NL192917C - Stable pharmaceutical preparation containing granulocyte colony stimulating factor. - Google Patents

Description

1 192917

Stable pharmaceutical preparation containing granulocyte colony stimulating factor

The present invention relates to a stable pharmaceutical preparation comprising as an active ingredient the physiologically active polypeptide granulocyte colony stimulating factor and a stabilizing or adsorption-preventing substance.

This preparation is known from the non-prepublished European patent application 0,215,126 of earlier grade. The known preparation uses mouse serum albumin.

To use granulocyte colony stimulating factor in a pharmaceutical preparation, it is desirable that loss or inactivation of the factor - the active ingredient - by adsorption to the wall of the container in which the preparation is applied, or by association, polymerization or oxidation of the factor is avoided.

This object is achieved by the preparation according to the present invention, characterized in that the preparation as stabilizing or adsorption-preventing substance comprises at least one substance selected from the group consisting of a pharmaceutically acceptable non-proteinaceous surfactant , a saccharide, a high molecular weight non-proteinaceous compound, and a protein, excluding mouse serum albumin.

British patent application No. 2,016,477 discloses a urine-isolated HGI glycoprotein which can be stabilized against viruses by the presence of albumin or urinary proteins. The HGI glycoprotein differs from the granulocyte colony stimulating factor.

European patent application 0,162,332 discloses a method for stabilizing a physiologically active substance, such as interferon, interieukin 2, lysozyme or an antibody complement, using a modified gelatin. Some problems associated with albumin can thus be avoided.

European patent application 0,178,576 discloses a method and a composition for stabilizing erythropoietin. Finally, European patent application 0,098,110 discloses a long-acting composition of a physiologically active polypeptide or glycoprotein, which is prepared by coupling to a plastic copolymer.

Chemotherapy is used as one of the methods of treating various infectious diseases, but chemotherapy has recently been found to cause some serious clinical problems, such as the emergence of drug-resistant organisms, alteration of the causative organisms, and significant side effects. To avoid the problems associated with chemotherapy involving the use of therapeutic agents such as antibiotics and bactericidal agents, attempts have been made to use a substance that activates the prophylactic powers of the host of an infectious organism, thereby provides a complete solution to the above-mentioned chemotherapy problems. It is believed that of the various prophylactic powers of the host, the phagocytic bactericidal activity of leukocytes has its strongest influence during the initial period of a bacterial infection, and it is therefore believed to be important to enhance the host's infectious protective powers. by promoting the growth of neutrophils and their differentiation to matured state. A granulocyte colony stimulating factor (G-CSF) is one of the very useful substances exhibiting such actions, and the holder of the present patent has previously filed a patent application relating to an anti-infective agent using G- CSF (Japanese Patent Application 23,777 / 1985).

As indicated above, chemotherapy as it is used today presents several unavoidable problems and intensive research has been conducted to utilize a drug capable of reducing the prophylactic actions of the host or person infected , to activate.

It is probably needless to mention that G-CSF itself has the ability to activate the prophylactic functions of the host and it has also been shown that the G-CSF has a greater therapeutic effect in clinical applications when used in combination with a substance which activates the host's prophylactic capabilities.

G-CSF is used in a very small amount and a pharmaceutical composition containing 0.1-500 µg (preferably 5-50 µg) G-CSF is usually administered at a dose rate of 1-7 times per week per adult. However, G-CSF tends to be adsorbed on the wall of the container such as an injection ampoule or syringe. Therefore, when the drug is used as an injection in a form as an aqueous solution, it will be adsorbed on the container wall such as an ampoule or a syringe. This results in failure of G-CSF to give full activity as a 192917 2 pharmaceutical, or necessitates processing of G-CSF in a more than necessary amount to compensate for any adsorption loss.

Furthermore, G-CSF is labile and highly subject to environmental factors such as temperature, humidity, oxygen and ultraviolet rays. Due to the action of such factors, G-CSF undergoes physical or chemical changes such as association, polymerization and oxidation and thus a large part of the activity is lost. These phenomena make it difficult to ensure complete completion of a therapeutic treatment by the administration of a very small amount of G-CSF in a very precise manner.

It is therefore necessary to develop a stable pharmaceutical preparation of G-CSF that is fully protected against a decrease in the activity of its active ingredient. This is the main object of the present invention to provide a stable pharmaceutical preparation of G-CSF.

The G-CSF present in the pharmaceutical composition of the present invention can be obtained by any of the methods described in the descriptions of Japanese Patent Applications 153.273 / 1984, 269.455 / 1985, 269.456 / 1985, 270.838 / 1985 and 270.839 / 1985. For example, a human G-CSF can be prepared by culturing a cell strain (CNCM reference number 1-315 or I-483) collected from tumor cells of patients with oral cavity cancer or by expressing a recombinant DNA (prepared using human G-CSF encoding gene) in an appropriate host cell (e.g. E. coli, C 127 cell or Chinese hamster ovary cells).

Any human G-CSF that has been purified to a high degree can be used as the G-CSF present in the pharmaceutical composition of the present invention. Preferably, human G-CSFs are substances obtained by separating the supernatant from the culture of a human G-CSF producing cell, and a polypeptide or glycoprotein having the human G-CSF activity obtained by transforming a host with a recombinant vector containing a gene encoding a polypeptide having the human G-CSF activity.

Two particularly preferred examples of human G-CSF are listed below: (1) human G-CSF with the following physicochemical properties: a) molecular weight: about 19,000 ± 1,000, measured by electrophoresis by a sodium dodecyl-30 sulfate - polyacrylamide gel, b) isoelectric point: with at least one of the three isoelectric points, pl = 5.5 ± 0.1, pi = 5.8 ± 0.1, and pl = 6.1 ± 0.1, c ) ultraviolet absorption: with a maximum absorbance at 280 nm and a minimum absorbance at 250 nm, d) amino acid sequence of the 21 residues with N: H2N-Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-35 Leu terminal -Pro-GIn -Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-GIn-Val- (2) human G-CSF with a polypeptide with the human granulocyte stimulating factor activity, represented by full or partial amino acid sequence as listed below, or a glycoprotein with both the listed polypeptide and a sugar chain moiety: (Met) Thr Pro Leu Gly Pro A la Ser Ser Leu Pro 40 Gin Ser Phe Leu Leu Lys Cys Leu Glu Gin Val

Arg Lys He Gin Gly Asp Gly Ala Ala Leu Gin

Glu Lys Leu (Val Ser Glu) m Cys Ala Thr Tyr Lys

Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly

His Ser Leu Gly He Pro Trp Ala Pro Leu Ser 45 Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly

Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu

Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly He

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu

Gin Leu Asp Val Ala Asp Phe Ala Thr Thr He 50 Trp Gin Gin With Glu Glu Leu Gly With Ala Pro

Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala

Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly

Val Leu Val Ala Ser His Leu Gin Ser Phe Leu

Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala 55 Gin Pro (provided m is 0 or n is 0 or 1).

For further details of the process for preparing these two types of G-CSF, 3 192917, reference is made to Japanese Patent Publications 153.273 / 1984, 269.455 / 1985, 269.456 / 1985, 270.838 / 1985 and 270.839 / 1985, all filed in the name of the holder of the present patent application.

Another method that can be used is to fuse a G-CSF producing cell with a self-profiling malignant tumor cell and culture the resulting hybridoma in the presence or absence of a mitogen.

The human G-CSF-containing solution obtained can be stored in a frozen state after further purification and, if necessary, evaporated by a technique known per se. Alternatively, the solution can be stored after being dehydrated by techniques such as freeze drying.

All of the human G-CSFs thus prepared can be processed as stated in the present invention to obtain stable G-CSF containing pharmaceutical compositions.

Specific examples of the surfactant used to obtain the stable G-CSF containing pharmaceutical composition of the present invention are listed below: nonionic surfactants with HLB of 6-18 such as sorbitan aliphatic acidic esters (such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate), glycerol aliphatic acid esters (such as glycerol monocaprilat, glycerol monomyristate, and glycerol monostearate), polyglycerol aliphatic esteryl-aceryl-butylcerylic-monostolinate-glycerol polyoxiethylene sorbitan monolaurate, polyoxiethylene sorbitan monooleate, polyoxiethylene sorbitan monostearate, polyoxiethylene sorbitan monopalminate, polyoxiethylene sorbitan trioleate, and polyoxiethylene sorbitan tristearearate), polyoxiethylene sorbitol phenyl etherethyl ether oxetrastyl ether 20 polyoxiethyleensorbitoltetraoleaat), polyethylene glycerin aliphatic acid esters (such as polyoxiethyieenglycerylmonostearaat), polyethyieenglycol-aliphatic acid esters (such as polyethylene glycol distearate), polyoxyethylene alkyl ethers (such as poiyoxiethyleenlaurylether), polyoxie-thyleenpolyoxipropyleenalkylethers (such as polyoxiethyleenpolyoxipropyleenglycolether, polyoxiethyleenpo-lyoxipropyleenpropylether, and polyoxiethyleenpolyoxipropyleencetylether), polyoxiethyieenalkylfenylethers 25 (as polyoxiethyleennonylfenylether) , polyoxyethylated castor oil, polyoxiethylated hardened castor oil (polyoxiethylated hydrogenated castor oil), polyoxiethylated beeswax derivatives (such as polyoxiethylated sorbitol beeswax), polyoxiethylene-lanolin (aliphatic ethyleneethylene-lanolin) -ethanolin-lanolin) non-ionic surfactants such as alkyl sulfuric acid salts with a C10-C18 alkyl group (such as sodium acetyl-30 sulfate, sodium lauryl sulfate and sodium leyl sulfate), polyoxiethylene alkyl ether sulfuric acid salts in which the average molar number of ethylene oxide addition is 2-4 and the alkyl group is 10-18 carbon atoms has (such as polyoxiethylene sodium lauryl sulfate), salts of alkyl sulfobamic acid esters in which the alkyl group has 8-18 carbon atoms (such as sodium lauryl sulfobamic acid ester); and natural surfactants such as lecithin, glycerophospholipid, sphingophospholipid (such as sphingomyelin) and sucrose aliphatic acid esters in which the aliphatic acid has 12-18 carbon atoms. These surfactants can of course be used independently or as a mixture.

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

The saccharide used in stabilizing the G-CSF-containing pharmaceutical composition 40 of the present invention can be selected from monosaccharides, oligosaccharides and polysaccharides as well as phosphate esters and nucleotide derivatives thereof, as long as they are pharmaceutically acceptable. Examples to be mentioned specifically are mentioned below, namely: trivalent and higher sugar alcohols such as glycerol, erythrytol, arabitol, xylitol, sorbitol and mannitol; acidic sugars such as glucuronic, iduronic, neuramic, galaturonic, gluconic, mannuronic, ketoglycolic, ketogalactonic, and ketogulonic acids; hyaluronic acid and its salts, chondroitin sulfate and its salts, heparin, inulin, chitin and its derivatives, chitosan and its derivatives, dextrin, dextran with an average molecular weight of 5,000-150,000 and alginic acid and its salts. All these saccharides can advantageously be used in an independent form or as a mixture.

The above-mentioned saccharides are preferably used in an amount of 1-10,000 50 parts by weight per part by weight of G-CSF.

Specific examples of the protein to be used in stabilizing G-CSF containing pharmaceutical compositions of the present invention include human serum albumin, human serum globulin, gelatin, acid-treated gelatin (having an average molecular weight of 7,000-100,000) alkaline treated gelatin (average molecular weight 7,000-100,000) 55 and collagen. It will be understood that these proteins can be used independently or as a mixture.

The above proteins are preferably used in amounts of 1-20,000 parts per 192917 4 parts by weight of G-CSF.

Typical examples of the high molecular weight compound to be used in stabilizing G-CSF containing pharmaceutical compositions of the present invention include: natural polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose and hydroxyethyl cellulose and synthetic polymers such as polyethylene cellulose (molecular weight = 300- 6,000), polyvinyl alcohol (molecular weight = 20,000-100,000) and polyvinylpyrrolidone (molecular weight = 20,000-100,000). It will be understood that these high molecular weight compounds can be used singly or in combination.

The high molecular weight compounds as mentioned above are preferably used in amounts of 1-20,000 parts by weight per part by weight of G-CSF.

In addition to the surfactant, saccharide, protein or high molecular weight compound as mentioned above, at least one substance selected from the group consisting of an amino acid, a sulfur-containing reducing agent and an antioxidant may also be included herein when preparing the G CSF-containing pharmaceutical preparation according to the invention. Examples of the amino acids to be used are glycine, threonine, tryptophan, lysine, hydroxilysine, histidine, arginine, cysteine, cystine and methionine. Examples of sulfur-containing reducing agents include: N-acetyl cysteine, N-acetyl homocysteine, thiotinic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, sulfonethione sulfonite, sodium sulfitulphite, sodium sulfulfite, sodium sulfulfite, sodium sulfulfite such as a thioalkanoic acid with 1-7 carbon atoms. Examples of antioxidants include erythorbic acid, dibutylhydroxitoluene, butylhydroxianisole, dk-oc-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid triminate, sodium dimethylate, tetraethyl acetate, propyl acetalate, propyl acetalate and sodium metaphosphate.

The above-mentioned amino acids, sulfur-containing reducing agents and antioxidants or mixtures thereof are preferably used in amounts of 1-10,000 parts by weight per part by weight of G-CSF.

To formulate the stable G-CSF-containing composition of the present invention in a suitable dosage form, one or more of the following may be incorporated herein: a diluent, solubilizing auxiliary, an isotonic, an excipient, a pH- modifying agent, a smoothing agent and a buffer.

The stabilized G-CSF-containing pharmaceutical composition of the present invention can be formulated in various ways for oral administration or for parenteral administration such as by injection, and different dosage forms can be used depending on the specific mode of administration.

Named dosage forms include: the forms intended for oral administration such as tablets, pills, capsules, granules and suspensions; solutions, suspension and freeze-dried preparations intended primarily for intravenous injection, intramuscular injection, subcutaneous injection and intracutaneous injection and the preparations intended for transmucosal administration such as rectal suppositories, nasal medicines and vaginal suppositories.

40 The detailed mechanism by which the above-mentioned substances stabilize or prevent adsorption of G-CSF is not yet clear. In the presence of a surfactant, the surface of G-CSF, which is a hydrophobic protein, would be coated with a surfactant to dissolve effectively preventing G-CSF present in a trace amount, adsorbed to the wall of the container or a syringe. A 45 saccharide or a high molecular weight hydrophilic compound would form a hydrated layer between G-CSF and the adsorbent surface of the container wall or syringe, effectively preventing adsorption of G-CSF. A protein would compete with G-CSF for adsorption on the wall of the container or syringe, effectively preventing the adsorption of G-CSF.

In addition to preventing G-CSF adsorption, the above-mentioned substances would also contribute to preventing the association or polymerization of the G-CSF molecules. In the presence of a surfactant, saccharide, protein or high molecular compound, the individual molecules of G-CSF are dispersed in these substances and the interaction between G-CSF molecules is sufficiently reduced to cause a significant decrease in the probability of their 55 association or polymerization. Furthermore, these substances would slow down the auto-oxidation of G-CSF under a high temperature or humidity, accelerate or prevent G-CSF from clumping or polymerizing as a result of its auto-oxidation. These influences of delaying the auto-oxidation of G-CSF or preventing association or polymerization would be further enhanced by the addition of an amino acid, a sulfur-containing reducing agent or an antioxidant.

The above problems are particularly noticeable in solutions for injection and in suspensions, but they can also occur during formulation of G-CSF in other dosage forms such as 5 tablets. Furthermore, the addition of surfactants, saccharides, proteins or high molecular weight compounds is also effective in the latter case.

By the invention, G-CSF is highly stabilized and maintains its activity over a long period of time as shown in the following examples. In order to achieve these results, the amount of each of these substances, especially the lower limit thereof, becomes critical and the following ranges are preferred: 1-10,000 parts by weight of surfactant, 1-10,000 parts by weight of saccharide, 1-20,000 parts by weight of protein and 1 -20,000 parts by weight of a high molecular weight compound per part by weight of G-CSF.

According to the present invention, a surfactant, a saccharide, a protein and a high molecular weight compound is used at a given concentration and this is not only effective in preventing adsorption on the wall of the container or syringe of G-CSF but also to improve the stability of a G-CSF containing pharmaceutical preparation. As a result, it becomes possible to ensure the administration of a small but very accurate dose of G-CSF to patients; because G-CSF is expensive, its efficient use will lead to a reduction in the cost of preparing G-CSF-containing pharmaceutical preparations.

The following examples are given only to illustrate the present invention, but are not to be construed as limiting. In these examples, the residual activity of G-CSF was determined by one of the following methods.

(a) Gentle agar method using mouse bone marrow cells. A horse serum in an amount of 0.4 ml was mixed with 0.1 ml of sample, 0.1 ml of suspension of C3H / He (female) mouse bone marrow cells (0.5 -1 x 105 core cells) and 0.4 ml of modified McCoy 5A culture solution containing 0.75% agar, poured into a plastic culture dish for tissue culture (diameter 35 mm) and grown for 5 days at 37 ° C in 5 % COj> / 95% air and at 100% humidity. The number of colonies formed were counted (one colony consisted of at least 50 cells) and the activity was determined with a unit which is the activity to form one colony.

The modified McCoy 5A culture solution used in method (a) was prepared as follows. Modified McCoy's 5A culture solution (double concentration).

Twelve grams of McCoy's 5A culture solution (Gibco), 2.55 g of MEM amino acid-vitamin mixture (Nissui Seiyaky Co., Ltd.), 2.18 g of sodium bicarbonate and 50,000 units of potassium penicillin G were dissolved in 500 ml of distilled water and the solution was filtered aseptically through a Millipore filter (0.22 µm).

(b) Reverse phase - high pressure - liquid chromatography

Using a reverse phase (4.6 mm x 300 mm; 5 µm) C8 column and a 40 n-propanol / trifluoroacetic acid mixture as mobile phase, the Test activity of G-CSF (injected in an amount equivalent to one pg) determined under the following gradient conditions: time (sec) solvent (A) solvent (B) gradient 45 0 100% 0%} linear 15 0% 100%} linear 25 100% 0%

Solvent (A): 30% n-propanol and 0.1% trifluoroacetic acid.

50 Solvent (B): 60% n-propanol and 0.1% tiifluoroacetic acid.

The assay was performed at a wavelength of 210 nm and the percentage of residual G-CSF activity was calculated using the following formula: 55 residual G-CSF _ the residual amount of G-CSF after the lapse of a certain time 1fin

activity (%) "the initial amount of G-CSF

192917 6

The residual amount of G-CSF determined by this method correlated very well with the result obtained when measured by the soft agar method (a) using mouse bone marrow cells.

Example I

To 5 µg of G-CSF, one of the stabilizers listed in Table A was added and the mixture was aseptically dissolved in 20 mM buffer solution (with 200 mM sodium chloride; pH is 7.4) to prepare a pharmaceutical composition containing 5 µg G -CSF per ml, which was then lyophilized. The time-dependent change of G-CSF activity was measured according to method (a) and the results are shown in Table A. The expression "activity (%)" in the table indicates the Test activity of G-CSF from the onset unit and is defined by the following formula: ji., * / »/ x activity unit over time ..., ΛΛ activity (%) = initial activity ^ unit - x 100

Freeze drying was performed as follows.

The G-CSF solution containing a stabilizing agent was placed in a sterile sulfa-treated glass vial, frozen at -40 ° C or below for 4 hours, subjected to initial drying by heating from -40 ° C to 0 ° C , over a period of 48 hours with a pressure increasing from 0.03 to 0.1 torr, then a second drying was performed by heating from 0 ° C to 20 ° C over a period of 12 hours at a pressure increasing from 0.03 to 0.08 torr, after which the interior of the glass vial was filled with sterile, dry gaseous nitrogen to achieve atmospheric pressure and then the vial was closed with a rubber stopper for freeze-drying, and then closed with an aluminum cap .

TABLE A

25 ----- - - —----— ----- stabilizer amount of activity (%) (parts by weight) - after storage at after storage at 4 ° C for 37 ° C for 30 6 months 1 month xylitol 10,000 92 86 mannitol 10,000 91 85 35 ---------------—---------- glucuronic acid 10,000 86 82 hyaluronic acid 2,000 92 89 40 dextran (mol. wt. 40,000) 2,000 95 90 heparin 5,000 85 80 chitosan 2,000 93 91 45 —----------- - ----- alginic acid 2,000 90 90 human serum albumin 1,000 98 99 50 human serum globulin 1,000 98 95 with alkaline substance treated gelatin 1,000 99 96 collagen 2,000 95 90 55 - polyethylene glycol (MW 4,000) 10,000 94 90 7 192917 TABLE A (continued) stabilizer amount of activity (%) (parts by weight) - 5 after storage at after storage at 4 ° C at 37 ° C for 6 months 1 month hydroxypropylcellulose 1,000 98 94 10 - sodium carboximethylceilulose 1,000 88 80 hydroxymethylceilulose 5,000 92 90 15 polyvinyl alcohol (mol. wt. 50,000) 2,000 96 95 polyvinylpyrro lidon (mol. wt. 50,000) 2,000 95 94 human serum albumin 2,000 20 mannitol 2,000 100 97 cysteine 100 human serum albumin 2,000 polyoxiethylene sorbitan monolaurate 100 99 96 25 manitol 2,000 human serum albumin 2,000 hydroxypropyl cellulose 500 98 92 dextran (mol. 40,000) 2,000 30 - polyoxiethylene sorbitan monolaurate 100 sorbitol 2,000 98 96 polyoxiethylated hardened castor oil 100 g4 g2 35 dextran (MW 40,000) 2,000 without addition - 74 58

40 Example II

To 10 µg of G-CSF, one of the stabilizers listed in Table B was added and the mixture was aseptically dissolved in 20 mM phosphate buffer solution (with 100 mM sodium chloride; pH is 7.4) to prepare a pharmaceutical composition pg G-CSF per ml. The preparation was aseptically placed in a sulfa-treated glass vial and closed to prepare a G-CSF solution. The time-dependent change in G-CSF activity in this solution was measured by the same method as used in Example I and the results are shown in Table B.

192917 8

TABLE B

stabilizing agent amount of activity (%) (parts by weight) -—-—— 5 after storage at after storage at after storage at 4 ° C for 4 ° C for room temperature-7 days 2 months ture for 1 month 10 mannitol 5,000 91 87 82 hyaluronic acid 2,000 93 87 70 dextran (MW 40,000) 2,000 96 95 85 15 -: -——— glycerol 10,000 90 90 88 neuramic acid 5,000 93 91 84 20 chitin 2,000 95 92 86 dextrin 2,000 90 92 87 human serum albumin 1,000 99 95 92 25 - Human Serum Globulin 1,000 98 94 90 Acid Treated Gelatin 2,000 97 96 87 30 Alkaline Treated Gelatin 500 99 95 92 Collagen 2,000 99 94 88 35 Polyethylene Glycol (Mol.

4,000) 10,000 94 89 90 hydroxypropylcellulose 2,000 98 95 92 40 sodium carboxymethylcellulose 2,000 92 91 80 hydroxyethylcellulose 4,000 92 94 90 polyvinyl alcohol (mol. Wt. 50,000) 4,000 97 93 90 45 - polyvinylpyrrolidone (mol.

50,000) 4,000 95 95 92 sorbitan monolaurate 400 97 96 95 50 - polyoxiethylene sorbitan monolaurate 400 100 96 94 polyoxiethylene sorbitan monosarate 400 98 97 94 9 192 917 TABLE B (continued) stabilizer amount of activity (%) (parts by weight) - 5 after storage at after storage at after storage at 4 ° C for 4eC for room temperature 7 days 2 months for 1 month 10 polyoxiethylene polyoxipropylene glygiolol ether 400 100 94 93 polyoxiethylated hardened castor oil 400 99 98 90 15 - sodium lauryl sulfate 2,000 97 93 87 lecithin 2,000 97 94 90 20 human serum albumin 2,000 mannitol 2,000 cysteine 100 100 99 497 human serum albumin 2,000 25 polyoxiethylene sorbitan monolaurate 100 99 97 95 mannitol 2,000 human serum albumin 1,000 30 hydroxypropylcellulose 500 99 97 95 dextran (MW 40,000) 2,000 polyoxiethylene sorbitan monolmitate 100 96 96 sorbitan 93 polyoxiethylated hardened castor oil 100 dextran (MW 40,000) 2,000 95 92 92 40 - without addition - 72 61 47

Example III

45 To 10 µg of G-CSF, one of the stabilizers as listed in Table C was added and the mixture was aseptically dissolved in a 20 mM phosphate buffer solution (with 100 mM sodium chloride; pH Is 7.4) to form a pharmaceutical preparation. prepare 10 µg of G-CSF per ml. One milliliter of the formulation was added to a sulfa-treated silicone-covered glass vial and stored at 4 ° C. The effectiveness of each stabilizing agent to prevent G-CSF adsorption was determined by measuring the Test activity of G-CSF in the solution after 0.5 and after 2 and after 24 hours. The measurement was performed according to method (b) using reverse phase - high pressure - liquid chromatography. The results are shown in Table C.

192 917 10

TABLE C

stabilizing agent quantity Test activity (%) (parts by weight at the beginning of 0.5 hours 2 hours 24 hours mannitol 5,000 100 93 90 91 hyaluronic acid 2,000 100 97 92 92 10 - dextran (MW 40,000) 2,000 100 98 95 96 glycerol 10,000 100 94 91 90 15 heparin 2,000 100 92 90 90 glucuronic acid 5,000 100 96 90 91 ketoglycolic acid 5,000 100 92 88 90 20 - human serum albumin 1,000 100 100 101 99 human serum globulin 1,000 100 98 100 98 25 alkaline treated gelatin 500 100 99 98 99 acid-treated gelatin 2,000 100 99 97 97 30 collagen 2,000 100 100 98 99 polyethylene glycol (mol. Wt.

4,000) 10,000 100 100 100 99 35 hydroxypropyl cellulose 2,000 100 100 100 99 sodium carboxymethyl cellulose 2,000 100 98 96 95 hydroxyl ethyl cellulose 4,000 100 96 93 92 40 - polyvinyl alcohol (mol.

50,000) 4,000 100 99 100 98 polyvinylpyrrolidone (mol. Wt.

45 50,000) 4,000 100 98 98 96 sorbitan monocaprylate 400 100 100 100 98 polyoxiethylene sorbitan 50 stearate 400 100 100 98 100 polyoxiethylated hardened castor oil 400 100 99 101 99 55 sodium lauryl sulfate 2,000 100 100 99 97

Claims (7)

11 192917 TABLE C (continued) stabilizing agent amount Test activity (%) (parts by weight - 0.5 hours 2 hours 24 hours iecithin 2,000 100 99 100 98 human serum albumin 2,000 10 mannitol 2,000 100 100 100 101 cysteine 100 human serum albumin 2,000 polyoxiethylene sorbitan monolaurate 100 100 100 98 99 mannitol 2,000 human serum albumin 1,000 hydroxypropyl cellulose dextran 500 100 101 99 100 20 (molecular weight 40,000) 2,000 polyoxiethylene sorbitan monolaurate 100 100 100 99 99 sorbitol 2,000 25 - polyoxiethylated 100 hardened castor oil 100 dextran (molecular weight 40,000) 2,000 30 without addition - 100 91 72 73 Stable pharmaceutical preparation comprising as active ingredient the physiologically active polypeptide granulocyte kionion-stimulating factor and a stabilizing agent or adsorption-preventing agent, characterized in that the preparation as a stabilizing agent or adsorption-preventing agent comprises at least one substance selected from the group consisting of a pharmaceutically acceptable non-proteinaceous surfactant, a saccharide, a high molecular weight compound, and a protein, 40 excluding mouse serum albumin. Stable pharmaceutical preparation according to claim 1, characterized in that the surfactant is selected from the group consisting of polyoxiethylene sorbitan monostearate, polyoxiethylene sorbitan monooleate and polyoxiethylene lauryl ether. Stable pharmaceutical preparation according to claim 1, characterized in that the saccharide is selected from the group consisting of mannitol and sorbitol. Stable pharmaceutical preparation according to claim 1, characterized in that the protein is selected from the group consisting of human serum albumin and gelatin treated with an acid or alkaline substance. Stable pharmaceutical preparation according to claim 1, characterized in that the preparation comprises a combination of human serum albumin, mannitol and cysteine. Stable pharmaceutical preparation according to claim 1, characterized in that the preparation comprises a combination of human serum albumin, mannitol and polyoxiethylene sorbitan monolaurate. Stable pharmaceutical preparation according to claim 1, characterized in that the preparation comprises a combination of polyoxiethylene sorbitan monolaurate and sorbitol.