric microspheres by coating protein drugs with starch and then dispersing them in an ethylacetate solvent in which a copolymer of polylactic acid-polyglycolic acid is dissolved and spray drying. In this method, the protein drug is coated with starch to block contact between the drug and the organic solvent.However, due to the nature of the viscous starch, it is difficult to control the size and shape of the coated protein drug. There is a possibility of causing a deviation, the drug release tendency of the polymer microspheres may depend on the size of the starch / protein particles, it is not easy to control the uniform distribution of the drug in the particles.

Korean Patent Laid-Open Publication No. 2004-18434 discloses spray-drying an aqueous BMP-2 or interferon drug with a protein drug stabilizer, 2-hydroxypropyl-beta-cyclodextrin, glycine (glycine) or mannitol in water phase, and then The obtained fine powder particles are dispersed in an acetonitrile solvent in which a polylactic acid-polyglycolic acid copolymer is dissolved, depressurized on a silicone oil, and acetonitrile is removed to produce a polymer microsphere. In addition, US Pat. No. 6,346,274 discloses polyhistidine (poly-L- in order to suppress irreversible aggregation of protein drugs, which often occurs during the manufacturing process by water-in-oil-in-water). stabilizers such as histidine, poly-L-arginine, bovine serum albumin, dextran, hydroxy-beta-cyclodextrin, Tween 20, and pluronic copolymers were used. However, in these documents, appropriate methods of protein drug stabilizers should be adopted and used according to the kind or properties of the protein drugs used, so these methods have limitations to universal techniques (VR Shinha, J. Control. Release, 90, 261-280, 2003).

On the other hand, US Patent No. 6,413,536 is a technique for physically mixing a sucrose acetate isobutyrate (SAIB) that is friendly and viscous to a bioactive drug and a bioactive drug and then lowering the viscosity of the SAIB using a solvent to inject into the body, And it is disclosed that the SAIB changed to high viscosity as the solvent injected through the natural outflow acts as a carrier in the body to induce a stable release of the bioactive drug.

However, no examples have been reported to extend the drug release period by using the SAIB in the formulation of drug-containing polymeric microspheres.

Accordingly, an object of the present invention is to provide a method for preparing a sustained release polymer microspheres which can induce stable release and uniform distribution of protein drugs in microspheres to minimize initial release of the drug and to continuously release the drug for a long time. will be.

According to the above object, the present invention,

(1) preparing a protein drug mixture by mixing and drying the protein drug, hydrophilic polymer and cyclodextrin derivative in an aqueous solution;

(2) preparing a primary emulsion (W / O, water-in-oil) by adding a viscous water-soluble aqueous polymer solution to an organic solvent containing a biodegradable polymer;

(3) dispersing the protein drug mixture in the primary emulsion (S / W / O, solid-in-water-in-oil); And

(4) Secondary emulsion (S / W / O / W, solid-in-water-in-oil-in-water) was prepared by adding the emulsion obtained in step (3) to an external aqueous continuous phase. And then separating the solids formed in the secondary emulsion

It includes, provides a method for producing a polymer microspheres.

Hereinafter, the present invention will be described in more detail.

In the polymer microsphere preparation according to the present invention, the protein drug is first coated with a cyclodextrin derivative, and then dispersed in an emulsion composed of an aqueous phase (containing a viscous water-soluble polymer) and an oil phase (containing a biodegradable polymer). It is characterized in that the secondary coating with a water-soluble polymer and the third coating with a biodegradable polymer. These primary and secondary coatings block contact between the protein drug and the organic solvent, reliably encapsulate the protein drug in the microspheres without chemical / physical denaturation, and effectively protect the initial over-release of the protein drug through three coats. It can be suppressed.

When the polymer microspheres preparation method according to the present invention in detail by dividing step by step as follows.

<Step (1)>

In step (1), the hydrophilic polymer and cyclodextrin derivatives are dissolved with protein drug in an aqueous solution such as distilled water, phosphate buffer, borate buffer and Tris-HCl buffer and then dried by lyophilization or spray drying. To prepare a protein drug micromix.

Representative examples of hydrophilic polymers used in the present invention include poly (beta-hydroxyethyl methacrylate), polyacrylamide, polyvinyl alcohol, polyacrylic acid ( polyacrylic acid and salts thereof, polyvinylpyrrolidone, polyethyleneglycol, hyaluronic acid, pullulan, pectin, lignin, starch, chitosan (chitosan), alginic acid, curdlan, gelatin, dextran, riban, glucan, polyhistidine, polylysine, these Derivatives thereof and mixtures thereof, preferably polyethylene glycol, hyaluronic acid, starch, pectin, alginic acid, chitosan, polyhistidine and polylysine, more preferably polyethylene glycol and You can use the Aru acid.

The hydrophilic polymer may be used in an amount of 0.01 to 10 parts by weight, preferably 0.2 to 3 parts by weight, based on 1 part by weight of protein drug.

In particular, polyethylene glycol is a biodegradable polymer that is commonly used, but is not particularly limited to one having a weight average molecular weight of 1,000 to 20,000, and may be used in an amount of 0.2 to 10 parts by weight based on 1 part by weight of protein drug. Polyethylene glycol is soluble in organic solvents such as dichloromethane, ethyl acetate, dimethyl sulfoxide, dimethyl formamide, chloroform, alcohol, acetone, and aqueous solutions, and thus breaks down the protein drug mixture more finely when the protein drug mixture is added to the water or oil phase It acts as a micronizing agent to evenly distribute the protein drug on the microspheres. In addition, polyethylene glycol, a crystalline polymer, forms a water channel, which is a main release path of a drug, in a copolymer of an amorphous polymer, polylactic acid-polyglycolic acid (a type of biodegradable polymer used in step (2)). It facilitates the continuous release of drug release.

In addition, hyaluronic acid is a natural high molecular material present in the form of an inorganic salt such as sodium, which may contribute to stabilization of protein drugs by interacting with a positively charged material to form a physical complex. Hyaluronic acid can be used in an amount of 0.01 to 0.5 parts by weight, preferably 0.05 to 0.2 parts by weight based on 1 part by weight of protein drug.

Representative examples of the cyclodextrin derivatives used in the present invention include 3-mono- O- methyl-cyclodextrin, 2,6-di-ortho-methyl-cyclodextrin (2 , 6-di- O- methyl-cyclodextrin), 2,3,6-tri-ortho-methyl-cyclodextrin (2,3,6-tri- O- methyl-cyclodextrin), 2-hydroxyethyl-cyclo 2-hydroxyethyl-cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3-hydroxypropyl-cyclodextrin, 6-ortho-glucosyl-cyclo Dextrin (6- O- Glucosyl-cyclodextrin), 6-ortho-maltosyl-cyclodextrin (6- O- maltosyl-cyclodextrin), 6-ortho-dimaltosyl-cyclodextrin (6- O- dimaltosyl-cyclodextrin ), 2,6-di-ortho-ethyl-cyclodextrin (2,6-di- O- ethyl-cyclodextrin), 2,3,6-tri-ortho-ethyl-cyclodextrin (2,3,6 -t ri- O- ethyl-cyclodextrin), 2,3-di-ortho-hexanoyl-cyclodextrin (2,3-di- O- hexanoyl-cyclodextrin), 2,3,6-tri-ortho-acetyl- Cyclodextrin (2,3,6-tri- O- acetyl-cyclodextrin), 2,3,6-tri-ortho-propanoyl-cyclodextrin (2,3,6-tri- O- propanoyl-cyclodextrin) , 2,3,6-tri-ortho-butanoyl-cyclodextrin (2,3,6-tri- O- butanoyl-cyclodextrin), 2,3,6-tri-ortho-hexanoyl-cyclodextrin ( 2,3,6-tri- O -hexanoyl-cyclodextrin) , 6- ortho-carboxymethyl-cyclodextrin (6- O -carboxymethyl-cyclodextrin), sulfated cyclodextrin (sulfated cyclodextrin), sulfo-butyl-cyclodextrin (sulfobutyl-cyclodextrin), derivatives thereof and mixtures thereof, preferably 2-hydroxypropyl-cyclodextrin, 2,6-di-ortho-methyl-cyclodextrin, 6-ortho-mal Tosyl-cyclodextrin, and sulfur Butyl-beta-cyclodextrin derivative-cyclodextrin sulfonate ether sodium (β-cyclodextrin sulfobutyl ether sodium, CAPTISOL (product name)) may be used. The cyclodextrin derivative may be used in an amount of 0.1 to 20 parts by weight based on 1 part by weight of protein drug. Cyclodextrin derivatives form complexes with protein drugs in the cavities present in the chemical structure to serve as protein drug stabilizers to prevent denaturation of protein drugs that may be caused by lyophilization and organic solvents.

Protein drugs used in the present invention are suitably composed of two or more amino acids and have a weight average molecular weight of 200 to 100,000. Representative examples of protein drugs include human growth hormone, insulin, bovine growth hormone, pig growth hormone, growth hormone releasing peptide, growth hormone releasing peptide, B cell factor, T cell factor, granulocyte-colony stimulating factor (G-CSF). ), Granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), erythropoietin, skeletal morphogenetic protein (bone) morphogenic protein, interferon, atriopeptin-III, monoclonal antibody, tumor necrosis factor (TNF), macrophage activating factor, interleukin, Tumor degenerating factor, insulin-like growth factor, epidermal growth factor, tissue plasminogen activity (tissue plasminogen activator), urokinase, protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin and its Fragment polypeptides, apolipoprotein-E, factor VII, factor VIII, factor IX, pancreatic polypeptide, protein C, C-reactive protein, renin inhibitors, collagenase inhibitors, superoxide dismutase, platelet derived growth factors , Epidermal growth factor, osteogenic growth factor, bone formation promoting protein, calcitonin, atriopeptin, cartilage inducing factor, connective tissue activator, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, Nerve growth factor, parathyroid hormone, secretin, jomatomedin, adrenocorticotropic hormone, glucagon, cholecystokinin, gastrin releasing peptide Tide, corticotropin releasing factor, thyroid stimulating hormone, monoclonal or polyclonal antibodies against various viruses, bacteria, toxins, and various virus-derived vaccine antigens, and also leuprorelin acetate. , Human leutenizing hormone releasing hormone analogues such as goserelin acetate, nafarelein acetate, buserelin acetate, and gonadorelin (humira), remicade, octreotide acetate, salts thereof, and mixtures thereof.

<Step (2)>

In step (2), a viscous water-soluble aqueous polymer solution (water phase) is added to an organic solvent (oil phase) containing a biodegradable polymer and vigorously stirred to prepare a primary emulsion (W / O, water-in-oil). do. At this time, the volume ratio of the aqueous phase and the oil phase may be in the range of 1: 3 to 1:30, preferably 1: 5 to 1:15.

Representative biodegradable polymers used in the present invention include poly (acryloyl hydroxyethyl) starch, copolymers of polybutylene terephthalate-polyethylene glycol, chitosan and derivatives thereof, Copolymers of polyorthoester-polyethylene glycol, copolymers of polyethylene glycol terephthalate-polybutylene terephthalate, poly sebacic anhydride, pullulan and derivatives thereof, starch and derivatives thereof, Cellulose acetate and its derivatives, polyanhydride, polycaprolactone, polycarbonate, polybutadiene, polyesters, polyhydroxybutyric acid ), Polymethyl methacrylate, polymethacrylic acid Polymethacrylic acid ester, polyorthoester, polyvinyl acetate, polyvinyl alcohol, polyvinyl butyral, polyvinyl formal, protein ( Albumin, casein, collagen, fibrin, fibrinogen, gelatin, homoglobin, transferrin, zein) and mixtures thereof, preferably polylactic acid, polyglycolic acid, copolymers of polylactic acid-polyglycolic acid and polycapro Lactones can be used. The biodegradable polymer is suitable to have an average molecular weight of 2,000 to 100,000, and it is excellent in biocompatibility and biodegradability since it is decomposed into water and carbon dioxide through the citric acid cycle, which is a normal biometabolism process when administered to the body. (SJ Holland et al., J. Controlled Release , 4, 155-180, 1986). The biodegradable polymer-containing organic solvent may include the biodegradable polymer at a concentration of 5 to 60% (w / v).

The organic solvent used in the present invention is required to have a miscibility that does not occur phase separation of the biodegradable polymer and hydrophobic surfactant, dichloromethane, ethyl acetate, dimethyl sulfoxide, dimethylformamide, chloroform, alcohol, acetone and It may be selected from the group consisting of a mixture thereof.

According to the present invention, hydrophobic surfactants and / or sucrose acetate isobutyrate (SAIB) can be added to the organic solvent containing the biodegradable polymer.

Representative examples of hydrophobic surfactants used in the present invention include fatty acids, olefins, alkyl carbons, silicones, sulfate esters, fatty alcohol sulfates. , Sulfated fat and oil, sulfonic acid salt, aliphatic sulfonate, alkylaryl sulfonate, ligmin sulfonate, phosphate ester (phosphoric acid ester), polyoxyethylene, polyglycerol, polyol, imidazoline, alkanolamine, hetamine, sulfomethamine , Phosphatids, sorbitan fatty acid esters and mixtures thereof, preferably sorbitan fatty acid esters, more preferably sorbitan wool You can use oleate (sorbitan monooleate). The hydrophobic surfactant can be added to the organic solvent at a concentration of 0.1 to 30% (v / v), preferably 0.1 to 20% (v / v).

SAIB is a viscous, biodegradable polymer that has affinity for protein drugs and can be added to organic solvents at a concentration of 5 to 60% (w / v). Among the biodegradable polymers described above, polylactic acid-polyglycolic acid Preference is given to using with the copolymer. The weight ratio of SAIB to biodegradable polymer may be 1: 0.1 to 1: 5, preferably 1: 0.2 to 1: 2. The use of viscous SAIB delays drug release of polymeric microspheres, allowing the drug to be released over a period of two months or longer.

Viscous water-soluble polymers used in the present invention are excellent in biocompatibility and harmless to the human body, including starch, cellulose, hemicellulose, pectin, lignin, chitosan ), Xanthan gum, alginic acid, pullulan, curdlan, gelatin, dextran, levan, hyaluronic acid, glucan glucan, collagen, salts thereof, and mixtures thereof. As the viscous water-soluble polymer, preferably starch, hyaluronic acid or gelatin, more preferably sweet potato starch, soluble starch or hyaluronic acid may be used. The water-soluble polymer aqueous solution may include the water-soluble polymer in a concentration of 0.1 to 10% (w / v). Viscous water-soluble polymers serve as viscous membranes that block the denaturation of protein drugs at the interface between organic solvents and water phases by imparting viscous properties to the aqueous micro-droplets on primary emulsions. Evenly enclosed can inhibit the early release of protein drugs and induce sustained release.

<Step (3)>

In step (3), the protein drug mixture prepared in step (1) is dispersed and vigorously stirred in a primary emulsion containing fine droplets of viscous water phase prepared in step (2) (S / W / O, The solid-in-water-in-oil protein drug mixture is transferred to microdroplets consisting of a viscous aqueous phase.

Polyethyleneglycol as a hydrophilic polymer in a protein drug mixture is dissolved in an organic solvent to refine the protein drug particles, and the micronized protein drug particles migrate into droplets consisting of a viscous aqueous phase and are evenly distributed in the droplets. In particular, the solidified protein drug mixture containing cyclodextrin, a drug stabilizer, has a primary denaturation blocking effect by inhibiting direct contact with an organic solvent and moves to droplets forming a viscous aqueous phase to prevent inflow of organic solvent. It will have a blocking effect. Since the protein drug particles are spread evenly in the droplets consisting of a viscous aqueous phase, it is possible to provide polymer microspheres having a uniform drug distribution.

<Step (4)>

In step (4), the emulsion obtained in step (3) is added to an external aqueous continuous phase to prepare a secondary emulsion (S / W / O / W, solid-in-water-in-oil-in-water). ), And then the desired polymer microspheres are prepared by separating the solid produced in the secondary emulsion by filtration or centrifugation.

External aqueous continuous phases used in the present invention include polyvinyl alcohol, methyl cellulose, cetyltrimethyl ammonium bromide, sodium dodecyl sulphate or polyoxyl. An aqueous solution of ethylene sorbitan monooleate may be used, and preferably an aqueous polyvinyl alcohol solution may be used. The concentration of the aqueous solution used as the external continuous phase may be 0.1 to 5% (w / v), preferably 0.3 to 2% (w / v). When an aqueous polyvinyl alcohol solution is used, the weight average molecular weight of the polyvinyl alcohol is 10,000 to 100,000, preferably 13,000 to 50,000, and the degree of hydrolysis is 75 to 95%, preferably 83 to 89%.

The polymeric microspheres prepared by the method of the present invention have an average particle diameter of 0.1 to 200 μm, preferably 1 to 100 μm, and may contain 1 to 40 wt% of the drug based on the total weight.

The sustained release polymer microspheres according to the present invention can stably and uniformly contain a protein drug, thereby minimizing the initial release of the drug and continually releasing the drug for a long time, that is, for more than one month, thereby injecting (muscle and subcutaneous injection) Aerosol formulations, graft tablets, implant preparations, and the like, which are intended to be absorbed into the nasal mucosa, bronchial mucosa, and pulmonary mucosa, together with conventional excipients.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1

To a 1 ml aqueous solution of phosphate (pH 5.1), polyethylene glycol (average molecular weight 2000), CAPTISOL (product name) (CyDex Inc.) and 100 mg of lysozyme, a drug were added, mixed and lyophilized to prepare a protein drug mixture. At this time, polyethylene glycol was dissolved at a rate of 2.5% (w / v) and CAPTISOL (product name) at a rate of 10% (w / v) to the aqueous phosphate solution.

The oily solution required for the preparation of the primary emulsion was dissolved in 500 mg of polylactic acid-polyglycolic acid (molar ratio of lactic acid: glycolic acid = 50:50) RG 502H (Boehringer Ingelheim Inc.) in 3 ml of dichloromethane. Then, water-soluble starch was dissolved in water as an internal aqueous phase to prepare 0.5 ml of a 2.5% (w / v) starch aqueous solution. The internal aqueous phase was poured into an oily solution and vigorously stirred to obtain a primary emulsion, and the protein drug mixture prepared above was dispersed therein to obtain an S / W / O solution.

An external aqueous continuous phase comprising 0.5% (w / v) polyvinyl alcohol and 0.9% (w / v) sodium chloride was uniformly stirred at 4,000 rpm with the S / W / O solution in a volume ratio of 1 to 200 It was added slowly and dispersed for 5 minutes in a homogenizer to prepare a S / W / O / W type secondary emulsion. The prepared secondary emulsion was centrifuged (3000 rpm, carried out for 2 minutes) to separate solids, and then put into a lyophilizer and dried for 48 hours to obtain a polymer microsphere.

Example 2

Polymeric microspheres were prepared in the same manner as in Example 1 except that CAPTISOL (product name) was dissolved in a ratio of 2.5% (w / v) in the aqueous solution of phosphate.

Example 3

CAPTISOL (Product Name) Polymeric microspheres were prepared in the same manner as in Example 1 except for using 2-hydroxypropyl-cyclodextrin instead.

Example 4

Polymeric microspheres were prepared in the same manner as in Example 1 except for using 2,6-di-ortho-methyl-cyclodextrin instead of CAPTISOL (product name).

Example 5

Polymeric microspheres were prepared in the same manner as in Example 1 except for using 6-ortho-maltosyl-cyclodextrin instead of CAPTISOL (product name).

Example 6

Polymeric microspheres were prepared in the same manner as in Example 1, except that 0.5 ml of an aqueous 0.25% (w / v) hyaluronic acid solution was used instead of an aqueous aqueous starch solution.

Example 7

Polymeric microspheres were prepared in the same manner as in Example 1, except that 0.5 ml of 1.5% (w / v) gelatin aqueous solution was used instead of the aqueous aqueous starch solution.

Example 8

Polymeric microspheres were prepared in the same manner as in Example 1, except that 0.5 ml of 0.25% (w / v) sweet potato starch aqueous solution was used instead of the aqueous aqueous starch aqueous solution.

Example 9

Copolymer of 400 mg polylactic acid-polyglycolic acid (molar ratio of lactic acid: glycolic acid = 50:50) A mixture of RG 502H and 0.25 ml of sorbitan monooleate 80 in 3 ml of dichloromethane Polymeric microspheres were prepared in the same manner as in Example 1 except that the dissolved oily solution was used.

Example 10

Copolymer of 400 mg polylactic acid-polyglycolic acid (molar ratio of lactic acid: glycolic acid = 50:50) Using an oily solution of a mixture of RG 502H and 0.10 ml of sorbitan monooleate 80 in 3 ml of dichloromethane Polymer microspheres were prepared in the same manner as in Example 1 except that.

Comparative Example 1

Polymeric microspheres were prepared in the same manner as in Example 1, except that the pure protein drug was not mixed with polyethylene glycol and CAPTISOL (product name), and the solidified protein drug was directly dispersed in the prepared oily solution without an internal aqueous phase. (solid-in-oil-in-water, S / O / W method).

Comparative Example 2

Polymeric microspheres were prepared in the same manner as in Example 1, except that pure protein drugs without polyethylene glycol and CAPTISOL (product name) were used, and an aqueous phosphate solution (pH 5.1) was used as the inner aqueous phase.

Comparative Example 3

Polymeric microspheres were prepared in the same manner as in Example 1 except for using pure protein drugs in which polyethylene glycol and CAPTISOL (product name) were not mixed.

Comparative Example 4

Polymeric microspheres were prepared in the same manner as in Example 1 except that CAPTISOL (Product Name) was not used.

Comparative Example 5

Polymer microspheres were prepared in the same manner as in Example 1 except that polyethylene glycol was not used.

Comparative Example 6

A polymeric drug mixture was prepared by mixing the same amount of water-soluble starch with lysozyme as in Example 1, and dispersing the protein drug mixture directly in the prepared oily solution without an internal aqueous phase. Was prepared.

Comparative Example 7

Polyethylene glycol, CAPTISOL (product name) and water-soluble starch of the same content as in Example 1 were mixed with lysozyme to prepare a protein drug mixture, except that the protein drug mixture was dispersed directly in the prepared oily solution without an internal aqueous phase. Polymeric microspheres were prepared in the same manner as in Example 1.

Example 11

Except for dissolving polyethylene glycol at a rate of 5% (w / v) with respect to an aqueous solution of phosphate, and additionally adding hyaluronic acid at a rate of 1% (w / v) to the aqueous solution of phosphate, which interacts with the drug. Polymeric microspheres were prepared in the same manner as in Example 1.

Example 12

Polymeric microspheres were prepared in the same manner as in Example 11, except that 0.25 ml of sorbitan monooleate 80 was additionally added to the oily solution as a hydrophobic surfactant.

Example 13

Polymeric microspheres were prepared in the same manner as in Example 11, except that 0.1 ml of sorbitan monooleate 80 was additionally added to the oily solution as a hydrophobic surfactant.

Example 14

Polymeric microspheres were prepared in the same manner as in Example 11, except that 0.05 ml of sorbitan monooleate 80 was additionally added to the oily solution as a hydrophobic surfactant.

Example 15

Hyaluronic acid, which interacts with the drug, is further added to the phosphate buffer containing polyethylene glycol and CAPTISOL (product name) at a rate of 0.5% (w / v), and a copolymer of 300 mg of polylactic acid-polyglycolic acid ( Molar ratio of lactic acid: glycolic acid = 50:50) using an oily solution of RG 502H and 100 mg of RG 503H (molar ratio of lactic acid: glycolic acid = 50:50) (Boehringer Ingelheim Inc.) in 3 ml of dichloromethane. Except for producing a polymer microspheres in the same manner as in Example 1.

Example 16

Polymeric microspheres were prepared in the same manner as in Example 15 except that CAPTISOL (product name) was dissolved in a ratio of 5.0% (w / v) in the aqueous solution of phosphate.

Example 17

Polymeric microspheres were prepared in the same manner as in Example 15 except that CAPTISOL (product name) was dissolved in a ratio of 2.5% (w / v) in the aqueous solution of phosphate.

Example 18

Dissolving polyethylene glycol at a rate of 5.0% (w / v) to an aqueous solution of phosphate, and using an oily solution in which 300 mg of RG 502H and 100 mg of sucrose acetate isobutyrate (SAIB) were dissolved in 3 ml of dichloromethane. Except for producing a polymer microspheres in the same manner as in Example 1.

Example 19

Polymeric microspheres were prepared in the same manner as in Example 18, except that 0.5 ml of sorbitan monooleate 80 was additionally added to the oily solution as a hydrophobic surfactant.

Example 20

Polymeric microspheres were prepared in the same manner as in Example 18, except that 0.25 ml of sorbitan monooleate 80 was additionally added to the oily solution as a hydrophobic surfactant.

Example 21

In 1 ml tertiary distilled water, polyethylene glycol (average molecular weight 2000), CAPTISOL (product name) and the drug 10 mg of gonadorelin (Gonadorelin, Cytoshop Inc., human leutenizing hormone releasing hormone homologue) Protein drug mixtures were prepared by addition, mixing and lyophilization. At this time, polyethylene glycol was dissolved at a rate of 0.5% (w / v) and CAPTISOL (product name) at a rate of 1% (w / v) to tertiary distilled water.

The oily solution required for the preparation of the primary emulsion was prepared by dissolving 100 mg of polylactic acid-polyglycolic acid copolymer RG 503H in 1 ml of dichloromethane and dissolving the water-soluble starch in water as an internal aqueous phase in 5.0% (w / v). 0.1 ml of starch aqueous solution was prepared. The internal aqueous phase was poured into an oily solution and vigorously stirred to obtain a primary emulsion, and the protein drug mixture prepared above was dispersed therein to obtain an S / W / O solution.

While stirring the external aqueous continuous phase containing 0.5% (w / v) polyvinyl alcohol and 0.9% (w / v) sodium chloride uniformly at 4,000 rpm, the S / W / O solution was added at a volume ratio of 1 to 200. Slowly added and dispersed in a homogenizer for 5 minutes to prepare a S / W / O / W type secondary emulsion. The prepared secondary emulsion was centrifuged (3000 rpm, carried out for 2 minutes) to separate solids, and then put into a lyophilizer and dried for 48 hours to obtain a polymer microsphere.

Example 22

Polymeric microspheres were prepared in the same manner as in Example 21, except that 10 mg of leuprorelin acetate (Cytoshop Inc.) was used as a protein drug.

Example 23

0.2 ml of tertiary distilled water containing polyethylene glycol and CAPTISOL (product name) at the same concentration as in Example 21, 2 mg of recombinant human growth hormone (WakO Inc.) as a protein drug, RG Polymeric microspheres were prepared in the same manner as in Example 21, except that RG 502H was used instead of 503H.

Example 24

Same as Example 21, except that polyethylene glycol was dissolved at a rate of 1% (w / v) in tertiary distilled water, and 0.05 ml of sorbitan monooleate 80 was further added to the oily solution as a hydrophobic surfactant. Polymeric microspheres were prepared by the method.

Example 25

Polymeric microspheres were prepared in the same manner as in Example 24, except that 10 mg of lurelin acetate was used as a protein drug.

Example 26

Except for using 0.2 ml of tertiary distilled water containing polyethylene glycol and CAPTISOL (product name) at the same concentration as in Example 21, using 2 mg of recombinant human growth hormone as protein drug, and using RG 502H instead of RG 503H. Prepared polymer microspheres in the same manner as in Example 24.

Example 27

Dissolve polyethylene glycol and CAPTISOL (product name) at a rate of 1% (w / v) and 0.25% (w / v), respectively, in tertiary distilled water, and 1% (w) of hyaluronic acid in tertiary distilled water. / v) was further added, and polymer microspheres were prepared in the same manner as in Example 21, except that an oily solution in which 75 mg of RG 502H and 25 mg of RG 503H was dissolved in 1 ml of dichloromethane was used. .

Example 28

Polymeric microspheres were prepared in the same manner as in Example 27, except that 10 mg of lurelin acetate was used as a protein drug.

Example 29

5 mg of recombinant human growth hormone was used as a protein drug, and 1% (w / v) of hyaluronic acid in the tertiary distilled water containing polyethylene glycol and CAPTISOL (product name) at the same concentration as in Example 21 was used. Polymer microspheres were prepared in the same manner as in Example 21, except that an oily solution in which 75 mg of RG 502H and 25 mg of RG 503H were dissolved in 1 ml of dichloromethane was added.

Example 30

Polymeric microspheres were prepared in the same manner as in Example 21, except that an oily solution in which 60 mg of RG 502H, 40 mg of SAIB, and 0.1 ml of sorbitan monooleate 80 was dissolved in 1 ml of dichloromethane was used.

Example 31

Polymeric microspheres were prepared in the same manner as in Example 30, except that 10 mg of lurelin acetate was used as a protein drug.

Example 32

Polymeric microspheres were prepared in the same manner as in Example 30, except that 0.2 ml of tertiary distilled water containing polyethylene glycol and CAPTISOL (product name) at the same concentration as in Example 21 were used, and 2 mg of recombinant human growth hormone was used as the protein drug. Was prepared.

Test Example 1 Experiment of Measuring Loading Efficiency of Drug

10 mg of the polymer microspheres prepared in the Examples and Comparative Examples were accurately weighed and placed in a test tube with a lid containing 0.5 ml of dichloromethane solution. To this was added 5 ml of 6 moles of hydrochloric acid and vigorously stirred for 1 hour, after which the solution was centrifuged at 5000 rpm for 5 minutes. 1 ml of supernatant was taken, transferred to another test tube, placed in a shaking water bath and shaken at a rate of 60 times per minute at a temperature of 37 ° C. for 24 hours. 6 ml of 1 mol of sodium hydroxide solution was added to the solution and shaken at a speed of 60 times per minute at a temperature of 37 ° C. for 24 hours in a shaking constant temperature bath. 50 μl of the obtained neutralized aqueous solution, 125 μl of 4% (w / v) sodium bicarbonate (pH 9.0), and 50 μl of 0.5% (w / v) trinitrobenzenesulfonic acid solution were mixed for 2 hours. After standing at room temperature, the amount of drug in the microspheres was calculated by measuring the drug concentration with a microplate reader at λ = 450 nm. Using the following equations (1) and (2) to calculate the encapsulation rate of the drug encapsulated in the polymer microspheres, the results are shown in Table 1 below. In addition, a scanning electron microscope (SEM) photograph of the polymer microspheres prepared in Example 1 is shown in FIG. 1.

Drug content (%) = (total amount of drug in microspheres / total amount of microspheres) × 100

Encapsulation efficiency (%) of drug = (total amount of drug enclosed in microspheres / total amount of drug used for manufacture) × 100

From Table 1, it can be seen that the polymer microspheres of the embodiment according to the present invention has a higher drug content and drug encapsulation efficiency than the polymer microspheres of the comparative example, such as having a drug encapsulation efficiency of 92% or more. This means that the high viscosity water soluble polymer used in the Examples effectively blocked the outflow of the protein drug to the external continuous phase.

Test Example 2 Investigation of the Distribution of Drugs in the Microspheres

Lysozyme labeled with rhodamine-B-isothiocyanate was encapsulated in polymeric microspheres in the same manner as in Example 1. Sectionalization of the microspheres for confocal micrographs was performed in the following manner. 3.75 g of gelatin was quantified, dissolved in 15 ml of distilled water, and 85 mg of glycerin was added thereto, followed by vigorous stirring. Subsequently, the solution was heated to about 40 ° C. to dissolve and stirred at room temperature with 10 mg of polymer microspheres, followed by vacuum drying for 1 hour, and cut into 10 μm micro-tombs to obtain microsphere cross sections. . The cross-sectioned specimens were vacuum dried for two days and the microsphere distribution of the drug was examined under confocal microscopy.

Confocal micrographs of the polymeric microspheres prepared in Example 1 are shown in FIG. 2. The photograph of FIG. 2 shows that lysozyme (red) labeled with rhodamine-B-isocyanate is evenly distributed throughout the polymeric microspheres.

Test Example 3: Insoluble aggregation degree (%) measurement experiment

10 mg of the polymer microspheres prepared in Examples and Comparative Examples were accurately weighed and dissolved in 0.5 ml of dichloromethane, and then centrifuged at 5000 rpm for 5 minutes to remove the suspension and naturally dried at room temperature for 1 hour. The precipitate was dissolved in 1 ml of an aqueous 10 mM phosphate solution, and then insoluble aggregates were obtained by centrifugation. The isolated aggregates were dissolved in 1 ml of 10 mM phosphate solution containing 6 moles of urea. 50 μl of this solution, 125 μl of 4% (w / v) sodium bicarbonate (pH 9.0) and 50 μl of 0.5% (w / v) trinitrobenzenesulfonic acid solution were mixed and left at room temperature for 2 hours. The concentration of undissolved aggregates was measured with a microplate reader at λ = 450 nm, and the insoluble aggregation rate of the drug in the microspheres was calculated using Equation 3 below, and the results are shown in Table 2 below.

% Of drug incoagulation (%) = (total amount of drug in drug coagulation in microspheres / total amount of drug enclosed in microspheres) × 100

From Table 2, it can be seen that the polymeric microspheres of the examples according to the present invention exhibit a significantly lower incidence of aggregation than the polymeric microspheres of the comparative example. This shows that the high viscosity water-soluble polymer used in the Examples prevented the invasion of the organic solvent into the protein drug to prevent denaturation of the protein drug.

For reference, from the results of Examples 1, 6 and 7, it can be seen that the excellent stabilizing effect on the protein drug in the order of water-soluble starch, hyaluronic acid and gelatin.

Test Example 4: In vitro of the drug ( in vitro Release experiment

Drug release experiments were conducted in vitro ( in vitro ) to confirm whether the protein drug is continuously released from the polymer microspheres prepared in Examples and Comparative Examples. 40 mg of polymer microspheres were placed in a test tube containing 10 ml of 0.033 mol of phosphate aqueous solution (pH 7.4, 0.01% sodium azide, 0.02% Tween 80), sealed, and placed in a shaker bath at 37 ° C. at 60 speeds per minute for 60 days. Abnormal drug was released continuously. 5 ml of the release containing the released drug was taken each hour and fresh phosphate solution was replenished. The concentration of the drug in the release solution was measured with a micro BCA reagent kit, and the cumulative release amount was calculated for each time, and is shown in FIGS. 3 to 13 as a graph of the rate of drug release over time.

From the graphs of FIGS. 3 to 13, the polymeric microspheres of the examples according to the invention continuously release the drug throughout the test due to the even distribution of the drug in the microspheres, whereas the polymeric microspheres of Comparative Example 7 (see FIG. 3) were initially It can be seen that the drug is released excessively.

For reference, closely examining the graphs of FIGS. 3 to 13, the higher the content of the cyclodextrin derivative in FIG. 3, the total amount of release increased. In FIG. 4, even if the type of cyclodextrin derivative is different, the release tendency of the drug is not significantly changed. Did. In addition, it can be seen from FIG. 5 that the total drug release amount is high in the order of sweet potato starch (Example 8), gelatin (Example 7), and hyaluronic acid (Example 6). At this time, the drug release amount is increased from about 15-20 days of release in FIGS. 3 to 7, which is determined by metabolism of the biodegradable polymer (ie, copolymer of polylactic acid-polyglycolic acid) after 15-20 days. It is interpreted that the release is relatively quick. In addition, in FIG. 7, the total drug release amount was reduced to around 65% by the electrical interaction between hyaluronic acid and lysozyme. In FIG. 8, it was confirmed that the total drug release increased due to the decrease of the hyaluronic acid content and the drug release occurred close to the 0 th order after 3 days. It can be seen from FIG. 9 that drug release is effectively delayed due to the use of high viscosity SAIB, and in FIG. 10, drug release is increased in about 13-17 days, and low molecular weight gonadorelin and lurelin acetate are compared to lysozyme. The release of microspheres was so easy that each drug showed a total drug release of at least 90%. In FIG. 11, the release of sorbitan monooleate 80 increased drug release at about 10 days, and the release of gonadorelin and lubrilin acetate was continued for 35 days. In FIG. 12, the drug release delay period is not clear, but the drug release begins to increase in about 10-15 days, and gonadorelin, lubrilin acetate, and recombinant human growth hormone have nearly zero drug release for 30 days. In FIG. 13, the use of highly viscous SAIB resulted in an increase in drug release at a relatively delayed level of about 25 days, with drug release close to zero in 60 days.

Test Example 5 In Vitro of Drug ( in vitro Denaturation experiment

10 mg of the polymer microspheres prepared in Examples 23, 26, 29 and 32 were placed in a test tube containing 10 ml of an aqueous solution of phosphate (pH 7.4, 0.01% sodium azide, 0.02% Tween 80), and the test tubes were taken 60 times per minute at 37 ° C. Shaken in a constant temperature bath. Subsequently, the test tube was centrifuged at 3000 rpm for 3 minutes to remove the supernatant suspension, and the microspheres obtained by lyophilization for 2 days were placed in a test tube containing 0.2 ml of dichloromethane, stirred for 10 seconds, and then centrifuged twice at 5000 rpm for 3 minutes. Dichloromethane was removed. 1 ml of ethanol was placed in the test tube and centrifuged twice at 5000 rpm for 3 minutes to remove remaining dichloromethane. The obtained drug was vacuum dried for 1 day and then subjected to SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Bio-rad electrophoresis system) test to observe whether the drug denaturation, and the results are shown in FIG.

As can be seen in Figure 14, the drug obtained by the extraction after the preparation of the polymer microspheres was analyzed by SDS-PAGE, the denaturation did not occur at all, was sealed in the polymer microspheres in a stabilized state.

Test Example 6: In vitro activity (%) investigation of drug

40 mg of the polymer microspheres prepared in Examples and Comparative Examples were placed in a test tube containing 10 ml of an aqueous solution of phosphate (pH 7.4, 0.01% sodium azide, 0.02% Tween 80), and the test tube was shaken at 37 ° C. at a speed of 60 times per minute. Shaking in the bath was filled with fresh phosphate buffer one day before the scheduled time and supplemented with fresh phosphate buffer after all drug release was recovered at that time. The concentration of the discharge liquid was measured by the method as described in Test Example 4. 150 μl of the eluate (or pure crude drug at the same concentration) was mixed with 100 μl of Micrococcus lysodeikticus (ATCC 4698) cell suspension (66 mM phosphate buffer, pH 6.2) at a concentration of 0.5 mg / ml. The absorbance (measured at 450 nm) of the mixed solution was monitored every 15 minutes at 15 second intervals and the degree of absorption over time was graphed to calculate the relative activity (%) through the initial linear slope using Equation 4 below. The results are shown in Table 3 below.

Relative activity of drug (%) = (initial linear slope for drug released from microspheres) / (initial linear slope for raw drug) × 100

From Table 3, it can be seen that the polymer microspheres of the embodiment according to the present invention exhibits drug activity in a longer term than the polymer microspheres of the comparative example, such as maintaining the protein drug activity at 95% or more for 28 days. In particular, the polymer microspheres (Example 18) including SAIB showing affinity with the protein drug showed better drug stability.

As such, the polymeric microspheres of the present invention can stably and uniformly contain the protein drug, thereby minimizing the initial release of the drug and continually releasing the drug for a long time, that is, for more than one month, thereby providing for injection (muscle and subcutaneous injection). It can be usefully used in preparations such as aerosol preparations, implantable tablets, implant preparations and the like which are absorbed into the nasal mucosa, bronchial mucosa and pulmonary mucosa together with the preparations and conventional excipients.

Claims (22)

(1) preparing a protein drug mixture by mixing and drying the protein drug, hydrophilic polymer and cyclodextrin derivative in an aqueous solution; (2) preparing a primary emulsion by adding a viscous water-soluble aqueous polymer solution to an organic solvent containing a biodegradable polymer; (3) dispersing the protein drug mixture in the primary emulsion; And (4) adding the emulsion obtained in step (3) to an external aqueous continuous phase to prepare a secondary emulsion, and then separating the solids produced in the secondary emulsion. A method for producing a polymer microspheres comprising a. The method of claim 1, In step (1) the hydrophilic polymer is poly (beta-hydroxyethyl methacrylate), polyacrylamide, polyvinyl alcohol, polyacrylic acid and salts thereof, polyvinylpyrrolidone, polyethylene glycol, hyaluronic acid, pullulan, pectin, A method for producing polymeric microspheres, characterized in that it is selected from the group consisting of lignin, starch, chitosan, alginic acid, curdlan, gelatin, dextran, ribene, glucan, polyhistidine, polylysine, derivatives thereof and mixtures thereof. The method of claim 1, In step (1), characterized in that the hydrophilic polymer is used in an amount of 0.01 to 10 parts by weight based on 1 part by weight of the protein drug, a method for producing polymer microspheres. The method of claim 2, The hydrophilic polymer is polyethylene glycol or hyaluronic acid, characterized in that the manufacturing method of the polymer microspheres. The method of claim 4, wherein Polyethylene glycol is used in an amount of 0.2 to 10 parts by weight based on 1 part by weight of protein drug, the method for producing polymer microspheres. The method of claim 4, wherein A method for producing polymeric microspheres, characterized in that hyaluronic acid is used in an amount of 0.01 to 0.5 parts by weight based on 1 part by weight of protein drug. The method of claim 1, In step (1) the cyclodextrin derivative is 3-mono-ortho-methyl-cyclodextrin, 2,6-di-ortho-methyl-cyclodextrin, 2,3,6-tri-ortho-methyl-cyclodextrin , 2-hydroxyethyl-cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3-hydroxypropyl-cyclodextrin, 6-ortho-glucosyl-cyclodextrin, 6-ortho-maltosyl-cyclodextrin, 6-Ortho-Dimaltosyl-cyclodextrin, 2,6-di-ortho-ethyl-cyclodextrin, 2,3,6-tri-ortho-ethyl-cyclodextrin, 2,3-di-ortho -Hexanoyl-cyclodextrin, 2,3,6-tri-ortho-acetyl-cyclodextrin, 2,3,6-tri-ortho-propanoyl-cyclodextrin, 2,3,6-tri-or So-butanoyl-cyclodextrin, 2,3,6-tri-ortho-hexanoyl-cyclodextrin, 6-ortho-carboxymethyl-cycle A process for producing polymeric microspheres, characterized in that it is selected from the group consisting of roddextrin, sulfated cyclodextrin, sulfobutyl-cyclodextrin, derivatives thereof and mixtures thereof. The method of claim 1, The cyclodextrin derivative in step (1), characterized in that used in an amount of 0.1 to 20 parts by weight based on 1 part by weight of protein drug, a method for producing polymer microspheres. The method of claim 1, In step (1) the protein drug is human growth hormone, insulin, bovine growth hormone, porcine growth hormone, growth hormone releasing peptide, growth hormone releasing peptide, B cell factor, T cell factor, granulocyte-colony stimulating factor, granulocyte macrophage-colonie Stimulating factor, macrophage colony stimulating factor, erythropoietin, skeletal morphogenetic protein, interferon, atriopeptin-III, monoclonal antibody, tumor necrosis factor, macrophage activator, interleukin, tumor degeneration factor, insulin-like growth factor, Surface growth factor, tissue plasminogen activator, urokinase, protein A, allergic inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin And fragment polypeptides thereof, apolipoprotein-E, factor VII, factor VIII, factor IX, pancreatic polypeptide, protein C, C-half Sex proteins, renin inhibitors, collagenase inhibitors, superoxide dismutase, platelet derived growth factors, epidermal growth factor, osteogenic growth factor, bone formation promoting protein, calcitonin, atriopeptin, cartilage inducing factor, binding Tissue activator, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factor, parathyroid hormone, secretin, jomatomedin, adrenocorticotropic hormone, glucagon, cholecystokinin, gastrin releasing peptide, corticosteroid Pin release factor, thyroid stimulating hormone, monoclonal or polyclonal antibodies, virus-derived vaccine antigens, leuurorelin acetate, gosirelin acetate, naparelin acetate, bushrelin acetate, gonadorelin, humira, remicade, jade Threotide acetate, salts thereof, and mixtures thereof Method of producing a polymer, characterized in that the microspheres selected from the group eojin. The method of claim 1, The biodegradable polymer in step (2) is polyacryloyl hydroxyethyl starch, copolymer of polybutylene terephthalate-polyethylene glycol, chitosan and its derivatives, copolymer of polyorthoester-polyethylene glycol, polyethylene glycol terephthalate Copolymers of polybutylene terephthalate, polyceva anhydride, pullulan and derivatives thereof, starch and derivatives thereof, cellulose acetate and derivatives thereof, polyanhydrides, polycaprolactones, polycarbonates, polybutadienes, poly From the group consisting of esters, polyhydroxybutyric acids, polymethyl methacrylates, polymethacrylic acid esters, polyorthoesters, polyvinyl acetates, polyvinyl alcohols, polyvinylbutyral, polyvinyl foams, proteins and mixtures thereof Characterized in that it is selected, a method for producing a polymeric microsphere. The method of claim 1, The biodegradable polymer in step (2), characterized in that it has an average molecular weight of 2,000 to 100,000, method for producing polymer microspheres. The method of claim 1, Step (2) characterized in that the concentration of the biodegradable polymer in the organic solvent is 5 to 60% (w / v), the method for producing a polymer microspheres. The method of claim 1, The hydrophobic surfactant is added to the organic solvent containing the biodegradable polymer in the step (2), characterized in that the manufacturing method of the polymer microspheres. The method of claim 13, Hydrophobic surfactants include fatty acids, olefins, alkylcarbons, silicones, sulfate esters, fatty acid alcohol sulfates, sulfated fats and oils, sulfonates, aliphatic sulfonates, alkylaryl sulfonates, ligmine sulfonates, phosphate esters, polyoxyethylene , Polyglycerol, polyol, imidazoline, alkanolamine, hetamine, sulfomethamine, phosphide, sorbitan fatty acid esters, and mixtures thereof. The method of claim 13, A hydrophobic surfactant is added at a concentration of 0.1 to 30% (v / v) with respect to the organic solvent. The method of claim 1, Sucrose acetate isobutyrate is further added to the organic solvent containing the biodegradable polymer in step (2). The method of claim 16, Sucrose acetate isobutyrate is added to the biodegradable polymer in a weight ratio of 1: 0.1 to 1: 5, characterized in that the manufacturing method of the polymer microspheres. The method of claim 1, Characterized in that the organic solvent in step (2) is selected from the group consisting of dichloromethane, ethyl acetate, dimethylsulfoxide, dimethylformamide, chloroform, alcohol, acetone and mixtures thereof. The method of claim 1, In step (2), the viscous water-soluble polymer is starch, cellulose, hemicellulose, pectin, lignin, chitosan, xanthan gum, alginic acid, pullulan, curdlan, gelatin, dextran, ribene, hyaluronic acid, glucan, collagen, salts thereof And characterized in that the mixture is selected from the group consisting of, a method for producing polymeric microspheres. The method of claim 1, Step (2) characterized in that the aqueous solution of viscous water-soluble polymer contains a water-soluble polymer at a concentration of 0.1 to 10% (w / v), the method for producing a polymeric microsphere. The method of claim 1, Process for producing a polymeric microsphere, characterized in that the external aqueous continuous phase in step (4) is an aqueous polyvinyl alcohol solution. A protein drug-containing sustained-release polymer microsphere obtained by the method of any one of claims 1 to 21.

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