PT85343B - METHOD FOR PREPARING A STABLE PHARMACEUTICAL COMPOSITION CONTAINING A STIMULATING FACTOR OF A GRANULOCYTE COLONY - Google Patents

Description

Descriptive memory referring to the CHUGAI SEIYAKU KABUSHIKI KAISHA, Japanese, industrial and commercial patent, with headquarters at No. 5-1, 5-chome, Ukima, Kita-ku, Tokyo, Japan, (invention: Minoru Machida, resi Japan), for PROCESS FOR THE PREPARATION OF A STABLE PHARMACEUTICAL COMPOSITION CONTAINING A STIMULATING FACTOR FROM A GRANULOCYTE COLONY.

DESCRIPTIVE MEMORY

The present invention relates to a process for preparing a pharmaceutical composition containing a granulocyte colony stimulating factor. In particular, the present invention relates to a stabilized pharmaceutical composition containing a granulocyte colony stimulating factor that is protected against the loss or inactivation of the active ingredient (i.e., the granulocyte colony stimulating factor) due to adsorption on the wall of a container in which the composition is placed, or, the combination, polymerization or oxidation of said ingredient.

Chemotherapy has been used as one of the processes to treat various infectious diseases, but chemotherapy has recently been found to lead to some • serious clinical problems such as organism production

resistant to drugs, changes in organisms causing disease and major side effects. To avoid these problems associated with chemotherapy involving the use of therapeutic agents such as antibiotics and bactericides, attempts have been made to use a substance that activates the prophylactic capabilities of the host of an infection-causing organism and thus gives rise to a complete solution to the aforementioned problems of chemotherapy. Of the host's various prophylactic capacities, it is believed that the phagocytic bactericidal action of leukocytes has the greatest influence on the initial period of infection by bacteria and it is therefore established as important to increase the protective capacities of host infections by promoting the growth of neutrophils and their differentiation in the mature state. A granulocyte colony stimulating factor (G-CSF) is one of the very useful substances that exhibit these actions and the same signatories of the present invention have previously filed a patent application for an infection-protecting agent using G-CSF (Japanese Patent Application) No. 23777/1985).

As mentioned above, chemo therapy as currently practiced involves several unavoidable problems and great efforts have been made to use a drug that is able to activate the prophylactic functions of the host or the person who has been infected.

Needless to say, G-CSF itself has the ability to activate the host's prophylactic functions and G-CSF has also been found to exhibit superior therapeutic effects in clinical applications if used in combination with a substance that activate the host's prophylactic capabilities.

Use G-CSF in a very small amount and generally a pharmaceutical composition containing 0.1-500 pg (preferably 5-50 pg) of G-CSF is administered at a dosage rate of 1 to 7 times per week per adult . However, G-CSF has a tendency to be adsorbed on the wall of your container such as an injection vial or syringe. So if the medicine is used as an injection in the form of an aqueous solution, it will be adsorbed on the wall of your container such as an ampoule or syringe. This phenomenon results in the failure of the G-CSF to fully reveal its activity as a pharmaceutical, pharmaceutical, or requires the incorporation of the G-CSF in an amount greater than necessary to compensate for its: possible loss by adsorption.

In addition, G-CSF is wooly and highly susceptible to environmental factors such as temperature, humidity, oxygen, and ultraviolet rays. By the action of these factors, G-CSF undergoes physical or chemical changes such as association, polymerization and oxidation and undergoes a great reduction in its activity. These phenomena make it difficult to ensure the complete establishment of a therapeutic action by administering a very small amount of the G-CSF in a very precise manner.

It is therefore necessary to develop a stable pharmaceutical composition of G-CSF that is fully protected against a drop in the activity of its effective component. This is the main object of the present invention which features a stable pharmaceutical composition of G-CSF.

The present inventors have conducted intensive studies in order to increase the stability of a pharmaceutical composition containing G-CSF and have found that this object can be effectively achieved by adding a pharmaceutically acceptable surfactant, saccharide, protein or compound.

So, the pharmaceutical composition is. containing G-CSF of the present invention. It is characterized by containing G-CSF and at least one substance chosen from the group consisting of a pharmaceutically acceptable surfactant, saccharide, protein and high molecular weight compound.

G-CSF to be incorporated into the pharmaceutical composition of the present invention can be obtained by any processes such as those described in the specifications of Japanese Patent Applications Nos. 153273/1984, 269455/1985, 269456/1985, 270838/1985 and 270839/1985. For example, human G-CSF can be prepared by culturing a cell strain (Number of

CNCM access 1-315 or 1-483) taken from tumor cells from cancer patients in the oral cavity, or expressing recombinant DNA (which has been prepared by the action of a gene encoding a human G-CSF) in a suitable host cell (e.g. E.coli, G 127 cell or ovary cells of a Chinese hamster).

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

Two particularly preferable examples of human G-CSF are given below:

(1) Human G-CSF having physicochemical properties

i) molecular weight: about 19 ooo + 1 ooo measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel;

ii) isoelectric point: having at least one of the three isoelectric points, pl = 5.5 + 0.1, pl = 5.8 + 0.1, and pl = 6.1 + 0.1;

iii) ultraviolet adsorption: having a maximum adsorption at 280 nm and a minimum adsorption at 250 nm;

iv) amino acid sequence of 21 residues of the N-terminus:

H ₂ N-Thr-Pro-Leu-Gly-Bro-Ala-Ser-Ser-Leu-Pro-Gin-Ser-Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln-Val- (2) G -Human CSF containing either a polypeptide with the activity of the human granulocyte stimulating factor which is represented by all or part of the following amino acid sequence, or a glycoprotein having either the said polypeptide or part of the sugar chain: ( Met) _n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu Glu Gin Vai Arg Lys lie Gin Gly Asp

Gly Tyr Wing Wing Lys Leu Cys Gin Glu Lys Leu (Gonna Be Glu) _m Cys Ala Thr Leu Vai Leu Leu Gly His His Pro Glu Glu To be Read Gly lie Pro Trp Allah Pro Read To be To be Cys Pro To be Gin Allah Read Gin Read Allah Gly Cys Read To be Gin Read His To be Gly Read Phe Read Tyr Gin Gly Read Read Gin Allah Read Glu Gly lie To be Pro Glu Read Gly Pro Thr Read Asp Thr Read Gin Read Asp Go Allah Asp Phe Allah Thr Thr lie Trp Gin Gin Met Glu Glu Read Gly Met Allah Pro Allah Read Gin Pro Thr Gin Gly Allah Met Pro Allah Phe Allah To be Allah Phe Gin Arg Arg Allah Gly Gly Go Read Go Allah To be His Read Gin To be Phe Read Glu Go To be Tyr Arg Go Read Arg His Read Allah Gin Pro (with the condition m is 0 or 1; and n be 0 or 1)

For details of the process for preparing these two types of G-CSF, see the specification of Japanese Patent Applications No. 153273/1984, 269455/1985, 269456/1985, 270838/1985, and '270839/1985, having been all presented by the signatories of the present invention.

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

The solution containing human G-CSF obtained can be stored in a frozen state after being purified and concentrated, if necessary, by any known technique. Alternatively, the solution can be stored after being dehydrated by means such as lyophilization.

All human G-CSF thus prepared can be processed as specified in the present invention in order to obtain pharmaceutical preparations containing stable G-CSF.

Typical examples of the surfactant that is used to prepare pharmaceutical compositions containing stable G-CSF of the present invention are shown below: nonionic surfactants with 6-18 HLB such as aliphatic acid sorbitan esters (for example sorbitan monocaprilate, sorbitan monolaurate and sorbitan monopalmitite), glycerin esters of aliphatic acids (eg

example glycerin monocaprylate, glycerin monomiristate, and glycerin monostearate) aliphatic acid polyglycerin esters (eg decaglyceryl monostearate, decaglyceryl distestate and decaglyceryl monolinoleate) polyoxyethylene / sorbitan acid polyoxyethylene esters sorbitan, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, monopal. polyoxyethylene sorbitan mitate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol esters of aliphatic acids (eg tetraethylene tetraethylene polyethylene sorbitol) ), polyethylene glycerin esters of aliphatic acids (eg polyoxyethylene glyceryl monostearate), polyethylene glycol esters of aliphatic acids (eg polyethylene glycol distearate) polyoxyethylene alkyl ethers (eg d and polyoxyethylene lauryl), polyoxyethylene polyoxypropylene alkyl ethers (e.g. polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, and polyoxyethylene polyoxypropylene cetyl ether) polyoxyethylene ethylene ethylene ethers, for example, polyoxyethyl ethylene ether, castile ether hardened polyoxyethylated beaver (eg polyoxyethylated hydrogenated castor oil), polyoxyethylated beeswax derivatives (eg polyoxyethylated beeswax with sorbitol), polyoxyethylene lanolin derivatives (eg polyoxyethylene lanolin), and polyoxyethylene aliphatic acid amides (eg polyoxyethylene stearic acid amide; non-ionic surfactants such as alkyl sulfuric acid salts having an alkyl group (eg sodium cetyl sulfate, sodium lauryl sulfate, and sodium oleyl sulfate), polyoxyethylene alkyl ether salts of sulfuric acid in which the The average molar number of ethylene oxide addition is 2 - 4 and the alkyl group has 10 - 18 carbon atoms (for example polyoxyethylene sodium lauryl sulfate), salts of the alkyl sulfosuccinate esters where the alkyl group has 8 - 18 carbon atoms (eg sodium lauryl sulfosuccinate ester) and natural surfactants such as lecithin, glycerophospholipid, sphingophospholipid (eg & & fingomyelin), and aliphatic acid sucrose esters in which the aliphatic acid has 12 - 18 atoms of carbon. These surfactants can obviously be used independently or in admixture.

The surfactants shown below are preferably used in amounts of 1 to 10,000 parts by weight per part by weight of G-CSF.

The saccharide to be used in the preparation of the pharmaceutical composition containing stable G-CSF of the present invention can be chosen from monosaccharides, oligosaccharides and polysaccharides, as well as phosphate esters and their nucleotide derivatives as long as they are pharmaceutically acceptable. Typical examples are given below: trivalent and high sugar alcohols such as glycerin, erythritol, arabi tol, xylitol, sorbitol and mannitol, acid sugars such as glucuronic acid, iduronic acid, neuraminic acid, galacturonic acid, gluconic acid, gluconic acid manurenic acid, ketoglycolic acid, ketogalactonic acid and ketogulonic acid, hyaluronic acid and its salts, chondroitin sulphate and its salts, heparin, inolina, chitin and its derivatives, chitosan and its derivatives, 5,000 to 150,000, and alginic acid and its salts. All of these saccharides can be used to advantage either independently or in mixture.

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

Typical examples of protein to be used in the preparation of the stable G-CSF containing pharmaceutical composition of the present invention include human serum albumin, human serum globulin, gelatin, acid-treated gelatin (average molecular weight 7,000 to 100,000), treated gelatin with alkaline substance (average molecular weight 7 000 to 100 000) and collagen. It goes without saying that these proteins can be used either independently or in a mixture.

The above proteins are »1

preferably used in amounts of 1-20,000 by weight per part by weight of G-CSF.

Typical examples of high molecular weight compounds to be used in the preparation of pharmaceutical compositions containing stable G-CSF of the present invention include: natural polymers such as hydroxypropylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose and hydroxymethylcellulose, and hydroxyethylcellulose; and synthetic polymers such as polyethylene glycol (molecular weight = 300-6,000), polyvinyl alcohol (molecular weight = 20,000-100,000), and polyvinylpyrrolidone (molecular weight = 20,000-100,000). It goes without saying that these high molecular weight compounds can be used either alone or in combination.

The above-mentioned high molecular weight compounds are desirably 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 described above, at least one member chosen from the group consisting of an amino acid, a sulfur reducing agent and an antioxidant may also be incorporated in the preparation of the composition pharmaceutical product containing G-CSF of the present invention. Illustrative amino acids include glycine, threonine, tryptophan, lysine, hydroxylisin, histidine, arginine, cysteine, cystine and methionine. Illustrative sulfur-reducing agents include: N-acetylcysteine, N-acetylchococellin, thiotic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium sulfate thios, sodium hydrogen sulfite, sodium sulfosite sodium, thiolactic acid, dithiothreitol, glutathione, and a moderate sulphurous reducing agent having a fridyl group such as, for example, a C4 -C4 thioalkanoic acid. Illustrative antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, df-0 <-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid lumate, lactic acid propylene and chelating agents such as ethylene diaminetetraacetate. sodium (EDTA), sodium pyrophosphate and sodium metaphosphate.

n

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

In order to formulate the stable G-CSF-containing composition of the present invention in a suitable dosage form, either an or. more of the following agents: a diluent, a solubilizing aid, an isotonic agent, an excipient, a pH modifier, a calming agent and a buffer.

The pharmaceutical composition of stabilized G-CSF of the present invention can be formulated either for oral administration or for parenteral administration such as injection applied in various ways, and various dosage forms can be used depending on the specific mode of administration. Typical dosage forms include: those which are indicated for oral administration such as tablets, pills, capsules, granules and suspensions; lyophilized solutions, suspensions and preparations with the main purpose of intravenous injection, intramuscular injection, subcutaneous injection; and those that are indicated for administration.transmucosal such as rectal suppositories, nasal medications and vaginal suppositories.

According to the present invention, at least one substance chosen from the group consisting of a surfactant, a saccharide, a protein or a high molecular weight compound is added to the pharmaceutical composition containing G-CSF in order to prevent it from being adsorbed on the container wall or in a syringe while simultaneously remaining stable over a long period of time.

The detailed mechanism by which the aforementioned substances stabilize G-CSF or prevent it from being adsorbed needs to be clarified. In the presence of a surfactant, the surface of G-CSF is a hydrophobic protein must be coated with the surfactant to become solubilized so as to prevent the G-CSF present in a trace amount from being effectively adsorbed on the

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container or syringe wall. A saccharide or a high molecular weight hydrophilic compound will form a hydrated layer between the G-CSF and the adsorbent surface of the wall of your container or syringe thus preventing the adsorption of the G-CSF effectively. A protein will compete with G-CSF for adsorption on the outside of its container or syringe, thereby effectively inhibiting the adsorption of G-CSF.

In addition to preventing the adsorption of G-CSF, the substances mentioned above will also help to prevent the association or polymerization of the G-CSF molecules. In the presence of a surfactant, saccharide, protein or with a high molecular weight rank, the individual G-CSF molecules will be dispersed in these substances and the interaction between them. G-CSF molecules are sufficiently reduced to cause a significant decrease in the likelihood of their association or polymerization. In addition, these substances will delay the oxidation of G-CSF which is accelerated to high temperature or humidity or will prevent the association or polymerization of G-CSF as a result of its auto-oxidation. These effects of delaying the auto-oxidation of G-CSF or preventing its association or polymerization will be further enhanced by the addition of an amino acid, a sulfur-reducing agent or an antioxidant.

The problems described above are particularly notable in solutions for injections and suspensions but also occur during the process of formulating G-CSF in other dosage forms such as tablets. The addition of surfactants, saccharides, proteins or high molecular weight compounds is also effective in the latter case.

By adding at least one substance chosen from the group consisting of a surfactant, saccharide, protein and high molecular weight compound, G-CSF is highly stabilized and maintains its activity for an extended period of time, as will be demonstrated in the examples that that follow. To achieve these results, the amount of each of these substances, in particular their lower limit, is critical and the following ranges are desirable: 1-10 000 parts by weight of surfactant, 1-10 000 parts by weight of i n

saccharide, 1-20,000 parts by weight of protein, and 1-20,000 parts by weight of a high molecular weight compound, per each parts by weight of G-CSF.

According to the present invention, a surfactant, a saccharide, a protein and / or a high molecular weight compound in a specified concentration is used and this is effective not only in preventing the adsorption of G-CSF on the wall of its container or syringe but also in increasing the stability of the pharmaceutical composition containing G-CSF. As a result, it is possible to ensure the administration of a small but very accurate dose of G-CSF to patients; since G-CSF is expensive, its effective use will lead to lower costs for the production of pharmaceutical compositions containing G-CSF.

The following examples are presented for the purpose of further illustrating the present invention but are not to be construed as limiting. In these examples, residual G-CSF activity was determined by one of the following procedures.

(a) Process of soft agar using cells from the rat spinal cord:

Horse serum (0.4 ml), 0.1 ml of the sample, 0.1 ml of a C3H / He rat bone marrow cell suspension (female) (0.5 - 1 x 10 cells) were mixed nuclear) and 0.4 ml of a modified McCoy 5A culture solution containing 0.75% agar, poured into a plastic tissue culture dish 0 35 mm), clotted, and cultured for 5 days at 379 ° C in a mixture of 5% CO ₂ /95% air and 100% humidity. The number of colonies formed (a colony consisting of at least 50 cells) was counted and the activity was determined with one unit being the activity to form a colony.

The modified McCoy 5A culture solution used in process (a) was prepared by the following procedures.

McCoy's modified culture solution (double concentration) Dissolved twice in 500 ml • of distilled water 12 g of McCoy SA (Gibco) culture solution, •

. 2.55 g of a MEM-vitamin amino acid medium (Nissui Seiyaku Co.,

Ltd.), 2.18 g of sodium bicarbonate and 50,000 units of potassium penicillin G and the solution was aseptically filtered through an M-illipore filter (0.22 µm).

(b) Reverse phase high performance liquid chromatography:

The residual activity of G-CSF (injected in an amount equivalent to 50%) was determined using a C8 reverse phase column (4.6 mm x 300 mm; 5 pm) and a mixture of n-propanol / trifluoroacetic acid as mobile phase. 1 pg) with the following gradient conditions:

Time (sec)

Solvent. THE'

Solvent B

Gradient

Solvent (A):

Solvent (B):

30%

60%

100%

0% linear

0%

100% linear

100%

0% linear n-propanol and n-propanol and trifluoroacetic acid trifluoroacetic acid detection at a length of ¹ nm was conducted and the percentage of residual G-CSF activity was calculated by the wave

210 following formula:

Residual activity = G-CSF (%) of residual amount of G-CSF after some time

------------------------------ x 100 starting amount of G-CSF

The amount of G-CSF determined by this process correlated very well with the result obtained by the soft agar process (a) using cells from the rat's spinal cord.

EXAMPLE 1

One of the stabilizing agents listed in Table 1 was added to 5 pg of G-CSF and the mixture was aseptically dissolved in a 20 mM buffer solution (containing 100 mM sodium chloride; pH 7.4) to obtain a far composition. medicine containing 5 µg of G-CSF per ml, which was then

affiliated. The time-dependent change of G-CSF activity was measured by process (a) and the results obtained are shown in Table 1. The term activity (%) in the table represents the residual activity of G-CSF in relation to the unit initial and is defined by the following formula:

activity unit after some time

Activity (%) = -----------------------------— x 100 unit of initial activity

Lyophilization was carried out by the following procedures:

The G-CSF solution containing a stabilizing agent was placed in a sterilized sulfated glass vial, cooled to -40 ° C or below for 4 hours or less, subjected to primary drying by heating between -40 ° C. C and 09 C over a period of 48 hours with the pressure being increased from 0.03 to 0.1 torr, and then secondary drying by heating from 09 C to 209 C over a period of 12 hours with the pressure increasing from 0 , 03 to 0.08 torr; then the interior of the flask was filled with sterile nitrogen gas to reach atmospheric pressure and the flask was closed with a lyophilizing rubber stopper, then sealed with an aluminum cap.

-2

Table 1

Stabilizing agent Quantity (parts by weight) Activity (%) After storage at 49C for 6 months After storage at 379 C for 1 month xylitol 10,000 92 86 mannitol 10,000 91 85 glucuronic acid 10,000 86 82 hyaluronic acid 2,000 92 89 dextran (p.m. 40,000) 2,000 95 90 heparin 5,000 85 80 chitosan 2,000 93 91 alginic acid 2,000 90 90 human serum albumin 1,000 98 99 human serum globulin 1,000 98 95 acid-treated gelatin 3 2 000 97 95 alkaline-treated gelatin 1,000 99 96 collagen 2,000 95 90 polyethylene glycol (p.m. 4,000) 10,000 94 90 hydroxypropyl cellulose 1,000 98 94 sodium carboxymethyl cellulose 1,000 88 80 hydroxymethylcellulose 5,000 92 90

Table 1 (cont.)

Stabilizing agent Quantity (parts by weight) Activity (%) After storage at 49C for 6 months After storage at 379 C for 1 month polyvinyl alcohol (50,000 p.m.) 2,000 96 95 polyvinylpyrrolidone · (50,000 p.m.) 2,000 95 94 human serum albumin mannitol cysteine 2,000 2,000 100. 100 97 human serum albumin polyoxyethylene sorbitan mannitol monolaurate 2,000 100 2,000 99 96 human serum albumin hydroxypropylcellulose dextran (p.m. 40,000) 2,000 500 2,000 98 92 polyoxyethylene sorbitan monolaurate sorbitol 100 2,000 98 96 polyoxyetide hardened castor oil dextran (p.m. 40,000) 100 2,000 94 92 not added - 74 58

Ί Γ

EXAMPLE 2

One of the stabilizing agents listed in Table 2 was added to 10 µg of G-CSF and the mixture was aseptically dissolved in a 20 mM phosphate buffer solution (containing 100 mM sodium chloride; pH 7.4) to obtain a pharmaceutical composition containing 10 µg of G-CSF per ml. The composition was aseptically loaded into a sulfated glass vial and sealed to obtain a solution of G-CSF. The time-dependent change in G-CSF activity in this solution was measured by the same procedure used in Example 1 and the results obtained in Table 2 are shown.

Table 2

Stabilizing agent Quantity (part by weight) Activity (%) After storage at 49C for 7 days After storing at 49C for 2 months After storing AT for 1 month mannitol 5,000 91 87 82 hyaluronic acid 2,000 93 87 70 dextran (p.m. 40,000) 2,000 96 95 85 glycerin 10,000 90 90 88 neuraminic acid 5,000 93 91 84 chitina 2,000 95 92 86 dextrin 2,000 90 92 87 human serum albumin 1,000 99 95 92 human serum globulin in 1,000 98 94 90 acid-treated gelatin 2,000 97 96 87 alkaline-treated gelatin 500 99 95 92

Ί ι

Table 2 (cont.)

Stabilizing agent Quantity (part by weight) Activity (%) After storing at 49C for 7 days After storage at 49C for 2 months After storing AT for 1 month collagen 2,000 99 94 88 polyethylene glycol (4,000 p.m.) 10,000 94 89 90 hydroxypropylcellulose 2,000 98 95 92 sodium carboxymethyl cellulose 2,000 92 91 80 hydroxyethylcellulose 4,000 92 94 90 polyvinyl alcohol (50,000 p.m.) 4,000 97 93 90 polyvinylpyrrolidone (50,000 p.m.) 4,000 95 95 92 sorbi tano monolaurate 400 97 96 95 polyoxyethylene sorbitan monolaurate 400 100 96 94 polyoxyethylene sorbite monostearate in 400 98 97 94 polyoxyethyl ether in polyoxypropylene glycol 400 100 94 93 hardened polyoxyethylated castor oil 400 99 98 90 Sodium lauryl sulfate 2,000 97 93 87 lecithin 2,000 97 94 90 human albumin serum mannitol cysteine 2,000 2,000 100 100 99 f 97

· ->

Table 2 (cont.)

Stabilizing agent Quantity (part by weight) Activity (%) After storing at 49C for 7 days After storing at 49C for 2 months After storing AT for 1 month hu 'bro serum albumin polyoxyethylene monolaurate mannitol 2,000 100 2,000 99 97 95 human serum albumin Bro hydroxypropyl cell if dextran (p.m.40,000) 1,000 500 2,000 99 97 95 polyoxyethylene sorbitan monopalmitate sorbitol 100 2,000 96 96 93 castor oil endure polyoxyethylated dextran (p.m.40 000) 100 2,000 95 92 92 not added - 72 61 47

EXAMPLE 3

10 µg of G-CSF, one of the stabilizing agents shown in Table 3, was added and the mixture was aseptically dissolved in a 20 mM phosphate buffer solution (containing 100 mM sodium chloride; pH 7.4) to obtain a pharmaceutical composition containing 10 µg of G-CSF per ml. 1 milliliter of the preparation was loaded into a silicon-treated and sulfated glass vial and left at 49 ° C. The effectiveness of each stabilizing agent in preventing the adsorption of G- • 4 - r was evaluated. -CSF measuring the residual activity of G-CSF in the solution in the past

0.5, 2 and 24 hours. The measurement was carried out by process (b) using reverse phase high performance liquid chromatography. The results obtained are shown in Table 3.

Claims (1)

Process for the preparation of a pharmaceutical composition containing a stimulating factor of a stable granulocyte colony, characterized by incorporating, in addition to the stimulating factor of a granulocyte colony, present as an active ingredient in an amount of 0.1-500 pg, preferably 5-50 pg, at least one substance chosen from the group consisting of a pharmaceutically acceptable surfactant, saccharide, protein and high molecular weight compound Process according to claim 1, characterized in that the surfactant is incorporated in an amount of 1 - 10,000 parts by weight per part by weight of the stimulating factor of a granulocyte colony. Process according to claim 1 or 2, characterized in that said surfactant is at least one member chosen from the group consisting of a nonionic surfactant, an anionic surfactant and a natural surfactant, the nonionic surfactant being one aliphatic acid sorbitan ester, an aliphatic acid glycerin ester, an aliphatic acid polyglycerin ester, an aliphatic acid polyoxyethylene sorbitan ester,. a polyoxyethylene sorbitol ester of aliphatic acid, an ester Polyoxyethylene Λ Λ aliphatic acid glycerin, an aliphatic acid polyethylene glycol ester., A polyoxyethylene alkyl ether, a polyoxyethylene polyoxypropylene alkyl ether, a polyoxyethylene alkyl phenyl ether, a polyoxyethylated hardened castor oil, a derivative of polyoxyethylated castor polyoxyethylated bees, a polyoxyethylene lanolin derivative, or an aliphatic acid polyoxyethylene amide, the anionic surfactant being. with an alkyl sulfate salt, a polyoxyethylenealkyl ether sulfate salt, or an alkyl sulfosuccinate ester salt, and the natural surfactant being lecithin, glycerophospholipid, sphingophospholipid, or an aliphatic acid sucrose ester. Process according to claim 1, characterized in that a saccharide is incorporated in an amount of 1 - 10,000 parts by weight per part by weight of the stimulating factor of a granulocyte colony. Process according to claim 1 or 4, characterized in that said saccharide is at least one member chosen from the group consisting of glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, glucuronic acid, iduronic acid, galacturonic acid, neuraminic acid, glycolic acid , manuronic acid, ketoglycolic acid, ketogalonic acid, ketogulonic acid, 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 5,000 - 150,000, and alginic acid and its salts. Ω The Process according to claim 1, characterized in that the protein is incorporated in an amount of 1 - 20,000 parts by weight per part by weight of the granulocyte colony stimulating factor. Process according to claim 1 or 6, characterized in that said protein is at least one member chosen from the group consisting of human serum albumin, human serum globulin, gelatin, acid-treated gelatin or alkaline substance with an average molecular weight of 7 00 (- 100 000, and collagen. Process according to claim 1, characterized in that the high molecular weight compound is incorporated in an amount of 1 - 20,000 parts by weight per part by weight of the stimulating factor of a colony of grantilocytes. Process according to claim 1 or 8, characterized in that said high molecular weight compound is at least one member chosen from the group consisting of hydroxypropyl cellulose, hydroxymethyl cellulose, carboxy • methyl cellulose, sodium hydroxyethyl cellulose, polyethylene glycol Ο Λ with a molecular weight of 300 - 6,000, polyvinyl alcohol with a molecular weight of 20,000 - 100,000 and polyvinylpyrrolidone with a molecular weight of 20,000 - 100,000. The applicant declares that the first applications for this patent were filed in Japan on July 18, 1986, under the numbers 169486/1986, 169487/1986, 169488/1986 and 169489/1986.