CA1297007C

CA1297007C - Stable pharmaceutical preparation containing granulocyte colony stimulating factor and process for producing the same

Info

Publication number: CA1297007C
Application number: CA000542239A
Authority: CA
Inventors: Minoru Machida
Original assignee: Chugai Pharmaceutical Co Ltd
Current assignee: Chugai Pharmaceutical Co Ltd
Priority date: 1986-07-18
Filing date: 1987-07-15
Publication date: 1992-03-10
Anticipated expiration: 2009-03-10
Also published as: YU47543B; FR2601591A1; SE503312C2; FR2601591B1; DK171308B1; IL83220A0; KR930004597B1; IE60290B1; NL8701640A; IT8767594A0; PT85343B; CH671157A5; NO872966D0; ES2010226A6; HK64893A; YU134287A; NL192917C; AU611856B2; GR871067B; BE1000253A3

Abstract

STABLE PHARMACEUTICAL PREPARATION CONTAINING
GRANULOCYTE COLONY STIMULATING FACTOR AND
PROCESS FOR PRODUCING THE SAME
Abstract of the Disclosure:
A stable granulocyte colony stimulating factor containing pharmaceutical preparation is disclosed. It contains, in addition to the granulocyte colony stimulating factor present as the effective ingredient, at least one substance selected from the group consisting of a pharmaceu-tically acceptable surfactant, saccharide, protein and high-molecular weight compound.

Description

STABLE PHARMACEUTICAL PREPARATION CONTAINING
GRANULOC~TE COI,ONY STIMUI.ATING FACTOR AND
PROCESS FOR PRODUCING THE SAME
The present invention relates to a pharmaceutîcal preparation containing a granulocyte colony stimulating factor. In particular, the present invention relates to a stabilized pharmaceutical preparation containing a granulo-cyte colony stimulating factor that is protected against loss or inactivation of the active component (i.e., granulo-cyte colony stimulating factor) due to adsorption on thewall of a container in which the preparation is put, or to association, polymerization or oxidation of said component.
Chemotherapy has been undertaken as one method for treating a variety of infectious diseases but it has recently been found that chemotherapy causes some serious clinical problems such as the generation of drug-resistant organisms, change of causative organisms, and high side effects. In order to-avoid these problems associated with chemotherapy involving the use of therapeutic agents such as antibiotics and bactericides, attempts are being made to use a substance that activates the prophylactic capabilities of the host of an infection-causing organism and thereby pro-viding a complete solution to the aforementioned problems of che therapy. Of the various prophylactic capabilities of 25 the host, the phagocytic bactericidal action of leucocytes is believed to cause the strongest influence in the initial period of bacterial infection and it is therefore assumed to be important to enhance the infection protecting capabili-ties of the host by promoting the growth of neutrophiles and their differentiation into the mature state. A granulocyte colony stimulating factor (G-CSF) is one of the very useful substances that exhibit such actions and the same assignee of the present invention previously filed a patent applica-tion on an infection protecting agent using G-CSF (Japanese Patent Application No. 23777/1985).
As mentioned above, chemotherapy as currently practiced involves various unavoidable problems and inten-sive efforts are being made to use a drug substance that is capable of activating the prophylactic functions of the host or the person who has been infected.
Needless to say, G-CSF displays by itself the ability to activate the prophylactic functions of the host and it has also been found that G-CSF exhibits greater therapeutic effects in clinical applications if it is used in combina-tion with a substance that activates the prophylactic capabilities of the host.
G-CSF is used in a very small amount and a pharma-ceutical preparation containing 0.1 - 500 ~S (preferably 5 - 50 ~g) of G-CSF is usually administered at a dose rate of 1 - 7 times a week per adult. However, G-CSF has a tendency to be adsorbed on the wall of its container such as an ampule for injection or a syringe. Therefore, if the drug is used as an injection in such a form as an aqueous solution, it will be adsorbed on the wall of its container such as an ampule or a syringe. This either results in the failure of G-CSF to fully exhibit its activity as a pharma-ceutical agent or necessitates the incorporation of G-CSF

~ 3--in a more-than-necessary amount making allowance for its possible loss due to adsorptionO
In addition, G-CSF is labile and highly s~sceptible to environmental factors such as temperature, humidity, oxygen and ultraviolet rays. By the agency of such factors, G-CSF undergoes physical or chemical changes such as associ-ation, polymerization and oxidation and suEfers a great loss in activity. These phenomenon make it difficult to ensure complete accomplishrnent of a therapeutic act by administer-ing a very small amount of G-CSF in a very exact manner.
It is therefore necessary to develop a stable pharmaceutical preparation of G-CSF that is fully protected against a drop in the activity of its effective component.
This is the principal object of the present invention which 15 provides a stable pharmaceutical preparation of G-CSF.
The present inventors conducted intensive studies in order to enhance the stability of a G-CSF containing pharmaceutical preparation and found that this object can effectively be attained by addition of a pharmaceutically 20 acceptable surfactant, saccharide~ protein or high-molecular weight compound.
` Therefore~ the stable G-CSF containing pharmaceutical preparation of the present invention is characterized by containing both G-CS~ and at least one substance selected 25 from the group of a pharmaceutically acceptable surfactant, saccharide, protein and high-molecular weight compound.
The G-CSF to be contained in the pharmaceutical prep-aration of the present invention can be obtained by any of ' ~ ' ;, .

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the methods such as those described in the specifications of Japanese Patent Application Nos. 153273/1984, 269455/1985, 269456/1985, 270838/1985 and 270839/1985. For example, a human G-CSF can be prepared either by ~ultivating a cell strain (CNCM Accession Number I-315 or I-483) collected from tumor cells of patients with oral cavity cancer, or by expressing a recombinant DNA (which has been prepared by the agency of a human G-CSF encoding gene) in an appropriate host cell (e.g. E. coli, C 127 cell or ovary cells of a Chinese hamster).
Any human G-CSF that has been purified to high degree may be employed as the G-CSF to be contained in the pharma ceutical preparation of the present invention. Preferable human G-CSFs are ones obtained by isolation from the super-natant of the culture of a human G-CSF producing cell, and a polypeptide or glycoprotein having the human G-CSF activity that is obtained by transforming a host with a recombinant vector haviny incorporated therein a gene coding for a polypeptide having the human G-CSF activity.
Two particularly preferable examples of humall G-CSF
are shown below:
(1) human G-CSF having the following physicochemical properties:
i) molecular weight: about 19,000 + 1,000 as measured by electrophoresis through a sodium dodecylsulfate - polyacrylamide gel;
ii) isoelectric point: having at least one of the ~hree isoelectric points, pI = 5.5 + 0.1, pI = 5.8 + 0.1, and pI = 6.1 ~ 0.1;

~2~ 7 iii) ultraviolet absorption: having a maximum absorption at 280 nm and a minimum absorption at 250 nm;
iv) amino acid sequence of the 2:L residues from N terminus:
H2N-Thr-~ro-Leu-Gly-Pro-A:La-Ser-Ser-Leu-Pro-Gln-Ser-Phe-Leu-Leu-Lys-Cys-Leu-GlU-Gln-Val (2) human G-CSF containing either a polypeptide having the human granulocyte stimulatillg factor activity which is represented by all or part oE the amino acid sequence shown below, or a glycoprotein having both said poly-peptide and a sugar chain portion:
(Met)nThr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu (Val Ser Glu~mCys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly ~ Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (provided that m is 0 or 1; and n is 0 or 1).

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For details of the method for preparing these two types of G-CSF, see the specification of Japanese Patent Application Nos. 153273/1984, 269455/1985~ 269456/1985, 270838/1985 and 270839/1985/ all having been filed by the assignee oF the present invention.
Another method that can be employed consists of perEorming fusion of a G-CSF producing cell with a self-proliFerating malignant tumor cell and c~31tivating the res~lting hybridoma in the presence or absence of mytogen.
The human G-CSF containing solution obtained may be stored in a frozen state after being further purified and concentrated~ as required, by any known technique. Alterna-tively, the solution may be stored after being dehydrated by such means as freeze-drying.
All of the human G-CSFs thus prepared can be processed as specified by the present invention in order to attain stable G-CSF containing pharmaceutical preparations.
Typical examples of the surfactant that is used to attain the stable G-CSF containing pharmaceutical prepara-tion of the present invention are listed below: nonionicsurfactants with HLB of 6 - 18 such as sorbitan aliphatic acid esters (e.g. sorbitan monocaprylate/ sorbitan mono-laurate and sorbitan monopalmitate~, glycerin aliphatic acid esters (e.g. glycerin monocaprylate, glycerin monomyristate, and glycerin monostearate), polyglycerin aliphatic acid esters (e.g. decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate), polyoxyethylene sorbitan aliphatic acid esters (e.g. polyoxyethylene --7~
sorbitan monolaurate, polyoxyethylene sorbi~an monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol aliphatic acid esters (e.g. polyoxyethylene sorbi-tol tetrastearate and polyoxyethylene sorbitol tetraoleate), polyethylene glycerin aliphatic acid esters (e.g. polyoxy-ethylene glyceryl monostearate), polyethylene glycol aliphatic acid estexs (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g.
polyoxyethylene polyoxypropylene glycol ether, polyoxy-ethylene polyoxypropylene propyl ether, and polyoxyethylene polyoxypropylene cetyl ether)! polyoxyethylene alkylphenyl ethers (e.g. polyoxyethylene nonylphenyl ether), polyoxy-ethylated castor oil, polyoxyethylated hardened castor oil (polyoxyethylated hydrogenated castor oil), polyoxyethylated beeswax derivatives (e.g. polyoxyethylated sorbitol beeswax), polyoxyethylene lanolin derivatives (e9g. polyoxyethylene lanolin), and polyoxyethylene aliphatic acid amides (e.g.
polyethylene stearic acid amide); ncnionic surfactants such as alkyl sulfuric acid salts having a C10 - Cl~ alkyl group (e.g. sodium cetyl sulfate, sodium lauryl sulfate and sodium oleyl sulfate), polyoxyethylene alkyl ether sulfuric acid salts wherein the average molar number of ethylene oxide addition is 2 - 4 and the alkyl group has 10 - 18 carbon atoms (e.g. polyoxyethylene sodium lauryl sulfate), salts of alkyl sulfosuccinate esters wherein the alkyl group has 8 ---8--18 carbon atoms (e.g. sodium lauryl sulfosuccinate ester);
and natural surfactants such as lecithin, glycerophospho-lipid, sphingophospholipid (e.g. sphingomyelin), and sucrose aliphatic acid esters wherein the aliphatic acid has 12 - 18 carbon atoms. These surfactants may of course be used either independently or in admixture.
The surfactants listed above are preferably used in amounts of 1 - 10,000 parts by weight per part by weight of G-CSFo The saccharide to be used in making the stable G-CSF
containing pharmaceutical preparation of the present inven-tion may be selected from among monosaccharides, oligo-saccharides, and polysaccharides, as well as phosphate esters and nucleotide derivatives thereof so long as they 15 are pharmaceutically acceptable. Typical examples are listed below: trivalent and higher sugar alcohols such as glycerin! erythritol, arabitol/ xylitol, sorbitol, and mannitol; acidic sugars such as glucuronic acid, iduronic acid, neuraminic acid, galacturonic acid, gluconic acid, 20 mannuronic acid, ketoglycolic acid, ketogalactonic acid and ketogulonic acid; hyaluronic acid and salts thereof, chon-droitin sulfate and salts thereo, heparin, inulin, chitin and derivatives thereof, chitosan and derivatives thereof, dextrin! dextran with an average molecular weights of 5,000 25 - 150,000, and alginic acid and salts thereof. All of these saccharides may be used with advantage either independently or in admixture.

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The saccharides listed above are preferably used in amounts of 1 10,000 parts by weight per part by weight of G~CSF.
Typical examples of the protein to be used in makiny the stable G~CSF containin~ pharmaceutical preparation of the present invention include hurnan serum albumin, human serum globulin, gelatin, acid-treated gelatin (average mol.
wt. = 7,000 - 100,000)! alkali-txeated gelatin (average mol.
wt. = 7,000 - 100,000), and collagen. Needless to say, these proteins may be used either independently or in admixture.
The proteins listed above are preferably used in amounts of 1 - 20,000 parts by weight per part by weight of G-CSF.
Typical examples of the high-molecular weigh compound to be used in making the stable C-CSF containing pharmaceu-tical preparation of the present invention include: natural polymers such as hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, and hydroxyethyl cellulose; and synthetic polymers such as polyethylene glycol (mol. wt. = 300 - 6,000)! polyvinyl alcohol (mol. wt.

= 20,000 - 100,000), and polyvinylpyrrolidone (mol. wt. =
.
20,000 - 100,000). Needless to say, these high-molecular weight compounds may be used either alone or in combinatlon.
The high-molecular weight compounds listed above are desirably used in amounts of 1 - 20,000 parts by weight per part by weight of G-CSF.

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-lU-In addition to the surfactant, saccharide, protein or high-molecular weight compound described above, at least one member selected from the group consisting of an amino acid, a sulfureous reducing agent and an antioxidant may also be incorporated in making the G-CSF containing pharmaceutical preparation of the present invention. Illustrative amino acids include glycine, threonine, tryptophan, lysine, hydroxylysine, histidine, arginine, cysteine, cystine, and methionine. Illustrative sulfureous reducing agents include: N-acetylcysteine, N-acetylhomocysteine, thioctic acid! thiodiglycol, thioethanolamine, thioglycerol, thio-sorbitol, thioglycolic acid and salts thereof, sodium thio-sulfate, sodium hydrogensulfite, sodium pyrosulfite, sodium sulfite, thiolactic acid, dithiothreitol, glutathione, and a 15 mild sulfureous reducing agent having a sulfhydryl group such as a Cl - C7 thioalkanoic acid. Illustrative anti-oxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, dQ-a-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, 20 L-ascorbic acid stearate, triamyl gallate, propyl gallate and chelating agents such as disodium ethylenediaminetetra-acetate (EDTA), sodium pyrophosphate and sodium metapho~phate.
The above-listed amino acids, sulfureous reducing 25 agents and antioxidants or mixtures thereof are preferably used in amounts of 1 - 10,000 parts by weight per part by weight of G-CSE'.

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For the purpose of Eorm~lating the stable G-CSF
containing preparation of the present in~ention in a suitable dosage form~ one or more the following agents may be incorporated: a diluent, a solubilizing aidl an isotonic agent, an excipient, a pH modifier, a soothing agent, and a buffer.
The stabilized G-CSF pharmaceutical preparation o~
the present invention may be formulated either for oral administratio~ or for parenteral administration such as by injection applied in various ways' and a variety of dosage forms may be employed depending upon the specific mode of administration. Typical dosage forms include~ those intended for oral administration such as tablets, pills, J. capsules, granules and suspensions; solutions, suspensions 15 and freeze-dried preparations principally intended for intravenous injection' intramuscular injection, subcutaneous injection and intracutaneous injection; and those intended for transmucosal administration such as rectal supposito-ries' nasal drugs, and vaginal suppositories.
According to the present invention, at least oné
substance selected from the group consisting of a surfac-tant, a saccharide! a protein or a high-molecular weight compound is added to a G-CSF containing pharmaceutical preparation so that it is prevented from being adsorbed on 25 the wall of its container or a syringe while at the same time, it remains stable over a prolonged period of time.
The detailed mechanism by which the substances mentioned above stabilized G-CSF or prevent it from being : : ' ' ., . - -, .
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~7'~'7 adsorbed is yet to be clarified. In the presence of a surfactant, the surface of G-CSF which is a hydrophobic protein would be covered with the surfactant to become solubilized so that the G-CSF present in a trace amount is effectively prevented from being adsorbed on the wall of its container or a syringe. A saccharide or hydrophilic high-molecular weight compound would form a hydrated layer between G-CSF and the adsorptive surface of the wall of its container or a syringe, thereby preventing adsorption of G-CSF in an effective manner. A protein would compete with G-CSF for adsorption on the wall of its container or a syringe, thereby effectively inhibiting adsorption of G-CSF.
Besides the prevention of ~-CSF adsorption! the substances mentioned above would also contribute to the prevention of association or polymerization of the molecules of G-CSF. In the presence of a surfactant9 saccharide, protein or high-molecular weight compound, the individual molecules of G-CSF are dispersed in these substances and the interaction between the G CSF molecules is sufficiently reduced to cause a significant decrease in the probability of their association or polymerization. In addition, these substances would retard the autoxidation of G-CSF that is accelerated under high temperature or humidity or prevent G-CSF from being associated or polymerized as a result of its autoxidation. These effects of retarding autoxidation of G-CSF or preventing it from being associated or polyme-rized would be further enhanced by addition of an amino acid, a sulfureous reducing agent or an antioxidant.
The problems described above are particularly notice-able in solutions for injection and in suspensions but they 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 this latter case.
Through the addition of at: least one substance selected from the group consisting of a surfactant, saccha-ride, protein and a high-molecular weight compound, G-CSF is highly stabilized and maintains its activity for a prolonged period of time, as will be demonstrated in the examples that follow. To attain these results, the amount of each of these substances, in particular its lower limit, is critical 15 and the following ranges are desirable: 1 - 10~0~0 parts by weight of surfactant, 1 - 10,000 parts by weight of saccha-ride, 1 - 20,000 parts by weight of proteinl and 1 20,000 parts by weight of high-molecular weight compound, per 1 part by weight of G-CSF.
According to the present invention, a surfactant, a saccharide, a protein and/or a high-molecular weight compound is used in a specified concentration and this is effective not only in preventing G-CSF from being adsorbed on the wall of its container or a syringe but also in enhancing the stability of a G-CSF containing pharmaceutical preparation. As a result, it becomes possible to ensure the administration of a small but highly precise dose of G-CSF to patients; since G-CSF is costly, its efficient `~ : - - , : , .
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utilization will lead to lower costs for the production of G-CSF con'aining pharmaceutical preparations.
The follo~ing examples are provided for the purpose of further illustrating the present invention but are in no sense to be taken as limiting. Xn these examples, the residual activity of G-CSF was determined by one of the following methods.
(a) Soft agar method usina mouse bone marrow cells:
A horse serum (0.4 ml), 0"1 ml of the sample, 0.1 ml of a C3H/He (female) mouse bone marrow cell suspension (0.5 - 1 x 105 nuclear cells), and 0.4 ml of a modified McCoy's 5A culture solution containing 0.75% of agar were mixed, poured into a plastic dish for tissue culture (35 mm0), coagulated, and cultured for 5 days at 37C in 5% CO2/95%
air and at 100% humidity. The number of colonies formed was counted (one colony consisting of at least 50 cells) and the activity was determined with one unit being the activity for forming one colony~
The modified McCoy's 5A culture solution used in the method ~a) was prepared by the followiny procedures.
Modified Mc~oy's_SA culture solution (double concentrationL
Twelve grams of McCoy's 5A culture solution (Gibco), 2.55 g of MEM amino acid-vitamin medium (Nissui Seiyaku ~o., Ltd.), 2~18 g of sodium bicarbonate and 50,000 uniks of potassium penicillin G were dissolved twice in 500 ml of distilled water and the solution was aseptically filtered through a Millipore filter (0~22 ~m)O

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~15-(b) Reverse-phase high-perEormance liquid chromatoqraphy:
Using a reverse-phase C8 column (4.6 mrn x 300 mm;
5 ~m) and an n-propanol/trifluoroacetic acid mixture as a mobile phase, the residual activity of G-CSF (injected in an amount equivalent to 1 ~g) was determined under the follow-ing gradient conditions:
Time (secL Solvent (A) Solvent (B) Gradient 0 100% o%
3 linear 0% 100%
. linear 100% o%
Solvent (A): 30% n-propanol and 0.1% trifluoroacetic acid Solvent (B): 60% n-propanol and 0.1% trifluoroacetic acid Detection was conducted at a wavelength of 210 nm and the percentage of the residual G-CSF activity was calculated by the following formula:

the residual amount of G-CSF
Residual G-CSF _ after the lapse of a given time x 100 activity (%3 the initial amount of G-CSF
The residual amount of G CSF as determined by this method correlated very well with the result attained in measurement by the soft agar method (a~ using mouse bone marrow cells.
Example l To 5 ~9 of G-CSF/ one of the stabilizing agents listed in Table 1 was added and the mixture was aseptically : dissolved in a 20 mM buffer solution (containing 100 mM
sodium chloride; pH 7.4) to make a pharmaceutical prepara-tion containing 5 ~g of G-CSF per ml! which was then freezer-dried. The time-dependent change in G-CSF activity ~7(31~

was measured by method (a) and the results are shown in Table 1. The term "activity (~)" in the table represents the residual activity of G-CSF relative to the initial unit and is defined by the following :Eormula:
activity unit after Activity (%) = inlitPal actiavgty u-nit e x 100 Freeze-drying was conducted by the following procedures:
l'he G-CSF solution containing a stabili~ing agent was put into a sterile sulfa-treated glass vial, frozen at -40C
or below for 4 hours, subjected to primary drying by heating from -40C to 0C over a period of 48 hours with the pres-sure increased Erom 0.03 to 0.1 torr, then to secondary during by heating from 0C to 20C for a period of 12 hours with the pressure increased from 0.03 to 0.08 torr; there-after, the interior of the vial was Eilled with a sterile 15 dry nitrogen gas to attain an atmospheric pressure and the vial was plugged with a freeze-drying rubber stopper, then sealed with an alum.inum cap.

Table l .. _ -- .
Activ. ty (%) Stabilizing agent (parts by After After weight) storage at 37C for 6 months 1 month ___ , .. __ .. _I
xylitol 10,000 92 86 . ~ _ mannitol 10,000 91 85 . ~ .. . . _ glucuronic acid 10,000 86 82 __ ......... . ~_ _ .
hyaluronic acid 2,000 92 89 .. . _. . __ .
dextran (m.w. 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 2,000 97 95 . ~ .
alkali-treated gelatin 1,000 99 g6 .. ..__ collagen 2,000 95 90 ... . _ . ____ polyethylene glycol 10,000 94 90 . ~ . .__ . __ . _ hydroxypropyl cellulose 1,000 98 94 _ __ ................................... _ sodium carboxymethyl 1,000 88 80 . _ - ~ .... _ __~ ._ hydroxymethyl cellulose 5,000 92 90 _ _ ___ _ (m.w. 50~000) 2~000 96 95 . ._ (m.w. 50,000) 2,000 95 94 . . ___ .. _ .
human serum albumin2 t mannitol 2,000 100 97 cystelne ~00 _ _ _ _ _ . ,.

3~3 Table 1 (cont'd) _ _ _ _ Activity (%) Stabilizing agent mo ntAfter After weight) storage at 37C for 6 months 1 month . .._ .
human serum albumin 2,000 polyoxyethylene 100 99 96 sorbitan monolaurate mannitol 2,000 . ~ . _ human serum albumin2,000 hydroxypropyl cellulose 500 98 92 dextran (m.w. 40,000)2,000 . .__ _ , __ polyoxyethylene 100 sorbitan monolaurate 98 96 sorbitol 2,000 _ polyoxyethylated 100 hardened castor oil dextran (m.w. 40,000)2,000 .. ___ . .. _ _ . _ not added _ 74 58 Example 2 To 10 ~g of G-CSF, one of the stabilizing agents listed in Table 2 was added and the mixture was aseptically dissolved in a 20 mM phosphate buffer solution tcontaining 5 100 mM sodlum chloride; pH, 7.4) to make a pharmaceutical preparation containing 10 ~g of G-CSF per ml. The prepara- !
tion was aseptically charged into a sulfa~treated glass vial and sealed to make a G~CSF solution. The time-dependent change in the activity o~ G-CSF in this solution was 10 measured by the same method as used in Example 1 and the results are shown in Table 2.

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Table 2 _ _--Activity (%) Amount After After After Stabilizing agent (parts storage storag~ storage weight) for ; for 2 forRl days months month . .__ .____ _ _ mannitol - 5,000 91 87 82 . .. __ _ . . __ . _ hyaluronic acid 2,000 93 87 70 . ~ . . . __ . . .
dextran (m.w. 40,000) 2,000 96 95 85 . .. .. _ .. __ . _ glycerin 10,000 90 90 88 _ . . _ _ . _~
neuraminic acid 5,000 93 91 84 .. __ _ _ . _ . _ _ . ._ chitin 2,000 95 92 86 _ . _ __ . . ._ __ dextrin 2,000 90 92 87 ... ._ _ .... _ .. . _ _ human serum albumin 1,000 99 95 92 _ ._ .. ._ . __ human serum globulin1,000 98 94 90 . _ _ . _ _ __ acid-treated gelatin2/000 97 96 87 ~~ ..... ._ . _ alkali-treated gelatin 500 99 95 92 _ . . .. _ . _ . . . __ collagen 2,000 99 94 88 . __ . .. .__ : (m.w. 4,000) 10!000 94 89 90 , . _ _ , . __ _ _ hydroxypropyl cellulose 2,000 98 95 92 _ . _ . . . _ . __ ~ ~ sodium carboxymethyl 2,000 92 91 80 . .. .__ _ ..
: hy~roxyethyl cellulose 4,000 92 94 90 _ - __ _.. ~_ __ _ __ (olyvinyl alcohol 4~000 97 93 90 . ~. , _ . _.
(m.w. 50,000) 4~000 95 95 92 . . ~ . _ __._ ~
sorbitan monolaurate 400 37 96 95 . __ ._ ~_ _~ .. __ sorbitan monolaurate __ 96 ___ _ , .

Table 2 (cont'd) . .__ _ . _ . ___ Activity (%) Amount After After After Stabilizing agent (part storage storage storage weight) for ; for 2 ~aOtrRl days months month _ _ _ polyoxyethylene 400 98 97 polyoxyethylene polyoxypropylene 400 100 94 93 glycol ether __ __ polyoxyethylated 400 99 98 90 .... __ . .__ . _ .
sodium lauryl sulfate 2,000 97 93 87 ._ ~ .. ._ _ _ lecithin 2,000 97 94 90 ._ human serum albumin 2,000 mannitol 2~000 100 99 97 ,cysteine 100 _ ~_ __ human serum albumin 2,000 polyoxyethylene 100 99 97 95 mannitol 2,000 __ _ __ _ human serum albumin 1,000 hydroxypropyl cellulose 500 99 97 95 dextran ~(m.w. 40,000)2,000 _ polyoxyethylene 100 sorbitan monopalmitate 96 96 93 sorbitol 2,000 .___ _. - . _ __ .. .
polyoxyethylated 10 n hardened castor oil 95 92 92 dextran (m.w. 40~,000) 2,000 _ _ ___ not added 72 61 47 ' - :
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Example 3 To 10 llg of G-CSF, one of the stabilizing ayents listed 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 make a pharmaceutical preparation containing 10 ~g of G-CSF per ml. One milli-liter of the preparation was charged into a sulfa-treated silicone-coated glass vial and left at 4C. The effective-ness of each stabilizing agent in preventing G-CSF adsorp-tion was evaluated by measuring the residual activity of G-CSF in the solution after 0.5, 2 and 24 hours. The measurement was conducted by method (b) using reverse-phase high~performance liquid chromatography. The results are shown in Table 3.

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' . . ' 1~970CD~d ~~2-Table 3 _ __ _ Amount Residual activity (~) Stabilizing agent (parts welght) initial 0.5 h ~ h 24 h . _ _ monnitol 5,000 100 93 90 91 _ _ hyaluronic acid 2,000 100 97 92 92 _ _ . _ dextran (m.w. 40,000) 2,000 100 98 95 96 ... , .. ... _ .. __ .
glycerin 10,000 100 94 91 90 _ .... _ _ ~ _ heparin 2,000 100 92 90 90 . . _ .. _ . . __ . __ __ glucuronic acid 5,000 100 96 90 91 . . __ . __ _ .
ketoglycolic acid 5,000 100 92 88 90 human serum albumin 1,000 100 100 101 99 human serum globulin 1,000 100 98 100 98 . . _ __. . _ ,~ . _ alkali-treated gelatin 500 100 99 98 99 . ___ _ . __ r __ acid-treated yelatin 2,000 100 99 97 97 .___ ... . .
collagen 2,000 100 100 98 99 (m.w. 4,000) 10,000 100 100 100 99 . . . _ hydroxypropyl cellulose 2,000 100 100 100 99 _ __ ____ _ _ _ ____- . __. _. ._ _____ .
sodium carboxymethyl 2,000 100 98 96 95 .~ ._ ..~ __ hydroxyethyl cellulose 4,000 100 96 93 92 _. __ ....... _ _ p(olyvinyl alcohol 4,000 100 99 100 98 _ . _ . . .. .
(m.w. 50,000) 4,000 100 98 98 96 .. ___.
sorbitan monocaprylate 400 100 100 100 98 . .. ____ . _ _ sorbitan monostearate 400 100 100 98 100 . .__ _ hardened castor oil 400 100 99 101 99 .
' Table 3 (cont'd) .. __~ _ .
Amount Residual activity (%) Stabilizing agent (parts weight) inltial 0.5 h 2 h 24 h sodium lauryl sulfate2!000 100 100 . 97 lecithin 2,000 100 99 100 98 h~man serum albumin2,000 mannitol 2,000 100 100 100 101 cysteine 100 __ . ... _ _ _ ..
human serum albumin 2,000 polyoxyethylene 100 100 100 98 99 mannitol 2,000 _ _ __ . . . _ .. _ human serum albumin 1,000 hydroxypropyl cellulose500 100 101 99 100 dextran (m.w. 40r000)2,000 _ __ polyoxyethylene 100 sorbitan monolaurate sorbitol ~ 2,000 _ polyoxyethylated 100 hardened castor oil dextran (m.w. 40,000) 2,000 __ ,. ....
not added ___ 100 91 72 73 .

.
,

Claims

1. A stable granulocyte colony stimulating factor containing pharmaceutical preparation that contains, in addition to the granulocyte colony stimulating factor present as the effective ingredient, at least one substance selected from the group consisting of a pharmaceutically acceptable surfactant/ saccharide, protein and high-molecular weight compound.

2. A stable granulocyte colony stimulating factor containing pharmaceutical preparation according to Claim 1 which contains the surfactant in an amount of 1 - 10,000 parts by weight per part by weight of the granulocyte colony stimulating factor.

3. A stable granulocyte colony stimulating factor con-taininq pharmaceutical preparation according to Claim 1 or 2 wherein said surfactant is at least one member selected from the group consisting of a nonionic surfactant, an anionic surfactant and a natural surfactant, the nonionic surfactant being a sorbitan aliphatic acid ester, a glycerin aliphatic acid ester! a polyglycerin aliphatic acid ester, a polyoxy-ethylene sorbitan aliphatic acid ester, a polyoxyethylene sorbitol aliphatic acid ester, a polyoxyethylene qlycerin aliphatic acid ester, a polyethylene glycol aliphatic acid ester! a polyoxyethylene alkyl ether, a polyoxyethylene polyoxypropylene alkyl ether, a polyoxyethylene alkylphenyl ether, a polyoxyethylated hardened castor oil, a polyoxy-ethylated beeswax derivative, a polyoxyethylene lanolin derivative, or a polyoxyethylene aliphatic acid amide, the anionic surfactant being an alkyl sulfate salt, a polyoxy-ethylene alkyl ether sulfate salt, or an alkyl sulfosucci-nate ester salt, and the natural surfactant being lecithin glycerophospholipid, sphingophospholipid, or a sucrose aliphatic acid ester.

4. A stable granulocyte colony stimulating factor containing pharmaceutical preparation according to Claim 1 which contains the saccharide in an amount of 1 - 10,000 parts by weight per part by weight of the granulocyte colony stimulating factor.

5. A stable granulocyte colony stimulating factor con-taining pharmaceutical preparation according to Claim 1 or 4 wherein said saccharide is at least one member selected from the group consisting of glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, glucuronic acid, iduronic acid, galacturonic acid, neuraminic acid, glyconic acid, man-nuronic acid, ketoglycolic acid, ketogalactonic acid, keto-gulonic acid, hyaluronic acid and salts thereof, chondroitin sulfate and salts thereof, heparin, inulin, chitin and derivatives thereof, chitosan and derivatives thereof, dextrin, dextran with an average molecular weight of 5,000 -150,000, and alginic acid and salts thereof.

6. A stable granulocyte colony stimulating factor containing pharmaceutical preparation according to Claim 1 which contains the protein in an amount of 1 - 20,000 parts by weight per part by weight of the granulocyte colony stimulating factor.

7. A stable granulocyte colony stimulating factor containing pharmaceutical preparation according to Claim 1 or 6 wherein said protein is at least one member selected from the group consisting of human serum albumin, human serum globulin, gelatin, acid- or alkali-treated gelatin with an average molecular weight of 7,000 100,000, and collagen.

8. A stable granulocyte colony stimulating factor containing pharmaceutical preparation according to Claim 1 which contains the high-molecular weight compound in an amount of 1 - 20,000 parts by weight per part by weight of the granulocyte colony stimulating factor.

9. A stable granulocyte colony stimulating factor containing pharmaceutical preparation according to Claim 1 or 8 wherein said high-molecular weight compound is at least one member selected from the group consisting of hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, poly-ethylene glycol 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.

10. A process for producing a stable pharmaceutical preparation containing granulocyte colony stimulating factor (G-CSF), which comprises the steps of:

a) mixing at a temperature of from 1 to 30° C the G-CSF
with at least one substance selected from the group consisting of a pharmaceutically acceptable surfac--tant in an amount of 1 - 10,000 parts by weight, a saccharide in an amount of 1 - 10,000 parts by weight, a protein in an amount of 1 - 20,000 parts by weight or a high molecular weight compound in an amount of 1 - 20,000 parts by weight per part of the G-CSF;
b) dissolving the mixture in a buffer solution;
c) sterilizing the solution; and d) charging a vessel with the solution.