KR101651101B1 - Delayed-release microspheres and a method for manufacturing the same - Google Patents

Abstract

The present invention relates to a sustained release microparticle and a method for producing the same, which comprises a physiologically active agent, a biocompatible polymer and a release retardant, wherein the release retardant is a sustained release microparticle having an HLB value of 10 to 20, ≪ / RTI > The sustained-release microparticles according to the present invention are excellent in the delayed release properties of physiologically active agents and can provide a short process time and a low residual solvent.

Description

TECHNICAL FIELD [0001] The present invention relates to a sustained-release microparticle and a method for manufacturing the same,

To a sustained-release microparticle and a method for producing the same.

The compounds may be prepared in the form of microparticles using a variety of known techniques. In particular, it is advantageous to encapsulate the biologically active ingredient within the biocompatible, biodegradable polymer within the microparticle to provide sustained or delayed release of the active ingredient. In such a production method, the active ingredient is mixed and dissolved in a solvent containing a biodegradable polymer, microspheres are prepared using a known method, and the solvent is removed to obtain a product.

At this time, a method of producing microparticles using two or more solvents without halogenated hydrocarbons has been disclosed, but the manufacturing time is too long and it is difficult to remove used solvents (see Korean Patent No. 10-0354270). Further, in order to remove the residual solvent in the above-mentioned patent, a method of washing the microparticles and extracting the solvent in the water-miscible organic solvent has been disclosed, but this also has a disadvantage that the working time is too long and the removal of the residual solvent is not complete Korean Patent No. 10-0432677).

In order to solve such problems, the present inventors have completed the process for producing microparticles and surfactants using the specific surfactants.

Disclosure of the Invention The present invention is to solve the above problems and to provide a bioactive drug, a biocompatible polymer and a release retarder that are excellent in the retardation of release of a bioactive agent and capable of providing a short process time and a low residual solvent, The present invention aims to provide a sustained-release microspheres having a HLB value of 5 to 35, which is a surfactant.

Also, there is provided a method for producing a microorganism, which comprises dissolving a physiologically active agent, a biodegradable polymer and a surfactant in an organic solvent and cooling them to form microparticles, wet-filtering the microparticles, stirring the mixture on a surfactant, wet- And a method for producing the sustained-release microparticles.

Various embodiments described herein are described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions, and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" the embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

In one embodiment of the present invention, the term "HLB value" (Hydrophile-Lipophile Balance) is a value indicating the degree of affinity of a surfactant for water and oil. To find a surfactant suitable for the purpose of use, It was a concept proposed by Wallace Griffin of the 1949 Atlas Powder Company to meet the needs. The HLB value is usually from 0 to 20, and the closer to 0 the lipophilic property is, and the closer to 20 the hydrophilic property is. However, it is reported that the HLB value is 20 or more recently.

In one embodiment of the present invention, the term "physiologically active drug" is a biologically active drug including, but not limited to, risperidone, paliperidone, aripiprazole, olanzapine, and the like as neuroleptic drugs have. Also, therapeutic agents for gastrointestinal tract such as aluminum hydroxide, calcium carbonate, magnesium carbonate, sodium carbonate and the like; Nonsteroidal contraceptive pill: Parasympathetic stimulant; Antipsychotic drugs such as haloperidol, bromperidol, fluphenazine, sulpiride, caripipramine, clocapramine, mosapramine, olanzapine, and sertindole; Chiropragmine HCL, clozapine, mezzanine, methiapine, leisure pin, thioridazine and the like; A psychostabilizer such as chlorodiaxanthide, diazepam, meflobamate, and themejem; Non-scientific (nasal) decongestants; Hypnotic sedatives such as codeine, phenobarbital, sodium pentobarbital, and sodium secobarbital; Steroids such as testosterone and testosterone propionate; Sulfonamide; Sympathetic excitement drug: vaccine; Vitamins and nutrients such as essential amino acids and essential fatty acids; Antimalaria agents such as 4-aminoquinoline, 8-aminoquinoline, and pyrimethamine; Anti-migraine agents such as marginal stone, penttermin, and sumatriptan; A therapeutic agent for Parkinson's disease such as L-dopa; Antipyretics such as atropine, metscopolamine bromide and the like; Anticholinergic drugs such as anti-inflammatory drugs, digestive drugs and enzymes; Jinhae medicines such as dextromethorphan and noscapine; Cardiovascular agents such as bronchodilators, hypertension therapeutic compounds, Rauwolfia alkaloids, coronary vasodilators, nitroglycerin, organic nitrides, pentaerythritol tetranitrate; Electrolyte replacement therapy such as potassium chloride; Ergot alkaloids such as caffeine-free or ergotamine, hydrated ergot alkaloids, dihydroergocristine methanesulfate, dihydroergone cornine methanesulfonate, dihydroergarko-tripeptide methanesulfate, and combinations thereof; Alkaloids such as atropine sulfate, belladonas, and hyosine hydrobromide; Analgesic medicine; Anesthetics such as codeine, dihydrocodeineone, mepiridine, morpholine and the like; Non-narcotic such as salicylate, aspirin, acetaminophen, d-proxiphen, etc .; It is not always necessary to use a drug such as cephalosporin, cephalosporin, chloramphenicol, gentamicin, kanamycin A, kanamycin B, penicillin, ampicillin, streptomycin A, antimycin A, chlorophenothiol, metronidazole, oxytetracycline penicillin G, My; Anticancer agents; Antipyretics such as mefenitone, phenobarbital and trimetadione; Anti-histamines such as chlorpinazine, dimehydrinate, diphenhydramine, perphenazine, and tripelenamine; Anti-inflammatory agents such as hormone preparations, hydrocortisone prednisolone, prednisone, non-hormone preparations, allopurinol, aspirin, indomethacin, phenylbutazone and the like; Cytotoxic agents such as prostaglandins, thiotepa, chlorambucil, cyclopospasmide, melphalan, nitrogen mildard, methotrexate and the like; Nessseria gonarrhea, Micobacterium tuberculosis, Herpes virus (Humonis, types 1 and 2), Candida albicans, Candida albicans (Candida albicans), Candida albicans Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Streptococcus ecoli, Micoplasma hominis, Hemophilus doublecray, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Trepone mapallidum, Brucella abortus, Brucella melitensis, Brucella melitensis, Brucella suis, Brucella canis, Campylobacter pterus, Campylobacter pterus testinis, Leptospira pomona, Brucella ovis, Equine Herpes virus, horse arthritis virus, IBR-IBP virus, BVD-MB virus, Clamydia psittaci, Trichomonas puotus (strains such as Strea monocytogenes, Brucella ovis, Equine Herpes virus, Tricomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa, But are not limited to, Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigentium, Aspergillus fumigatus, ), Absidia ramosa, Trypanosoma equiperdum, Barbesia cavalli, Clostridium Microbial antigens such as tetany; An antibody that neutralizes the microorganisms, a ribonuclease, a neuraminidase, a trypsin, a glucocogenphosphorylase, a sperm lactdehydegenase, a sperm hyaluronidase, an adenosine triphosphatase, an alkaline phosphatase, But are not limited to, an enzyme, an enzyme, an enzyme, an enzyme, an enzyme, an alkaline phosphatase, an aminopeptidase, trypsin, chymotrypsin, amylase, muramadinease, acrosomal proteinase, diesterase, glutamate dehydrogenase, Enzymes such as glycosphosphatase, lipase, ATP-aza alpha peptate gamma glutamyl transpeptidase, sterol-3-beta-all-dehydrogenase and DPN-di-aflorease, diethylstilbestrol, Estrogen such as 17-beta-estradiol, estrone, ethidyl estradiol, mestanol; But are not limited to, norepinephrine, norethindrone, norgestrel, ethynodiol diacetate, linestrenol, medroxyprogesterone acetate, dimestysterone, megestroate, acetate, chlormadinone acetate, nogeustimate, , Progesterone such as melengesterol, norethynol; And sanitizer compounds such as nonylphenoxy polyoxyethylene glycol, benzethonium chloride, chlorindanol, and the like.

In one embodiment of the present invention, the "release control agent" is a component capable of delaying or suppressing the release of the physiologically active agent encapsulated in the microspheres. It is intended to delay or suppress the excessive release of the physiologically active agent initially, ≪ / RTI > release of the bioactive agent. For example, but not limited to, polysorbate (Tween), sorbitan ester (Span), poloxamer, Gelucire, and the like can be used. More specifically, Tween 80, Poloxamer 188, Poloxamer 407, Gelucire 44/14, Span 20, PEG 4000, Span 80, Gelucire 43/01 and the like.

In one embodiment of the invention, "microparticles" or "microparticles" are solid particles dispersed or dissolved in biodegradable, biocompatible polymers that act as matrix particles and include solid particles comprising the active agent.

In one embodiment of the invention, "biocompatibility" means that the substance of interest is not toxic to the body, is pharmaceutically acceptable, is not carcinogenic, and does not cause inflammation significantly in body tissues. Thus, a "biocompatible polymer" refers to, for example but not limited to, polylactide, polyglycolide, poly (lactide-co-glycolide) and poly (lactide-co-glycolide) glucose. In the present invention, the proportion of the poly (lactide glycolide) copolymer of the biocompatible polymer may be 50:50 to 95: 5. For example, Resomer RG502H, RG503H, RG504H, RG752H, RG752S, RG753S, RG755S, RG756S, RG757S, RG858S, R202H, R203H, R207H or 5050 DLG, 6535 DLG, 7525 DLG, 8515 DLG from Lakeshare Co., , 955 DLG, and the like. In one embodiment, the biocompatible polymer may comprise from 42.0 to 82.0% by weight, or from 52.0 to 72.0% by weight, or from 57.0 to 67.0% by weight, based on the total weight of the microsphere.

In one embodiment of the present invention, risperidone, a sustained-release formulation of risperidone, is initially administered with an oral preparation, which is administered daily for 15 days while administering a sustained-release formulation. Therefore, if the drug is released early in the case of the western medicine, the concentration of the drug in the blood may increase, which may cause toxicity and side effects. Therefore, the drug should not be released for 15 days after the administration, and then released. Therefore, it is possible to manufacture microparticles having a lag time during a certain period of time, and at the same time shortening the processing time, which is a disadvantage of the conventional production method, and easily removing the residual solvent

In one embodiment of the present invention, the physiologically active drug and the biocompatible polymer are dissolved in a non-aqueous solution, the release retardant is mixed, and the mixture is added to an aqueous medium to prepare a microparticle. . ≪ / RTI >

Hereinafter, the present invention will be described in detail. More specifically, the present invention relates to a method for producing a biocompatible polymer, comprising: i) dissolving a physiologically active agent and a biocompatible polymer in an organic solvent; Ii) mixing a release retardant with the non-aqueous solution of step i); Iii) introducing the solution formed in step ii) into an aqueous medium to form microparticles; And iv) removing the organic solvent. In the above embodiment, the non-aqueous solvent which can be used for dissolving the biocompatible polymer in step i) has a volatilization temperature of 85 占 폚 or less and is not particularly limited so long as it can dissolve the biodegradable polymer. For example, methylene chloride, chloroform, acetonitrile, ethyl acetate and the like can be used. Further, in the above embodiment, in the step iv), any method which is commonly used can be used for removing the organic solvent. To remove the organic solvent are, for stirring, heating, such as a nitrogen purged (N ₂ purge) it can be used example but is not limited to this.

In one embodiment of the invention, the release retardant may comprise from 0.1 to 10.0 parts by weight, or from 0.1 to 5.0 parts by weight, based on 100 parts by weight of the microparticle.

In one embodiment of the invention, the sustained release microspheres are provided which comprise a biologically active agent, a biocompatible polymer and a release retardant, wherein the release retardant is a surfactant having an HLB value of 5 to 35. In this embodiment, the release-retarding agent is selected from the group consisting of polysorbate (Tween), sorbitan ester (Span), poloxamer and Gelucire, Wherein the biocompatible polymer is at least one selected from the group consisting of polylactide, polyglycolide, poly (lactide-co-glycolide) and poly (lactide-co-glycolide) Providing a sustained-release microparticle having an intrinsic viscosity of 0.5 to 1.9 dL / g, wherein the biocompatible polymer is provided with a sustained release microparticle containing 0.1 to 10.0 parts by weight per 100 parts by weight of the microparticle, Wherein the amount of the physiologically active agent is 25 to 51 parts by weight based on the total weight of the microparticle, and the physiologically active agent is at least one selected from the group consisting of risperidone, Provide any one of a sustained-release microspheres selected from the group consisting of pyrrolidone, aripiprazole, and olanzapine.

In one embodiment of the invention, there is provided an injection or pharmaceutical composition comprising said microparticle.

In one embodiment of the present invention, there is provided a method for producing a biocompatible polymer, comprising dissolving a biologically active agent and a biocompatible polymer in an organic solvent; Adding a release retardant and mixing to prepare a mixture; Introducing the mixture into an aqueous medium to form a microsphere; And removing the organic solvent. The present invention also provides a method for producing sustained-release microparticles. In this embodiment, there is provided a process for the preparation of a sustained release microparticle further comprising wet filtration and lyophilization of the microparticles after removal of the organic solvent, wherein the release retardant is a surfactant having an HLB value of 5 to 35, The present invention provides a method for producing sustained-release microparticles, which provides a method for producing a sustained-release microparticle in which the wet filtration is carried out with a bovine mesh having a size of 20 to 200 탆, and a wet filtration is further carried out with a bovine mesh having a size of 20 to 60 탆 do.

The sustained-release microparticles according to the present invention are excellent in the delayed release properties of physiologically active agents and can provide a short process time and a low residual solvent.

1 is a photograph of a surface of commercially available microspheres taken by SEM (see Comparative Example 7)
2 is a SEM photograph of the surface of one of the microparticles according to one embodiment of the present invention. (Microparticles containing release inhibitor)
FIG. 3 through FIG. 7 are graphs showing the dissolution rate at 37 ° C. according to one embodiment of the present invention and a comparative example.

Hereinafter, the components and technical features of the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention and are not intended to limit the scope of the invention.

Manufacture of microspheres

43% (w / w) (manufactured by Aurobindo, Inc.) of a total solid content including a biodegradable polymer having a ratio of lactide to glycolides of 75:25 to 95:15 by Boehringer Gelheim or Lakeshare and risperidone, (W / w), and the ethanol is dissolved in a solvent of 12% (w / w) relative to the total weight of the non-aqueous solvent, and the concentration of the biocompatible polymer is 15% ). The mixture was completely dissolved in the solvent by stirring without solid matter.

Then, a release retardant such as Tween 80 or Poloxamer 188 was added so as to be 1 part by weight (w / w) based on 100 parts by weight of the polymer (w / w) and sufficiently stirred to prepare a dispersion.

The dispersion was slowly added to a 0.5% polyvinyl alcohol (Mn = 30,000 to 70,000; Sigma) aqueous solution at 10 DEG C using an L4R mixer (Silverson, UK) inline unit at 3,000 rpm to prepare microspheres.

Thereafter, the temperature was raised to 40 ° C., the organic solvent was volatilized for 4 hours, and the solution was cooled to 10 ° C. for 1 hour. The prepared microspheres were washed with the injection water and then subjected to the primary wet filtration with a 25 μm and 160 μm thick extrudate. The mixture was further stirred with a 3% (w / w) Tween 20 solution, followed by secondary wet filtration with a 45 um sized bovine embryo, followed by lyophilization and refrigeration. Various microspheres were prepared by varying the type and ratio of the biodegradable polymer and the release retardant (see Table 1).

Manufacturing example Biodegradable polymer Release Delay% HLB value One RG757S Tween 80 15 2 RG757S Poloxamer 188 29 3 RG858S: RG756S = 60: 40 Tween 80 15 4 RG858S: RG756S = 80: 20 Poloxamer 188 29 5 7525 9E Poloxamer 188 29 6 8515 7E Gelucire 44/14 14 7 8515 7E Gelucire 50/13 13 8 8515 7E Gelucire 43/01 One 9 8515 7E Poloxamer 188 29 10 RG858S Tween 80 15 11 RG858S PEG4000 18.5 12 RG858S Span 80 4.3 13 8515 7E: 7525 9E = 70: 30 Tween 80 15 14 8515 7E: 7525 9E = 70: 30 Poloxamer 188 29

Microparticles were prepared in the same manner as in Example 1 except that the release retardant was not included, and the biodegradable polymer was used in the same manner as in any of Preparation Examples 1 to 14 of Table 1 (see Table 2).

Comparative Example Biodegradable polymer One RG757S 2 RG858S: RG756S = 60: 40 3 7525 9E 4 8515 7E 5 RG858S 6 8515 7E: 7525 9E = 70: 30 7 Risperdal Constar

Identify the features and effects of microspheres

Micropartial morphometry

Approximately 10 mg of microspheres were fixed on an aluminum stub and platinum coated for 3 minutes under a vacuum of 0.1 torr and a high voltage of 10 kV to observe the surface of the microspheres. And the surface of the microspheres was observed using an image analysis program. The measurement results are shown in Fig.

Measurement of the rate of risperidone in the microspheres

Approximately 110 mg of the microspheres were completely dissolved in the mobile phase, filtered through a 0.45 μm syringe filter and used as a sample solution, and the content of risperidone encapsulated in the microspheres was measured by HPLC. The column used was C18 ODS 3 mu m and 4.0 x 100 mm, the injection amount was 20 mu L, and the detection wavelength was 275 nm. The mobile phase was measured using a pH 6.7 ammonium acetate buffer: acetonitrile = 75:25. The measurement results are shown in Table 3, indicating that about 90% or more of the risperidone was contained in the solution.

Drug inclusion rate (%) Production Example 1 88.6 Production Example 2 90.2 Production Example 3 89.5 Production Example 4 91.1 Production Example 5 91.2 Production Example 6 93.5 Production Example 7 90.7 Production Example 8 89.9 Production Example 9 87.2 Production Example 10 85.9 Production Example 11 83.5 Production Example 12 88.1 Production Example 13 87.5 Production Example 14 83.9 Comparative Example 1 86.2 Comparative Example 2 85.3 Comparative Example 3 89.1 Comparative Example 4 85.6 Comparative Example 5 87.1 Comparative Example 6 84.3

Microsphere in vitro  Long term dissolution test

Approximately 20 mg of the microspheres of Production Example 1-14 and Comparative Example 1-7 were taken at 37.0 ° C in a 200 ml test tube in a pH 7.4 HEPES solution, and the supernatant was taken at 1 day, 8 days, 15 days, and the elution amount of risperidone was measured by HPLC , And the measurement results and graphs are shown in Tables 4 and 3, respectively.

Dissolution rate of risperidone (%) Days 1 day 8 days 15th Production Example 1 0.1 1.2 5.4 Production Example 2 0.2 1.9 4.1 Production Example 3 0.3 0.6 5.0 Production Example 4 0.2 0.5 1.0 Production Example 5 0.3 1.6 4.8 Production Example 6 0.3 1.1 6.5 Production Example 7 0.3 1.3 6.9 Production Example 8 0.2 39.8 50.0 Production Example 9 0.4 5.2 11.4 Production Example 10 0.1 1.9 4.6 Production Example 11 0.7 1.7 13.6 Production Example 12 0.5 41.4 69.1 Production Example 13 0.3 0.7 1.8 Production Example 14 0.7 2.4 3.9 Comparative Example 1 0.4 15.0 - Comparative Example 2 0.2 4.5 30.8 Comparative Example 3 0.3 79.6 - Comparative Example 4 0.2 26.2 - Comparative Example 5 0.1 0.6 12.6 Comparative Example 6 0.2 46.0 - Comparative Example 7 1.0 3.0 7.2

* Confirmation of dissolution rate inhibition rate from 37 ℃ to 15 days

From the above results, it was found that the dissolution rate of the active ingredient was significantly delayed to 15 days in the samples using the release retardant having the HLB value of 5 to 35 as compared with the other preparation examples and the comparative example.

Residual solvent test of microspheres

About 100 mg of microspheres were placed in a GC vial and dissolved in 10 mL of internal standard solution, and residual solvent was measured in the microspheres using GC. The column used was filled with G25 series, and the inner diameter was 25 m x 0.32 mm, the film thickness was 0.5 mu m, the injection amount was 1 mu l, and the temperature was 250 DEG C using a flame ionization detector.

Methylene chloride
(ppm) ethanol
(ppm) Ethyl acetate
(ppm) Benzyl alcohol
(ppm) Production Example 1 71 215 Production Example 2 61 230 Production Example 3 N / D 220 Production Example 4 N / D 230 Production Example 5 55 205 Production Example 6 35 210 Production Example 7 43 220 Production Example 8 N / D 235 Production Example 9 78 235 Production Example 10 90 225 Production Example 11 49 210 Production Example 12 72 215 Production Example 13 N / D 290 Production Example 14 90 260 Comparative Example 1 63 225 Comparative Example 2 75 230 Comparative Example 3 85 230 Comparative Example 4 50 310 Comparative Example 5 83 294 Comparative Example 6 76 276 Comparative Example 7 - 100 1236 1437

(N / D: Not Detected, not detected)

Blood drug concentration test

The prepared microspheres and commercially available preparations were diluted in 0.5 mL of the suspension, and a dose of 900 ug / kg was administered intramuscularly (IM) as risperidone, and about 0.5 mL of a blood sample was collected at a predetermined time. 100 uL of the collected blood was added with 100 uL of acetonitrile, and proteins were separated and centrifuged to analyze the concentration of risperidone using LC / MS / MS. The measurement results are shown in Fig.

Claims (14)

A biologically active agent, a biocompatible polymer and a release retardant,
The biocompatible polymer has PLA: PGA = 70:30 to 89:11,
The physiologically active agent is contained in an amount of 25 to 51 parts by weight based on 100 parts by weight of the microparticle,
The release-retarding agent is contained in an amount of 0.1 to 10.0 parts by weight based on 100 parts by weight of the microparticle,
For the sustained release microparticles having an HLB value of 5 to 35,
Wherein the microparticle is Tween 80 or Poloxamer 188 when the biocompatible polymer is PLA: PGA = 70:30 to 79:21,
Wherein the microparticle is a sustained release microparticle wherein the release retardant is Tween 80, Gelucire 44/14, Gelucire 50/13, or PEG 4000 when the biocompatible polymer is PLA: PGA = 80:20 to 89:11. delete The method according to claim 1,
Wherein the biocompatible polymer is at least one selected from the group consisting of polylactide, polyglycolide, poly (lactide-co-glycolide) and poly (lactide-co-glycolide) The method of claim 3,
Wherein the biocompatible polymer has an intrinsic viscosity of 0.5 to 1.9 dL / g. delete delete The method according to claim 1,
The physiologically active agent is any one selected from the group consisting of risperidone, paliperidone, aripiprazole, and olanzapine. Dissolving a physiologically active agent and a biocompatible polymer in an organic solvent;
Adding a release retardant and mixing to prepare a mixture;
Introducing the mixture into an aqueous medium to form a microsphere; And
Removing the organic solvent,
The biocompatible polymer has PLA: PGA = 70:30 to 89:11,
The physiologically active agent is contained in an amount of 25 to 51 parts by weight based on 100 parts by weight of the microparticle,
The release-retarding agent is contained in an amount of 0.1 to 10.0 parts by weight based on 100 parts by weight of the microparticle,
For the sustained release microparticles having an HLB value of 5 to 35,
Wherein the microparticle is Tween 80 or Poloxamer 188 when the biocompatible polymer is PLA: PGA = 70:30 to 79:21,
Wherein the microspheres are Tween 80, Gelucire 44/14, Gelucire 50/13, or PEG 4000 when the biocompatible polymer is PLA: PGA = 80:20 to 89:11. 9. The method of claim 8,
Further comprising, after removing the organic solvent, wet filtration and lyophilization of the microspheres. delete 10. The method of claim 9,
Wherein the wet filtration is carried out with a mortar of 20 to 200 mu m in size. 12. The method of claim 11,
Wherein the wet filtration is further carried out with a sizing of 20 to 60 um. delete An injection comprising the microparticle of any one of claims 1, 3 and 4.

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