CN101536984B - Injection-use recombinant human Endostatin porous sustained-release microsphere and preparation method thereof - Google Patents
Injection-use recombinant human Endostatin porous sustained-release microsphere and preparation method thereof Download PDFInfo
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- CN101536984B CN101536984B CN2008100198765A CN200810019876A CN101536984B CN 101536984 B CN101536984 B CN 101536984B CN 2008100198765 A CN2008100198765 A CN 2008100198765A CN 200810019876 A CN200810019876 A CN 200810019876A CN 101536984 B CN101536984 B CN 101536984B
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Abstract
The invention relates to a recombinant human Endostatin porous sustained-release microsphere for injection, and a preparation method thereof, in particular to an injection-use porous sustained-release microsphere which takes a biodegradable polymer of lactic acid and hydroxyacetic acid as a matrix and comprises recombinant human Endostatin and a pore-forming agent, and a preparation method thereof.
Description
Technical field
The present invention relates to a kind of injection-use recombinant human Endostatin porous sustained-release microsphere and preparation method thereof; Being specifically related to a kind of is substrate with biodegradable lactic-co-glycolic acid polymer, comprises injection porous sustained-release microsphere of recombinant human vascular endothelial inhibin and porogen and preparation method thereof.
Background technology
The Folkman professor of U.S. Harvard Medical School proposes " tumor growth dependence angiogenic growth " theory in the sixties in 20th century; Be that solid tumor is in growth and transfer process; Crucial effects has been played in the generation of blood vessel; This moment, tumor cell can send some promotion blood vessels to the outgrowth cytokine of tumor tissues (aFGF, bFGF, VEGF etc.).Other discovers; Some endogenetic angiogenesis inhibitors such as Angiostatin, Endostatin can the growth of line artery in tumor tissues, make growth of tumor and transfer be in stagnation (O ' Relly, M.S.; Et al.Cell.79:315-328,1994; O ' Relly, M.S., et al.Cell.88:277-285,1997); Organize opening that medium vessels generates phenotype and close dynamic equilibrium (Folkman, J.Nat.MED.1:27-31,1995 depended between regional angiogenesis stimulating factor of tissue local and the inhibitive factor; Hanahan, D., et al.Cell.86:353-364,1996).Folkman professor last century the seventies proposed to suppress " dying of hunger tumor therapy " theory of tumor growth with blood vessel endothelium chalone (Endostatin).But, limited its large-scale application in tumor patient because Endostatin exists problems such as being easy to deposition and renaturation difficulty in expressing the preparation process.
People such as professor Luo Yongzhang are through modifying the nucleotide coding sequence of human Endostatin; Produce the recombinant human vascular endothelial inhibin that N-terminal has 9 additional aminoacid sequences (be named as:
Chinese name: grace degree
) (ZL 00107569.1), its aminoacid sequence is:
(M) GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRA FLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFD GKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQ SAASCHHAYIVLCIENSFMTASK, wherein the Met of its N-terminal is deleted by part sometimes when by escherichia coli expression.
The human Endostatin Endostar of reorganization keeps all biological activitys of endogenous Endostatin; Solved an Endostatin difficult problem in process of production simultaneously; Expression is higher, and curative effect is stronger, and does not cause immunogenicity in the body because of additional N terminal sequence; Its injection has obtained the approval list marketing of national drug food Surveillance Authority, unites the NP chemotherapy regimen clinically and is used for the nonsmall-cell lung cancer patient.
But as extrinsic protein, the human Endostatin of reorganization is easy to by immune system recognition in vivo, and then is degraded, so the interior half-life of body is very short, and needs of patients is frequently injected, and clinical compliance is very poor.Have in the recent period simultaneously and discover that the drug effect that low dose in the animal body continues to give Endostatin is better than intermittent administration (Minoru Kuroiwa, etal.J.Pediatr.Surg.38:1499-1505,2003; Oliver Kisker, et al.Cancer Res.61:7669-7674,2001).With the lactic-co-glycolic acid polymer be substrate the biodegradable microsphere last century the eighties be used in the Injectable sustained release preparation of polypeptide drugs; The Lupron Depot of Takeda company for example, the Trelstar Depot of Debiopharm company and the Sandostatin Depot of Novartis company etc.Polypeptide drugs are at muscle or subcutaneous along with the continuous biodegradation of polymeric material slowly is discharged in the body; In the long period, keep effective blood drug level in the body; Do not wait thoughtful 6 month from one deenergized period; Effect is remarkable, the disease of or lifelong administration long-term for needs, and compliance of patients improves greatly.Along with development of molecular biology, increasing macro-molecular protein medicine also need overcome the defective of medicine itself by this slow release platform.
The degradable controlled release microball prepn generally is made up of medicine and degradable polyalcohol group matter, adopts the preparation of solvent evaporation method or intra-liquid desiccation method, microsphere features smooth surface or the atomic little pore that has the little solvent volatilization to stay.Because Endostar molecular weight big (21kD), microsphere prepares in the process possibly form polymer again; In vivo after the injection; The inner medicine of microsphere is kept release after being difficult in prominent releasing; Because the speed of microsphere surface degraded is lower than the speed of internal material degraded; Only discharging later stage material generation severely degrade and collapsing under the diffusing situation and could finally discharge fully, this is very unfavorable for constant blood drug level in the maintenance body.
Summary of the invention
The objective of the invention is the problem that exists in above-mentioned conventional degradable controlled release microsphere and the method for preparing in order to solve; Biodegradable microsphere that a kind of surface is covered with uniform small pores and preparation method thereof is provided; The human Endostatin porous microsphere of resulting reorganization is sealed the productive rate height; Dashing forward, it is little to release, and initial stage Endostar can discharge through hole equably in release; Discharge the later stage, porous structure can also promote release medium to inner the flowing of microsphere, quickens the degraded and the corrosion of microsphere, and protein is discharged fully.
But the human Endostatin porous sustained-release microsphere preparation of the reorganization among the present invention can be used for subcutaneous injection, intramuscular injection or intratumor injection.Said preparation is adapted at preparing the purposes of the injectable drug that is used for lasting release recombinant human vascular endothelial inhibin.
The present invention includes a kind of injection-use recombinant human Endostatin porous sustained-release microsphere, it is characterized in that comprising recombinant human vascular endothelial inhibin, biodegradable adjuvant lactic-co-glycolic acid polymer and porogen.Each weight percentages of components wherein: recombinant human vascular endothelial inhibin accounts for that 1%wt to 30%wt, porogen account for 1%wt to 20%wt, the lactic-co-glycolic acid polymer accounts for 50%wt to 98%wt; Sustained-release micro-spheres volume mean diameter is between 0.1 μ m to 300 μ m, between preferred 10 μ m to the 200 μ m.Surface aperture uniform distribution, pore size is between 0.01 μ m to 4 μ m.
The polymer of lactic-co-glycolic acid described in the present invention is formed through polymerization by hydroxyacetic acid and lactic acid; Polymer can be the copolymerization or the homopolymer of lactic acid and hydroxyacetic acid acid, also can refer to single polylactic acid; Polymerization can be random, block or graft type; The end of polymer can be ester group or carboxyl, and they can have D-, the optical configuration of L-or DL.
Lactic-co-glycolic acid polymer as porous sustained-release microsphere substrate among the present invention can be the different lactic-co-glycolic acid mixture of polymers of one or more molecular weight and monomer ratio.
When used polymer was copolymer or the homopolymer of lactic-co-glycolic acid, the mol ratio of lactic acid/hydroxyacetic acid was 40: 60 to 100: 0, preferred 50: 50 to 85: 15; Polymer weight average molecular weight size is 5000 to 150000 dalton, is preferably 5000 to 100000 dalton.The polymer weight average molecular weight should record (GPC) according to the gel chromatography of dialysing.The size of molecular weight has determined polymer degradation speed in vivo, and then has determined the deenergized period of medicine, can select one thoughtful six months deenergized periods, preferred one thoughtful three months.
Porogen described in the present invention can disperse or migrate to the uniform hole of generation among the foreign minister in microsphere forms; Comprise sugar and polyalcohols (as: mannitol, trehalose, chitosan, sucrose, glucose etc.); Inorganic salts (as: sodium chloride, potassium chloride, sodium acetate, sodium phosphate etc.); Amino acids (as: L-arginine, L-lysine etc. contain the aminoacid and the salt thereof of base); High molecular polymer class (as: poloxamer 188, Polyethylene Glycol PEG, polyvinylpyrrolidone PVP etc.), one or more in the fatty acid glycerine esters (like Oleum Glycines, Semen Maydis oil, medium chain triglyceride MCT).
Wherein said molecular weight polyethylene glycol scope is 1000 to 8000 dalton; The polyvinylpyrrolidonemolecules molecules weight range is 2000 to 20000 dalton.
Wherein said medium chain triglyceride MCT is higher than 95% sad (C by content
8H
16O
2) and capric acid (C
10H
20O
2) mixture that the triglyceride of two kinds of satisfied fatty acid is formed, comprise by the C8 that describes with following general formula and six kinds of chemical compounds (1,2,3,4,5,6) of C10 satisfied fatty acid triglyceride.Wherein C8 and C10 can be the alkyl of straight or branched.The operable commercial product of MCT has the Captex 355 of ABITEC company, the LABRAFC CC of Gattefosse company, the LIPOID MCT of Lipoid company.
Recombinant human vascular endothelial inhibin described in the present invention is recombinant human vascular endothelial inhibin Endostar.
The method for preparing of recombinant human Endostatin porous sustained-release microsphere preparation of the present invention, it comprises
(1) preparation contains the water in oil Emulsion of porogen, and wherein interior water is the buffer that contains recombinant human vascular endothelial inhibin, and oil phase is the organic solvent that contains the lactic-co-glycolic acid polymer; Porogen can be dispersed in interior water or the oil phase;
(2) biphase mixed and dispersed is become to join the outer aqueous phase that contains hydrophilic emulsifier behind the breast stir, make the organic solvent volatilization in the water in oil emulsion, the polymer formation microsphere that is separated; The temperature range of volatilization process control is at 0 to 40 degree centigrade; Pressure is controlled at 0.01 to 101kpa;
(3) the thus obtained microsphere washing is dry, get the sustained release microsphere agents finished product.
In step (2); In order to control the particle diameter of microsphere; Can adopt 4000 rev/mins to 20000 rev/mins high speed machine to disperse (like homogenizer, colloid mill, propeller agitator), perhaps adopt the probe of 50W to 200W power ultrasonic, dispersion or ultransonic time were controlled at 10 seconds to 10 minutes.
Because recombinant human vascular endothelial inhibin is only stable under certain pH value; Recombinant human vascular endothelial inhibin is dissolved in the buffer in the above-mentioned method for preparing, and wherein buffer can be a kind of in acetate buffer, phosphate buffer, Tris buffer, the glycine-hydrochloride buffer.The pH value of buffer is between 2 to 9; Preferably between 3 to 8.The concentration of recombinant human vascular endothelial inhibin in buffer is 1 to 500mg/ml, and preferred 10 to 300mg/ml.。
Organic solvent is one or both mixing in dichloromethane, ethyl acetate, acetonitrile, the methanol in the method for preparing of the present invention.The mixed volume ratio is 0.1: 9.9 to 9.9: 0.1; The composition of two kinds of solvent can be dichloromethane+ethyl acetate, dichloromethane+acetonitrile, dichloromethane+methanol, ethyl acetate+acetonitrile, ethyl acetate+methanol; The concentration of lactic-co-glycolic acid polymer in organic solvent is 1 to 500mg/ml.
In solvent evaporates, in the process that microsphere forms, the three-phase system of W/O/W need maintain a relatively stable state.At first, in order finally to form emulsion, must keep certain ratio between the three-phase, described in the method for preparing of the present invention in the water in oil Emulsion volume ratio of oil phase and interior water be 99: 1 to 51: 49; Preferred 95: 5 to 60: 40.Water in oil Emulsion is 0.1: 99.9 to 30: 70 with the volume ratio of outer water.Preferred 0.1: 99.9 to 20: 80.Secondly, for the surface energy that makes Emulsion reduces, outer aqueous phase should add certain hydrophilic emulsifier; Hydrophilic emulsifier described in the method for preparing of the present invention is one or more among polyvinyl alcohol, tween 20, tween 60, the tween 80, and wherein polyvinyl alcohol degree of polymerization scope is 100 to 3000 dalton, and the alcoholysis degree scope is 85 to 89%.The percetage by weight scope of the shared outer water of hydrophilic emulsifier is at 0.01%wt to 5%wt, preferred 0.05%wt to 3%wt.The 3rd; In the solvent evaporates process, can adopt usually used any conventional method to disperse multiphase system, said method comprises uses blade mechanism agitator, magnetic stirring apparatus or rotary evaporator; Evaporating solvent under the condition of normal pressure or decompression, pressure is controlled at 0.01-101kpa.The control temperature range can overall process constant temperature at 0 to 40 degree centigrade in the volatilization process, also can adopt the mode of temperature programming, to shorten the time of solvent evaporates.Mixing time was controlled at 2 to 24 hours.
After microsphere among the present invention is collected, need carry out necessary dried, remove and anhydrate or other volatile material such as dichloromethane.Drying steps can adopt lyophilization processing or vacuum drying treatment, and the persistent period of drying stage can be for example 2 hours to 5 days.
Advantage of the present invention has:
The recombinant human Endostatin porous sustained-release microsphere preparation of one the present invention preparation can slowly discharge recombinant human vascular endothelial inhibin for a long time in vivo, significantly reduces patient's administration number of times, improves compliance of patients.Through selecting or mixing the deenergized period that the molecular weight that uses the different carriers polymeric material can conveniently be regulated medicine.
The structure of two porous surfaces can make that macro-molecular protein discharges uniformly in the microsphere, and the three-phase of avoiding conventional sustained-release micro-spheres occurring discharges.Discharging the later stage simultaneously, loose structure helps release medium to the microsphere internal penetration, quickens the corrosion and the degraded of microsphere, makes drug release complete; Whole dispose procedure approaches constant speed more.When clinical practice, help reducing the blood medicine fluctuation that the irregular in vivo release of medicine causes, in deenergized period, obtain more stable therapeutic effect.
The polyhydric alcohol that uses in three perforating agents can effectively prevent proteic gathering, reduces the polymeric generation of the big volume of non-activity; Also help proteinic release.
Description of drawings
The SEM stereoscan photograph contrast of Fig. 1 porous microsphere and atresia microsphere
The release in vitro behavior curve of Fig. 2 porous microsphere and atresia microsphere.
Fig. 3 contains the SEM stereoscan photograph on the porous microsphere surface of different porogen.
The SEM stereoscan photograph of porous microsphere in dispose procedure among Fig. 4 embodiment three.
The release in vitro behavior curve of porous microsphere among Fig. 5 embodiment four.
The specific embodiment
The present invention makes more detailed description through following examples, but can not be with its protection domain that is construed as limiting the invention.
Embodiment one
Prescription one and the identical technology of prescription two employings: precision takes by weighing Endostar and is dissolved in the phosphate buffer of 80ml pH 8.0 and forms water.PLGA and PEG 4000 be dissolved in the 240ml dichloromethane form organic facies.With above-mentioned biphase mixing, homogenizer 10000rpm high speed dispersion 5 minutes forms water in oil Emulsion; Then this colostrum being poured onto 3.2L contains 3% polyvinyl alcohol 1788 (degree of polymerization is about 1700; Alcoholysis degree 88%) outer aqueous phase; Under 20 degrees centigrade, mechanical agitation normal pressure solvent flashing 2 hours uses the filtering with microporous membrane of 0.8 μ m to obtain microsphere; And with pure water washing three times, lyophilization obtained finished microballoon products in 24 hours.
Precision takes by weighing two crowdes of finished microballoon products 20mg respectively, with 1ml dichloromethane dissolving, adds phosphate buffer (PBS) the extraction medicine of 10ml PH=7.4 again, and (test kit comes from Thermo Scientific, is called BCA with the method for BCA with the PBS solution after the extraction
TMProteinAssay Kit) mensuration protein concentration wherein calculates drug loading and seals productive rate.One the drug loading of wherein writing out a prescription is 18.25%, seal productive rate is 91.25%, and the drug loading of prescription two is 18.69%, seal productive rate is 93.45%.Can find out that from the result porogen is to the not influence of envelop rate of microsphere.Wherein albumen weighs ÷ medicine carrying microballoons gross weight, seals the protein content that actual protein content ÷ calculates according to theoretical inventory in productive rate=microsphere in drug loading=medicine carrying microballoons.
Thus obtained microsphere is sticked on the copper little platform, with gold/palladium parcel, in scanning electron microscope (scanning electronicmicroscopy SEM), observe form, the surface of visible porous microsphere has been covered with uniform aperture, and the atresia microsphere features smooth surface.There were significant differences on form for both.(Fig. 1)
Precision takes by weighing each batch finished microballoon products 50mg, and parallel 3 parts, place the test tube that contains 10ml phosphate buffer (0.1M pH 7.0), put into 37 degree water bath with thermostatic control joltings.Get supernatant 1ml in the test tube at each time point respectively, replenish the fresh phosphate buffer of equal volume simultaneously.The content of Endostar is measured with the BCA method in the sample.The total release percentage of medicament and drug release time mapping (Fig. 2).As can be seen from the figure in 35 days deenergized period, the release of porous microsphere is obviously than even (the linearly dependent coefficient R of atresia microsphere
2=0.844 vs 0.464), the later stage discharges slowly that phenomenon improves, and this loose structure with microsphere surface is relevant.Discharging the later stage (35 days), porous microsphere also discharges (90.33%vs 73.07%) fully than atresia microsphere accumulative total.
Embodiment two
Precision takes by weighing Endostar and different porogen and is dissolved in glycine-hydrochloride buffer of 100ml pH 3.0 and obtains water.PLGA is dissolved in the 158ml dichloromethane forms organic facies.With biphase mixing, ultrasonic 10 seconds of 200W probe forms water in oil emulsion; Then this colostrum is poured onto the outer aqueous phase that 1.032L contains 0.1%tween 80, under 0 degree centigrade, mechanical agitation normal pressure solvent flashing 24 hours; Use the filtering with microporous membrane of 0.8 μ m to obtain microsphere; And with pure water washing three times, vacuum drying 5 days obtains finished microballoon products.
Precision takes by weighing finished microballoon products 20mg, and the method for pressing among the embodiment one is measured protein content in the microsphere, calculates drug loading and seals productive rate, and three the drug loading of wherein writing out a prescription is 1.80%, seal productive rate is 90.00%; Prescription four drug loading is 1.75%, seal productive rate is 87.50%; Prescription five drug loading is 1.63%, seal productive rate is 81.50%.
Above-mentioned three prescription thus obtained microspheres are sticked on the copper little platform, with gold/palladium parcel, in scanning electron microscope (scanningelectronic microscopy SEM), observe the pore-size distribution of form microsphere surface, visible three prescription microsphere surfaces all have little pore size distribution; Void shape that dissimilar perforating agents forms and appearance not of uniform size, pore size: prescription three＜prescription four＜prescription five (Fig. 3), the range estimation pore-size distribution is in 0.1 to 4 mu m range; Along with the increase in aperture, proteic envelop rate slightly descends.
Embodiment three
Precision takes by weighing Endostar and is dissolved in the tris buffer of 100ml pH 5.0 and obtains water.MCT and PLGA are dissolved in (volume ratio dichloromethane: form organic facies ethyl acetate=1: 1) in the 1.9L mixed solvent.With above-mentioned biphase mixing, homogenizer 4000rpm high speed dispersion 10 minutes forms water-in-oil emulsion; Then this colostrum being poured onto 20L contains 1%PVA0486 (degree of polymerization is about 400; Alcoholysis degree 86%) outer aqueous phase heats up since 0 ℃ of speed according to 1 ℃/min, remains at last under 30 ℃; Mechanical agitation solvent flashing 6 hours, Whole Process Control pressure is at 20kpa.Use the filtering with microporous membrane of 0.8 μ m to obtain microsphere, and with pure water washing three times, vacuum drying obtained finished microballoon products in 2 hours.
Precision takes by weighing finished microballoon products 20mg, and the method for pressing among the embodiment one is measured protein content in the microsphere, calculates drug loading and seals productive rate, and six the drug loading of wherein writing out a prescription is 26.21%, seal productive rate is 87.36%; It is 25.827 μ m that the Mastersizer2000 laser particle analyzer of use Ma Erwen records the microsphere volume mean diameter.
Precision takes by weighing finished microballoon products 50mg, and totally 2 parts, place the test tube that contains 10ml phosphate buffer (0.1M pH 7.0), put into 37 degree water bath with thermostatic control joltings.7 days and 14 days sample is taken out pure water washing three times, vacuum drying 24 hours respectively.Thus obtained microsphere is sticked to respectively on the copper little platform with discharging preceding microsphere sample, with gold/palladium parcel, the form (Fig. 4) of in scanning electron microscope (scanning electronic microscopy SEM), observing microsphere in the dispose procedure; Degraded and corrosion from inside to outside taken place in the substrate of visible porous microsphere in dispose procedure from figure.In the time of 14 days, the corrosion of microsphere is very serious, and microsphere surface has formed the cavity of bulk, helps discharging the stripping (14 days cumulative release albumen 95.40%) of late protein.
This be because, 1 has adopted low-molecular-weight PLGA, the ratio of hydroxyacetic acid also increases to some extent, the degradation speed of material is very fast.2 porous structures have increased release medium to the inner infiltration of microsphere, have quickened inner degraded and the corrosion of microsphere.
Embodiment four
The accurate respectively Endostar, trehalose, L-tyrosine of taking by weighing is dissolved in the sodium-acetate buffer of 60ml pH 5.0 and obtains water.PLGA is dissolved in (volume ratio dichloromethane: acetonitrile=9: 1) form organic facies in the mixed solvent of 140ml.With above-mentioned biphase mixing, under airtight condition, used colloid mill circulation homogenizing 2 minutes, form O/W type colostrum; Then this colostrum is poured onto 19.8L and contains the 0.5%PVA 0486 outer aqueous phase of (degree of polymerization is about 400, alcoholysis degree 86%), under 20 degrees centigrade; Mechanical agitation normal pressure solvent flashing 8 hours; Use the filtering with microporous membrane of 0.8 μ m to obtain microsphere, and with pure water washing three times, lyophilization obtained finished microballoon products in 48 hours.
Precision takes by weighing finished microballoon products 20mg, and the method for pressing among the embodiment one is measured protein content in the microsphere, calculates drug loading and seals productive rate, and seven the drug loading of wherein writing out a prescription is 24.03%, seal productive rate is 80.10%.
Precision takes by weighing finished microballoon products 50mg, and parallel 3 parts, place the test tube that contains 10ml phosphate buffer (0.1M pH 7.0), put into 37 degree water bath with thermostatic control joltings.Get supernatant 1ml in the test tube at each time point respectively, replenish the fresh phosphate buffer of equal volume simultaneously.The content of Endostar is measured with the BCA method in the sample.The total release percentage of medicament and drug release time mapping (Fig. 5).As can be seen from the figure write out a prescription seven in trimestral deenergized period, discharge evenly (R
2=0.9401), 90 days cumulative release 81.49%.
Claims (7)
1. injection-use recombinant human Endostatin porous sustained-release microsphere, said sustained-release micro-spheres is characterised in that and comprises:
(1) percentage by weight that recombinant human vascular endothelial inhibin, said recombinant human vascular endothelial inhibin account for said sustained-release micro-spheres is 1%wt to 30%wt;
(2) biodegradable adjuvant lactic-co-glycolic acid polymer, the percentage by weight that said lactic-co-glycolic acid polymer accounts for said sustained-release micro-spheres is 50%wt to 98%wt;
(3) porogen, said porogen are selected from one or more in PEG4000, medium chain triglyceride, trehalose, poloxamer 188, mannitol, the sodium chloride, and the percentage by weight that said porogen accounts for said sustained-release micro-spheres is 1%wt to 20%wt;
The mean diameter of said sustained-release micro-spheres is between 0.1 μ m to 300 μ m, and the pore size of surperficial aperture is between 0.01 μ m to 4 μ m, and said recombinant human vascular endothelial inhibin is recombinant human vascular endothelial inhibin Endostar.
2. microsphere according to claim 1, the mol ratio that it is characterized in that the lactic acid/hydroxyacetic acid of said lactic-co-glycolic acid polymer is 40: 60 to 85: 15, polymer weight average molecular weight size is 5000 to 150000 dalton.
3. the method for preparing of the described injection-use recombinant human Endostatin porous sustained-release microball prepn of claim 1, it comprises:
(1) preparation contains the water in oil Emulsion of porogen; Wherein interior water is the buffer that contains recombinant human vascular endothelial inhibin, and said buffer is selected from a kind of in acetate buffer, phosphate buffer, Tris buffer or the glycine-hydrochloride buffer; Oil phase is the organic solvent that contains the lactic-co-glycolic acid polymer, and said organic solvent is selected from one or both mixing in dichloromethane, ethyl acetate, acetonitrile or the methanol; In water or the oil phase, the volume ratio of oil phase and interior water was 99: 1 to 51: 49 in the said water in oil Emulsion in porogen was dispersed in;
(2) biphase mixed and dispersed is become to join the outer aqueous phase that contains hydrophilic emulsifier behind the breast stir; Make the organic solvent volatilization in the water in oil emulsion; The polymer formation microsphere that is separated, said hydrophilic emulsifier is selected from one or more among polyvinyl alcohol, tween20, tween 60, the tween 80; The temperature range of volatilization process control is at 0 to 40 degree centigrade; Pressure is controlled at 0.01 to 101kpa, and water in oil Emulsion is 0.1: 99.9 to 30: 70 with the volume ratio of outer water;
(3) the thus obtained microsphere washing is dry, get the sustained release microsphere agents finished product.
4. method for preparing according to claim 3, wherein the pH value of the said buffer of step (1) is between 2 to 9; The concentration of recombinant human vascular endothelial inhibin in buffer is 1mg/ml to 500mg/ml.
5. method for preparing according to claim 3 is characterized in that the said mixed organic solvents volume ratio of step (1) is 0.1: 9.9 to 9.9: 0.1; The concentration of lactic-co-glycolic acid polymer in organic solvent is 1mg/ml to 500mg/ml.
6. method for preparing according to claim 3, the percetage by weight scope that it is characterized in that the shared outer water of said hydrophilic emulsifier is at 0.01%wt to 5%wt.
7. method for preparing according to claim 6, wherein polyvinyl alcohol degree of polymerization scope is 100 to 3000 dalton, and the alcoholysis degree scope is 85 to 89%.
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| CN103055302B (en) * | 2013-01-08 | 2014-05-14 | 福建医科大学附属协和医院 | Ultrasonic biological effect mediated recombinant human endostatin controlled release preparation |
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