CN101797232B - Method for preparing small-granularity recombination human vascular endothelium inhibin slowly-released particle for injection - Google Patents
Method for preparing small-granularity recombination human vascular endothelium inhibin slowly-released particle for injection Download PDFInfo
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- CN101797232B CN101797232B CN 200910231612 CN200910231612A CN101797232B CN 101797232 B CN101797232 B CN 101797232B CN 200910231612 CN200910231612 CN 200910231612 CN 200910231612 A CN200910231612 A CN 200910231612A CN 101797232 B CN101797232 B CN 101797232B
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Images
Abstract
The invention relates to a preparation method for recombination human vascular endothelium inhibin slowly-released particle for injection, in particular to a method by adopting secondary emulsification process to prepare small-granularity recombination human vascular endothelium inhibin slowly-released particle. The method adopts a particular secondary emulsification process to prepare the small-granularity bio-degradable particle. One emulsification step is innovatively added, so the granularity of the finished product is effectively reduced; and the process has strong controllability and is applicable to the industrial production.
Description
Technical field
The present invention relates to the preparation method of the small-granularity recombination human vascular endothelium inhibin slowly-released microgranule of a kind of injection, relate in particular to a kind of special second emulsifying technology that adopts, with biodegradable lactic-co-glycolic acid polymer is substrate, comprises the preparation method of the Injectable sustained release microgranule of recombinant human vascular endothelial inhibin and/or porogen.
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 (aFGF of tumor tissues, bFGF, VEGF etc.).Some endogenetic angiogenesis inhibitors such as Endostatin can by the growth of a kind of endothelial cell specific pattern line artery in tumor tissues (O ' Relly, M.S., et al.Cell.88:277-285,1997), organize the opening of medium vessels generation phenotype and close the dynamic equilibrium (Folkman that will depend between tissue local zone angiogenesis stimulating factor and the inhibitive factor, J.Nat.MED.1:27-31,1995; 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).Experiment showed, the growth that can in multiple transplanted tumor model, significantly suppress humanized and animal derived malignant tumor after the Endostatin administration and transfer (O ' Relly, M.S., et al.Cell.79:315-328,1994; Yamaguchi, N., et al.theEMBO journal.18:4414-4423,1999; Schuch, G., et al.Leukemia.19:1312-1317,2005; Yokoyama, Y.and Ramakrishnan.S., Cancer.104:321-331,2005); Endostain is the fragment of a 20kDa of C-terminal of collagen protein 18, and its aminoacid sequence is:
(M) HSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKP
People such as professor Luo Yongzhang are by 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 partially removed (its sequence is respectively SEQ ID NO.1 and SEQ ID NO.2) sometimes when by escherichia coli expression.
The human Endostatin Endostar of reorganization keeps all biological activitys of endogenous Endostatin, solved Endostatin renaturation problem of difficult in process of production simultaneously, expression is higher, curative effect is stronger, and do 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 in vivo by immune system recognition, and then is degraded, so the interior half-life of body is very short.The grace degree that has gone on the market
Injection needed dropleting medicine-feeding two hours every day in 14 days administration cycle, and patient's clinical compliance is very poor; Simultaneously phase ii clinical trial found that accumulated dose and instillation number of times can influence peak concentration and paddy concentration level, between individuality during medicine the curve difference opposite sex very big, this has influenced the stable curative effect of medicine to a certain extent.Have in the recent period and discover that the drug effect that low dose in the animal body continues to give Endostatin is better than intermittent administration (Minoru Kuroiwa, et al.J.Pediatr.Surg.38:1499-1505,2003; Oliver Kisker, etal.Cancer Res.61:7669-7674,2001), this mainly is to have increased the medicine holdup time in vivo, reduces the big ups and downs of blood drug level.Therefore be necessary to study the preparation that a kind of Endostatin of allowing can slowly discharge in vivo for a long time.
Because good biodegradability and biocompatibility, with the lactic-co-glycolic acid polymer be substrate the biodegradable microsphere last century the eighties be widely used in the Injectable sustained release preparation of polypeptide drugs, for example Lupron of Takeda company
The Trelstar of Debiopharm company
Sandostatin with Novartis company
Deng.In addition, protein medicine microsphere also has the successfully case of listing, for example the growth hormone microsphere of Ipsen company.It is shorter that these medicines all are faced with the interior half-life of body, needs the defective of frequent drug administration.After being prepared into the biodegradable microsphere, polypeptide and pharmaceutical grade protein 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, deenergized period from week to six month is not waited, effect is remarkable, the disease of or lifelong administration long-term for needs, 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 method for preparing polylactic acid-glycolic guanidine-acetic acid microgranule commonly used at present comprises: emulsion solvent evaporates-extraction (multi-emulsion method), cold nebulization extraction method, supercritical fluid technology etc.
Wherein the cold nebulization extraction method successfully applies to develop recombinant human somatropin's microsphere, and its product is by FDA's approval listing.This method can be avoided the influence of high temperature to protein stability effectively, the microsphere envelop rate height of preparation, prominent releases for a short time, is particularly suitable for the preparation of protein medicine microsphere.But complex process, the drawbacks limit that equipment cost is higher development and the application of this method in pharmaceuticals industry circle.
Characteristics such as that supercritical fluid technology has is safe, nontoxic, pollution-free, production process gentleness are applicable to the preparation of protein medicine microsphere.And the microsphere goods have evenly, easily control, the high advantage of productive rate.The research that utilizes supercritical fluid technology to prepare microball preparation is just day by day goed deep into, and gradually to industrialized development.
Multi-emulsion method be present search time the earliest, a kind of method for preparing microsphere widest in area, most comprehensive in content.This method technology is simple, the microsphere drug loading height of preparation, protein is distribution uniform in microsphere, medicine be easy to discharge and prominent release little.Multi-emulsion method prepares the microsphere process and is made up of following four steps usually:
A) protein solution (interior water) or micropowder add and contain in the organic facies of polymeric material dispersion formation colostrum;
B) with in above-mentioned colostrum adding and the immiscible continuous phase of organic facies, disperse to form emulsion, organic solvent volatilizees or is extracted to continuous phase, makes microsphere curing;
C) microsphere is collected, is washed and be dry.
Traditional multi-emulsion method Effect Factors for Sythetic Technology is numerous, the difficult quality control of product, the particle diameter of microgranule is an example, the dispersive intensity of emulsifying, emulsifying temperature, the triphasic ratio of emulsion, the speed of solvent evaporates or extraction all can influence the size of diameter of particle and distribute, and then influences preparation drug release behavior and the compliance of patient when administration in vivo.Therefore adopt the protein sustained-release microparticle of improved multi-emulsion method prepared small particle diameter and distribution homogeneous that its necessity is arranged.
Summary of the invention:
A technical problem of the present invention's design is the preparation method that discloses the small-granularity recombination human vascular endothelium inhibin slowly-released microparticle compositions of a kind of injection, and it is short that the characteristics of microgranule long-acting slow-release can overcome the existing listing preparation half-life, needs the defective of frequent drug administration; It is complete that simultaneously relative smaller particle size helps protein release in vivo, and particle size distribution makes clinical administration convenient uniformly.
Technical conceive of the present invention:
Pharmaceutical grade protein discharges the influence that is subjected to a lot of different factors from polylactic-co-glycolic acid (PLGA) microsphere: the polarity of protein itself and size; The hydrophilic of PLGA, degree of crystallinity and degradation speed; The outward appearance of microsphere itself and particle size distribution or the like (James M., et al.Adv.Drug Deliv.Rev.28:5-24,1997).The typical module of pharmaceutical grade protein from microsphere discharges is that in the long cycle, albumen discharges slowly after prominent releasing in 24 hours, and burst size and not exclusively.Studies show that the prominent albumen of releasing mainly is attached to microsphere surface or loose that part (van de Weert et al.J.Control.Release.68:31-40,2000) that is adsorbed in the hydrophilic aperture.And the later stage of sustained-release micro-spheres discharge depend primarily on drug molecule see through the diffusion velocity in hydrophilic duct and the degradation speed of PLGA material (O ' Hagan et al.Int.J.Pharm.103:31-45,1994; Cohen, S., et.al., Pharmaceuticalresearch.8:713-720,1991).Because the Endostatin molecular weight is relatively large, after prominent releasing, be wrapped in inner albumen and be difficult to pass fine and close microsphere substrate and discharge, caused the later stage to discharge slowly and not exclusively, often can not reach clinical effective treatment concentration, also design of influence prescription and dosage are determined simultaneously.
To the objective of the invention is the problem that exists in above-mentioned conventional degradable controlled release microsphere and the preparation method in order solving, a kind of biodegradable microgranule of small particle diameter and novel preparation method thereof to be provided.After the mean diameter of microgranule reduces, can reduce the distance of protein diffusion effectively, increase the release ratio surface area of microparticulate systems.Relend the effect that helps porogen in addition and make sparse tiny aperture, original surface become close and increase, the macro-molecular protein medicine is evenly discharged completely by these apertures.On the other hand, be difficult for very fast gathering after the small particle diameter microparticulate, also can use small size syringe needle, made things convenient for clinical use and administration, the misery of bringing to patient when reducing injection greatly.
But the human Endostatin porous sustained-release microparticle formulation 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.
Based on above-mentioned technical conceive, the inventor proposes following technical scheme:
The second emulsifying preparation method of recombinant human Endostatin porous sustained-release microparticle formulation of the present invention comprises the steps:
A. an emulsifying prepares water in oil colostrum, and wherein interior water is the buffer that contains recombinant human vascular endothelial inhibin; Oil phase is the organic solvent that contains the lactic-co-glycolic acid polymer; If the adding porogen then according to the characteristic of its hydrophilic and oleophilic, is dissolved in it in interior water or the oil phase in advance; With biphase mixing high speed dispersion, finally form the water-in-oil type colostrum of uniform particle diameter.
B. second emulsifying prepares the emulsion of W/O/W, in 10 seconds to 30 seconds after colostrum forms, add the outer aqueous phase of the second emulsifying that contains hydrophilic emulsifier immediately, high speed dispersion forms trickle and the Water-In-Oil oil-in emulsion of homogeneous joins a large amount of outer aqueous phases that contains hydrophilic emulsifying agent with this emulsion, under the condition of normal pressure or decompression, stir, control certain temperature, along with the volatilization of the organic solvent in the emulsion, the polymer dissolution degree reduces rapidly and then the formation microgranule that is separated.
C. the collection of microgranule and drying after solvent evaporates is finished, at last with behind the gained particulate collecting, are washed and drying, get the sustained-release microparticle preparation finished product.
Because recombinant human vascular endothelial inhibin is only stable under certain pH value, recombinant human vascular endothelial inhibin should be dissolved in the buffer in the above-mentioned preparation method, 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; Preferred 10 to 300mg/ml.
Organic solvent is one or both mixing in dichloromethane, ethyl acetate, acetonitrile, the methanol in the preparation method of the present invention.When using two kinds of organic solvents, 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; Preferred 10-400mg/ml.
In the preparation method of the present invention, for the surface energy that makes Emulsion reduces, outer aqueous phase should add certain hydrophilic emulsifier; Hydrophilic emulsifier described in the preparation method of the present invention is one or more among polyvinyl alcohol, tween 20, tween 60, the tween80, wherein polyvinyl alcohol degree of polymerization scope 100 to 3000Da; The alcoholysis degree scope is 85 to 89%; Preferred polyvinyl alcohol specification is 05-88 or 04-86.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.
An emulsifying in the preparation method of the present invention, can adopt the high speed machine of 4000rpm/min to 20000rpm/min to disperse (as the high shear disperser, homogenizer, colloid mill, propeller agitator), perhaps adopt the probe ultra-sonic dispersion of 50W to 200W power, the emulsive time is controlled at 10s to 10min.
Second emulsifying in the preparation method of the present invention can adopt high speed machine to disperse, and the rotating speed of mechanical dispersion is controlled at 6500rpm/min to 13500rpm/min.The emulsifying jitter time is controlled within 2 to 60s; Preferred 2 to 30s, and long jitter time easily causes the probability of breakdown of emulsion to increase the homogeneous of the too short then uncontrollable particle diameter of jitter time.
In order finally to form emulsion, must keep certain ratio between the three-phase, described in the preparation method 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.The volume ratio of the outer water of water in oil Emulsion and second emulsifying is 0.1: 99.9 to 30: 70.Preferred 0.1: 99.9 to 20: 80.
The emulsion of this uniformly emulsify is transferred within 1 to 5min the outer aqueous phase that contains hydrophilic emulsifying agent in a large number carries out solvent evaporates and microgranule solidifies, the volume of outer water should satisfy and volatilizees at liquid level after the organic solvent that allows in the emulsion is partly dissolved and passes apace water.So its volume should be 3-1000 times of the emulsion volume; Preferred 3-100 doubly.In the solvent evaporates process, can adopt usually used any conventional method to disperse multiphase system, described method comprises uses blade mechanism agitator, magnetic stirring apparatus or rotary evaporator, evaporating solvent under the condition of normal pressure or decompression, and pressure is controlled at 0.01-101kpa.The control temperature range is at 0 to 40 ℃ in the volatilization process, and preferred 4～30 ℃, can overall process constant temperature, also can adopt the mode of temperature programming, to shorten the time of solvent evaporates.Mixing time is controlled at 1 to 24h; Preferred 2 to 16h.
Particulate collecting among the present invention can adopt filtering method, and the microgranule after the collection is by a large amount of water for injection washings, with the polyvinyl alcohol of removing remained on surface and the albumen that is not wrapped.Need then microgranule is carried 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 2h to 5d.
Injection-use recombinant human Endostatin sustained-release microparticle among the present invention is characterized in that comprising recombinant human vascular endothelial inhibin, biodegradable adjuvant lactic-co-glycolic acid polymer and/or porogen.Each weight percentages of components wherein: recombinant human vascular endothelial inhibin accounts for that 1%wt to 30%wt, porogen account for 0%wt to 30%wt, the lactic-co-glycolic acid polymer accounts for 40%wt to 99%wt, sustained-release microparticle is a spheroidal particle, volume average particle size is between 0.1 μ m to 100 μ m, between preferred 10 μ m to the 60 μ m.The microparticle surfaces that adds porogen is distributed with uniform aperture, and pore size is between 0.01 μ m to 4 μ m.
Human Endostatin Endostatin or its analog of recombinant human vascular endothelial inhibin described in the present invention for obtaining by gene expression.Analog can refer to add, and lacks or change 2 to arrive a plurality of amino acid residue human Endostatin mutants, and preferred Endostatin N-terminal has the recombinant human vascular endothelial inhibin Endostar of 9 additional aminoacid sequences.
The polymer of lactic-co-glycolic acid described in the present invention is formed by polymerization by hydroxyacetic acid and lactic acid; Polymer can be the copolymerization or the homopolymer of lactic acid and hydroxyacetic 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 sustained-release microparticle 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 the copolymer of lactic-co-glycolic acid or homopolymer, the mol ratio of lactic acid/hydroxyacetic acid was 40: 60 to 100: 0, preferred 50: 50 to 85: 15; The ratio of polymer adopts nuclear magnetic resonance spectroscopy to measure (NMR).Polymer weight average molecular weight size is 5000 to 150000Da, is preferably 5000 to 100000Da.The polymer weight average molecular weight should record according to the gel chromatography (GPC) of dialysing.
The characteristic of polymer has determined polymer degradation speed in vivo, and then influences the deenergized period of medicine.Polymer ends is that the degradation speed of carboxyl is the polymer of ester group faster than end; The ratio of hydrophilic hydroxyacetic acid is high more, and the molecule of polymer is low more, and degradation speed is fast more.By selecting different polymer or different mixture of polymers, can select one thoughtful six months the deenergized periods of medicine, 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 the microgranule forming process, 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. contains the aminoacid and the salt thereof of base), high molecular polymer class (as: poloxamer 188, Polyethylene Glycol PEG, polyvinylpyrrolidone PVP etc.), the fatty acid glycerine esters is (as Oleum Glycines, Semen Maydis oil, medium chain triglyceride MCT) one or more in.
Wherein said molecular weight polyethylene glycol scope 1000 to 8000Da; The polyvinylpyrrolidonemolecules molecules weight range 2000 to 20000Da.
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 formed of the triglyceride of two kinds of satisfied fatty acid.Wherein C8 and C10 can be the alkyl of straight or branched.The operable commercial product of MCT has Captex 355, the LABRAFCCC of Gattefosse company, the LIPOID MCT of Lipoid company of ABITEC company.
The recombinant human vascular endothelial inhibin microgranule of the present invention's preparation need be suspended in the injection solvent when injection, the injection solvent need have certain viscosity can guarantee in the short period that helping of microgranule select state, also must possess suitable osmotic pressure and pH value simultaneously, the untoward reaction that produces when injecting to alleviate.Therefore injecting solvent is made up of water for injection, suspending wetting agent (as: sodium carboxymethyl cellulose CMC-Na, Tween 80, Polyethylene Glycol PEG, hydroxypropyl emthylcellulose HPMC, polyvinylpyrrolidone PVP, dextran etc.), osmotic pressure regulator (as: mannitol, sorbitol, lactose, glucose, sodium chloride etc.), buffer salt or acid (as: sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium acetate, glacial acetic acid etc.).
Advantage of the present invention has:
The injection-use recombinant human Endostatin sustained-release microparticle 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.By selecting or mixing and use different prescriptions and technological parameter can conveniently regulate the deenergized period of medicine.
A kind of biodegradable microgranule of small particle diameter that two the present invention have adopted special second emulsifying prepared.Increased to this process innovation property by a step emulsifying step, reduced the particle diameter of finished product very effectively little; This process controllability is strong, is fit to commercial production.After the mean diameter of microgranule reduces, can reduce the distance of protein diffusion effectively, increased microparticulate systems the release ratio surface area this help discharging early stage to spread leading drug release.Relending the effect that helps porogen has in addition increased the infiltration of later stage moisture, has effectively accelerated the degraded of substrate.This helps discharging the leading drug release of substrate degradation in later stage.By preparation technology's washing, can effectively reduce prominent the releasing that the albumen by surface adsorption causes again.Therefore, the sustained-release microparticle for preparing among the present invention can improve the three-phase release of conventional sustained-release microparticle.Obtain comparatively ideal release mode in vivo and in vitro.
Description of drawings
The SEM stereoscan photograph contrast of the microgranule of emulsifying of Fig. 1 single and second emulsifying preparation.(annotate: A-1 (* 50), A-2 (* 200) are the prescription one of single emulsification method preparation; B-1 (* 150), B-2 (* 1,000) are the prescription two of second emulsifying method preparation.)
The particle size distribution figure of the microgranule of emulsifying of Fig. 2 single and second emulsifying preparation.(annotate: A is the prescription one of single emulsification method preparation; B is the prescription two of second emulsifying method preparation)
Fig. 3 contains the SEM stereoscan photograph on the small porous particle surface of different porogen.(annotate: C is the prescription three that contains trehalose, and D is the prescription six that contains MCT)
The release in vitro behavior curve of microsphere among Fig. 4 embodiment three.
Release behavior curve in the body of microsphere among Fig. 5 embodiment three.
The specific embodiment
The present invention is for a more detailed description by following examples, but the protection domain that it can not be construed as limiting the invention.
Embodiment one
Prescription one and prescription two employing identical prescriptions but adopt different prepared: precision takes by weighing Endostar and is dissolved in the phosphate buffer of 56ml pH 7.4 and forms water.PLGA is dissolved in the 365ml dichloromethane forms organic facies.With above-mentioned biphase mixing, homogenizer 10000rpm high speed dispersion 5min forms water in oil Emulsion; Prescription one directly is poured onto this colostrum 7.5L and contains the 0.3% polyvinyl alcohol 1788 outer aqueous phase of (degree of polymerization is about 1700, alcoholysis degree 88%), under 20 ℃, and mechanical agitation normal pressure solvent flashing 2h; Second prescription disperses (IKA homogenizer T20) 20s with the outer water of polyvinyl alcohol 1788 second emulsifyings of colostrum and 800ml 2% in the 13500rpm/min high speed shear earlier.Again this emulsion is poured onto the outer aqueous phase that 6.7L contains 0.097% polyvinyl alcohol 1788, under 20 ℃, mechanical agitation normal pressure solvent flashing 2h; Two prescription thus obtained microspheres all use the filtering with microporous membrane of 0.8 μ m to obtain microgranule, and wash three times with pure water, and lyophilization 24h obtains the microgranule finished product.
Precision takes by weighing two crowdes of microgranule finished product 20mg respectively, with the dissolving of 1ml dichloromethane, adds phosphate buffer (PBS) the vortex mixed 3min of 10ml pH=7.4 again, the centrifugal 10min of 4000rpm/min.Extract supernatant, (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 16.34%, seal productive rate is 96.12%, and the drug loading of prescription two is 15.87%, seal productive rate is 93.35%.Second emulsifying technology in order to reduce the particle diameter of emulsion droplet, has adopted the dispersion of high energy in first step emulsifying, reduced the envelop rate of medicine thereby the seondary effect of bringing is a structure of destroying emulsion.But the present invention has adopted suitable jitter time and the triphasic ratio of emulsion, reduces to minimum to the destruction of emulsion high speed dispersion.As can be seen from the results, though adopted second emulsifying, the envelop rate of microgranule and drug loading significantly do not descend.Wherein albumen weighs ÷ medicine carrying microgranule gross weight, seals the protein content that actual protein content ÷ calculates according to theoretical inventory in productive rate=microgranule in drug loading=medicine carrying microgranule.
The gained microgranule is sticked on the copper little platform, with gold/palladium parcel, observe form in scanning electron microscope (scanning electronicmicroscopy SEM), the microspherulite diameter of visible second emulsifying prepared is significantly less than the single emulsifying process, and surface area increases greatly.And the microsphere surface of second emulsifying preparation is slick and sly more, does not have fold (Fig. 1).Get two prescriptions respectively and measure its volume average particle size (Fig. 2) with laser particle analyzer (Sympatec HELOS Sucell), the volume average particle size of prescription one is 135.10 μ m, and X90 is 189.81 μ m; The volume average particle size 22.22 μ m of prescription two, X90 is 35.60 μ m.The microgranule volume average particle size only prepares the sixth of microgranule mean diameter behind the second emulsifying for the single emulsifying process.
Embodiment two
Precision takes by weighing Endostatin 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.Porogen joins according to different character that (trehalose, PEG 4000, L-arginine are dissolved in aqueous phase in water and the organic facies, Lipoid MCT joins in the organic facies), with biphase mixing, the ultrasonic 120s of 200W probe, form water in oil emulsion, then this colostrum is poured onto the outer aqueous phase of second emulsifying that 1.7L contains 0.4%Tween 80, under 6500rpm/min intensity, high speed shear is disperseed (IKA homogenizer T20) 30s.Resulting emulsion is added the outer aqueous phase that 18L contains 0.05%Tween 80, under the 0-4 degrees celsius, mechanical agitation normal pressure solvent flashing 24 hours, use the filtering with microporous membrane of 0.8 μ m to obtain microgranule, and with pure water washing three times, vacuum drying 5 days obtains the microgranule finished product.
Precision takes by weighing microgranule finished product 20mg, and the method for pressing among the embodiment one is measured protein content in the microgranule, calculates drug loading and seals productive rate, as shown in the table: PEG 4000 and L-arginine hydrophilic are strong, in the second emulsifying process, Endostatin reveals more serious, therefore causes envelop rate to descend.And the L-arginine is the micromolecule porogen, and the effect that stops Endostatin to reveal is poorer.Trehalose is a natural polymer, has certain viscosity in water, helps keeping the stable of colostrum; And MCT is dissolved in the organic solvent, the proteic leakage of aqueous phase in also can not causing.Therefore Endostatin's did not seal when these two kinds of porogen did not influence second emulsifying and prepare microgranule.
To write out a prescription three and the prescription six gained microgranules stick on the copper little platform, with gold/palladium parcel, in scanning electron microscope (scanningelectronic microscopy SEM), observe the pore-size distribution of form microparticle surfaces, as seen after adopting second emulsifying technology, contain microparticle surfaces that trehalose the obtains sparse and dark hole that distributing, and the microparticle surfaces that adds MCT has been covered with and uniform hole tiny.
Embodiment three
Prescription seven and prescription eight employing identical prescriptions but adopt different prepared: 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 500ml mixed solvent.With above-mentioned biphase mixing, homogenizer 4000rpm high speed dispersion 10 minutes, form water-in-oil emulsion, prescription seven directly is poured onto this colostrum 20L and contains 1%PVA 0486 (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.
Prescription eight contains the outer aqueous phase of second emulsifying of 3%PVA0486 at 9500rpm/min high speed shear dispersion (IKA homogenizer T20) 7s with colostrum and 2L.After forming emulsion, add the outer aqueous phase that 18L contains 0.78%PVA 0486, heat up, remain at last under 30 ℃ since 0 ℃ of speed according to 1 ℃/min, mechanical agitation solvent flashing 6 hours, Whole Process Control pressure is at 20kpa.
More than the collection of preparation gained microgranule all uses the filtering with microporous membrane of 0.8 μ m to obtain, and washs three times with pure water, and vacuum drying obtained the microgranule finished product in 2 hours.
Precision takes by weighing microgranule finished product 20mg, and the method for pressing among the embodiment one is measured protein content in the microgranule, calculates drug loading and seals productive rate, and seven the envelop rate of wherein writing out a prescription is 87.34%, drug loading 21.30%; The envelop rate of prescription eight is 86.05%, drug loading 20.99%.Measuring its volume average particle size with laser particle analyzer (Sympatec HELOS Sucell) is respectively: 7 95.34 μ m write out a prescription; 8 19.94 μ m write out a prescription.
Precision takes by weighing each batch microgranule finished product 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 in the release medium is measured with the BCA method.Total release percentage and drug release time mapping (Fig. 4) with medicine.As can be seen from the figure in 35 days deenergized period, the Endostar microgranule that adopts second emulsifying prepared prescription eight relatively and the microgranule of single emulsifying process preparation prescription seven, prominent the releasing (22.58% vs 14.32%) that does not have significantly increasing medicament, but because particle diameter obviously reduces, releasable porous surface is long-pending to be increased greatly, the drug accumulation rate of release is accelerated, and discharges (79.04% vs 32.22%) more fully.
Other gets 50 of male SD rats (200-220g), is divided into two groups at random.Precision takes by weighing each batch finished microballoon products 100mg/ part, inject solvent (containing 5% mannitol, 0.5%CMC-Na, 0.1% tween 80) suspendible with 2ml after, subcutaneous injection is gone into the SD rat back, 25 of each prescription injections.Respectively at putting to death 5 of every group of rats on the the 0th, 1,10,22,30 day after the injection.Remaining microsphere under the bark fetching, after the method for pressing among the embodiment one was extracted, HPLC measured the surplus of Endostar.The result as shown in Figure 5, the prescription eight that adopts the second emulsifying prepared as can be seen in vivo the Endostar surplus reduce speed much larger than prescription seven.After 30 days, prescription eight can only be recovered to 9.78% in the injection site, and the prescription seven of big particle diameter also remains 57.75% Endostar in the injection site.Illustrate and adopted second emulsifying technology, the release in vivo of Endostar microgranule is also more complete.
Claims (1)
1. the preparation method of the injection small-granularity recombination human vascular endothelium inhibin slowly-released microparticle formulation of a volume average particle size between 1 μ m to 100 μ m is characterized in that it comprises the following steps:
A. emulsifying for the first time prepares water in oil Emulsion, 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 is dispersed in interior water or the oil phase; Described buffer pH value scope is 2 to 9; Described organic solvent is one or both mixing in dichloromethane, ethyl acetate, acetonitrile, the methanol; The volume ratio of described oil phase and interior water is 99:1 to 51:49;
B. biphase mixing dispersion and emulsion is formed colostrum, add again and contain hydrophilic emulsifying agent second emulsifying water and carry out dispersion and emulsion formation second time emulsion, subsequently this emulsion is added the outer aqueous phase that contains hydrophilic emulsifier, under the condition of normal pressure or decompression, stir, make the organic solvent volatilization in the water in oil emulsion, the polymer formation microgranule that is separated, volatilization process keep temperature range at 0 to 40 ℃, treat that microgranule is in the fluid drying molding; Described hydrophilic emulsifier is one or more among polyvinyl alcohol, tween 20, tween 60, the tween 80; The described emulsifying second time dispersion and emulsion time is controlled within 2 to 60s; The volume ratio of described colostrum and second emulsifying water is 1:1 to 1:10; Described emulsion is 0.1:99.9 to 30:70 with the volume ratio of outer water;
C. with gained particulate collecting, washing, drying, get the sustained-release microparticle preparation finished product.
2. preparation method according to claim 1 is characterized in that described recombinant human vascular endothelial inhibin is Endostatin.
3. preparation method according to claim 1 is characterized in that described recombinant human vascular endothelial inhibin is Endostar.
4. preparation method according to claim 1 is characterized in that each weight percentages of components in the described sustained-release microparticle: recombinant human vascular endothelial inhibin accounts for that 1%wt to 30%wt, porogen account for 0%wt to 30%wt, the lactic-co-glycolic acid polymer accounts for 40% wt to 99%wt.
5. preparation method according to claim 4, the sustained-release microparticle volume average particle size that it is characterized in that recombinant human vascular endothelial inhibin are between 10 μ m to the 60 μ m.
6. preparation method according to claim 1 is characterized in that described buffer is a kind of in acetate buffer, phosphate buffer, Tris buffer, the glycine-hydrochloride buffer; The pH value scope of buffer is 3 to 8; The concentration of recombinant human vascular endothelial inhibin in buffer is 1 mg/ml to 500 mg/ml.
7. preparation method according to claim 1, it is characterized in that the described emulsifying second time disperses to use high speed machine to shear, the rotating speed of mechanical dispersion is controlled at 6500rpm/min to 13500rpm/min, and emulsification times is controlled within 2 to 30s.
8. preparation method according to claim 1, when it is characterized in that described organic solvent is two kinds of organic solvents mixing, blended combination is selected from a kind of in dichloromethane+ethyl acetate, dichloromethane+acetonitrile, dichloromethane+methanol, ethyl acetate+acetonitrile, the ethyl acetate+methanol, and the mixed volume ratio is 0.1:9.9 to 9.9:0.1; The concentration of lactic-co-glycolic acid polymer in organic solvent is 1 mg/ml to 500 mg/ml.
9. preparation method according to claim 1, the percetage by weight scope that it is characterized in that the shared outer water of described hydrophilic emulsifier is at 0.01%wt to 5%wt.
10. preparation method according to claim 10, it is characterized in that wherein polyvinyl alcohol degree of polymerization scope 100 to 3000Da; The alcoholysis degree scope is 85 to 89%.
11. preparation method according to claim 1, the temperature range that it is characterized in that described volatilization process control is at 4 to 30 ℃.
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| WO2015175545A1 (en) | 2014-05-12 | 2015-11-19 | The Johns Hopkins University | Highly stable biodegradable gene vector platforms for overcoming biological barriers |
| AU2016211696B2 (en) | 2015-01-27 | 2018-05-10 | The Johns Hopkins University | Hypotonic hydrogel formulations for enhanced transport of active agents at mucosal surfaces |
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