CN100349612C - Sustained releasing microspheric preparation of glucokinase mutant and its making method - Google Patents
Sustained releasing microspheric preparation of glucokinase mutant and its making method Download PDFInfo
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- CN100349612C CN100349612C CNB2005100238449A CN200510023844A CN100349612C CN 100349612 C CN100349612 C CN 100349612C CN B2005100238449 A CNB2005100238449 A CN B2005100238449A CN 200510023844 A CN200510023844 A CN 200510023844A CN 100349612 C CN100349612 C CN 100349612C
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Abstract
The present invention belongs to the field of biologic medical preparation and a formulation of a preventive sustained release microsphere for non-vein application, particularly to a lactic acid hydroxyacetic acid segmented copolymer (PLGA) formulation of a preventive sustained release microsphere and a preparation method thereof. In the present invention, a staphylokinase mutant is packed by using biologic degradable polymer materials PLGA as carrier materials by a multiple emulsion solvent evaporation method; emulsification is carried out by ultrasonic agitation or high-speed agitation; organic solvent is volatilized so as to form a polymer sustained release microsphere under low speed agitation. The sustained release microsphere of the present invention has the advantages of smooth surface, regular particles without adhesion, high drug loading dosage and high encapsulation efficiency; besides, the average particle diameter is from 70 mu m to 120 mu m; the sustained release period keeps more than 30 days, and the present invention is suitable for non-vein application.
Description
Technical field
The invention belongs to field of biological pharmacy, the preventative sustained release microsphere agents that relates to a kind of non-vein administration, especially a kind of lactic-co-glycolic acid block copolymer (PLGA) sustained release microsphere agents that comprises glucokinase mutant (staphylokinase variant) and preparation method thereof.
Background technology
(staphylokinase is the excretory a kind of albumen of staphylococcus aureus Sak) to natural Sbphylokinase, is made up of 136 aminoacid.Sak itself is not a kind of enzyme, in human plasma, form 1: 1 complex with plasminogen (Plg), this complex is activated by the fibrinolysin (Plm) of clot surface trace and is SakPlm, the substrate specificity of Plm changes among the SakPlm, form plasminogen activator efficiently, activate remaining Plg and form Plm.Plm catalysis thrombosis main matrix fibrin degradation, thereby thrombus.α 2-antiplasmin in the blood plasma (α 2-antiplasmin, α 2-AP) can suppress free Plm in the blood plasma rapidly.α 2-AP has descended 100 times to the inhibition speed that combines fibrinous SakPlm, therefore, the Sak plasminogen activation has the fibrin specificity of height, this fibrin specificity has been avoided the activation of systemic Plg, has improved the efficient (Collen D et al.Nature Medicine.4.279-284 (1998)) of thrombolytic.Clinical effectiveness shows that to patients of acute myocardial infarction, r-Sak has equal efficiency of thrombolysis with rt-PA at least, and r-Sak has stronger fibrin selectivity.R-Sak has a good application prospect as thrombolytic drug.
Sak is a foreign protein.Among most of crowds, the basic antibody horizontal of Sbphylokinase is lower, clinical use also seldom causes severe allergic reaction, but a lot of patient's second weekly assemblies after medication produce the neutralizing antibody of high titre, and this high titer antibody level can keep the several months, limited clinically the repeated use (Declerck PJ et al.thromb Haemost.71.129-133 (1994)) to Sak.Research is arranged on the basis of genetic engineering Sbphylokinase research, structure has reduced immunogenicity, has the novel mutation body Sak (K35R of thrombolytic and antiplatelet aggregation concurrently, DGR), be the medicine (Su HB et al.ActaBiochim Biophys Sin.36 336-42 (2004)) of very promising prevention and treatment thrombotic disease.But DGR is the same with Sak, and molecular weight is little, and the half-life weak point has only 3 minutes in the body, and whole body is used will need be difficult to carry out long term administration with the administration of intravenous drip mode clinically by quick inactivating.Therefore, research and development for a long time slow release DGR can keep its bioactive slow releasing preparation significant again.Characteristics such as PLGA is little good slow release framework material in the preparation of asking, and has safety non-toxic, and is biodegradable, the PLGA microsphere of the recombinant human growth factor by FDA approval listing, proves that PLGA is the carrier material of good encapsulating protein and polypeptide drug at present.Through consulting document, still there is not glucokinase mutant sustained-release micro-spheres and correlational study thereof report at present.
Summary of the invention
The preventative sustained-release micro-spheres that the purpose of this invention is to provide a kind of non-vein administration, especially a kind of PLGA sustained-release micro-spheres that comprises glucokinase mutant and preparation method thereof.
The present invention adopts the multiple emulsion solvent evaporation method, seal glucokinase mutant with Biodegradable polymer material lactic-co-glycolic acid block copolymer (PLGA) for carrier material, carry out emulsifying by ultrasonic or high-speed stirred, under stirring at low speed, make the organic solvent volatilization, form the polymer sustained-release micro-spheres.
Described sustained-release micro-spheres is base starting material with PLGA, smooth surface, regular particles does not have adhesion, mean diameter between 70-120 μ m, drug loading and envelop rate height, slow-release period is suitable for the non-vein administration more than 30 days.
Described glucokinase mutant comprises various glucokinase mutants and the external trim thereof that produces by gene mutation, as: the natural Sbphylokinase that from staphylococcus, extracts, recombinant glucokinase mutant (recombinantstaphylokinase, r-Sak), the polyethylene glycol modified product of recombinant glucokinase mutant.
Technical scheme of the present invention realizes by following method and step:
1. preparation microsphere
Adopt W/O/W multiple emulsion solvent evaporation method, PLGA is dissolved in makes oil phase in the organic facies, aqueous phase solution or suspension add above-mentioned oil phase in getting, and the volume ratio of wherein interior water and oil phase is 5～20%, will form colostrum after its homogenize; Colostrum is added dropwise in the outer water disperse medium, fully homogenize, under the room temperature stirring at low speed 4-6 hour, promptly get the PLGA microsphere that contains glucokinase mutant, normal freeze-drying is preserved.Aforesaid operations is finished under 37 ℃ or room temperature.
The homogenize mode of described preparation microsphere comprises high-speed stirred and ultrasonic, and ultrasonic time is 20-40 second, and mixing time is 3-5 minute.Common ultrasonic cell disruptor of the present invention, its power are 1200 watts, and using power is good with low-power.
The PLGA molecular weight that the present invention selects for use is 4.0 * 10
3-5.5 * 10
4, wherein (PLA: mass percent PLG) is 50: 50-85: 15 for polylactic acid and polyglycolic acid.
Described organic solvent is the mixed liquor of dichloromethane (DCM) or dichloromethane and acetone (AC), and wherein the volume ratio of DCM and AC is 75: 35-100: 0.
Water is the buffer (pH4-9) that contains glucokinase mutant in the described microsphere, wherein can add 0.1-7% polyvinyl alcohol (PVA), 0.1-10% trehalose, 0.5-10% magnesium hydroxide, 0.5-10% magnesium carbonate, 0.5-10%NaCl or their mixture separately, to increase the stability of glucokinase mutant when sealing and/or discharge.
The concentration of described outer water disperse medium PVA is 0.5-5%, can add the inorganic salt that concentration is 0-10%, and to increase the envelop rate of glucokinase mutant, the pH of outer water is identical with interior water.
2. the sign of microsphere
Mensuration is loaded with the PLGA envelop rate of DGR:
Accurately take by weighing the PLGA microsphere, add 0.5ml dichloromethane dissolving carrier material, 0.5ml 0.01M hydrochloric acid, abundant extracting protein is measured the aqueous phase protein content with the Bradford method from organic facies; DGR amount/DGR inventory * 100% in envelop rate=microsphere.
Measure the activity of DGR in the PLGA microsphere:
Dry microspheres is dissolved in the dichloromethane, the centrifugal organic facies that is dissolved with PLGA of removing, phosphate buffer dissolution precipitation with 0.2ml pH7.4, after centrifugal, isolate cleer and peaceful precipitation, precipitation is with the dissolving with hydrochloric acid of 0.01M, with the content of the DGR in Bradford protein assay mensuration precipitation and the supernatant, simultaneously, measure the activity of DGR in the supernatant with casein gel slab solusphere method.
The granularmetric analysis instrument is measured the particle size distribution of microsphere, the configuration of surface of scanning electron microscope scanning microsphere.
Mensuration is loaded with the PLGA release in vitro curve of DGR:
Get pH7.4PBS buffer 1ml in centrifuge tube, the PLGA microsphere is suspended wherein, put on 37 ℃, 50rpm shaking table, sampling every day, each sampling is with the fresh original buffer of buffer replacement; Fibrinolytic in the working sample is measured the back and is calculated the active percentage rate that discharges of accumulative total, draws external release curve.
Casein gel slab solusphere method is measured the fibrinolytic of DGR:
Defatted milk powder is dissolved in 0.05M pH 7.4PB, in 60 ℃ of water-baths; Agarose is dissolved in 0.05M pH 7.4PB, and heating for dissolving is mixed with last solution after being cooled to 60 ℃, adds NaN3 and plasminogen, and mixing waters on flat board.Beat diameter 3-4mm aperture with card punch behind the gel formation.In each hole, add equal-volume standard substance or sample doubling dilution liquid, 37 ℃ of wet box incubated overnight.Measure each hole solusphere diameter, make standard curve with the logarithm of active logarithm of standard substance and solusphere diameter, the active unit of calculation sample.
Mensuration is loaded with the interior release profiles of PLGA microsphere of DGR:
4 of rabbit, the about 2.5kg of body weight, every intramuscular injection PLGA microsphere 180mg, microsphere suspends with the normal saline solution that contains 2% sodium carboxymethyl cellulose, the time of setting from rabbit ear arterial blood extracting 0.4ml, anticoagulant heparin, 3000rpm * 5min is centrifugal, get blood plasma, measure DGR activity in the blood plasma with the chromophoric substrate method.
Chromophoric substrate method method is measured the fibrinolytic of DGR: the anti-DGR polyclonal antibody of the rabbit bag behind the purification of usefulness 0.1ml dilution in 1: 100 is by 96 plates, 4 ℃ are spent the night, PBS washing 3 times, add blood plasma to be measured and contain the normal rabbit blood plasma of Sak standard substance, 37 ℃ of incubations 2 hours, PBS washing 3 times, every pipe add 0.1ml chromophoric substrate S
2390With Fibrinolysin (human) original mixture, S
2390Be respectively 0.3mM, 0.5 μ M with the final concentration of human plasminogen, 37 ℃ of incubations, 405nm measures absorbance, makes A
405-activity curve is sought the range of linearity.
The result shows that the present invention adds after the PVA by interior aqueous phase, can obviously improve the stability of DGR in the microsphere, makes in the thus obtained microsphere to be in activated state more than the DGR90%, and does not add the microsphere of PVA, has only about 70%DGR to be in activated state; Interior aqueous phase adds Ma (OH)
2Can improve the stability of DGR when discharging, reduce proteic inactivation; After outer aqueous phase adds inorganic salt, can significantly improve the envelop rate of DGR.
The prepared microsphere features smooth surface of the present invention, outward appearance is even, and regular particles does not have adhesion, mean diameter is at 70-105 μ m, drug loading, envelop rate height, and slow-release period is more than 30 days, biodegradable, good biocompatibility can be used for non-vein form administrations such as subcutaneous, muscle.
Description of drawings
Fig. 1 is the electromicroscopic photograph that is loaded with the PLGA microsphere of DGR.
Fig. 2 is the particle size distribution figure of gained PLGA microsphere.
Fig. 3 is the vitro drug release curve that is loaded with the PLGA microsphere of DGR.
Fig. 4 is the drug disposition release profiles that is loaded with the PLGA microsphere of DGR.
The specific embodiment
Below by embodiment technical scheme of the present invention is further described
Embodiment 1:
With 0.2ml concentration is that the DGR solution (0.02M phosphate buffer, pH 7.4) of 10mg/ml is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
The thus obtained microsphere mean diameter is 95.1 μ m, and envelop rate is 7.09%, and drug loading is 0.068%.
Embodiment 2:
With 0.2ml concentration is that the DGR solution (0.02M phosphate buffer, pH 7.4) of 10mg/ml is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA and 2.5% sodium chloride with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
The thus obtained microsphere mean diameter is 93.7 μ m, and envelop rate is 45.4%, and drug loading is 0.0.46%.Active DGR accounts for 75.0% in the microsphere, and insoluble aggregate accounts for 25%.
Embodiment 3:
With 0.2ml concentration is that the DGR solution (contain the 0.02M phosphate buffer of 2%PVA, pH 7.4) of 10mg/ml is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
The thus obtained microsphere mean diameter is 104.3 μ m, and envelop rate is 12.5%, and drug loading is 0.119%.
Embodiment 4:
With 0.2ml concentration is that the DGR solution (contain the 0.02M phosphate buffer of 2%PVA, pH 7.4) of 10mg/ml is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA and 2.5%NaCl with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
The thus obtained microsphere mean diameter is 75.4 μ m, and envelop rate is 78.1%, and drug loading is 0.74%, and the electron-microscope scanning result shows the smooth surface of microsphere, has uniform aperture.Active DGR accounts for 91.4% in the microsphere, and insoluble aggregate accounts for 8.6%.
Embodiment 5:
With 0.2ml concentration is that the DGR solution (contain the 0.02M phosphate buffer of 2%PVA, pH 7.4) of 40mg/ml is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA and 2.5%NaCl with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
The thus obtained microsphere mean diameter is 98.7 μ m, and envelop rate is 83.9%, and drug loading is 3.8%.Active DGR accounts for 67.6% in the microsphere, and insoluble aggregate accounts for 32.4%.
Embodiment 6:
With 0.2ml concentration is that the DGR solution (contain the 0.02M phosphate buffer of 2%PVA, pH 7.4) of 40mg/ml is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA and 2.5%NaCl with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
The thus obtained microsphere mean diameter is 86.1 μ m, and envelop rate is 85.5%, and drug loading is 3.4%.Active DGR accounts for 96% in the microsphere, and insoluble aggregate accounts for 4%.
Embodiment 7:
With 0.2ml concentration is the polyethyleneglycol modified glucokinase mutant SY160-P5 (Circulation.2000 of 10mg/ml; 102:1766-1772.) solution (0.02M phosphate buffer, pH 7.4) is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH7.4) that 75ml contains 2%PVA with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
Embodiment 8:
With 0.2ml concentration is the polyethyleneglycol modified glucokinase mutant SY161-P5 (Circulation.2000 of 10mg/ml; 102:1766-1772.) solution solution (0.02M phosphate buffer, pH 7.4) is emulsifiable in the dichloromethane solution of 2.5mlPLGA, is ultrasonic emulsification 30s under the condition of 80W at power, obtains the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
Embodiment 9:
With 0.2ml concentration is the wild type recombinant glucokinase (number of patent application: 94112105.4) solution (0.02M phosphate buffer of 10mg/ml, pH 7.4) be emulsifiable in the dichloromethane solution of 2.5mlPLGA, be ultrasonic emulsification 30s under the condition of 80W at power, obtain the W/O emulsion.This emulsion is injected in the 0.02M phosphate buffer (pH 7.4) that 75ml contains 2%PVA with microscale sampler, rotating speed stirring 1min with 600rpm makes W/O/W emulsion, in this emulsion impouring 225ml 0.02M phosphate buffer, 300rpm stirs 6h under the room temperature, organic solvent is sent out, and microsphere solidifies the centrifugal collection in back, distilled water washing three times, lyophilization, low-temperature preservation.
Claims (8)
1, the sustained release microsphere agents that contains glucokinase mutant, it is characterized in that by the lactic-co-glycolic acid block copolymer being that carrier material is sealed glucokinase mutant, make the polymer sustained release microsphere agents, described sustained-release micro-spheres particle diameter is at 70-120 μ m, and described glucokinase mutant comprises glucokinase mutant and the external trim thereof that produces by gene mutation.
2, the described preparation method that contains the sustained release microsphere agents of glucokinase mutant of claim 1, it is characterized in that adopting the multiple emulsion solvent evaporation method, carry out emulsifying, under stirring at low speed, make the organic solvent volatilization by ultrasonic or high-speed stirred, form the polymer sustained-release micro-spheres, comprise the steps:
(1) the lactic-co-glycolic acid block copolymer is dissolved in makes oil phase in the organic facies;
(2) get interior aqueous phase solution or suspension and add above-mentioned oil phase, the volume ratio of wherein interior water and oil phase is 5～20%, to form colostrum after its homogenize, water is the pH of buffer 4-9 that contains glucokinase mutant in the described microsphere, wherein adds 0.1-7% polyvinyl alcohol, 0.1-10% trehalose, 0.5-10% magnesium hydroxide, 0.5-10% magnesium carbonate, 0.5-10%NaCl or their mixture separately;
(3) colostrum is added dropwise in the outer water disperse medium, homogenize, stirring at room 4-6 hour, must contain the PLGA microsphere of glucokinase mutant, normal freeze-drying is preserved.
3, by the described method of claim 2, the homogenize mode that wherein prepares microsphere is a high-speed stirred, and mixing time is 3-5 minute.
4, by the described method of claim 2, the homogenize mode that wherein prepares microsphere is ultrasonic, and ultrasonic time is 20-40 second.
5, by the described method of claim 2, wherein said lactic-co-glycolic acid block copolymer amount is 4.0 * 10
3-5.5 * 10
4, wherein the mass percent of polylactic acid and polyglycolic acid is 50: 50-85: 15.
6, by the described method of claim 2, wherein said organic solvent is the mixed liquor of dichloromethane or dichloromethane and acetone, and wherein the volume ratio of dichloromethane and acetone is 75: 35-100: 0.
7, by the described method of claim 2, the concentration of wherein said outer water disperse medium is 0.5-5%, wherein adds the inorganic salt that concentration is 0-10%.
8,, it is characterized in that described glucokinase mutant is the natural Sbphylokinase that extracts or the polyethylene glycol modified product of recombinant glucokinase mutant or recombinant glucokinase mutant from staphylococcus by the described sustained release microsphere agents that contains glucokinase mutant of claim 1.
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| CN104587449B (en) * | 2015-01-06 | 2017-01-11 | 中国人民解放军第二军医大学 | Reactive-oxygen-species sensitive nanoparticle capable of promoting vascularization of surface of wound and preparation method thereof |
| CN105031629B (en) * | 2015-07-07 | 2019-08-23 | 中国人民解放军军事医学科学院基础医学研究所 | SAK-HV oral controlled-release microballoon and the preparation method and application thereof |
| CN105288613B (en) * | 2015-11-25 | 2018-08-31 | 河北师范大学 | A kind of nano particle vaccine preparation and preparation method thereof containing recombination hepatitis B surface antigen |
| CN111686809A (en) * | 2020-06-21 | 2020-09-22 | 复旦大学 | Carbonyl reductase/isopropanol dehydrogenase co-immobilized catalyst and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0330180A1 (en) * | 1988-02-24 | 1989-08-30 | Biomaterials Universe, Inc. | Polylactic acid type microspheres containing physiologically active substance and process for preparing the same |
| WO1991012882A1 (en) * | 1990-02-22 | 1991-09-05 | Medgenix Group S.A. | Microspheres for the controlled release of water-soluble substances and process for preparing them |
| CN1069827C (en) * | 1995-06-09 | 2001-08-22 | 浙江大学 | Polypeptide protein micro-beads medicine prepn. method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0330180A1 (en) * | 1988-02-24 | 1989-08-30 | Biomaterials Universe, Inc. | Polylactic acid type microspheres containing physiologically active substance and process for preparing the same |
| WO1991012882A1 (en) * | 1990-02-22 | 1991-09-05 | Medgenix Group S.A. | Microspheres for the controlled release of water-soluble substances and process for preparing them |
| CN1069827C (en) * | 1995-06-09 | 2001-08-22 | 浙江大学 | Polypeptide protein micro-beads medicine prepn. method |
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