CN108403643A - Protein and peptide drugs continuous release microsphere and preparation method thereof - Google Patents
Protein and peptide drugs continuous release microsphere and preparation method thereof Download PDFInfo
- Publication number
- CN108403643A CN108403643A CN201810522338.1A CN201810522338A CN108403643A CN 108403643 A CN108403643 A CN 108403643A CN 201810522338 A CN201810522338 A CN 201810522338A CN 108403643 A CN108403643 A CN 108403643A
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- Prior art keywords
- protein
- preparation
- peptide drugs
- continuous release
- drug
- Prior art date
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- Granted
Links
- 239000004005 microsphere Substances 0.000 title claims abstract description 94
- 239000003814 drug Substances 0.000 title claims abstract description 87
- 229940079593 drug Drugs 0.000 title claims abstract description 85
- 238000002360 preparation method Methods 0.000 title claims abstract description 67
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 63
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 52
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 39
- 229920000642 polymer Polymers 0.000 claims abstract description 33
- 239000006210 lotion Substances 0.000 claims abstract description 23
- 229920001184 polypeptide Polymers 0.000 claims abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 14
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- 239000003223 protective agent Substances 0.000 claims abstract description 10
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- 239000003960 organic solvent Substances 0.000 claims abstract description 7
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- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 claims description 10
- 229960003773 calcitonin (salmon synthetic) Drugs 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 108010068072 salmon calcitonin Proteins 0.000 claims description 9
- IEJIGPNLZYLLBP-UHFFFAOYSA-N dimethyl carbonate Chemical compound COC(=O)OC IEJIGPNLZYLLBP-UHFFFAOYSA-N 0.000 claims description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 6
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 229920000382 poly(ethylene glycol) methyl ether-block-poly(L-lactide-co-glycolide) Polymers 0.000 claims description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
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- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
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- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 108010010803 Gelatin Proteins 0.000 claims description 2
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- 108010050904 Interferons Proteins 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 2
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims description 2
- 239000008273 gelatin Substances 0.000 claims description 2
- 229920000159 gelatin Polymers 0.000 claims description 2
- 235000019322 gelatine Nutrition 0.000 claims description 2
- 235000011852 gelatine desserts Nutrition 0.000 claims description 2
- 229940079322 interferon Drugs 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
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- 230000002035 prolonged effect Effects 0.000 claims description 2
- -1 sorbierite Substances 0.000 claims description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims 1
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- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
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- 238000010008 shearing Methods 0.000 description 4
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- 230000008023 solidification Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012074 organic phase Substances 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- NIQCNGHVCWTJSM-UHFFFAOYSA-N Dimethyl phthalate Chemical compound COC(=O)C1=CC=CC=C1C(=O)OC NIQCNGHVCWTJSM-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000011805 ball Substances 0.000 description 2
- 210000003022 colostrum Anatomy 0.000 description 2
- 235000021277 colostrum Nutrition 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
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- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- IMROMDMJAWUWLK-UHFFFAOYSA-N Ethenol Chemical compound OC=C IMROMDMJAWUWLK-UHFFFAOYSA-N 0.000 description 1
- 108010022355 Fibroins Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 108010000817 Leuprolide Proteins 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 108010016076 Octreotide Proteins 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001142 circular dichroism spectrum Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- FBSAITBEAPNWJG-UHFFFAOYSA-N dimethyl phthalate Natural products CC(=O)OC1=CC=CC=C1OC(C)=O FBSAITBEAPNWJG-UHFFFAOYSA-N 0.000 description 1
- 229960001826 dimethylphthalate Drugs 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 229960004338 leuprorelin Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229960002700 octreotide Drugs 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960002668 sodium chloride Drugs 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011882 ultra-fine particle Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/23—Calcitonins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Endocrinology (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a kind of protein and peptide drugs continuous release microspheres and preparation method thereof.The preparation method includes the following steps：(1) protein or polypeptide water soluble drug and protein protective agent are dissolved in water, obtain water phase drug solution；Polymer carrier is dissolved in organic solvent, oil phase Polymer Solution is obtained；(2) the water phase drug solution is added in the oil phase Polymer Solution, W/O lotions is prepared into using cell crushing instrument or high speed shear instrument；(3) the W/O lotions are at the uniform velocity fed by peristaltic pump in ultrafine dust preparation system and the protein and peptide drugs continuous release microsphere is prepared.The preparation method prescription is simple, and mild condition, preparation process is simple, and parameter is controllable, and reproducibility is good, and production efficiency is high.The protein and peptide drugs microballoon prepared with this method, entrapment efficiency is high, and burst release rate is low, and it is lasting that drug release rate is stablized, and microsphere features smooth surface, ball-type are complete, uniform particle diameter.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, more particularly to a kind of protein and peptide drugs continuous release microsphere and its preparation side
Method.
Background technology
Protein and peptide drugs are the large biological molecules being most widely used at present, because it is with potency height, bio-compatible
Property the good, features such as dosage is low, preventing, serving as more and more important role in diagnosing and treating disease.As having in organism
The active material of standby metabolic function, polypeptide drugs are related to the every field such as hormone, nerve, cell growth and reproduction.With
Traditional small-molecule chemical drug is compared, effect is clear, toxic side effect is small, pharmacological action is strong in protein and peptide drugs, and not
Good reaction is few, safety higher, therefore its research and development has become one of the hot spot in medical research and development field.
The stability of protein and peptide drugs is poor, and oral administration biaavailability is low, and Half-life in vivo is short, thus clinically leads
Frequent drug administration by injection is usually required in order to reach curative effect based on the dosage form of injection and freeze-dried powder, lead to that patient's is suitable
Answering property is low.In order to improve the bioavilability of protein and peptide drugs, administration frequency is reduced, the exploitation of durative action preparation is drug
One Main way of novel form and new industrial research development.Wherein, it after long-acting injection microsphere preparation is by single-dose, can tie up
The internal effective blood drug concentration a few days to several months etc. is held, the treatment requirement of various disease is met.Up to the present, pass through FDA
The protein and peptide drugs microballoon of approval includes that Leuprorelin microballoon, music score of Chinese operas Rayleigh microballoon, Octreotide microballoon and growth hormone are micro-
Ball etc..
In existing research, W/O/W multi-emulsion methods are to prepare to carry the most common method of polypeptide drugs microball preparation.It should
Method is that water-solubility protein polypeptide drugs are dissolved in inner aqueous phase, and the carriers such as PLGA are dissolved in organic phase, and organic phase breast is added in inner aqueous phase
Change forms w/o type colostrum, then colostrum is transferred to formation W/O/W type emulsions in the outer aqueous phase containing emulsifier, and emulsion is in large volume
It is slowly stirred in dispersed phase, so that organic phase is gradually volatilized and cure balling-up.This process steps is cumbersome, and due to the addition of outer aqueous phase,
It is easy to cause protein and peptide drugs to migrate out from inner aqueous phase, leads to the shortcomings of microsphere encapsulation rate is low, burst release rate is high.
Invention content
Based on this, the present invention provides a kind of preparation methods of protein and peptide drugs continuous release microsphere, utilize party's legal system
Standby microballoon, entrapment efficiency is high, and acquisition time is long, and burst release rate is low.
Specific technical solution is as follows：
A kind of preparation method of protein and peptide drugs continuous release microsphere, includes the following steps：
(1) protein or polypeptide water soluble drug and protein protective agent is soluble in water, obtain water phase drug solution；It will be high
Molecular vehicle material is dissolved in organic solvent, obtains oil phase Polymer Solution；
(2) the water phase drug solution is added in the oil phase Polymer Solution, is cut using cell crushing instrument or high speed
It cuts instrument and is prepared into W/O lotions；
(3) the W/O lotions are at the uniform velocity fed by peristaltic pump and the albumen is prepared in ultrafine dust preparation system
Polypeptide drug continuous release microsphere.
In wherein some embodiments, protein or polypeptide water soluble drug described in step (1) are selected from cow's serum
One or more of albumin, salmon calcitonin, vascular endothelial growth factor, thymopeptide-5 and interferon.
In wherein some embodiments, the protein protective agent described in step (1) is selected from mannitol, sorbierite, gelatin, sugarcane
One or more of sugar, trehalose, lactose, maltose and fructose.
In wherein some embodiments, the polymer carrier described in step (1) is selected from mPEG-PLGA, carboxyl end
The mixture of any one or more in the PLGA at end and the PLGA of C-terminal.
In wherein some embodiments, the molecular weight of the PLGA is 20000-50000Da；Point of the mPEG-PLGA
Son amount is 30000-60000Da.
In wherein some embodiments, the organic solvent described in step (1) is selected from dichloromethane, dimethyl carbonate and second
One or more of acetoacetic ester.
In wherein some embodiments, it is 1.5-2.5 that the organic solvent described in step (1), which is volume ratio,:1 dichloromethane
The mixture of alkane and dimethyl carbonate.
In wherein some embodiments, protein or polypeptide are water-soluble in the water phase drug solution described in step (1)
A concentration of 5-30mg/mL of drug.
In wherein some embodiments, protein or polypeptide are water-soluble in the water phase drug solution described in step (1)
A concentration of 15-25mg/mL of drug.
In wherein some embodiments, a concentration of 10- of protein protective agent in the water phase drug solution described in step (1)
30mg/mL。
In wherein some embodiments, a concentration of 15- of protein protective agent in the water phase drug solution described in step (1)
25mg/mL。
In wherein some embodiments, polymer carrier in the oil phase Polymer Solution described in step (1)
A concentration of 20-80mg/mL.
In wherein some embodiments, polymer carrier in the oil phase Polymer Solution described in step (1)
A concentration of 30-70mg/mL.
In wherein some embodiments, polymer carrier in the oil phase Polymer Solution described in step (1)
A concentration of 40-60mg/mL.
In wherein some embodiments, polymer carrier in the oil phase Polymer Solution described in step (1)
A concentration of 45-55mg/mL.
In wherein some embodiments, the volume of water phase drug solution and oil phase Polymer Solution described in step (2)
Than being 1:10-40.
In wherein some embodiments, the volume of water phase drug solution and oil phase Polymer Solution described in step (2)
Than being 1:15-35.
In wherein some embodiments, the volume of water phase drug solution and oil phase Polymer Solution described in step (2)
Than being 1:18-25.
In wherein some embodiments, the power of cell crushing instrument is 200-700W in step (2), is crushed time 30-
90s；The shear velocity of high speed shear instrument is 8000-12000rpm, shear time 30-90s.
In wherein some embodiments, the power of cell crushing instrument is 300-500W in step (2).
In wherein some embodiments, the power of cell crushing instrument is 350-450W in step (2).
In wherein some embodiments, the liquid supply speed of peristaltic pump is 4-15mL/min in step (3), prepared by ultrafine dust
The rotation dish rotating speed of system is 7000-14000rpm.
In wherein some embodiments, the liquid supply speed of peristaltic pump is 8-12mL/min in step (3), prepared by ultrafine dust
The rotation dish rotating speed of system is 8000-13000rpm.
In wherein some embodiments, the rotation dish rotating speed of ultrafine dust preparation system is 8500-12500rpm.
The present invention also provides a kind of protein and peptide drugs continuous release microspheres.
Specific technical solution is as follows：
A kind of protein and peptide drugs continuous release microsphere, is prepared by above-mentioned preparation method.
The protein and peptide drugs continuous release microsphere and preparation method thereof of the present invention has the following advantages and beneficial effect：
The preparation method of protein and peptide drugs continuous release microsphere provided by the invention is simple, mild condition, preparation process letter
Single, parameter is controllable, and reproducibility is good, and production efficiency height is, it can be achieved that serialization large-scale production.In the protein and peptide of the present invention
In prolonged drug method for preparing microsphere, directly by the protein containing protein protective agent or polypeptide drug aqueous solution and macromolecule
Carrier organic solution is prepared into W/O lotions using cell crushing instrument or high speed shear instrument, then the W/O lotions are fed UPPS systems
It is formed by curing microballoon.This method is compared with microballoon prepared by traditional W/O/W multi-emulsion methods, no outer aqueous phase, can avoid drug from Nei Shui
Mutually leak out to outer aqueous phase, improve entrapment efficiency, reduce porosity, and effectively reduce protein or polypeptide drug be gathered in it is micro-
Ball surface layer and caused by burst drug release phenomenon.In addition, heretofore described preparation method is mild, it can protected protein polypeptide medicine
The structure of object.Therefore, the preparation method of protein and peptide drugs injectable microsphere provided by the invention, can efficiently solve tradition
Protein and peptide drugs microball preparation burst release rate is high, encapsulation rate is low, medicines structure destructible, the problems such as drug release mode is uncontrollable.
The protein and peptide drugs microballoon prepared with the method for the invention, for entrapment efficiency up to 90% or more, burst release rate is low (first
Day release rate is 3%-6%), it is lasting that drug release rate is stablized, and the sustained release phase was up to 30-120 days, and microsphere features smooth surface, ball
Type is complete, uniform particle diameter.This method is suitable for the biologics of protein, polypeptide, oligopeptides, vaccine isoreactivity sensitivity, has and faces
The actual application value of bed.
By selecting specific polymer carrier type and molecular weight, the concentration of cooperation control pharmaceutical aqueous solution, height
The concentration of molecular vehicle organic solution and the proportioning of the two, the specific preparation condition of W/O lotions, the drug of UPPS is for liquid speed
Degree, preparation parameters, the reasonable cooperation of each parameter in a certain range such as rotation dish rotating speed keep prepared protein and peptide drugs long
Imitating microballoon has higher encapsulation rate and lower burst release rate.
Description of the drawings
Fig. 1 is the preparation process schematic diagram of the protein and peptide drugs long-acting injection microsphere of the present invention；
Fig. 2 is embodiment 1, the scanning electron microscope (SEM) photograph of bovine serum albumin microspheres prepared by comparative example 1 and comparative example 2；
Fig. 3 is the grain size distribution of bovine serum albumin microspheres prepared by embodiment 1；
Fig. 4 is the scanning electron microscope (SEM) photograph of bovine serum albumin microspheres prepared by embodiment 2 and embodiment 4；
Fig. 5 is the grain size distribution of salmon calcitonin microsphere prepared by embodiment 3；
Fig. 6 is the grain size distribution of bovine serum albumin microspheres prepared by comparative example 1；
Fig. 7 is the grain size distribution of bovine serum albumin microspheres prepared by comparative example 2；
Fig. 8 is the bovine serum albumin microspheres In-vitro release curves pair that in embodiment 6 prepared by UPPS methods and W/O/W multi-emulsion methods
Than figure；
Fig. 9 is the circular dichroism spectrogram that bovine serum albumin(BSA) drug secondary structure is detected in embodiment 7.
Specific implementation mode
It below will invention is further described in detail by specific embodiment and in conjunction with attached drawing.
UPPS：Ultrafine dust preparation system, apparatus structure is with use referring to document Wen X, Peng X, Fu H, et
al.Preparation and in vitro evaluation of silk fibroin microspheres produced
by a novel ultra-fine particle processing system[J].Int J Pharm,2011.416:195-
201.
UPPS prepares the principle of microballoon：Solution, suspension or emulsion are supplied to by feed liquor system in rotation dish system top
At heart nozzle, preparation solution reaches rotation dish by nozzle, disperses to form droplet in the case where revolving dish high speed rotation, the solvent of droplet is in air-flow
It volatilizes, cures up to microballoon in.
Embodiment 1：The preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the present embodiment includes the following steps：
(1) bovine serum albumin(BSA) is dissolved in the aqueous solution containing 20mg/mL mannitol, is obtained dense containing bovine serum albumin(BSA)
Degree is the water phase (W) of 20mg/mL, by Poly(D,L-lactide-co-glycolide (PLGA, the LA of carboxyl terminal:GA=50:50, molecule
Amount 40000Da) it is dissolved in dichloromethane and dimethyl carbonate 2:1(v:V) in the mixed solvent, it is 50mg/ to obtain polymer concentration
The oil phase (O) of mL；
(2) (grease volume in oil phase (PLGA organic solutions) is added in the water phase of preparation (Bovine Serum Albumin in Aqueous Solution)
Than being 20:1), using cell crushing instrument ice bath, power 400W, when a length of 30s under conditions of, by the two emulsification be prepared into
W/O lotions；
(3) above-mentioned W/O lotions are at the uniform velocity fed into UPPS by peristaltic pump with 10mL/min, (rotating speed is in high speed rotation dish
Shearing atomization forms droplet under the action of 12000rpm), and the solvent of droplet volatilizes in airflow field, and droplet solidification obtains ox blood
Pure protein microsphere is denoted as microballoon 1.
Bovine serum albumin microspheres manufactured in the present embodiment are placed on the metal objective table for posting conductive tape, thin gold is sprayed
Scanning electron microscope sample is made, the pattern of microballoon is observed under awkward silence at a meeting scanning electron microscope (SEM), as a result sees the A figures in Fig. 2,
Microsphere features smooth surface manufactured in the present embodiment it can be seen from A figures in Fig. 2, ball-type is complete, and regular particles are without being adhered.
Precision weighs bovine serum albumin microspheres 5mg manufactured in the present embodiment and is placed in 7mL centrifuge tubes, and lysate is added
After 2mL (lysate group becomes the NaOH solution of the 0.1mol/L containing 0.5% (w/v) lauryl sodium sulfate) vortex mixed, put
Enter 37 DEG C, vibrated in the gas bath oscillator of 100rpm and wait for that microballoon dissolves for 24 hours, centrifuge 5min under 12000rpm, take supernatant according to
Micro-BCA methods measure the content of bovine serum albumin(BSA) in microballoon, then calculate its entrapment efficiency.Experimental result is shown, is implemented
Bovine serum albumin microspheres encapsulation rate prepared by example 1 reaches 103.15 ± 2.18.
Using laser particle size analyzer (Mastersizer2000) to bovine serum albumin microspheres manufactured in the present embodiment into
Row granularity and the measurement of distribution (result is shown in Fig. 3).The result shows that bovine serum albumin microspheres grain size manufactured in the present embodiment is equal
One, it is in normal distribution, d (0.1)=11.37 ± 1.42 μm, d (0.5)=15.92 ± 2.01 μm, d (0.9)=23.01 ± 4.00
μm。
Embodiment 2：The preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of the present embodiment includes the following steps：
(1) bovine serum albumin(BSA) is dissolved in the aqueous solution containing 20mg/mL mannitol, is obtained dense containing bovine serum albumin(BSA)
Degree is the water phase (W) of 20mg/mL, and carboxyl terminal is by Poly(D,L-lactide-co-glycolide (PLGA, molecular weight 20000Da, LA:GA
=50:50) it is dissolved in dichloromethane and dimethyl carbonate 2:1(v:V) in the mixed solvent, it is 50mg/mL to obtain polymer concentration
Oil phase (O)；
(2) (grease volume in oil phase (PLGA organic solutions) is added in the water phase of preparation (Bovine Serum Albumin in Aqueous Solution)
Than being 20:1), using cell crushing instrument ice bath, power 400W, when a length of 30s under conditions of, by the two emulsification be prepared into
W/O lotions；
(3) above-mentioned W/O lotions are at the uniform velocity fed into UPPS by peristaltic pump with 10mL/min, (rotating speed is in high speed rotation dish
Shearing atomization forms droplet under the action of 12000rpm), and the solvent of droplet volatilizes in airflow field, and droplet solidification obtains serum
Albumin microsphere is denoted as microballoon 2.
The scanning electron microscope (SEM) photograph of bovine serum albumin microspheres manufactured in the present embodiment such as B figures (the same implementation of assay method in Fig. 4
Example 1), for B in Fig. 4 figure as can be seen that major part microsphere features smooth surface, ball-type is complete, and regular particles are without being adhered.
Embodiment 3：The preparation of salmon calcitonin microsphere
The preparation method of the salmon calcitonin microsphere of the present embodiment includes the following steps：
(1) salmon calcitonin is dissolved in the aqueous solution containing 20mg/mL mannitol, it is a concentration of obtains calcitonin containing salmon
Poly(D,L-lactide-co-glycolide (PLGA, molecular weight 40000Da) is dissolved in dichloromethane and carbon by the water phase (W) of 20mg/mL
Dimethyl phthalate 2:1(v:V) in the mixed solvent obtains the oil phase (O) that polymer concentration is 50mg/mL；
(2) (grease body in oil phase (PLGA organic solutions are added) is added in the water phase of preparation (salmon calcitonin aqueous solution)
Product is than being 20:1), using cell crushing instrument ice bath, power 400W, when a length of 30s under conditions of, by the two emulsify prepare
At W/O lotions；
(3) above-mentioned W/O lotions are at the uniform velocity fed into UPPS by peristaltic pump with 10mL/min, (rotating speed is in high speed rotation dish
Shearing atomization forms droplet under the action of 12000rpm), and the solvent of droplet volatilizes in airflow field, and droplet solidification obtains salmon
Calcitonin microsphere.
The granularity distribution result of salmon calcitonin microsphere manufactured in the present embodiment is shown in Fig. 5 (assay method is with embodiment 1), d
(0.1)=10.70 ± 0.44 μm, d (0.5)=125.46 ± 1.03 μm, d (0.9)=23.31 ± 1.63 μm.
Embodiment 4：The preparation of bovine serum albumin microspheres
Substantially with embodiment 1, difference is to polymerize in oil phase the preparation method of the bovine serum albumin microspheres of the present embodiment
Object concentration is different, and polymer concentration is respectively 20mg/mL, 40mg/mL, 60mg/mL, 80mg/mL.Thus obtained microsphere is denoted as micro- respectively
Ball 3-6.
Each microballoon manufactured in the present embodiment is observed under awkward silence at a meeting scanning electron microscope (SEM) as described in Example 1
The pattern of microballoon, as a result：For microballoon 3 since PLGA concentration is too low in irregular shape, microballoon collapses (the A figures in such as Fig. 4), microballoon 4
It is complete with 5 ball-type of microballoon, regular particles without being adhered, microballoon 6 since PLGA excessive concentrations lead to filiform occur, part microballoon
(the C figures in such as Fig. 4) in irregular shape.
Embodiment 5：The preparation of bovine serum albumin microspheres
For the preparation method of the bovine serum albumin microspheres of the present embodiment substantially with embodiment 1, difference is grease volume ratio
Difference, grease volume are respectively 5:1、10:1、30:1、40:1、60:1.Thus obtained microsphere is denoted as microballoon 7-11 respectively.
Each microballoon manufactured in the present embodiment is observed under awkward silence at a meeting scanning electron microscope (SEM) as described in Example 1
The pattern of microballoon, as a result：Microballoon 7 causes drying incomplete, is adhered between microsphere particle, bad dispersibility since watr-proportion is excessively high；
8,9,10 surface of microballoon is smooth, and ball-type is complete, and nothing is adhered between particle；Microballoon 11 fails to form microballoon since oil phase ratio is excessively high.
Embodiment 6：The preparation of bovine serum albumin microspheres
For the preparation method of the bovine serum albumin microspheres of the present embodiment substantially with embodiment 1, difference is that high speed revolves dish
Rotating speed is different, and the rotating speed that high speed revolves dish is respectively 6000rpm, 9000rpm, 15000rpm.Thus obtained microsphere is denoted as microballoon 12- respectively
14。
Each microballoon manufactured in the present embodiment is observed under awkward silence at a meeting scanning electron microscope (SEM) as described in Example 1
The pattern of microballoon, as a result：Microballoon 12 and 13 surfaces are smooth, and ball-type is complete, and nothing is adhered between particle；Microballoon 14 is due to revolving dish rotating speed mistake
It is big to cause part that fragment shape is presented.
Embodiment 7：The preparation of bovine serum albumin microspheres
For the preparation method of the bovine serum albumin microspheres of the present embodiment substantially with embodiment 1, difference is liquid supply speed not
Together, liquid supply speed is respectively 3mL/min, 20mL/min.Thus obtained microsphere is denoted as microballoon 15-16 respectively.
Each microballoon manufactured in the present embodiment is observed under awkward silence at a meeting scanning electron microscope (SEM) as described in Example 1
The pattern of microballoon, as a result：15 surface of microballoon is smooth, and ball-type is complete, but particle diameter distribution is uneven；Microballoon 16 is too fast due to liquid supply speed
Solvent is caused to fail to volatilize completely, ball-type is irregular and particle aggregation.
Embodiment 8：The preparation of bovine serum albumin microspheres
For the preparation method of the bovine serum albumin microspheres of the present embodiment substantially with embodiment 1, difference is cell crushing instrument
Power it is different, the power of cell crushing instrument is respectively 100W, 200W, 500W, 700W.Thus obtained microsphere is denoted as microballoon 17- respectively
20。
Each microballoon manufactured in the present embodiment is observed under awkward silence at a meeting scanning electron microscope (SEM) as described in Example 1
The pattern of microballoon, as a result：When the power of cell crushing instrument is 100W, molding microballoon, power 200W, 500W and 700W are had no
When, microsphere features smooth surface, ball-type is complete, and nothing is adhered between particle.
The encapsulation rate of bovine serum albumin microspheres in each embodiment is tested by the assay method of embodiment 1, as a result 1 institute of table
Show：
The encapsulation rate of 1 bovine serum albumin microspheres of table
As shown in Table 1, the encapsulation rate of bovine serum albumin microspheres mainly with the concentration of PLGA, grease phase volume ratio, revolve dish
Rotating speed is related with the cell crushing instrument power of W/O lotions is formed.In a certain range, PLGA concentration is higher, the encapsulation rate of drug
Higher, when a concentration of 40-80mg/mL of PLGA, microsphere encapsulation rate is up to 90% or more.The difference of grease phase volume ratio can influence W/
The property of O lotions, and then the encapsulation rate of microballoon is influenced, when volume ratio is 10:1-30:When 1, microsphere encapsulation rate is up on 90%.
It is high almost without the microsphere encapsulation rate for influencing and obtaining on microsphere encapsulation rate when rotation dish rotating speed is 6000-12000rpm, and turn up
When to 15000rpm, encapsulation rate declines, and causes microballoon is broken to cause this is because rotating speed is excessively high.When the power of cell crushing instrument is
When 200-700W, microsphere encapsulation rate is higher, and when power is 400-500W, encapsulation rate is up to 90%.
Comparative example 1：The preparation of bovine serum albumin(BSA) W/O/W microballoons
(1) bovine serum albumin(BSA) is dissolved in the aqueous solution containing 20mg/mL mannitol, is obtained dense containing bovine serum albumin(BSA)
Degree is the water phase (W1) of 20mg/mL, and Poly(D,L-lactide-co-glycolide (PLGA, molecular weight 40000Da) is dissolved in dichloromethane
With dimethyl carbonate 2:1(v:V) in the mixed solvent obtains the oil phase that polymer concentration is 50mg/mL.It weighs a certain amount of poly-
Suitable ultra-pure water is added in vinyl alcohol (PVA), and 90 DEG C of heating stirrings to PVA are completely dissolved, and stops heating, stands cooling, prepares
At the PVA aqueous solutions of a concentration of 50mg/mL, it is placed in 4 DEG C of refrigerator outer aqueous phases to be used as (W2).
(2) (grease body in oil phase (PLGA organic solutions) is added in the water phase of preparation (W1, Bovine Serum Albumin in Aqueous Solution)
Product is than being 20:1), using cell crushing instrument ice bath, power 400W, when a length of 30s under conditions of, by the two emulsify prepare
At W/O lotions；Then outer aqueous phase (the PVA aqueous solutions of W2,50mg/mL) is added, the volume ratio of oil phase and outer aqueous phase is 1:10, profit
With cell crushing instrument ice bath, power 300W, when a length of 30s under conditions of, by the two emulsification be prepared into W/O/W lotions；
(3) above-mentioned W/O/W lotions are transferred in the sodium-chloride water solution of 500mL, 50mg/mL, in the stirring of 600rpm
It is stirred overnight under rate with volatile organic solvent, solidified microsphere；Microballoon is collected by centrifugation, milli-Q water is used in combination three times；By gained
Microballoon be freeze-dried up to bovine serum albumin(BSA) W/O/W microballoons, is denoted as microballoon 21.
The scanning electron microscope result of bovine serum albumin(BSA) W/O/W microballoons prepared by this comparative example is shown in Fig. 2 B, and (assay method is the same as real
Apply example 1).Compared with microballoon prepared by embodiment 1, the microspherulite diameter prepared by comparative example is smaller, and size is inhomogenous.W/O/W
The particle diameter distribution of microballoon is shown in Fig. 6 (assay method is with embodiment 1), compared with microballoon prepared by embodiment 1, prepared by comparative example
Microspherulite diameter wider distribution, and two peaks, d (0.1)=1.78 ± 0.15 μm, d (0.5)=10.66 ± 1.79 μm, d is presented
μm (0.9)=148.62 ± 8.71；Its entrapment efficiency is 75.39 ± 0.58% (assay method is with embodiments 1), compared with embodiment
The encapsulation rate of 1 bovine serum albumin microspheres prepared is low.
Comparative example 2：The preparation of bovine serum albumin microspheres
The preparation method of the bovine serum albumin microspheres of this comparative example includes the following steps：
(1) bovine serum albumin(BSA) particle is prepared：Bovine serum albumin(BSA) and PEG are added in the phosphate buffer of pH 7.4
Polyethylene glycol (5：1, w/w) mixed solution, is formed.After -80 DEG C of precoolings are stayed overnight, vacuum drying obtains solid powder.It will obtain
Hybrid solid powder with dichloromethane fully wash remove PEG after, be dried to obtain bovine serum albumin(BSA) BSA particles (S).
(2) S/O suspensions are prepared：By carboxyl terminal Poly(D,L-lactide-co-glycolide (PLGA, molecular weight 40000Da,
LA:GA=50:50) it is dissolved in dichloromethane and dimethyl carbonate 2:1(v:V) in the mixed solvent, obtaining polymer concentration is
The oil phase (O) of 50mg/mL.(ox blood in oil phase (PLGA organic solutions) is added in the solid phase (bovine serum albumin(BSA) particle) of preparation
Pure albumen：PLGA=1:50, w/w), using cell crushing instrument ice bath, power 400W, when a length of 30s under conditions of, will
The two mixing is prepared into S/O suspensions；
(3) above-mentioned S/O suspensions are at the uniform velocity fed into UPPS by peristaltic pump with 10mL/min, (rotating speed is in high speed rotation dish
Shearing atomization forms droplet under the action of 12000rpm), and the solvent of droplet volatilizes in airflow field, and droplet solidification obtains ox blood
Pure protein microsphere is denoted as microballoon 22.
The scanning electron microscope result of bovine serum albumin microspheres prepared by this comparative example is shown in Fig. 2 C (same embodiments of assay method
1).Compared with microballoon prepared by embodiment 1, the microballoon sphere prepared by comparative example collapses, rough, in irregular shape.
The particle diameter distribution of microballoon prepared by this comparative example is shown in Fig. 7 (assay method is with embodiment 1), compared with microballoon prepared by embodiment 1,
Microspherulite diameter prepared by comparative example is not in normal distribution, d (0.1)=7.123 ± 4.58 μm, d (0.5)=17.14 ± 1.56 μ
M, d (0.9)=27.46 ± 0.12 μm；Its entrapment efficiency is 73.57 ± 2.83 (assay method is with embodiments 1), compared with embodiment
The encapsulation rate of 1 bovine serum albumin microspheres prepared is low.
Embodiment 9：Detect the tablets in vitro curve of bovine serum albumin microspheres
The bovine serum albumin microspheres that precision weighs the preparation of embodiment 1 are placed in each 20mg of microballoon prepared in comparative example 1
In 2mL sample centrifuge tubes, phosphate buffer PBS (pH=7.4) 1mL is added, sets in gas bath oscillator, in 37 DEG C, 100rpm
Under conditions of constant temperature oscillation, take out centrifuge tube in preset time point, centrifuge 5min under the conditions of 10000rpm, draw whole supernatants
Liquid simultaneously measures its medication amount；In addition fresh PBS (pH=7.4) 1mL are supplemented in sample centrifuge tube, set gas bath oscillator relaying
Persistent oscillation.The sample of release in vitro uses the content of Micro BCA kit measurement bovine serum albumin(BSA)s (result is shown in Fig. 7).It is real
It tests the results show that its slow-release time of the microballoon of the preparation of embodiment 1 is about 110 days, first day burst size is only 4.83 ± 0.16%, right
W/O/W microballoon first day burst sizes prepared by ratio 1 reach 20.01 ± 0.45%, and burst release rate is high.
The tablets in vitro curve of the bovine serum albumin microspheres of other each embodiment and comparative examples is detected in the same way,
Its slow-release time, the results are shown in Table 2 for first day burst size and cumulative release amount.
The tablets in vitro result of 2 bovine serum albumin microspheres of table
| Microballoon is numbered | Slow-release time (day) | First day burst size (%) | Cumulative release amount (%) |
| 1 | 110 | 4.83±0.16 | 90.41±5.00 |
| 2 | 85 | 3.58±0.22 | 90.95±2.17 |
| 3 | 110 | 16.91±2.37 | 86.24±2.82 |
| 4 | 110 | 5.28±1.33 | 92.53±2.56 |
| 5 | 110 | 5.89±0.97 | 96.78±8.37 |
| 6 | 110 | 3.32±0.24 | 39.43±1.01 |
| 7 | 110 | 18.43±0.74 | 93.98±0.64 |
| 8 | 110 | 12.12±0.32 | 93.81±9.33 |
| 9 | 110 | 4.19±0.14 | 83.79±0.65 |
| 10 | 110 | 3.55±0.30 | 84.06±0.64 |
| 11 | 85 | 1.67±0.70 | 46.73±2.46 |
| 12 | 110 | 22.33±0.31 | 87.22±7.79 |
| 13 | 110 | 3.75±0.35 | 91.60±2.51 |
| 14 | 110 | 15.29±0.50 | 87.74±2.46 |
| 15 | 110 | 3.64±0.31 | 90.41±2.78 |
| 16 | 110 | 5.81±0.53 | 87.45±1.74 |
| 17 | 30 | 35.52±0.42 | 89.09±1.06 |
| 18 | 110 | 8.39±0.29 | 80.69±2.56 |
| 19 | 110 | 3.81±0.26 | 82.15±0.64 |
| 20 | 110 | 3.52±0.15 | 75.14±6.99 |
| 21 | 110 | 20.01±0.45 | 94.47±2.84 |
| 22 | 110 | 30.82±1.20 | 79.82±0.98 |
As shown in Table 2, bovine serum albumin microspheres release mainly with the concentration of PLGA, grease phase volume ratio, rotation dish turn
Speed is related with the cell crushing instrument power of W/O lotions is formed.When a concentration of 40-60mg/mL of PLGA, for 24 hours burst release rate be less than 6% and
Cumulative release amount is up to 80% or more.The difference of grease phase volume ratio can influence the property of W/O lotions, and then influence releasing for microballoon
It puts, when volume ratio is 20:1-40:When 1, burst release rate is less than 5% for 24 hours and Cumulative release amount is up to 80% or more.Revolving dish rotating speed is
When 9000-12000rpm, burst release rate is less than 5% for 24 hours and Cumulative release amount is up to 80% or more.The power of cell crushing instrument can shadow
The property of W/O lotions, such as particle size, emulsion intercalation method etc. are rung, and then influences the mouldability of microballoon and the release of microballoon,
When it is 400-500W, burst release rate is less than 5% for 24 hours and Cumulative release amount is up to 80% or more.In addition, comparative example 1 and comparative example 2
Method for preparing microsphere it is different from embodiment 1, encapsulation rate and releasing effect are much worse than embodiment 1.
Embodiment 10：Circular dichroism detector detects the secondary structure of drug in microballoon
The microballoon that precision weighs the preparation of embodiment 1 is appropriate, is respectively placed in 7mL centrifuge tubes, and it is molten that 0.5mL dichloromethane is added
Solve microballoon, add ultra-pure water, vortex mixed 20min fully extracts bovine serum albumin(BSA) in water phase, under 10000rpm from
Supernatant is transferred in another centrifuge tube by heart 5min, and pure using ox blood in Micro BCA kit measurement supernatants
The concentration of albumen；A certain amount of bovine serum albumin(BSA) bulk pharmaceutical chemicals are separately taken, standard reference material, reference substance concentration are configured to ultra-pure water
It is consistent with above-mentioned prepare liquid concentration.
The secondary structure of protein drug in each sample is detected using circular dichroism detector：Sample is added in 1mm cuvettes, so
Cuvette is placed in circular dichroism spectrometer afterwards, setting wavelength scanning range is 180-260nm, and the circular dichroism for recording each sample is set a song to music
Line, as a result display such as Fig. 9.The results show that bovine serum albumin(BSA) drug shows and ox blood in microsphere sample prepared by embodiment 1
Circular dichroism spectra spectrogram as pure protein raw materials medicine phases illustrates that microballoon prepared by embodiment 1 being capable of protected protein secondary structure.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of preparation method of protein and peptide drugs continuous release microsphere, which is characterized in that include the following steps：
(1) protein or polypeptide water soluble drug and protein protective agent is soluble in water, obtain water phase drug solution；By macromolecule
Carrier material is dissolved in organic solvent, obtains oil phase Polymer Solution；
(2) the water phase drug solution is added in the oil phase Polymer Solution, utilizes cell crushing instrument or high speed shear instrument
It is prepared into W/O lotions；
(3) the W/O lotions are at the uniform velocity fed by peristaltic pump and the polypeptide is prepared in ultrafine dust preparation system
Class prolonged drug microballoon.
2. the preparation method of protein and peptide drugs continuous release microsphere according to claim 1, which is characterized in that step (1)
Described in protein or polypeptide water soluble drug be selected from bovine serum albumin(BSA), salmon calcitonin, vascular endothelial growth factor,
One or more of thymopeptide-5 and interferon；And/or
Protein protective agent described in step (1) be selected from mannitol, sorbierite, gelatin, sucrose, trehalose, lactose, maltose and
One or more of fructose；And/or
Polymer carrier described in step (1) is selected from the PLGA of mPEG-PLGA, the PLGA of carboxyl terminal and C-terminal
One or more of；And/or
Organic solvent described in step (1) is selected from one or more of dichloromethane, dimethyl carbonate and ethyl acetate.
3. the preparation method of protein and peptide drugs continuous release microsphere according to claim 2, which is characterized in that the PLGA
Molecular weight be 20000-50000Da, the molecular weight of the mPEG-PLGA is 30000-60000Da.
4. according to the preparation method of claim 1-3 any one of them protein and peptide drugs continuous release microspheres, which is characterized in that
A concentration of 5-30mg/mL of protein or polypeptide water soluble drug in water phase drug solution described in step (1)；And/or
A concentration of 10-30mg/mL of protein protective agent in water phase drug solution described in step (1)；And/or
A concentration of 20-80mg/mL of polymer carrier in oil phase Polymer Solution described in step (1).
5. the preparation method of protein and peptide drugs continuous release microsphere according to claim 4, which is characterized in that step (1)
Described in water phase drug solution in a concentration of 15-25mg/mL of protein or polypeptide water soluble drug；And/or
A concentration of 15-25mg/mL of protein protective agent in water phase drug solution described in step (1)；And/or
A concentration of 40-60mg/mL of polymer carrier in oil phase Polymer Solution described in step (1).
6. according to the preparation method of claim 1-3 any one of them protein and peptide drugs continuous release microspheres, which is characterized in that
The volume ratio of water phase drug solution and oil phase Polymer Solution described in step (2) is 1:10-40.
7. the preparation method of protein and peptide drugs continuous release microsphere according to claim 6, which is characterized in that step (2)
Described in water phase drug solution and oil phase Polymer Solution volume ratio be 1:15-35.
8. according to the preparation method of claim 1-3 any one of them protein and peptide drugs continuous release microspheres, which is characterized in that
The power of cell crushing instrument is 200-700W in step (2), is crushed time 30-90s；The shear velocity of high speed shear instrument is
8000-12000rpm, shear time 30-90s.
9. according to the preparation method of claim 1-3 any one of them protein and peptide drugs continuous release microspheres, which is characterized in that
The liquid supply speed of peristaltic pump is 4-15mL/min in step (3), and the rotation dish rotating speed of ultrafine dust preparation system is 7000-
14000rpm。
10. a kind of protein and peptide drugs continuous release microsphere, which is characterized in that by claim 1-9 any one of them preparation side
Method is prepared.
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| CN114210277A (en) * | 2021-12-17 | 2022-03-22 | 上海交通大学 | Multi-element environment-responsive polysaccharide microsphere and preparation and application methods thereof |
| CN114984870A (en) * | 2022-03-15 | 2022-09-02 | 齐欣 | Recombinant protein microsphere and preparation method and application thereof |
| DE102022107247A1 (en) | 2022-03-28 | 2023-09-28 | Universität des Saarlandes, Körperschaft des öffentlichen Rechts | CONTINUOUS PRODUCTION OF PARTICLES |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109806385A (en) * | 2019-01-31 | 2019-05-28 | 浙江圣兆药物科技股份有限公司 | A kind of leuprorelin acetate microball preparation and preparation method thereof |
| CN109806385B (en) * | 2019-01-31 | 2022-04-08 | 浙江圣兆药物科技股份有限公司 | Leuprorelin acetate microsphere preparation and preparation method thereof |
| CN114210277A (en) * | 2021-12-17 | 2022-03-22 | 上海交通大学 | Multi-element environment-responsive polysaccharide microsphere and preparation and application methods thereof |
| CN114984870A (en) * | 2022-03-15 | 2022-09-02 | 齐欣 | Recombinant protein microsphere and preparation method and application thereof |
| DE102022107247A1 (en) | 2022-03-28 | 2023-09-28 | Universität des Saarlandes, Körperschaft des öffentlichen Rechts | CONTINUOUS PRODUCTION OF PARTICLES |
| WO2023186839A1 (en) * | 2022-03-28 | 2023-10-05 | Universitaet Des Saarlandes | Continuous production of particles |
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