CN101642559B - Pharmaceutical composition containing micronized human vascular endostatin - Google Patents
Pharmaceutical composition containing micronized human vascular endostatin Download PDFInfo
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- CN101642559B CN101642559B CN 200810122763 CN200810122763A CN101642559B CN 101642559 B CN101642559 B CN 101642559B CN 200810122763 CN200810122763 CN 200810122763 CN 200810122763 A CN200810122763 A CN 200810122763A CN 101642559 B CN101642559 B CN 101642559B
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- human endostatin
- solid form
- endostatin
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- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Images
Abstract
The invention relates to a pharmaceutical composition containing micronized solid human vascular endostatin serving as an active component and a preparation method for the composition. The pharmaceutical composition contains a medicinal carrier and the micronized solid human vascular endostatin, wherein the medicinal carrier can be a lacti-glycolic acid polymer. The pharmaceutical composition can also contain 0.5 to 30 weight percent of protective agent.
Description
Technical field
The present invention relates to contain the human Endostatin pharmaceutical composition of micronizing solid form, relate in particular to a kind of active good human Endostatin preparation of drug combination method that keeps.
Background technology
Discover that solid tumor needs the generation of blood vessel that nutrition is provided in growth and transfer process; Tumor cell can send some signals and promoted blood vessel to the tumor tissues hypertrophy this moment; 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), the Folkman professor of U.S. Harvard Medical School 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; Producing the recombinant human vascular endothelial inhibin that N-terminal has 9 additional aminoacid sequences (is named as: Endostar; Chinese name: the 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 grace degree of reorganization keeps all biological activitys of endogenous Endostatin; Solved an Endostatin difficult problem in process of production simultaneously; Purge process is simple, and purity is high, and activity is kept; The list marketing of its injection is united the NP chemotherapy regimen clinically and is used for the nonsmall-cell lung cancer patient.
The reorganization human Endostatin as extrinsic protein, be easy to by immune system recognition in vivo, and then be degraded, thus body in the half-life very short, needs of patients is frequently injected, clinical compliance is very poor.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; For example like the Lupron Depot of Takeda company, the Trelstar Depot of Switzerland 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, 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.
Sustained release microsphere agents generally adopts the method preparation of W/O/W, and pharmaceutical grade protein pre-exists in interior aqueous phase, under high speed shear or ultransonic effect, forms colostrum with the organic solvent that contains the lactic-co-glycolic acid polymer, and redispersion is to outer aqueous phase.Protein receives tensile influence on oil-water interfaces, structure is easy to change, and causes the forfeiture of its BA.Solid-state protein does not then receive the influence of oil-water interfaces; It can well keep active in preparation, therefore, the albumen solid state is just being become at present the focus of preparation research and development; Yet; The technology of existing production solid-state protein can't be produced the micronized protein of particle diameter less than 3um, thus can not satisfy the needs of some novel form research and development, such as the less microsphere of particle diameter.The method of existing micronized protein has had a lot, and overall conclusion can be divided into following several kinds: spray drying method, freeze-drying, atomizing freeze drying method, supercritical fluid technology.Spray drying is to utilize nebulizer that drug solution is separated into tiny droplet; And in the heated drying medium rapidly evaporating solvent form the process of dry powder; The grain size of micropowder that this technology makes is even; But this method need be used hot-air, is not suitable for dry temperature-resistant protein and polypeptide drugs.The atomizing freeze drying method combines the atomization steps in the spray drying with lyophilization, step is that the pharmaceutical aqueous solution that atomizes is sprayed in the liquid nitrogen, is dried with cryodesiccated step, obtains the uniform drug powder of particle diameter.This method batch processing amount is big, but cost is higher, and the grain size of micropowder that obtains is bigger.Supercritical fluid technology is the technology of newly-developed; The character that can be used for supercritical fluid is extracted solvent; Desiccation protein, still, the problem that still there are many needs researchs in this technology and solve: generally less like albumen and the dissolubility of polypeptide drug in organic solvent; How increase the dissolubility of medicine through changing solvent composition, and the relation between different operating conditions and the heterogeneity micropowder that finally obtains.
Summary of the invention
To the objective of the invention is the problem that exists in the existing protein microsphere method for preparing in order solving, a kind of active method for preparing that keeps good human blood-vessel endothelia inhibin sustained-released microsphere preparation to be provided.
But the human Endostatin medicinal composition microsphere preparation among the present invention can be used for subcutaneous injection or intramuscular injection.Said preparation is suitable for continuing to discharge the purposes of human Endostatin.
The invention discloses a kind of pharmaceutical composition that comprises pharmaceutically suitable carrier and human Endostatin, it is characterized in that this active component human Endostatin is the micronizing solid form.
The human Endostatin of above-mentioned micronizing solid form can prepare through following steps:
(1) in containing the buffer salt solution of human Endostatin, add soluble metallic salt, the pH that adjusts this solution then obtains proteic fine particle deposition to proteic isoelectric point, IP (pI), and protein solution becomes suspension;
(2) with behind suspension and the freeze drying protectant mix homogeneously, lyophilizing;
(3) the solid powder agglomates after the lyophilizing is used solvent wash, remove freeze drying protectant, obtain micronized protein.
The freeze drying protectant of the method for preparing of above-mentioned micronized human vascular endostatin is a Polyethylene Glycol, in lyophilizing, plays space figuration and stable effect, and the human Endostatin protein body that precipitates can be adsorbed on the skeleton of Polyethylene Glycol.
The method for preparing molecular weight polyethylene glycol scope of above-mentioned micronized human vascular endostatin is 1000 to 8000 dalton.Polyethylene Glycol has good suspension stability, and molecular weight ranges keeps it in lyophilizing, not assemble at the equal good stabilize proteins granule of 1000 to 8000 dalton, does not sink.
The volume ratio of suspension and freeze drying protectant is 1: 99～99: 1 in the method for preparing of above-mentioned micronized human vascular endostatin, step (2), preferred 15: 85～90: 10; Human Endostatin protein concentration in the suspension is 0.01mg/ml～500mg/ml, and the concentration of freeze drying protectant is 1%～50% (w/v).The concentration of freeze drying protectant can not be less than 1%, otherwise suspension stability is very poor, and albumen precipitation can sink to assembling very soon; The excessive concentration of freeze drying protectant also is not suitable for because after the lyophilizing in order to obtain the albumen micropowder, need washing with an organic solvent to remove this protective agent, if the ratio that protective agent accounts for is too high, need to consume a large amount of organic solvents, clean repeatedly, make troubles to operation.Keep the concentration of 1%～50% (w/v) more suitable.
The method for preparing of above-mentioned micronized protein, the cleaning solvent in the step (3) is the solvent of solubilized Polyethylene Glycol, for example: acetone, ethyl acetate, dichloromethane, acetonitrile.Ethyl acetate, dichloromethane, acetonitrile.
Pharmaceutically suitable carrier of pharmaceutical composition of the present invention is the lactic-co-glycolic acid polymer.Pharmaceutically suitable carrier lactic-co-glycolic acid polymer 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, and they can have D-, the optical configuration of L-or DL.
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 99: 1, preferred 50: 50 to 85: 15; Polymer weight average molecular weight size is 5000～150000 dalton, is preferably 10000 to 80000 dalton.The size of molecular weight has determined polymer degradation speed in vivo, and then has determined can select a thoughtful February deenergized period deenergized period of medicine.The polymer weight average molecular weight should record (GPC) according to the gel chromatography of dialysing.
The preferred recombinant human vascular endothelial inhibin Endostar of the human Endostatin of pharmaceutical composition of the present invention.
Said pharmaceutical composition is a solid composite medicament, and significant solid composite medicament is: microsphere and implant.
Preferred solid composite medicament is a microsphere in the scope of the invention.Because the common administering mode of microsphere is a drug administration by injection, convenient, need not after the injection to take out.And implant need carry out the minor operation implantation and take out, and causes suffering to patient.
Pharmaceutical composition of the present invention, human Endostatin account for the 1%wt to 30%wt of compositions, preferred 10%wt to 20%wt.The volume average particle size of microsphere is between 0.1um to 300um.
In compositions, can also add protective agents such as some sugar that play the skeleton supporting role, polyhydric alcohol or polymer, play space figuration and Stabilization, wherein the polyalcohols material can also effectively prevent proteic gathering, reduces the polymeric generation of non-activity.Protective agent accounts for the 0.5%wt to 30%wt of compositions.
The protective agent of pharmaceutical composition of the present invention comprises one or more in polyhydric alcohol (sorbitol, xylitol, mannitol), saccharide (trehalose, chitosan, glucosan, sucrose, lactose, glucose), the high molecular polymer (poloxamer 188, Polyethylene Glycol, polyvinylpyrrolidone).
The Polyethylene Glycol weight average molecular weight range of pharmaceutical composition of the present invention is 1000 to 8000 dalton; The polyvinylpyrrolidone weight average molecular weight range is 2000 to 20000 dalton.
The Pharmaceutical composition outward appearance of the present invention's preparation is spherical, porous surface or atresia, and inside is alveolate texture.
The Pharmaceutical composition of the present invention's preparation can suppress the generation of the new vessels of mammal malignant tumor, is used to treat all kinds of cancers.
Advantage of the present invention has:
One adopts the human blood-vessel endothelia inhibin sustained-released microsphere preparation of method preparation of the present invention keeping its active while, 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.Molecular weight through selecting the different carriers polymeric material can conveniently be regulated release rate of drugs.
Two with human Endostatin solution and the common lyophilization of protective agent; Form micronized human Endostatin solid; This pharmaceutical grade protein of human Endostatin is kept the integrity of protein multilevel hierarchy by skeleton backing material (protective agent) protection.The protein Preparation of this solid form is become microsphere, eliminated the problem that contingent oil water interfacial tension makes the protein active forfeiture.
Three become solid-state with protein Preparation, improved the stability that albumen is prepared to microsphere.
Description of drawings
The particle size distribution figure of microsphere among Fig. 1 embodiment one
The SEM stereoscan photograph of microsphere among Fig. 2, Fig. 3 embodiment one
The particle size distribution figure of microsphere among Fig. 4 embodiment two
(Mw=30000, lactic acid: the Endostar microsphere that hydroxyacetic acid=75: 25) prepares is the release behavior curve outward with PLGA among Fig. 5 embodiment two.
Among Fig. 6 embodiment three Endostar is processed the microsphere front and back to the HUVEC cell-proliferation activity.
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
Precision is measured 50ml recombinant human vascular endothelial inhibin Endostar protein solution (9.1462mg/ml; PH=5.5); Add 0.004% zinc acetate, after dissolving fully to zinc salt, in this interval of PH to 8.8-9.3 with sodium hydroxide solution (1mol/L) accent protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-8000 solution of 50% (w/v), and magnetic agitation 5min makes it mix homogeneously.The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Dissolve lyophilized products with dichloromethane; PEG-8000 dissolves in dichloromethane, centrifugal after, obtain containing the micronized protein of dichloromethane in centrifuge tube bottom; The room temperature drying under reduced pressure it; Obtain exsiccant micronized protein, (Mw=50000, lactic acid: hydroxyacetic acid=50: 50) 2g is dissolved in the mixed solvent of 3ml ethyl acetate and 7ml acetone and forms organic facies with PLGA.The above-mentioned composition powder is suspended in the organic facies, and 10000rpm high speed dispersion 20s forms S/O two-phase disperse system; Then this colostrum is poured onto in the soybean oil that 1L contains 0.2% soybean lecithin, under 20 degrees centigrade, mechanical agitation normal pressure solvent flashing 6 hours; Use the filtering with microporous membrane of 0.8 μ m to obtain microsphere; And use petroleum ether, and filter microsphere, drying obtains finished microballoon products.
Precision takes by weighing finished microballoon products 20mg, with 1ml dichloromethane dissolving, adds phosphate buffer (PBS) the extraction medicine of 10ml PH=7.4 again, and (test kit comes from ThermoScientific, is called BCA with the method for BCA with the PBS solution after the extraction
TMProtein Assay Kit) mensuration protein concentration wherein, wherein Endostar accounts for the 1.27%wt of microsphere total amount, and sealing productive rate is 99.21%, uses Mastersizer 2000 laser particle analyzers to record the microsphere volume mean diameter and is 70.461um.(Fig. 1).
Thus obtained microsphere is observed form in scanning electron microscope (scanning electronic microscopy), visible microspherulite diameter homogeneous, microsphere outward appearance rounding, porous surface.Microsphere is wrapped up the back section under scanning electron microscope, observe in hard paraffin, visible its internal structure is loose.(Fig. 2, Fig. 3)
Embodiment two
Precision is measured 50ml recombinant human vascular endothelial inhibin albumen Endostar solution (9.1462mg/ml; PH=5.5); Add 0.004% zinc acetate, after dissolving fully to zinc salt, in this interval of PH to 8.8-9.3 with sodium hydroxide solution (1mol/L) accent protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-8000 solution of 50% (w/v), and magnetic agitation 5min makes it mix homogeneously.The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Dissolve lyophilized products with dichloromethane, PEG-8000 dissolves in dichloromethane, centrifugal after, obtain containing the micronized protein of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronized protein.With 2ml dichloromethane dissolving said composition powder, centrifugal, supernatant discarded (containing PEG6000) becomes protein powder subsequent use the albumen precipitation vacuum drying.(Mw=30000, lactic acid: hydroxyacetic acid=75: 25) 2g is dissolved in the mixed solvent of 5ml dichloromethane and 7ml acetonitrile and forms organic facies with PLGA.Protein powder is suspended in the organic facies 10000rpm high speed dispersion 40s; Form S/O two-phase disperse system, then this colostrum is poured onto in the liquid paraffin that 2L contains 0.5% sorbester p18, under 20 degrees centigrade; Mechanical agitation normal pressure solvent flashing 6 hours uses the filtering with microporous membrane of 0.8 μ m to obtain microsphere, and uses petroleum ether; Filter microsphere, drying obtains finished microballoon products.
Precision takes by weighing finished microballoon products 20mg, with 1ml dichloromethane dissolving, adds phosphate buffer (PBS) the extraction medicine of 10ml PH=7.4 again, and (test kit comes from ThermoScientific, is called BCA with the method for BCA with the PBS solution after the extraction
TMProtein Assay Kit) mensuration protein concentration wherein, wherein Endostar accounts for the 2.13%wt of microsphere total amount, and sealing productive rate is 99.39%, uses Mastersizer 2000 laser particle analyzers to record the microsphere volume mean diameter and is 81.488um.(Fig. 4).
Precision takes by weighing finished microballoon products 50mg, and parallel 3 parts, place the centrifuge tube (the pipe lid of screwing, the back stereomutation prevents to evaporate) 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.Discharge 11.25%, the 15 day on the 1st day and discharge release 89.96% in 49.88%, the 30 day.(Fig. 5)
Embodiment three:
The microsphere of getting embodiment two preparations carries out the biological activity determination of Endostar
1, the HUVEC cell is cultivated based on 37 ℃ with the ECM that adds FBS, ECGS, P/O Solution, 5%CO
2Incubator in cultivate, inoculate after treating that cell state is good and getting into exponential phase.
2, cell is used 0.25% trypsinization, and the centrifugal 5min of 1000rpm abandons supernatant, and with culture medium suspendible again, microscopically is used the blood cell counting plate living cell counting.Transferring cell density is 5000/ml, and every hole adds 160 μ l cell suspension, puts 37 ℃ of 5%CO
2Cultivate in the incubator.
The extract that obtains during 3, with Endostar stock solution and Endostar microsphere assay is diluted to 2.5mg/ml in advance with the phosphate buffer of pH7.4; By final concentration in the hole is 500,250,125,62.5,31.25,15.625,0 μ g/ml; Every hole adds 40 μ l injection volumes; Each gradient establish 3 parallel, 37 ℃ of 5%CO
2Cultivate 96h in the incubator.
4, add 5mg/ml MTT working solution, every hole 20 μ l put 37 ℃ of 5%CO
2Cultivate 4h in the incubator, discard cell conditioned medium, every hole adds DMSO 200 μ l.Place 10min, use ELIASA 490nm wavelength to measure the OD value down.
5, obtain cell inhibitory rate according to the OD value, computing formula is suppression ratio (IR)=(matched group OD value mean-experimental group OD value mean)/(a matched group OD value mean-blank OD value mean).(Fig. 6)
As can be seen from the figure, discharged the medicine in the liquid and discharged the BA that medicine in the liquid has all kept 90% above Endostar stock solution on the 30th day on the 15th of microsphere the day.
Embodiment four
Precision is measured 50ml recombinant human vascular endothelial inhibin Endostar protein solution (9.1462mg/ml; PH=5.5); Add 0.004% zinc acetate, after dissolving fully to zinc salt, in this interval of PH to 8.8-9.3 with sodium hydroxide solution (1mol/L) accent protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-8000 solution of 50% (w/v), and magnetic agitation 5min makes it mix homogeneously.The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Dissolve lyophilized products with dichloromethane, PEG-8000 dissolves in dichloromethane, centrifugal after, obtain containing the micronized protein of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronized protein.(Mw=80000, lactic acid: hydroxyacetic acid=65: 35) 2g is dissolved in the mixed solvent of 5ml dichloromethane and 6ml acetone and forms organic facies with PLGA.Protein powder is suspended in the organic facies 10000rpm high speed dispersion 3min; Form S/O two-phase disperse system, then this colostrum is poured onto in the liquid paraffin that 2L contains 0.1% sorbester p17, under 20 degrees centigrade; Mechanical agitation normal pressure solvent flashing 6 hours uses the filtering with microporous membrane of 0.8 μ m to obtain microsphere, and uses petroleum ether; Filter microsphere, drying obtains finished microballoon products.
Embodiment five
Precision is measured 50ml recombinant human vascular endothelial inhibin Endostar protein solution (102.8mg/ml; PH=6.5); Add 0.001% magnesium sulfate, after dissolving fully to magnesium salt, in this interval of PH to 8.8-9.3 with sodium hydroxide solution (1mol/L) accent protein solution.At this moment, a large amount of albumen precipitations, solution presents milky, and adding 50ml concentration is the PEG-6000 solution of 20% (w/v), and magnetic agitation 5min makes it mix homogeneously.The above-mentioned solution for preparing is divided in the cillin bottle of specification 30ml, and the loading amount specification is the 5ml/ bottle, lyophilizing.Freeze-drying time is one day.Dissolve lyophilized products with dichloromethane, PEG-6000 dissolves in dichloromethane, centrifugal after, obtain containing the micronized protein of dichloromethane in centrifuge tube bottom, the room temperature drying under reduced pressure it, obtain exsiccant micronized recombinant human vascular endothelial inhibin albumen.。(Mw=30000, lactic acid: hydroxyacetic acid=50: 50) 2g is dissolved in the mixed solvent of 5ml ethyl acetate and 6ml acetonitrile and forms organic facies with PLGA.Protein powder is suspended in the organic facies; 10000rpm high speed dispersion 10min; Form S/O two-phase disperse system, then this colostrum is poured onto in the liquid paraffin that 2L contains 0.2% sorbester p37 temperature programmed control; Rise to 40 degrees centigrade by 20 degrees centigrade in 1 hour, slowly be cooled to 10 degrees centigrade by 40 degrees centigrade in back five hours.Mechanical agitation normal pressure solvent flashing 6 hours uses the filtering with microporous membrane of 0.8 μ m to obtain microsphere, and uses petroleum ether, filters microsphere, and drying obtains finished microballoon products.
Claims (14)
1. one kind comprises pharmaceutically suitable carrier and active component is the pharmaceutical composition of human Endostatin, it is characterized in that said pharmaceutically suitable carrier is the lactic-co-glycolic acid polymer; Said active component is the human Endostatin of micronizing solid form, and its preparation comprises following steps:
(1) in containing the buffer salt solution of human Endostatin, add soluble metallic salt, the PH that adjusts this solution then obtains proteic fine particle deposition to proteic isoelectric point, IP, and protein solution becomes suspension;
(2) with behind suspension and the freeze drying protectant mix homogeneously, lyophilizing;
(3) the solid powder agglomates after the lyophilizing is used solvent wash, remove freeze drying protectant, obtain micronized protein.
2. pharmaceutical composition according to claim 1 is characterized in that the human Endostatin of micronizing solid form accounts for the 1%wt-30%wt of compositions.
3. pharmaceutical composition according to claim 2 is characterized in that the human Endostatin of micronizing solid form accounts for the 10%wt-20%wt of compositions.
4. according to each pharmaceutical composition in the claim 1 to 3, wherein human Endostatin is recombinant human vascular endothelial inhibin Endostar.
5. pharmaceutical composition according to claim 1 is characterized in that the volume ratio of suspension described in the human Endostatin method for preparing step (2) of micronizing solid form and freeze drying protectant is 1:99 to 99:1; The recombinant human vascular endothelial inhibin protein concentration is 0.01mg/ml to 500mg/ml in the suspension.
6. pharmaceutical composition according to claim 5 is characterized in that the volume ratio of suspension described in the human Endostatin method for preparing step (2) of micronizing solid form and freeze drying protectant is 15:85 to 90:10.
7. pharmaceutical composition according to claim 1; It is characterized in that freeze drying protectant is a Polyethylene Glycol described in the human Endostatin method for preparing step (2) of micronizing solid form; Molecular weight is 1000 to 8000 dalton, and the concentration of freeze drying protectant is 1% to 50% (w/v).
8. pharmaceutical composition according to claim 1 is characterized in that cleaning solvent described in the human Endostatin method for preparing step (3) of micronizing solid form is selected from a kind of in acetone, ethyl acetate, dichloromethane, the acetonitrile.
9. pharmaceutical composition according to claim 8 is characterized in that cleaning solvent described in the human Endostatin method for preparing step (3) of micronizing solid form is selected from a kind of in ethyl acetate, dichloromethane, the acetonitrile.
10. pharmaceutical composition according to claim 1, the mol ratio that it is characterized in that the lactic acid/hydroxyacetic acid of lactic-co-glycolic acid polymer is 40:60 to 99:1, polymer weight average molecular weight size is 5000 to 150000 dalton.
11. pharmaceutical composition according to claim 10, the mol ratio that it is characterized in that the lactic acid/hydroxyacetic acid of lactic-co-glycolic acid polymer is 50:50 to 85:15, and polymer weight average molecular weight size is 10000 to 80000 dalton.
12., it is characterized in that said pharmaceutical composition also comprises 0.5%wt to 30%wt protective agent according to claim 1 or 4 described pharmaceutical compositions.
13. pharmaceutical composition according to claim 12 is characterized in that protective agent is selected from one or more in sorbitol, xylitol, mannitol, trehalose, chitosan, glucosan, sucrose, lactose, glucose, poloxamer 188, Polyethylene Glycol or the polyvinylpyrrolidone.
14. pharmaceutical composition according to claim 13, wherein the Polyethylene Glycol weight average molecular weight range is 1000 to 9000 dalton; The polyvinylpyrrolidone weight average molecular weight range is 2000 to 20000 dalton.
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