CN101642559B - Pharmaceutical composition containing micronized human vascular endostatin - Google Patents

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

Pharmaceutical composition containing micronized human vascular endostatin

technical field

The invention relates to a pharmaceutical composition containing human vascular endostatin in micronized solid form, in particular to a preparation method of a human vascular endostatin pharmaceutical composition with good activity.

Background technique

Studies have found that during the growth and metastasis of solid tumors, angiogenesis is needed to provide nutrition. At this time, tumor cells will send some signals to promote the proliferation of blood vessels to tumor tissues. Some endogenous angiogenesis inhibitors such as Angiostatin and Endostatin can hinder The growth of blood vessels in tumor tissue makes tumor growth and metastasis stagnate (O'Relly, M.S., et al. Cell.79:315-328, 1994; O'Relly, M.S., et al Cell.88:277- 285, 1997), Professor Folkman of Harvard Medical School in the United States proposed the theory of "starving tumor therapy" by using endostatin (Endostatin) to inhibit tumor growth in the 1970s. However, due to the problems of easy precipitation and difficult renaturation of Endostatin in the process of expression and preparation, its large-scale application in tumor patients is limited.

Professor Luo Yongzhang and others modified the nucleotide coding sequence of human endostatin to produce recombinant human endostatin with 9 additional amino acid sequences at the N-terminus (named: Endostar, Chinese name: Endostatin) ( ZL 00107569.1), its amino acid sequence is:

(M) GGSHHHHHHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDPNGRRLTESYCETWRTEAPSATGQASSLGGRLLGQSAASCHHAYIVLCIENS when expressed by the end part of E.

The recombinant human endostatin Endostatin maintains all the biological activities of endogenous Endostatin, and at the same time solves the difficulties in the production process of Endostatin. The purification process is simple, the purity is high, and the activity is well preserved. Its injection has been marketed and sold clinically. Combined with NP chemotherapy for patients with non-small cell lung cancer.

Recombinant human endostatin, as an exogenous protein, is easily recognized by the immune system in vivo and then degraded. Therefore, the half-life in vivo is very short, and patients need frequent injections, resulting in poor clinical compliance. Biodegradable microspheres based on lactic acid-glycolic acid polymers have been used in sustained-release preparations for injections of polypeptide drugs in the 1980s, such as Lupron Depot from Takeda, Trelstar Depot from Debiopharm and Novartis from Switzerland. The company's Sandostatin Depot et al. Polypeptide drugs are slowly released into the body with the continuous biodegradation of polymer materials in the muscles or subcutaneously, maintaining the effective blood drug concentration in the body for a long time, and the release cycle ranges from one week to 6 months. The patient's compliance has been greatly improved. With the development of molecular biology, more and more macromolecular protein drugs also need to rely on this sustained release platform to overcome the defects of the drug itself.

Sustained-release microsphere preparations are generally prepared by the method of water-in-oil-in-water. Protein drugs first exist in the internal water phase, and form colostrum with organic solvents containing lactic acid-glycolic acid polymers under the action of high-speed shear or ultrasound. and then dispersed into the external aqueous phase. Proteins are affected by tension at the oil-water interface, and their structure is easily changed, resulting in the loss of their biological activity. The solid protein is not affected by the oil-water interface, and it can maintain its activity well during the preparation. Therefore, the solidification of protein is now becoming a hot spot in the development of formulations. However, the existing technology for producing solid protein cannot produce Micronized protein with a particle size of less than 3um cannot meet the needs of some new dosage form development, such as microspheres with a smaller particle size. There are many existing methods for micronizing protein, which can be generally classified into the following categories: spray drying method, freeze drying method, spray freeze drying method, and supercritical fluid technology. Spray drying is the process of using an atomizer to disperse the drug solution into fine droplets, and quickly evaporate the solvent in a hot drying medium to form a dry powder. The particle size of the micropowder produced by this technology is uniform, but this method requires the use of hot air. Suitable for drying protein and peptide drugs that are not sensitive to temperature. The spray freeze-drying method combines the atomization step in spray drying with freeze-drying. The step is to spray the atomized drug aqueous solution into liquid nitrogen, and dry it by freeze-drying steps to obtain drug powder with uniform particle size. This method has a large batch capacity, but the cost is relatively high, and the particle size of the obtained micropowder is relatively large. Supercritical fluid technology is a newly developed technology that can use the properties of supercritical fluid to extract solvents and dry proteins. However, there are still many problems that need to be studied and solved in this technology: such as the solubility of proteins and peptide drugs in organic solvents Generally small, how to increase the solubility of the drug by changing the solvent composition, and the relationship between different operating conditions and the final micropowder with different properties.

Contents of the invention

The object of the present invention is to solve the problems existing in the existing protein microsphere preparation method, and provide a preparation method of human vascular endostatin sustained-release microsphere preparation with good activity.

The microsphere preparation of human endostatin pharmaceutical composition in the present invention can be used for subcutaneous injection or intramuscular injection. The formulation is suitable for use in the sustained release of human endostatin.

The invention discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and human endostatin, which is characterized in that the active ingredient human endostatin is in the form of micronized solid.

The above-mentioned human vascular endostatin in micronized solid form can be prepared by the following steps:

(1) Add soluble metal salts to the buffered saline solution containing human endostatin, and then adjust the pH of the solution to the isoelectric point (pI) of the protein to obtain fine particle precipitation of the protein, and the protein solution becomes a suspension ;

(2) After mixing the suspension and the freeze-drying protective agent uniformly, freeze-dry;

(3) washing the lyophilized solid powder block with a solvent to remove the lyoprotectant to obtain micronized protein.

The lyoprotectant used in the preparation method of micronized human vascular endostatin is polyethylene glycol, which plays a role in shaping and stabilizing the space during lyophilization, and the precipitated human vascular endostatin protein particles can be adsorbed on the polymer on the backbone of ethylene glycol.

The preparation method of the above-mentioned micronized human endostatin has a polyethylene glycol molecular weight ranging from 1000 to 8000 Daltons. Polyethylene glycol has good suspension stability, and the molecular weight ranges from 1000 to 8000 Daltons, which can stabilize protein particles well and keep them from aggregating and sinking during lyophilization.

In the preparation method of the above micronized human endostatin, the volume ratio of the suspension to the lyoprotectant in step (2) is 1:99 to 99:1, preferably 15:85 to 90:10; in the suspension The concentration of the human endostatin protein is 0.01 mg/ml-500 mg/ml, and the concentration of the lyoprotectant is 1%-50% (w/v). The concentration of the lyoprotectant should not be less than 1%, otherwise the suspension stability will be poor, and the protein precipitate will sink and aggregate quickly; the concentration of the lyoprotectant is too high, because it is necessary to use The protective agent is removed by washing with an organic solvent. If the proportion of the protective agent is too high, a large amount of organic solvent will be consumed and repeated cleaning will bring inconvenience to the operation. It is more appropriate to keep the concentration of 1%-50% (w/v).

In the above preparation method of micronized protein, the washing solvent in step (3) is a solvent that can dissolve polyethylene glycol, for example: acetone, ethyl acetate, dichloromethane, acetonitrile. Preference is given to ethyl acetate, dichloromethane, acetonitrile.

The pharmaceutically acceptable carrier of the pharmaceutical composition of the present invention is a lactic acid-glycolic acid polymer. The pharmaceutically acceptable carrier lactic acid-glycolic acid polymer is formed by polymerization of glycolic acid and lactic acid; the polymer can be a copolymer or homopolymer of lactic acid and glycolic acid, and can also refer to a single polylactic acid; the polymerization can be random , block or graft type, they can have D-, L- or DL optical configuration.

When the polymer used is a copolymer or homopolymer of lactic acid-glycolic acid, the mol ratio of lactic acid/glycolic acid is 40:60 to 99:1, preferably 50:50 to 85:15; the weight average molecular weight of the polymer is 5,000 to 150,000 Daltons, preferably 10,000 to 80,000 Daltons. The size of the molecular weight determines the degradation rate of the polymer in the body, which in turn determines the release period of the drug. The release period can be selected from one week to two months. The polymer weight average molecular weight should be determined according to gel dialysis chromatography (GPC).

The human endostatin in the pharmaceutical composition of the present invention is preferably recombinant human endostatin Endostar.

The pharmaceutical composition is a solid pharmaceutical composition, and meaningful solid pharmaceutical compositions are: microspheres and implants.

Preferred solid pharmaceutical compositions within the scope of the present invention are microspheres. Because microspheres are usually administered by injection, it is convenient and does not need to be taken out after injection. The implants need to be implanted and taken out by minor surgery, which causes pain to the patient.

In the pharmaceutical composition of the present invention, human endostatin accounts for 1%wt to 30%wt of the composition, preferably 10%wt to 20%wt. The volume average particle size of the microspheres is between 0.1um and 300um.

In the composition, some protective agents such as sugars, polyols or polymers can also be added to support the skeleton, and play a role in shaping and stabilizing the space. Among them, polyols can also effectively prevent protein aggregation and reduce inactive production of polymers. The protectant comprises from 0.5%wt to 30%wt of the composition.

The protective agent of the pharmaceutical composition of the present invention includes polyalcohol (sorbitol, xylitol, mannitol), carbohydrate (trehalose, chitosan, dextran, sucrose, lactose, glucose), macromolecular polymer One or more of the substances (poloxamer 188, polyethylene glycol, polyvinylpyrrolidone).

The polyethylene glycol weight average molecular weight of the pharmaceutical composition of the invention ranges from 1000 to 8000 daltons; the polyvinylpyrrolidone weight average molecular weight ranges from 2000 to 20000 daltons.

The pharmaceutical composition prepared by the invention is spherical in appearance, porous or non-porous on the surface, and has a honeycomb structure inside.

The pharmaceutical composition prepared by the invention can inhibit the formation of new blood vessels of malignant tumors in mammals, and is used for treating various cancers.

Advantage of the present invention has:

The human endostatin slow-release microsphere preparation prepared by the method of the present invention can slowly release recombinant human endostatin in the body for a long time while maintaining its activity, greatly reducing the number of administrations of patients and improving the compliance of patients . The drug release rate can be easily adjusted by selecting the molecular weight of different carrier polymer materials.

2. Freeze-dry the human endostatin solution and the protective agent together to form a micronized human endostatin solid. Human endostatin, a protein drug, is protected by a skeleton support material (protective agent) to maintain a protein hierarchical structure integrity. The protein in solid form is prepared into microspheres, which eliminates the problem that the protein activity may be lost due to the oil-water interfacial tension.

Third, the protein is prepared into a solid state, which improves the stability of the protein prepared into microspheres.

Description of drawings

The particle size distribution figure of microsphere in Fig. 1 embodiment one

The SEM scanning electron micrograph of microsphere in Fig. 2, Fig. 3 embodiment one

The particle size distribution figure of microsphere in Fig. 4 embodiment two

Figure 5 is the in vitro release behavior curve of Endostar microspheres prepared with PLGA (Mw=30000, lactic acid:glycolic acid=75:25) in Example 2.

Fig. 6 The proliferative activity of HUVEC cells before and after making Endostar into microspheres in Example 3.

Detailed ways

The present invention is described in more detail through the following examples, but they cannot be construed as limiting the protection scope of the present invention.

Embodiment one

Precisely measure 50ml of recombinant human endostatin Endostar protein solution (9.1462mg/ml, PH=5.5), add 0.004% zinc acetate, until the zinc salt is completely dissolved, regulate the protein with sodium hydroxide solution (1mol/L) The pH of the solution is in the range of 8.8-9.3. At this point, a large amount of protein precipitated, and the solution was milky white. Add 50ml of PEG-8000 solution with a concentration of 50% (w/v), and stir magnetically for 5 minutes to make it evenly mixed. The above-prepared solution was divided into 30ml vials, the filling specification was 5ml/bottle, and freeze-dried. Dissolve the freeze-dried product with dichloromethane, dissolve PEG-8000 in dichloromethane, and after centrifugation, obtain micronized protein containing dichloromethane at the bottom of the centrifuge tube, dry it under reduced pressure at room temperature, and obtain dry micronized protein. 2 g of PLGA (Mw=50000, lactic acid:glycolic acid=50:50) was dissolved in a mixed solvent of 3 ml ethyl acetate and 7 ml acetone to form an organic phase. Suspend the powder of the above composition in the organic phase, disperse at a high speed of 10000rpm for 20s to form an S/O two-phase dispersion system, then pour the colostrum into 1L of soybean oil containing 0.2% soybean lecithin, at 20 degrees Celsius, mechanically Stir under normal pressure to evaporate the solvent for 6 hours, filter through a 0.8 μm microporous membrane to obtain microspheres, wash with petroleum ether, filter the microspheres, and dry to obtain finished microspheres.

Accurately weigh 20 mg of the finished microsphere, dissolve it in 1 ml of dichloromethane, then add 10 ml of phosphate buffered saline (PBS) with pH=7.4 to extract the drug, and use the BCA method for the extracted PBS solution (the kit is from ThermoScientific, named BCA ^TM Protein Assay Kit) was used to determine the protein concentration, wherein Endostar accounted for 1.27%wt of the total amount of microspheres, and the encapsulation yield was 99.21%. The volume average particle size of the microspheres measured by Mastersizer 2000 laser particle size analyzer was 70.461um . (figure 1).

Observing the morphology of the obtained microspheres in a scanning electron microscope (scanning electronic microscopy), it can be seen that the particle size of the microspheres is uniform, the appearance of the microspheres is round, and the surface is porous. After the microspheres were wrapped in solid paraffin, the microspheres were sectioned and observed under a scanning electron microscope, which showed that the internal structure was loose. (Figure 2, Figure 3)

Embodiment two

Accurately measure 50ml of recombinant human endostatin protein Endostar solution (9.1462mg/ml, PH=5.5), add 0.004% zinc acetate, until the zinc salt is completely dissolved, regulate the protein with sodium hydroxide solution (1mol/L) The pH of the solution is in the range of 8.8-9.3. At this point, a large amount of protein precipitated, and the solution was milky white. Add 50ml of PEG-8000 solution with a concentration of 50% (w/v), and stir magnetically for 5 minutes to make it evenly mixed. The above-prepared solution was divided into 30ml vials, the filling specification was 5ml/bottle, and freeze-dried. Dissolve the lyophilized product with dichloromethane, and dissolve PEG-8000 in dichloromethane. After centrifugation, micronized protein containing dichloromethane is obtained at the bottom of the centrifuge tube, and dried under reduced pressure at room temperature to obtain dry micronized protein. The composition powder was dissolved with 2 ml of dichloromethane, centrifuged, the supernatant (containing PEG6000) was discarded, and the protein precipitate was vacuum-dried into protein powder for later use. 2 g of PLGA (Mw=30000, lactic acid:glycolic acid=75:25) was dissolved in a mixed solvent of 5 ml of dichloromethane and 7 ml of acetonitrile to form an organic phase. Suspend the protein powder in the organic phase, disperse at a high speed of 10000rpm for 40s, to form an S/O two-phase dispersion system, then pour the colostrum into 2L of liquid paraffin containing 0.5% Span 60, and mechanically stir at 20 degrees Celsius The solvent was evaporated under normal pressure for 6 hours, and the microspheres were obtained by filtering with a 0.8 μm microporous membrane, washed with petroleum ether, filtered, and dried to obtain the finished microspheres.

Accurately weigh 20 mg of the finished microsphere, dissolve it in 1 ml of dichloromethane, then add 10 ml of phosphate buffered saline (PBS) with pH=7.4 to extract the drug, and use the BCA method for the extracted PBS solution (the kit is from ThermoScientific, named BCA ^TM Protein Assay Kit) was used to determine the protein concentration, wherein Endostar accounted for 2.13%wt of the total amount of microspheres, and the encapsulation yield was 99.39%. The volume average particle size of the microspheres measured by Mastersizer 2000 laser particle size analyzer was 81.488um . (Figure 4).

Precisely weigh 50mg of the finished microspheres, put them in 3 parallel portions, put them in a centrifuge tube containing 10ml of phosphate buffer (0.1M pH 7.0) (tighten the cap of the tube to prevent the volume from changing after the liquid evaporates), put it in a constant temperature water bath at 37 degrees Shake. Take 1ml of the supernatant in the test tube at each time point, and supplement the same volume of fresh phosphate buffer at the same time. The content of Endostar in the samples was determined by BCA method. The cumulative release percentage of the drug was plotted against the release time. Released 11.25% on day 1, 49.88% on day 15, and 89.96% on day 30. (Figure 5)

Embodiment three:

Get the microsphere prepared by embodiment two to carry out the biological activity assay of Endostar

1. HUVEC cells were cultured with ECM supplemented with FBS, ECGS, and P/O Solution in an incubator at 37°C and 5% CO ₂ , and inoculated after the cells were in good condition and entered the logarithmic growth phase.

2. Digest the cells with 0.25% trypsin, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend with the medium, and count live cells with a hemocytometer under a microscope. Adjust the cell density to 5000 cells/ml, add 160 μl of cell suspension to each well, and culture in a 5% CO ₂ incubator at 37°C.

3. Pre-dilute the endostar stock solution and the extract obtained during the determination of the content of Endostar microspheres with pH 7.4 phosphate buffer to 2.5mg/ml, and the final concentration in the well is 500, 250, 125, 62.5, 31.25, 15.625 , 0 μg/ml, add 40 μl drug volume to each well, set 3 parallels for each gradient, and culture in 37° C. 5% CO ₂ incubator for 96 hours.

4. Add 5 mg/ml MTT working solution, 20 μl per well, culture in a 5% CO ₂ incubator at 37°C for 4 hours, discard the cell supernatant, and add 200 μl of DMSO to each well. Leave it for 10 min, and measure the OD value with a microplate reader at a wavelength of 490 nm.

5. Calculate the cell inhibition rate according to the OD value, and the calculation formula is inhibition rate (IR)=(the average OD value of the control group-the average OD value of the experimental group)/(the average OD value of the control group-the average OD value of the blank) . (Figure 6)

It can be seen from the figure that the drug in the release liquid of the microspheres on the 15th day and the drug in the release liquid on the 30th day both maintained more than 90% of the biological activity of the Endostar stock solution.

Embodiment Four

Precisely measure 50ml of recombinant human endostatin Endostar protein solution (9.1462mg/ml, PH=5.5), add 0.004% zinc acetate, until the zinc salt is completely dissolved, regulate the protein with sodium hydroxide solution (1mol/L) The pH of the solution is in the range of 8.8-9.3. At this point, a large amount of protein precipitated, and the solution was milky white. Add 50ml of PEG-8000 solution with a concentration of 50% (w/v), and stir magnetically for 5 minutes to make it evenly mixed. The above-prepared solution was divided into 30ml vials, the filling specification was 5ml/bottle, and freeze-dried. Dissolve the lyophilized product with dichloromethane, and dissolve PEG-8000 in dichloromethane. After centrifugation, micronized protein containing dichloromethane is obtained at the bottom of the centrifuge tube, and dried under reduced pressure at room temperature to obtain dry micronized protein. 2 g of PLGA (Mw=80000, lactic acid:glycolic acid=65:35) was dissolved in a mixed solvent of 5 ml of dichloromethane and 6 ml of acetone to form an organic phase. Suspend the protein powder in the organic phase, disperse at a high speed of 10,000rpm for 3min, to form an S/O two-phase dispersion system, then pour the colostrum into 2L of liquid paraffin containing 0.1% Span 80, and stir mechanically at 20°C The solvent was evaporated under normal pressure for 6 hours, and the microspheres were obtained by filtering with a 0.8 μm microporous membrane, washed with petroleum ether, filtered, and dried to obtain the finished microspheres.

Embodiment five

Accurately measure 50ml of recombinant human endostatin Endostar protein solution (102.8mg/ml, PH=6.5), add 0.001% magnesium sulfate, and after the magnesium salt is completely dissolved, use sodium hydroxide solution (1mol/L) to regulate protein The pH of the solution is in the range of 8.8-9.3. At this point, a large amount of protein precipitated, and the solution was milky white. Add 50 ml of PEG-6000 solution with a concentration of 20% (w/v), and stir magnetically for 5 minutes to make it evenly mixed. The above-prepared solution was divided into 30ml vials, the filling specification was 5ml/bottle, and freeze-dried. Freeze-drying time is one day. Dissolve the freeze-dried product with dichloromethane, dissolve PEG-6000 in dichloromethane, and after centrifugation, obtain micronized protein containing dichloromethane at the bottom of the centrifuge tube, dry it under reduced pressure at room temperature, and obtain dry micronized recombinant human blood vessels endostatin protein. . Dissolve 2 g of PLGA (Mw=30000, lactic acid:glycolic acid=50:50) in a mixed solvent of 5 ml ethyl acetate and 6 ml acetonitrile to form an organic phase. Suspend the protein powder in the organic phase and disperse at a high speed of 10,000rpm for 10 minutes to form an S/O two-phase dispersion system, then pour the colostrum into 2L of liquid paraffin containing 0.2% Span 85, and control the temperature within 1 hour Raise from 20 degrees Celsius to 40 degrees Celsius, and then slowly cool down from 40 degrees Celsius to 10 degrees Celsius in the next five hours. The solvent was evaporated under normal pressure by mechanical stirring for 6 hours, and the microspheres were obtained by filtering with a 0.8 μm microporous membrane, washed with petroleum ether, filtered, and dried to obtain the finished microspheres.

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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