HU198627B - Process for producing stable pharmaceutical composition comprising granulocyte colony stimulating factor - Google Patents

Description

The present invention relates to a pharmaceutical composition comprising granulocyte colony stimulating factor. More particularly, the present invention relates to the preparation of a stabilized pharmaceutical composition comprising the granulocyte colony stimulating factor, wherein the active component (i.e., the granulocyte colony stimulating factor) is protected against loss or inactivation by adsorption of the composition to the container wall; polymerization or oxidation.

Chemotherapy is used to treat a variety of infectious diseases, but nowadays it is often seen that chemotherapy can cause serious clinical problems such as the development of drug-resistant organisms, the exchange of disease-causing organisms, and serious side effects. To eliminate the problems associated with chemotherapy, including those involving the use of pharmaceutical agents such as antibiotics and bactericides, they are experimenting with substances that activate the host's preventive ability against an infectious organism to provide the best possible solution to the above-mentioned problems of chemotherapy provide. Of the various host preventive capabilities, leukocytes consider phagocytic bactericidal activity to be the most potent influence on the initial period of bacterial infection and therefore attach great importance to enhancing the host's defense against infection by promoting their growth and differentiation to neutrophils. Granulocyte colony stimulating factor (G-CSF) is one of the very useful substances that exhibit such activity; Applicants of the present invention have previously filed a patent application for an anti-infective agent using G-CSF (Japanese Patent Application Serial No. 23,777/1985).

As mentioned above, chemotherapy carries a variety of unavoidable problems and intense efforts are being made to employ therapeutic agents capable of activating the preventive functions of the host or the infected person.

Needless to emphasize, G-CSF alone demonstrates the ability to activate host preventive functions and has also been found to have a greater therapeutic effect in clinical use when used in combination with another agent that activates the host. preventive abilities.

G-CSF is used in very small amounts, so generally 0.1 to 500 µg (preferably 5-50 µg) of G-CSF is administered 1 to 7 times per week in adult doses.

However, G-CSF tends to adsorb its container, e.g. on the wall of the injection ampoule or syringe. Therefore, when used as an aqueous solution for injection, the drug is administered in a container, i. adsorbed on the wall of the ampoule or syringe. Thus, G-CSF loses its ability to exhibit its full activity as a pharmaceutical agent, or requires the administration of a greater than desired amount of G-CSF, thus compensating for any loss due to adsorption.

In addition, G-CSF is unstable and highly sensitive to environmental factors such as temperature, humidity, oxygen and ultraviolet rays. As a result of these factors, G-CSF undergoes physical and chemical changes such as association, polymerization, and oxidation, and thus suffers a large loss of activity. These phenomena make it difficult to fully achieve therapeutic activity by administering very small amounts of G-CSF in a very controlled manner.

It is therefore necessary to develop a stable pharmaceutical formulation of G-CSF which is fully protected against a decrease in the activity of the active substance. It is a primary object of the present invention that the present invention provides a stable pharmaceutical formulation of G-CSF.

The present inventors have been conducting intensive studies to increase the stability of G-CSF-containing pharmaceutical formulations and have discovered that this task can be effectively accomplished using pharmaceutically acceptable surfactants, saccharides, proteins and / or high molecular weight compounds. .

Accordingly, the pharmaceutical composition of the present invention containing stable G-CSF is characterized in that it comprises, in addition to G-CSF, at least one of pharmaceutically acceptable surfactants, saccharides, proteins, and high molecular weight compounds.

G-CSF, which is contained in the pharmaceutical compositions of the present invention, can be prepared by any of the methods described in, for example, 153 273/1984, 269 455/1985,

259456/1985, 270,838/1985, and 270,839/1985, e.g. human G-CSF can be made either by culturing a cell line (CNCM 1-315 or 1-483) isolated from a tumor in patients with oral cancer, or by recombinant DNA (a gene encoding human G-CSF). expression) in a suitable host cell (e.g., F. coli, C 127 cells, or Chinese hamster ovary cells).

Any human G-CSF that has been highly purified may be used as G-CSF in the therapeutic application of the present invention.

EN 198627 Β in preparations. Preferred human G-CSFs are those isolated from the supernatant of a culture of a human G-CSF producing strain and polypeptides or glycoprolines having human G-CSF activity and obtained from a host transformed with a recombinant vector which is a human G- CSF. Contains a gene encoding a polypeptide having CSF activity.

Examples of two particularly preferred G-CSFs are given below.

(1) Human G-CSF having the following physicochemical properties:

i.) molecular weight: approx

000 ± 1000, sodium dodecyl sulphate polyacrylamide gel elec trophoresis gels:

ii. ) isoelectric point: has at least one of the following three isoelectric points: pl = 5.5 ± 0.1, pl = 5.8 ± 0.1, and pl = 6.1 ± 0.1;

iii. ) ultraviolet adsorption: has a maximum

280 nm and a minimum of 250 nm;

arc. The 21 residues have the amino acid sequence

Starting from the N-terminal:

HzN-Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln-Val (2) Human G-CSF comprising either a polypeptide having human granulocil stimulating factor activity, which polypeptide is characterized in all or part of the amino acid sequence shown below, or comprising a glycoprotein having the aforementioned polypeptide structure and a sugar moiety:

(Met) n Thr Pro Leu Gly Pro below Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu Glu Gin With Arg Lys Ile Gin Gly asp Gly below below Leu Gin Glu Lys Leu (With Ser Glu). Cys below Thr Tyr Lys Leu Cys His Pro Glu Glu Leu With Leu Leu Gly His Ser Leu Gly Ile Pro Trp below Pro Leu Ser Ser Cys Pro Ser Gin below Leu Gin Leu below Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin below Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu asp Thr Leu Gin Leu asp With below asp Phe below Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met below Pro below Leu Gin Pro Thr Gin Gly below Met Pro below Phe below Ser below Phe Gin Arg Arg below Gly Gly With Leu With below Ser His Leu Gin Ser Phe Leu Glu With Ser Tyr Arg With Leu Arg His Leu below Gin Pro -

(provided that m is 0 or 1; and n is 0 or 1).

For details on methods for making these two types of G-CSF, see Japanese Patent Application Nos. 153,273/1984, 269,455/1985, 269,456 / 1985, 270,838/1985, and 270,839/1985, each of which is hereby incorporated by reference. submitted.

Another method that may be employed involves fusing G-CSF producing cells with self-proliferating malignant tumor cells and culturing the resulting hybridoma in the presence or absence of mitogen.

The resulting solution of human G-CSF can be stored frozen, if necessary, after further purification and concentration by any known technique. Alternatively, the solution may be administered e.g. may be stored after freeze-drying dehydration.

Each of the human G-CSFs thus prepared can be processed according to the description of the present invention to provide a stable pharmaceutical composition containing G-CSF.

Typical surfactants that may be used to formulate the stable G-CSF pharmaceutical formulation of the present invention include, but are not limited to, nonionic surfactants with 6-18 HLBs, such as aliphatic carboxylic acid esters of sorbitan (e.g. esters of sorbitan monocaprilat, sorbitan monolaurate and sorbitan monopalmitate), glycine (jf), aliphatic carboxylic acid esters (e.g. glycerol)

HU 198627 Β monocaprylate, glycerol monomyristate, and glycerol monostearate), aliphatic carboxylic acid esters of polyglycerol (e.g., decaglycerylrone monostearate, decaglyceryl distearate and decaglyceryl monolinoleate), polyoxyethylene sorbitan polyoxylate, aliphatic carboxylate. esters of aliphatic carboxylic acid ester of polyoxyethylene sorbitan mono-stearate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyethylene sorbitan monopalnite, polyoxyethylene sorbitan thyroleate and polyoxyethylene sorbitan tristearate, aliphatic carboxylic acid esters of glycerol (e.g., polyoxyethylene glyceryl monostearate), esters of polyethylene glycol aliphatic carboxylic acid (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene, ethers (e.g., polyoxyethylene-polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, and polyoxyethylene-polyoxypropylene-cetyl ether); polyoxyethylated, hydrogenated snowflake) and natural surfactants such as. lecithin. These surfactants can, of course, be used alone or in admixture.

The above-mentioned surfactants are preferably used in an amount of from 1 to 10,000 parts by weight / G-CSF.

Useful saccharides in the preparation of pharmaceutical compositions containing stable G-CSF include monosaccharides, oligosaccharides and polysaccharides, and their phosphate esters and nucleotide derivatives, as long as they are pharmaceutically acceptable. Typical examples are listed below: straight-chain and higher sugar alcohols, e.g. glycerol, erythritol, arabic, xylitol, sorbitol and mannitol; acid sugars such as sugar. glucuronic acid, iduronic acid, neuraminic acid, galacturonic acid, gluconic acid, mannuronic acid, ketoglycolic acid, ketogalactonic acid and ketogulonic acid; hiarulonic acid and its salts; chondroitin sulfate and its derivatives; chitosan and its derivatives; dextrin; dextrene with an average molecular weight of 5,000-15,000; and alginic acid and its salts. All of these saccharides can be used alone or in a mixture.

The saccharides listed above are preferably used in an amount of 1-10,000 parts by weight per liter of G-CSF.

Typical examples of useful proteins for the preparation of stable G-CSF-containing pharmaceutical compositions of the present invention include: human serum albumin, human serum globulin, gelatin, acid-treated gelatin (average molecular weight: 7,000-100,000), alkali-treated gelatin ( average molecular weight: 7,000-100,000) and collagen. Needless to say, these proteins can also be used in materials or mixtures.

The above-listed proteins are preferably used in an amount of from 1 to 20,000 parts by weight per liter of G-CSF.

Typical examples of high molecular weight materials that can be used in the preparation of stable G-CSF-containing pharmaceutical compositions of the present invention include: natural polymers, e.g. hydroxypropyl cellulose, hydroxymethyl cellulose; sodium carboxymethylcellulose, and hydroxyethylcellulose; and synthetic polymers, e.g. polyethylene glycol (molecular weight: 300-600), polyvinyl alcohol (molecular weight: 20,000-100,000), and polyvinylpyrrolidone (molecular weight: 20,000-100,000). Needless to say, these high molecular weight materials can be used alone or in combination.

The high molecular weight materials listed above are desirably used in an amount of from 1 to 20,000 parts by weight of G-CSF.

In addition to the surfactants, saccharides, proteins, or high molecular weight materials described above, at least one additional substance may be used in the preparation of the G-CSF-containing pharmaceutical compositions of the present invention: amino acids, sulfur reducing agents, and antioxidants. Examples of amino acids include glycine, threonine, tryptophan, lysine, hydroxylysine, histidine, arginine, cysteine, cystine and methionine. Sulfur reducing agents include, for example, N-acetyl cysteine, N-acetyl homocysteine, thioctanoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, sodium bisulfite, sodium sulfite, such as thio-lactic acid, dithiothreifc, glutathione, and a mild sulfur-containing reducing agent having a mercapto moiety, e.g. a C 1-7 thioalkanoic acid. Antioxidants include, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, dl-oC-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, triamyl gallate, , and chelating agents such as. disodium ethylene diamine tetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.

The amino acids, sulfur-reducing agents, and antioxidants or mixtures thereof listed above are used in an amount of from 1 to 10,000 parts by weight per liter of G-CSF.

One or more of the following agents may be used to formulate a stable G-CSF-containing composition of the present invention in a suitable dosage form: diluents, solubilizing auxiliaries, isotonic agents, binders, pH modifiers, tranquilizers, and buffers.

The stabilized G-CSF-containing pharmaceutical compositions of the present invention may be formulated for oral or parenteral administration (e.g., by injection via multiple routes) and a wide variety of dosage forms may be used depending upon the particular route of administration. Typical dosage forms include:

EN 198627 Β intended for oral administration in the form of tablets, pills, capsules, granules and suspensions; solutions, suspensions and freeze-dried formulations, in particular intended for intravenous injection, subcutaneous injection and intracutaneous injection, and for administration through the mucous membrane, such as suppositories, nasal medications, and suppositories.

According to the present invention, at least one substance is added to the pharmaceutical composition containing G-CSF, such as surfactants, saccharides, proteins, or high molecular weight substances, thereby preventing the composition from being adsorbed to the wall of the container or the wall of the syringe. the formulation should be stable for a prolonged period of time.

The detailed mechanism by which the above-mentioned substances stabilize G-CSF or prevent it from adsorbing is not yet clear. The surface of G-CSF, which is a hydrophobic protein, is coated and solubilized by surfactants, thus preventing even trace amounts of G-CSF from being adsorbed to the surface of its container or syringe. The saccharides or hydrophilic high molecular weight materials can form a hydrated layer between the G-CSF and the adsorptive surface of the container or syringe wall, thus preventing the adsorption of G-CSF. Proteins may compete with G-CSF for adsorption of the container or syringe wall, thereby inhibiting G-CSF adsorption.

In addition to preventing G-CSF adsorption, the above-mentioned materials may also be involved in preventing G-CSF molecules from bonding or polymerizing. In the presence of surfactants, saccharides, proteins, or high molecular weight compounds, each of the G-CSF molecules is dispersed in these materials and the interaction between the G-CSF molecules is reduced accordingly, thus reducing the likelihood of their bonding or polymerization. In addition, these materials may inhibit G-CSF autooxidation, which is accelerated at high temperatures and humidity, and prevent G-CSF from bonding or polymerizing as a result of autooxidation. The inhibition of G-CSF autooxidation, coupling and polymerization can be further enhanced by the addition of an amino acid, a sulfur-containing reducing agent, or an autooxidant.

The problems described above occur mainly in solutions and suspensions for injection, but also occur in procedures for the formulation of G-CSF, when other dosage forms, e.g. pills. Addition of surfactants, saccharides, proteins or high molecular weight substances is also effective in the latter case.

The addition of at least one of surfactants, saccharides, proteins, and high molecular weight substances stabilizes G-CSF to a great extent and maintains its activity for an extended period, as shown in the following examples. To achieve these results, the amount of each of these materials, especially at the lower end, is critical, and the following ranges are desirable: 1-10,000 parts by weight of surfactant, ΙΙΟ000 parts by weight of saccharide, 1-20,000 parts by weight of protein and 1-20,000 parts by weight of high molecular weight material. , 1 part by weight based on GCSF.

According to the present invention, surfactants, saccharides, proteins and / or high molecular weight substances are used in a specific concentration and are effective not only to adsorb to the wall of the G-CSF container or syringe, but also to prevent the G-CSF from being adsorbed to the wall. of a pharmaceutical composition containing. As a result, it is possible to provide a small but very accurate dose of G-CSF; since G-CSF is a costly substance, its effective use results in lower costs for the preparation of pharmaceutical compositions containing G-CSF.

The following examples are provided to illustrate the present invention in more detail, but are not intended to limit the scope of the invention. The residual activity of G-CSF in these specimens is determined by one of the following methods.

(a) Soft agar method using mouse bone marrow cells

Equine serum (0.4 ml), 0.1 ml sample, 0.1 ml C3H / He (female) mouse bone marrow cell suspension (0.5-1.10 ^5- nuclear cells), and 0.4 ml modified McCoy-5A culture medium. A solution containing 0.75% agar was mixed, poured into a tissue culture plastic dish (35 mmef), coagulated and cultured for 5 days at 37 ° C in 5% CO2 / 95% air at 100% humidity. The number of colonies formed is counted (one colony contains at least 50 cells) and the activity is determined by counting the activity that forms a colony as a unit.

The modified McCoy 5A culture solution used in Method (a) is prepared by the following procedure.

HU 198627 Β

Modified McCoy 5A culture solution (double concentration) g McCoy 5A culture solution (Gibco), 2.55 g MEM amino acid nutrient medium (Nissui Seiyaku Co., Ltd.), 2.18 g sodium hydrogen carbonate, and 50,000 units of potassium G-penicillin are dissolved in 500 ml of double-distilled water and the solution is aseptically filtered through a Millipore filter (0.22 m).

(b) Reverse Phase, High Performance Liquid Chromatography

Using a reversed phase C8 column (4.6 mm x 300 mm; 5 m) and a mobile phase mixture of n-propanol / trifluoroacetic acid, the residual activity of G-CSF (injected in 1 µg equivalent) was as follows: is defined as:

Time (Sec) (A) Solvent (B) Solvent Gradient 0 100% 0% 1 linear 15 0% 100% ³ linear 25 100% 0% J linear

Solvent A: 30% n-propanol and 0.1% trifluoroacetic acid Solvent B: 60% n-propanol and 0.1% trifluoroacetic acid

Detection is performed at 210 nm and the percentage of residual G-CSF activity is calculated using the formula:

the remaining amount of G-CSF has elapsed over time

After the rest

G-CSF activity% = - x 100 is the initial amount of G-CSF

The residual amount of G-CSF determined by this method is very consistent with the results obtained with soft bone agar (a) using mouse bone marrow cells.

Example 1

One of the stabilizers listed in Table 1 is added to Mg G-CSF and the mixture is aseptically dissolved in 20 mM buffer (containing 100 mM sodium chloride, pH 7.4) to 5 mg G-CSF / ml. containing a pharmaceutical composition which is then freeze-dried. The change in G-CSF activity over time is measured by method (a) and the results are shown in Table 1. The activity (%) in the table represents the residual activity of G-CSF relative to the starting unit and is defined by the following formula:

activity unit after a period of time

Activity (%) = -χ 100 baseline activity10 units

A solution containing G-CSF containing a stabilizer is placed in a sterile, sulfated glass vial, frozen at -40 ° C or lower for 4 hours, and in the first stage of drying at -40 ° C for 48 hours. while raising the pressure from 4 Pa to 13 Pa, the second step raises the temperature from 0 ° C to 20 ° C in 12 hours while increasing the pressure from 13 Pa to 35 Pa then fill the inside of the vial with sterile dry nitrogen gas to bring it to atmospheric pressure and stopper the vial with a freeze-dried rubber stopper and then seal with an aluminum cap.

TABLE 1

stabilization Quantity Activity (%) zélószer (Phr) 4 ° C snow 37 ° C snow moderation moderation in existence 6 in existence 1 months months through the end end supported lake Rolas Rolas after after xylitol 10,000 92 86 mannitol 10,000 91 85 glucuronic acid 10,000 86 82 hyaluronic acid 2000 92 89 dextran (molecule- weight: 40,000 2000 95 90 heparin 5000 85 80 chitosan 2000 93 91 alginic acid 2000 90 90 human rum albumin 1000 98 99

-611

HU 198627 Β

Stahilisation- Volume Activity (%) Detergent -—

4 ° C heat 37 ° C heat moderation moderation leten 6 leten 1 months months over, over through the end supported zell Rolas Rolas after Illán

human serum globulin 1000 98 95 acid-treated gelatin 2000 97 95 alkali-treated gelatin 1000 99 96 collagen 2000 95 90 polyethylene glycol (molecular weight: 40,000) 10,000 94 90 hydroxypropylmethylcellulose phthalate, hydroxypropyl cellulose 1000 98 94 Sodium arm- boximetil- -cellulose 1000 88 80 hydroxy-me- methyl cellulose 5000 92 90 polyvinyl alcohol (molecular weight: 50,000) 2000 96 95 polyvinylpyrrolidone (molecular weight: 50,000) 2000 95 94 human serum albumin 2000 mannitol 2000 100 97 cysteine 100

human serum albumin hydroxypropylcellulose dextren (molecular weight: 40,000) 2000 500 2000 98 92 polyoxyethylene ethylene -szorbi- sorbitan mono- laureate 100 98 96 sorbitol 2000 polyoxylated, hardened snow fat dextren 100 (molecules No end caps: 40,000) additive 2000 94 92 without - 74 58

Example 2 To a µg G-CSF is added one of the stabilizers listed in Table 2 and the mixture is aseptically dissolved in 20 mM phosphate buffer (containing 100 mM sodium chloride, pH 7.4) to give pg of the pharmaceutical composition containing G-CSF / inl. The change in G-CSF activity over time was measured by the same method as that used in Example 1; the results are shown in Table 2.

human serum albumin 2000 polyoxyethylene sorbitan monolaurate 100 99 96 mannitol 2000

13 HU 198627 B 14 Second SPREADSHEET stabilizer Quantity Activity (%, (Phr) Storage at 4 ° C for 7 days Storage at 4 ° C for 2 months Storage at room temperature for 1 month mannitol 5000 91 87 82 hyaluronic acid 2000 93 87 70 dextran (molecular weight: 40,000) 2000 96 95 85 glycerol 10,000 90 90 88 neuraminic 5000 93 91 84 chitin 2000 95 92 86 dextrin 2000 90 92 87 human serum albumin 1000 99 95 92 human serum globulin 1000 98 94 90 acid-treated gelatin 2000 97 96 87 alkali-treated gelatin 500 99 95 92 collagen 2000 99 94 88 polyethylene glycol (molecular weight: 40,000) 10,000 94 89 90 hydroxypropyl cellulose 2000 98 95 92 Sodium carboxymethyl -cellulose 2000 92 91 80 hydroxyethyl cellulose 4000 92 94 90 polyvinyl alcohol (molecular weight: 40,000) 4000 97 93 90 polyvinylpyrrolidone (molecular weight: 40,000) 4000 95 95 92 sorbitan monolaurate 400 97 96 95 polyoxyethylene szorbi- sorbitan monolaurate 400 100 96 94 polyoxyethylene sorbitan monostearate 400 98 97 94 polyoxypropylene glycol ether 400 100 94 93

-815

HU 198627 Β

Sfabilizálószer Quantity (Phr) Activity (%) Storage at 4 ° C for 7 days Storage at 4 ° C for 2 months Storage at room temperature for 1 month polyoxylated, hard- - made snowman 400 99 98 90 nótrium lauryl sulfate 2000 97 93 87 lecithin 2000 97 94 90 human serum albumin 2000 mannitol 2000 100 99 97 cysteine 100 human serum albumin 2000 polyoxyethylene szorbi- sorbitan monolaurate 100 99 97 95 mannitol 2000 human serum albumin 1000 hydroxypropyl fibrous pulp 500 99 97 95 dextran (molecular 40,000) 2000 polyoxyethylene szorbi- sorbitan monopalmitate 100 96 96 93 sorbitol 2000 polyoxyethylated, snowman 100 95 92 92 dextran (molecular 40,000) 2000 without additives - 72 61 47

Example 3 To pg-G-CSF, one of the stabilizers listed in Table 3 was added and the mixture was aseptically dissolved in 20 mM phosphate buffer (containing 100 mM sodium chloride, pH 7.4) to give 10 Mg. Prepare a pharmaceutical composition containing G-CSF / ml. 1 ml of the composition is filled into a sulfak-treated, silicone lined glass vial and allowed to stand at 4 ° C. The effectiveness of each stabilizer in preventing G-CSF adsorption was evaluated by measuring the residual G-CSF activity in the solution at 0.5; 2; and 24 hours later. The measurement is carried out by Method (b) using reverse phase high performance liquid chromatography. The results are shown in Table 3.

-917

HU 198627 Β

TABLE 3

stabilizer Quantity (Phr) initial Residual activity (%) 0.5 h 2 H 24 hrs mannitol 5000 100 93 90 91 hyaluronic acid 2000 100 97 92 92 dextran (molecular weight: 40,000) 2000 100 98 95 96 glycerol 10,000 100 94 91 90 heparin 2000 100 92 90 90 glucuronic 5000 100 96 90 91 ketoglikolsav 5000 100 92 88 90 human serum albumin 1000 100 100 101 99 human serum globulin 1000 100 98 100 98 alkali-treated gelatin 500 100 99 98 99 acid-treated gelatin 2000 100 99 97 97 collagen 2000 100 100 98 99 polyethylene glycol (molecular weight 4000) 10,000 100 100 100 99 hydroxypropyl cellulose 2000 100 100 100 99 Sodium carboxymethyl -cellulose 2000 100 98 96 95 hydroxyethyl cellulose 4000 100 96 93 92 polyvinyl alcohol (molecular weight: 50,000) 4000 100 99 100 98 polyvinylpyrrolidone (molecular weight: 50,000) 4000 100 98 98 96 sorbitan monocaprilat 400 100 100 100 98 polyoxyethylene szorbi- Ten monostearate 400 100 100 98 100 polyoxyethylated cured snow grease 400 100 99 101 99 sodium laureth sulfate 2000 100 100 99 97 lecithin 2000 100 99 100 98 human serum albumin 2000 mannitol 2000 100 100 100 101 cysteine 100

-1019

HU 198627 Β

Claims (1)

Patent Claim Point A method granulocyte preparing a stable pharmaceutical composition comprising a colony stimulating factor, wherein the colony stimulating factor 1 part by weight of G-1 to 10,000 parts by weight of surfactant, 1 to 10,000 parts by weight of saccharide, 1 to 20,000 parts by weight of protein and / or 1 to 20,000 parts by weight of ^{high-4- (} Mixed with ¹ molecular weight substance to form a pharmaceutical composition wherein at least one nonionic surfactant and / or natural surfactant is used as the surfactant, the aliphatic carboxylic acid ester of sorbitan, the aliphatic carboxylic acid ester of glycerol, the aliphatic carboxylic acid carboxylic acid ester, polyoxyethylene sorbitol aliphatic carboxylic acid ester, 50 polyoxyethylene glycerol aliphatic carboxylic acid ester, polyethylene glycol aliphatic carboxylic acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether, lecithin as the natural surfactant, preferably glycerol, erythritol, arabic, xylitol, sorbitol, mannitol, glucuronic acid, iduronic acid, galacturonic acid, neuraminic acid, gluconic acid, mannuronic acid, ketoglycolic acid, ketogalactonic acid as the saccharide. ketoglylic acid and / or salts thereof, hyaluronic acid and / or salts, chondroitin sulfate and / or salts, heparin, inulin, chitin and / or derivatives thereof, chitosan and / or derivatives thereof, dextrin, dextran having an average molecular weight of 5000-150,000 and / or or alginic acid and / or salts thereof, human serum albumin, human serum globulin, gelatin, acid or alkali treated protein having an average molecular weight of 7000-100,000 and / or collagen, high molecular weight hydroxypropyl cellulose, hydroxymethylcellulose sodium carboxymethylcellulose, hydroxyethylcellulose, polyethylene glycol having an average molecular weight of 300-6000, polyvinyl alcohol having an average molecular weight of 20,000-100,000 and / or polyvinylpyrrolidone having an average molecular weight of 20,000-100,000.