CN115389640B - A method for determining the content of insulin liposome drug - Google Patents
A method for determining the content of insulin liposome drug Download PDFInfo
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- CN115389640B CN115389640B CN202110570014.7A CN202110570014A CN115389640B CN 115389640 B CN115389640 B CN 115389640B CN 202110570014 A CN202110570014 A CN 202110570014A CN 115389640 B CN115389640 B CN 115389640B
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- insulin
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- methanol
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 143
- 239000002502 liposome Substances 0.000 title claims abstract description 95
- 102000004877 Insulin Human genes 0.000 title claims abstract description 74
- 108090001061 Insulin Proteins 0.000 title claims abstract description 74
- 229940125396 insulin Drugs 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000003814 drug Substances 0.000 title claims abstract description 23
- 229940079593 drug Drugs 0.000 title claims abstract description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000003960 organic solvent Substances 0.000 claims abstract description 33
- 239000012528 membrane Substances 0.000 claims abstract description 26
- 239000002253 acid Substances 0.000 claims abstract description 21
- 239000000203 mixture Substances 0.000 claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000007791 liquid phase Substances 0.000 claims abstract description 10
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 150000003904 phospholipids Chemical class 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims 4
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 18
- 229920001184 polypeptide Polymers 0.000 abstract description 2
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 35
- 239000002904 solvent Substances 0.000 description 15
- 150000002632 lipids Chemical class 0.000 description 13
- 230000000887 hydrating effect Effects 0.000 description 11
- UHNSRFWQBVXBSK-UHFFFAOYSA-N methanol;2,2,2-trifluoroacetic acid Chemical compound OC.OC(=O)C(F)(F)F UHNSRFWQBVXBSK-UHFFFAOYSA-N 0.000 description 10
- 239000004094 surface-active agent Substances 0.000 description 10
- 239000012736 aqueous medium Substances 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 8
- 238000001035 drying Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 238000011282 treatment Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- -1 insulin phospholipid Chemical class 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 150000007513 acids Chemical class 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 230000010494 opalescence Effects 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000007813 chromatographic assay Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229940042880 natural phospholipid Drugs 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000935 solvent evaporation Methods 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 description 1
- 101001011741 Bos taurus Insulin Proteins 0.000 description 1
- 239000004380 Cholic acid Substances 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 206010024604 Lipoatrophy Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010005991 Pork Regular Insulin Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 235000019416 cholic acid Nutrition 0.000 description 1
- 229960002471 cholic acid Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of pharmacy and discloses a method for measuring the content of insulin liposome medicaments, which is characterized by comprising the steps of (1) adding a membrane breaker composition into an insulin liposome, uniformly mixing, and (2) detecting the content of the insulin medicaments by adopting a high-efficiency liquid phase method, wherein the membrane breaker composition comprises acid and an organic solvent, the acid is one or more of TFA, HCl, H 2SO4, and the organic solvent is one or more of methanol, ethanol and isopropanol. The method for measuring the content of the insulin liposome drug is convenient, is easy to quantitatively analyze, has no damage to chromatographic columns and liquid phase systems, and lays a foundation for accurately measuring related preparations of hydrophilic protein polypeptide drugs.
Description
Technical Field
The invention relates to the field of pharmacy, in particular to a method for measuring the content of insulin liposome medicines.
Background
Insulin is used as a first-choice medicament for treating insulin-dependent diabetes mellitus and severe non-insulin-dependent diabetes mellitus, is limited by the problems of large molecular weight, poor stability, low bioavailability and the like, generally needs to be injected for administration, has low patient compliance, and can generate adverse reactions such as subcutaneous lipoatrophy, subcutaneous induration and the like after long-term frequent injection, thereby bringing great pain to patients. Thus, drug development has focused on developing safe, effective non-injection modes to replace insulin injections either entirely or partially. Because of the self-characteristics of insulin, the insulin is directly administrated by a non-injection route and hardly absorbed, and the bioavailability of the non-injection administration can be improved by using a pharmaceutical carrier for presentation. In the pharmaceutical microparticle carrier, liposome, especially flexible liposome, has the advantages of enhanced permeation, increased solubility, high encapsulation efficiency, small adverse reaction and the like, and can penetrate a pore canal with a size much smaller than that of the liposome in a complete form for drug delivery due to the inclusion of the edge activator, so that the liposome becomes an effective and efficient carrier in insulin non-injection preparation research.
The reliable analysis method is a quality evaluation means of prescription process research. At present, the main method for measuring the content of insulin in the insulin liposome is to take insulin liposome suspension, rupture a membrane by sodium cholate solution, extract lipid by chloroform, vortex, centrifuge after mixing evenly, take supernatant for measurement (Zhang Li, rong, zhang Chunxia. Measurement of the content and encapsulation efficiency of insulin liposome medicine, chinese pharmacist .2010,(03):364-365;Na Zhang,Qi N.Ping,Gui H.Huang,et,al.Investigation of lectin-modified insulin liposomes as carriers for oral administration,Pharmaceutical Nanotechnology,2005,294(1-2):247-259), has more complicated pretreatment process of insulin liposome samples, takes longer time, and involves mixing of solutions in the treatment process, so that certain deviation exists in the measurement result of the samples. 451-453), the organic component of the mobile phase in the method can not thoroughly crack the liposome with higher lipid concentration, and the liposome is adopted for direct sample injection analysis, so that the chromatographic column and a liquid phase system are easily blocked for a long time, and the service life is influenced.
Disclosure of Invention
The present inventors have found during the course of the study that insulin liposomes can be subjected to membrane disruption treatment with a composition comprising a specific acid and a specific organic solvent. Based on the above, the technical problem solved by the invention is to provide a method for measuring the content of the drug in the insulin liposome.
In order to solve the technical problems of the invention, the invention provides the following technical scheme:
The invention provides a method for measuring the content of a drug in an insulin liposome, which is characterized by comprising the steps of (1) adding a membrane breaker composition into the insulin liposome, uniformly mixing, and (2) detecting the content of the insulin drug by adopting a high-performance liquid phase method, wherein the membrane breaker composition comprises an acid and an organic solvent, the acid is one or more of TFA, HCl, H 2SO4, and the organic solvent is one or more of methanol, ethanol and isopropanol.
In one embodiment of the invention, the insulin is selected from at least one of natural insulin, porcine insulin, bovine insulin, recombinant human insulin and various medium and long acting insulins, and the lipid material is selected from at least one of natural phospholipid, synthetic phospholipid, cholesterol, cholic acid and salts thereof.
In one embodiment of the present invention, the insulin liposome comprises a liposome prepared directly from insulin and lipid, and/or a liposome prepared from an insulin-phospholipid complex and lipid, wherein the phospholipid in the insulin-phospholipid complex can be at least one of natural phospholipid or synthetic phospholipid.
Optionally, the liposome is a surfactant-containing flexible liposome.
Alternatively, the insulin liposome is prepared by any one of the following methods:
(1) Preparation of insulin phospholipid complexes
Referring to the patent 'a lipid complex of insulin, a preparation method thereof and a solvent evaporation method in a preparation' (application number CN 201110196733.3) for preparing the insulin phospholipid complex;
(2) The preparation of the flexible liposome of the insulin-phospholipid complex can be selected from any one of the following methods:
The method (A) comprises dissolving insulin-phospholipid complex and surfactant in organic solvent, volatilizing solvent, drying to obtain film, and hydrating the film. The preparation method comprises the steps of (1) dissolving the insulin-phospholipid complex and a surfactant in an organic solvent, volatilizing the solvent, and drying to obtain a film, wherein the film is hydrated, namely adding a proper amount of aqueous medium into the dried film, hydrating for a certain time at a certain temperature, and homogenizing to obtain a liposome solution, thus obtaining the insulin-phospholipid complex flexible liposome;
the method (B) comprises dissolving insulin-phospholipid complex in organic solvent, volatilizing solvent, drying to obtain film, and hydrating the film with aqueous medium containing surfactant. Dissolving a surfactant in a proper amount of aqueous medium, adding the surfactant into the dried film, hydrating for a certain time at a certain temperature, and homogenizing to obtain a liposome solution, thus obtaining the flexible liposome of the insulin-phospholipid complex;
the method (C) comprises dissolving insulin-phospholipid complex and surfactant in organic solvent, adding aqueous medium, homogenizing to obtain milk, and removing organic solvent. Specifically, the insulin-phospholipid complex prepared in the step (1) and a surfactant are taken and dissolved in an organic solvent, then an aqueous medium is added, homogenization treatment is carried out to form milk, the organic solvent is removed by decompression and rotary evaporation, and a liposome solution is obtained, thus obtaining the insulin-phospholipid complex flexible liposome;
The method (D) comprises dissolving insulin-phospholipid complex in organic solvent, mixing with aqueous medium containing surfactant, homogenizing to obtain emulsion, and removing organic solvent. Specifically, the insulin-phospholipid complex prepared in the step (1) is taken to be dissolved in an organic solvent, then a surfactant is dissolved in an aqueous medium, the two are mixed and homogenized to form milk, the organic solvent is removed by reduced pressure rotary evaporation, and the liposome solution is obtained, so that the insulin-phospholipid complex flexible liposome is obtained.
(3) Preparation of insulin common flexible liposome
The insulin phospholipid complex in the step (2) is replaced by insulin to prepare the insulin common flexible liposome.
(4) Preparation of insulin-phospholipid complex common liposome
Dissolving insulin-phospholipid complex and lipid material in organic solvent, volatilizing solvent, drying to obtain film, and hydrating the film. The preparation method comprises the steps of (1) dissolving the insulin-phospholipid complex and a lipid material in an organic solvent, volatilizing the solvent, drying to obtain a film, hydrating the film, adding a proper amount of aqueous medium into the dried film, hydrating for a certain time at a certain temperature, and homogenizing to obtain a liposome solution, thus obtaining the insulin-phospholipid complex common liposome.
(5) Preparation of insulin common liposome
Dissolving insulin and lipid materials in organic solvent, volatilizing solvent, drying to obtain film, and hydrating the film. The preparation method comprises the steps of dissolving insulin and lipid materials in an organic solvent, volatilizing the solvent, drying to obtain a film, hydrating the film, adding a proper amount of aqueous medium into the dried film, hydrating for a certain time at a certain temperature, and homogenizing to obtain a liposome solution, thus obtaining the insulin common liposome.
In one embodiment of the invention, the insulin liposome contains 0.004-2% of insulin drug by weight and 0.012-50% of lipid by weight.
In one embodiment of the present invention, the volume ratio of the insulin liposome to the membrane breaker composition is 1 (5-50), preferably 1 (25-50).
In one embodiment of the present invention, the weight percentage of the organic solvent in the film breaker composition is 75% -100%, preferably 90% -100%.
In one embodiment of the invention, the acid in the breaker composition is TFA, in weight percent 0.01-0.5%, preferably 0.01-0.1%.
In one embodiment of the invention, the acid in the breaker composition is H 2SO4, 0.01-0.1% by weight.
In one embodiment of the invention, the acid in the breaker composition is HCl, 0.1-0.5% by weight.
In one embodiment of the invention, the organic solvent is methanol.
In one embodiment of the invention, the insulin liposome is a flexible liposome.
The method of the invention has the advantages of convenient use, effective membrane rupture and medicine dissolution, easy quantitative analysis, no damage to chromatographic columns and liquid phase systems, and lays a foundation for accurate determination of hydrophilic protein polypeptide medicines and related preparations.
The disclosed temperature or content ranges may be expressed as upper and lower limits, respectively, as one or more lower limits, or as one or more upper limits, a given range being defined by selecting a lower limit and an upper limit, the selected lower and upper limits defining boundaries of a particular range, all ranges that may be defined in this manner being inclusive and combinable, i.e., any lower limit may be combined with any upper limit to form a range, e.g., ranges of 1-40 and 0.6-50 are listed for a particular parameter, understood as ranges of 0.6-40 and 1-50 are contemplated, and, further, all ranges of 1-30, 1-40, 1-50, 0.5-30, 0.5-40, 0.5-50 are contemplated if minimum range values of 1 and 0.5 are listed, and if maximum range values of 50,40 and 30 are listed.
The invention relates to weight percentages based on the total weight of the composition, unless otherwise indicated.
Beneficial technical effects
The invention adopts the composition containing acid and organic solvent to rupture membrane and then carries out liquid phase measurement, the method can accurately quantify the medicine of the insulin liposome without damaging a chromatographic column and a liquid phase system, and simultaneously solves the defects of complex pretreatment operation and deviation in measurement in the prior art, ensures the effective rupture membrane of the insulin liposome and accurate measurement of content results.
Drawings
FIG. 1 is a chromatogram of the solvent (0.01% TFA-methanol) in the chromatographic assay of insulin flexible liposome content.
Fig. 2 is a chromatogram of a blank flexible liposome (blank solution) in an insulin flexible liposome content chromatographic assay.
FIG. 3 is a chromatogram of insulin (control solution) in the assay for flexible liposome content of insulin.
FIG. 4 is a chromatogram of flexible liposome (test solution) of insulin phospholipid complex in the content chromatography of flexible liposome of insulin.
Detailed Description
The invention is further described in detail below by means of the figures and examples. The features and advantages of the present invention will become more apparent from the exemplary description. Any embodiment described herein as exemplary is not necessarily to be construed as preferred or advantageous over other embodiments.
In addition, the technical features described below in the different embodiments of the present invention may be combined with each other as long as they do not collide with each other.
EXAMPLE 1 preparation of liposomes
A. the insulin-phospholipid complex is prepared by a solvent evaporation method. 60mg of insulin is weighed and dissolved in 6mL of 0.1% TFA methanol solution, 600mg of phospholipid is weighed and dissolved in 54mL of dichloromethane, the two solutions are uniformly mixed, reduced pressure rotary evaporation is carried out at 37 ℃, when the residual amount of the solvent is about the original methanol volume, 1 time of dichloromethane is added, and the mixture is dried and placed in a vacuum drying oven, and dried overnight, thus obtaining the insulin.
B. The preparation method of the flexible liposome of the insulin-phospholipid complex comprises the steps of weighing 600mg of phospholipid, which is equivalent to 60mg of phospholipid complex 660mg,Tween20 400mg of insulin, placing the flexible liposome into a 100mL round bottom flask, adding 20mL of methylene dichloride to dissolve all the flexible liposome completely, performing rotary evaporation to form a film under the water bath condition of 37 ℃, drying the film for 2 hours in vacuum, weighing 100mg of sodium cholate, adding a proper amount of PBS (pH 7.4) with 0.02mol/L to prepare PBS solution of sodium cholate, adding the solution into the prepared lipid film, and hydrating the lipid film for 30 minutes. Homogenizing, and filtering with 0.22 μm filter membrane.
EXAMPLE 2 Membrane rupture solvent composition
(1) Selection of organic solvents
1ML of the flexible liposome of the insulin-phospholipid complex prepared in example 1 was precisely removed, and methanol, ethanol, isopropanol and acetonitrile containing 0.1% TFA were added to a 50mL volumetric flask, respectively, and the opalescence of the liposome was observed while gently shaking.
TABLE 1 results of membrane rupture treatments with different organic solvents containing 0.1% TFA
(2) Selection of acids
1ML of the flexible liposome of the insulin-phospholipid complex prepared in example 1 was removed by precision pipetting in a 50mL volumetric flask, and 0.1% TFA, HCl, H 2SO4、HAC、H3PO4 in methanol was added to observe dissolution of the insulin liposome in various acids in methanol. The results were as follows:
TABLE 2 results of membrane rupture treatment of methanol solutions containing different acids
1ML of the flexible liposome of the insulin-phospholipid complex prepared in the example 1 is precisely removed, and 0.01%, 0.1% and 0.5% of TFA, HCl, H 2SO4% of methanol solution are respectively added into a 50mL volumetric flask to observe the membrane rupture condition of the liposome.
TABLE 3 influence of acid content on rupture of membranes
1ML of the flexible liposome of the insulin-phospholipid complex prepared in example 1 was precisely removed, and 0.01%, 0.1% and 0.5% of TFA, HCl, H 2SO4 methanol solutions were added to a 50mL volumetric flask, respectively, to observe dissolution. Stock solutions were then prepared at a concentration of 100ug/ml, and subjected to liquid phase analysis at 0h, 3h, 6h, 9h and 12h, respectively, for evaluation of solution stability. The results were as follows:
TABLE 4 influence of acid content on stability of solution after membrane rupture treatment
| TFA | HCl | H2SO4 | |
| 0.01% | 12H of stabilization | \ | Low response value and poor solution stability |
| 0.1% | 9H stabilization | Low response value and poor solution stability | Low response value and poor solution stability |
| 0.5% | Unstable state | Low response value and poor solution stability | \ |
(3) Concentration of organic solvent
1ML of the flexible liposome of the insulin-phospholipid complex prepared in example 1 was precisely removed, and 0.1% aqueous TFA methanol solution containing 50%, 75% and 90% methanol was added to each flask to observe opalescence of the liposome.
TABLE 5 influence of organic solvent concentration on rupture of membranes
(4) Acid concentration
1ML of the flexible liposome of the insulin-phospholipid complex prepared in example 1 was precisely removed, and 0.01%, 0.1% and 0.5% TFA methanol and 90% aqueous methanol were added to a 50mL volumetric flask, respectively, to prepare a stock solution having a concentration of 100ug/mL, and the stock solution was subjected to liquid phase analysis at 0h, 3h, 6h, 9h and 12h, respectively, to evaluate the stability of the solution.
TABLE 6 influence of organic solvent concentration on solution stability after Membrane rupture treatment
| Methanol solution | 90% Aqueous methanol solution | |
| 0.01% | 12h | 12h |
| 0.1% | 9h | 9h |
| 0.5% | Poor solution stability | Poor solution stability |
Example 3
Based on the example 1, 0.8mg, 200mg and 400mg of insulin are used respectively, the preparation of the flexible liposome of the insulin-phospholipid complex is carried out without changing the other prescription processes, so that the flexible liposome of the complex with the weight percentages of insulin being 0.004%, 1% and 2% are obtained, and in the preparation of the flexible liposome of the insulin-phospholipid complex, 2.4mg, 2.5g and 5g of phospholipids are adopted, and the preparation of the flexible liposome of the insulin-phospholipid complex is carried out without changing the other prescription processes, so that the flexible liposome of the complex with the weight percentages of phospholipids being 0.012%, 25% and 50% are obtained.
1ML of the insulin-phospholipid complex flexible liposome prepared in the embodiment is precisely removed, quantified in 5, 25 and 50mL volumetric flasks, 0.01%, 0.1% and 0.5% of TFA methanol solution are respectively added to dilute to the scale, 1mL of insulin flexible liposome is precisely removed, PBS is used for diluting to the corresponding volume, insulin with the corresponding mass is weighed and dissolved in the methanol solution without acid with the corresponding volume, and the membrane rupture dissolution condition is observed. The results were as follows:
(1) Insulin-phospholipid complex flexible liposome and solvent volume ratio of 1:5
* Note that the opalescence in the table is lighter than that of the flexible insulin nanovesicles diluted by PBS, and the generated precipitates are less than those generated by dissolving the corresponding insulin in methanol solution without acid.
(2) Insulin-phospholipid complex flexible liposome and solvent volume ratio of 1:25
(3) Insulin-phospholipid complex flexible liposome and solvent volume ratio of 1:50
EXAMPLE 4 drug content assay of insulin phospholipid Complex Flexible Liposome
Chromatographic conditions
Column-Agilent ZORBAX 300SB-C18 (250X 4.6mm,5 μm);
mobile phase 0.2mol/L sulfate buffer (28.4 g anhydrous sodium sulfate, 2.7mL phosphoric acid, water to 800mL, adjusting pH to 2.3 with ethanolamine, water to 1000 mL) -acetonitrile (74-26);
Column temperature of 40 ℃, flow rate of 1.00mL/min, detection wavelength of 214nm;
sample injection amount is 20.0 mu L, solvent is 0.01% TFA-methanol
Content determination
Accurately weighing insulin 10mg, placing into a 10mL volumetric flask, dissolving with 0.01% TFA methanol solution, diluting to scale, shaking, accurately transferring from 0.6mL to 10mL volumetric flask, diluting to scale with 0.01% TFA-methanol, shaking, and preparing into reference solution with concentration of 60 μg/mL. 1mL of the flexible liposome of the insulin-phospholipid complex prepared in the example 1 was precisely removed, placed in a 50mL volumetric flask, added with 0.01% TFA-methanol to the scale, and shaken well to obtain a sample solution. A blank flexible liposome solution was prepared without insulin according to the method of example 1, 1mL of the blank flexible liposome was precisely measured, placed in a 50mL volumetric flask, diluted to scale with 0.01% TFA-methanol, and shaken well to obtain a blank solution. Sample analysis was performed under chromatographic conditions of Agilent ZORBAX300SB-C18 (250×4.6mm,5 μm), mobile phase of 0.2mol/L sulfate buffer (28.4 g anhydrous sodium sulfate, dissolved in water, 2.7mL of phosphoric acid, 800mL of water, pH adjusted to 2.3 with ethanolamine, and water to 1000 mL) -acetonitrile (74:26), column temperature of 40deg.C, flow rate of 1.00mL/min, detection wavelength of 214nm, sample amount of 20.0. Mu.L, and chromatogram as shown.
The method has the advantages that the content of the flexible liposome of the insulin-phospholipid complex is determined by the method, the solvent and auxiliary materials do not interfere with the determination at the wavelength of 214nm, the peak area and the concentration are in good linear relation within the range of 6-180 ug/mL, the quantitative limit is 1.500ug/mL, samples with three concentrations of low (6 ug/mL), medium (60 ug/mL) and high (180 ug/mL) are continuously sampled for 5 times, the RSD is in the range of 0.05% -1.84%, the stability of the solution is good within 12 hours at room temperature, the average recovery rate of the samples with three levels of 80%, 100% and 120% is 98.86%, the RSD is 1.01% (n=9), and the RSD of the content of 6 samples is repeatedly determined to be 1.11%.
The invention has been described above in connection with preferred embodiments, which are, however, exemplary only and for illustrative purposes. On this basis, the invention can be subjected to various substitutions and improvements, and all fall within the protection scope of the invention.
Claims (4)
1. A method for measuring the content of insulin liposome medicine is characterized by comprising the following steps of
(1) Membrane rupture, namely adding a membrane rupture agent composition into the insulin liposome, mixing uniformly, and
(2) Adopts a high-performance liquid phase method to detect the content of insulin drugs,
The membrane breaker composition is an acid-containing organic solvent or an acid-containing organic solvent aqueous solution, wherein the acid in the membrane breaker composition is TFA, the weight percentage of the acid in the membrane breaker composition is 0.01-0.1%, the organic solvent is selected from more than one of methanol, ethanol and isopropanol, the organic solvent aqueous solution is 75% methanol aqueous solution or 90% methanol aqueous solution, the insulin drug weight content in the insulin liposome is 0.004-2%, and the phospholipid weight content in the insulin liposome is 0.012-50%.
2. The method for determining the drug content of the insulin liposome according to claim 1, wherein the volume ratio of the insulin liposome to the membrane breaker composition is 1 (5-50).
3. The method for determining the content of insulin liposome drugs according to any one of claims 1 to 2, wherein the organic solvent is methanol.
4. The method of determining the drug content of insulin liposomes according to any one of claims 1 to 2, wherein the insulin liposomes are flexible liposomes.
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