AU611856B2

AU611856B2 - Stable pharmacuetical preparation containing granulocyte colony stimulating factor and process for producing the same

Info

Publication number: AU611856B2
Application number: AU75665/87A
Authority: AU
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: 1991-06-27
Also published as: SE8702907L; DK368387D0; NL192917C; YU134287A; SE8702907D0; AT402259B; NO872966L; CA1297007C; HK64893A; CN1033738C; NO171828C; CN87104963A; DE3723781A1; KR880001297A; YU47543B; NL192917B; GB2193631B; BE1000253A3; HU198627B; IT8767594A0

Description

PHILLIPS ORMONDE AND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia ii

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STUART TAYLOR P17/11/79

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Application Number: Lodged: Class Int. Class 6118.56 Complete Specification Lodged: Accepted: Published: Priority a aet

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Related Art: *0 0 a a Fit APPLICANT'S REFERENCE: FP/C-1-995

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t Name(s) of Applicant(s): Chugai Seiyaku Kabushiki Kaisha Address(es) of Applicant(s): 5-1, Ukima, Kita-Ku,, Tokyo,,

JAPAN.

Address for Service is: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mart: Attorneys 367 Collins St.ret Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: STABLE PHARMACEUTICAL PREPARATION CONTAINING GRANULOCYTE COLONY STIMULATING FACTOR AND PROCESS FOR THE SAME Our Ref 61008 POF Code: 1594/64971 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): a j a 6003q/1 1 x XJ-I^JJLL Vivi± -(iqJL. O4 rll rld. I Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia STABLE PHARMACEUTICAL PREPARATION CONTAINING GRANULOCYTE COLONY STIMULATING FACTOR AND PROCESS FOR PRODUCING THE SAME The present invention relates to a pharmaceutical preparation containing a granulocyte colony stimulating CC -csw) factor. In particular, the present invention relates to a stabilized pharmaceutical preparation containing a granulocyte colony stimulating factor that is protected against loss or inactivation of the active component granulocyte colony stimulating factor) due to adsorption on the wall of a container in which the preparation is put, or to t association, polymerization or oxidation of said component.

Chemotherapy has been undertaken as one method for a°o treating a variety of infectious diseases but it has recently been found that chemotherapy causes some serious oo 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 j a substance that activates the prophylactic capabilities of the host of an infection-causing organism and thereby providing a complete solution to the aforementioned problems of chemotherapy. Of the various prophylactic capabilities of 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 capabilities of the host by promoting the growth of neutrophiles and -2their 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 application 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 intensive efforts are being made to use a drug substance that is capable of activating the prophylactic functions of the host ti 4 or the person who has been infected.

Needless to say, G-CSF displays by itself the ability tD oo to activate the prophylactic functions of the host and it has also been found that G-CSF exhibits greater therapeutic o 15 effects in clinical applications if it is used in combinaoC tion with a substance that activates the prophylactic o capabilities of the host.

G-CSF is used in a very small amount and a pharmaceutical preparation containing 0.1 500 gg (preferably 5 50 gg) 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 pharmaceutical agent or necessitates the incorporation of G-CSF

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-3- S* 0 *a 0e 0 0 00 00 0 0 o0 00 o o 0 01 Sa 00 0 o 00 0 00 0 00 0 00 0 00 iU o li 4 0 in a more-than-necessary amount making allowance for its possible loss due to adsorption.

In addition, G-CSF is labile and highly susceptible 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 association, polymerization and oxidation and suffers a great loss in activity. These phenomenon make it difficult to ensure complete accomplishment of a therapeutic act by administer- 10 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-CSF and at least one substance selected from the group of a pharmaceutically acceptable surfactant, saccharide, protein and high-molecular weight compound.

The G-CSF to be contained in the pharmaceutical preparation of the present invention can be obtained by any of ~i -4the 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 cultivating a cell strain (CNCM Accession Number 1-315 or 1-483) 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 coli, C 127 cell or ovary cells of a L 0 10 Chinese hamster).

00 Any human G-CSF that has been purified to high degree may be employed as the G-CSF to be contained in the pharma- :0 ceutical preparation of the present invention. Preferable human G-CSFs are ones obtained by isolation from the super- °o 15 natant of the culture of a human G-CSF producing cell, and a oo polypeptide or glycoprotein having the human G-CSF activity .0 that is obtained by transforming a host with a recombinant vector having incorporated therein a gene coding for a polypeptide having the human G-CSF activity.

Two particularly preferable examples of human G-CSF are shown below: 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 three isoelectric points, pi 5.5 0.1, pi 5.8 0.1, and pI 6.1 0.1; i

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I- iii) ultraviolet absorption: having a maximum absorption at 280 nm and a minimum absorption at 250 nm; iv) amino acid sequence of the 21 residues from N terminus:

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2 N-Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser- Phe-Leu-Leu-Lys-Cys-Leu-Glu-Gln-Valhuman G-CSF containing either a polypeptide having the human granulocyte stimulating factor activity which is represented by all or part of the amino acid sequence 0 shown below, or a glycoprotein having both said polypeptide and a sugar chain portion: 1 1 S0o oa ao 0 at a O o a 0 a1 a o 9 a at 0 0 0 4 9 0 0 a a a o I a a (Met) nThr Gin Ser Arg Lys Glu Lys Leu Cys His Ser Ser Cys Cys Leu Tyr Gin Ser Pro Gin Leu Trp Gln Ala Leu Phe Ala Val Leu Glu Val Gin Pro Phe Ile Leu His Leu Pro Ser Gly Glu Asp Gin Gin Ser Val Ser Leu Gln (Val Pro Gly Ser Gin Leu Leu Val Met Pro Ala Ala Tyr Leu Gly Ser Glu Ile Gin Leu Leu Gly Ala Glu Thr Phe Ser Arg Lys Cys Asp Gly Glu)mCys Glu Leu Pro Trp Ala Leu has Ser Gin Ala Pro Thr Asp Phe Glu Leu Gin Gly Gin Arg His Leu Val Leu Pro Leu Gly Pro Ala Ser Leu Ala Ala Val Ala Gin Gly Leu Leu Ala Gly Ala Arg Gin Arg Ser Leu Glu Gin Ala Leu Thr Tyr Leu Leu Pro Leu Leu Ala Leu Phe Glu Gly Asp Thr Thr Thr Met Ala Met Pro Ala Gly Ser Phe His Leu Pro Val Gln Lys Gly Ser Gly Leu Ile Leu Ile Pro Ala Gly Leu Ala (provided that m is 0 or 1; and n is 0 or 1).

-6- 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 performing fusion of a G-CSF producing cell with a selfproliferating malignant tumor cell and cultivating the resulting hybridoma in the presence or absence of mytogen.

t it T The human G-CSF containing solution obtained may be stored in a frozen state after being further purified and .i O concentrated, as required, by any known technique. Alternaasa tively, the solution may be stored after being dehydrated by such means as freeze-drying.

15 All of the human G-CSFs thus prepared can be o 4.

o. 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- 20 tion of the present invention are listed below: nonionic surfactants with HLB of 6 18 such as sorbitan aliphatic acid esters sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate), glycerin aliphatic acid esters glycerin monocaprylate, glycerin monomyristate, and glycerin monostearate), polyglycerin aliphatic acid esters decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate), polyoxyethylene sorbitan aliphatic acid esters polyoxyethylene -7sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene eorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitol aliphatic acid esters polyoxyethylene sorbitol tetrastearate and polyoxyethylene sorbitol tetraoleate), polyethylene glycerin aliphatic acid esters polyoxyethylene glyceryl monostearate), polyethylene glycol aliphatic acid esters polyethylene glycol distearate), polyoxyethylene alkyl ethers polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g.

polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, and polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers polyoxyethylene nonylphenyl ether), polyoxyethylated castor oil, polyoxyethylated hardened castor oil (polyoxyethylated hydrogenated castor oil), polyoxyethylated beeswax derivatives polyoxyethylated sorbitol beeswax), polyoxyethylene lanolin derivatives polyoxyethylene lanolin), and polyoxyethylene aliphatic acid amides (e.g.

polyethylene stearic acid amide); nonionic surfactants such as alkyl sulfuric acid salts having a C 1 0

C

1 8 alkyl group 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 polyoxyethylene sodium lauryl sulfate), salts of alkyl sulfosuccinate esters wherein the alkyl group has 8 I I j, I e -8- 0 00 os 0 000 0 0 00 00 0 0000 0 00 00 0 I, (0 00 000 0 00~ 00 0 00 00OI 00J 0 00 0 00l 0 00 00 0l 000 *C I 18 carbon atoms sodium lauryl sulfosuccinate ester); and natural surfactants such as lecithin, glycerophospholipid, sphingophospholipid 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-CSF.

10 The saccharide to be used in making the stable G-CSF containing pharmaceutical preparation of the present invention may be selected from among monosaccharides, oligosaccharides, 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, chondroitin sulfate and salts thereof, heparin, inulin, chitin and derivatives thereof, chitosan and derivatives thereof, dextrin, dextran with an average molecular weights of 5,000 150,000, and alginic acid and salts thereof. All of these saccharides may be used with advantage either independently or in admixture.

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S a 9,9 4 a, 9a o 99$, 7 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 making the stable G-CSF containing pharmaceutical preparation of the present invention include human serum albumin, human serum globulin, gelatin, acid-treated gelatin (average mol.

wt. 7,000 100,000), alkali-treated gelatin (average mol.

wt. 7,000 100,000), and collagen. Needless to say, 10 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.

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6 a *4 1 to be used in making the stable G-CSF containing pharma utical preparation of the present invention include- natural polymers such as hydroxypropyl cellulose, hy rxymethyl cellulose, sodium carboxymethyl cellulo and hydroxyethyl cellulose; and synthetic polymers bch as polyethylene glycol (mol. wt. 300 6,00 polyvinyl alcohol (mol. wt.

20,000 100,000), andolyvinylpyrrolidone (mol. wt. 20,000 100,000). eedless to say, these high-molecular weight compoun may be used either alone or in combination.

Th igh-molecular weight compounds listed above are desi bly used in amounts of 1 20,000 parts by weight per rLtby weight of G-CSF.

1 9a A high-molecular weight compound as used herein the claims and descriptions and in making the stable G-CSF containing pharmaceutical preparation of the present invention are natural polymers, 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 combination.

a 0 0o f o 00 0 0 0 000 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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9000 00« 40 0 L i 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 10 include: N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thio- 0p44 sulfate, sodium hydrogensulfite, sodium pyrosulfite, sodium sulfite, thiolactic acid, dithiothreitol, glutathione, and a 15 mild sulfureous reducing agent having a sulfhydryl group ,o such as a C C thioalkanoic acid. Illustrative antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, d9-a-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, 4,4 o 20 L-ascorbic acid stearate, triamyl gallate, propyl gallate 444444 Sand chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.

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

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0 40 4&u~ 4 4 4L 1 4 rr 44,4 -11- For the purpose of formulating the stable G-CSF containing preparation of the present invention in a suitable dosage form, one or more the following agents may be incorporated: a diluent, a solubilizing aid, an isotonic agent, an excipient, a pH modifier, a soothing agent, and a buffer.

The stabilized G-CSF pharmaceutical preparation of the present invention may be formulated either for oral administration or for parenteral administration such as by 10 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, capsules, granules and suspensions; solutions, euspensions 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 suppositories, nasal drugs, and vaginal suppositories.

20 According to the present invention, at least one substance selected from the group consisting of a surfactant, 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 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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-12adsorbed 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 G-CSF adsorption, the S 15 substances mentioned above would also contribute to the prevention of association or polymerization of the molecules of G-CSF. In the presence of a surfactant, 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. The. effects of retarding autoxidation of G-CSF or preventing it from being associated or polymerized would be further enhanced by addition of an amino -13- .c- 00 98 o 00o 0 0 0 006 0 009i 0 l1 0 0 9 a 0 «g 000 0 40 0 0 £9 £4 0 t 1 *t s I 1 t c acid, a sulfureous reducing agent or an antioxidant.

The problems described above are particularly noticeable 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, saccharide, 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 and the following ranges are desirable: 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 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 ii

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4 4 44 r ,I 444, r 44 a+ o+« oi+* r cr 4 4 4I 044 4 I4 4 4 4 4 4 4 f i t i 1 1 i I. -14utilization will lead to lower costs for the production of G-CSF containing pharmaceutical preparations.

The following examples are provided for the purpose of further illustrating the present invention but are in no sense to be taken as limiting. In these examples, the residual activity of G-CSF was determined by one of the following methods.

Soft agar method using 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 1 x 10 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 mm coagulated, and cultured for 5 days at 37 0 C in 5% CO 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 was prepared by the following procedures.

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 medium (Nissui Seiyaku Co., Ltd.), 2.18 g of sodium bicarbonate and 50,000 units 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 im).

i~c~U i i 7 9944 rt 9 9 4( 94 Reverse-phase high-performance liquid chromatography: Using a reverse-phase C8 column (4.6 mm x 300 mm; 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 following gradient conditions: Time (sec) Solvent Solvent Gradient 0 100% 0% linear 0% 100% linear linear 25 100% 0% linear Solvent 30% n-propanol and 0.1% trifluoroacetic acid Solvent 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 activity the initial amount of G-CSF x 100 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 using mouse bone marrow cells.

20 Example 1 To 5 pg 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 preparation containing 5 pg of G-CSF per ml, which was then freezer-dried. The time-dependent change in G-CSF activity 9 o 999 0, 94 9 49 9 99~ 40 9 9 9r 444 4 4 44 I~ 9 944 -16was measured by method 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 formula: activity unit after Activity the lapse of a given time 100 initial activity unit Freeze-drying was conducted by the following procedures: The G-CSF solution containing a stabilizing agent was put into a sterile sulfa-treated glass vial, frozen at -40 0

C

or below for 4 hours, subjected to primary drying by heating 10 from -40 0 C to 0°C over a period of 48 hours with the pressure increased from 0.03 to 0.1 torr, then to secondary during by heating from 0'C to 20 0 C for a period of 12 hours with the pressure increased from 0.03 to 0.08 torr; thereafter, the interior of the vial was filled with a sterile dry nitrogen gas to attain an atmospheric pressure and the vial was plugged with a freeze-drying rubber stopper, then sealed with an aluminum cap.

I 4 4 4 o 4o .sr 4 o 4 a a 4a 4 0 44 44 I 0 44 44 a 4a o 44s 4 44 04 a 00; I a: -17- Table 1 Activity Amount After After Stabilizing agent (parts by s storage at storage at weight) 4 0 C for 37 0 C for 6 months 1 month xylitol 10,000 92 86 mannitol 10,000 91 glucuronic acid 10,000 86 82 hyaluronic acid 2,000 92 89 dextran 40,000) 2,000 95 heparin 5,000 85 chitosan ?,000 93 91 alginic acid 2,000 90 human serum albumin 1,000 98 99 human serum globulin 1,000 98 acid-treated gelatin 2,000 97 alkali-treated gelatin 1,000 99 96 collagen 2,000 95 90 polyethylene glycol 10,000 94 4,000) hydroxypropyl cellulose 1,000 98 94 sodium carboxymethyl 1,000 88 cellulose hydroxymethyl cellulose 5,000 92 polyvinyl alcohol 2,000 96 50,000) polyvinylpyrrolidone 2,000 95 94 50,000) human serum albumin 2,000 mannitol 2,000 100 97 cysteine 100 i ii: Ir 1:

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1'> I7 t -18- 4 4 *a 4 444 4 4 44 *a 4 44,, o I4 os 4 44"s 4 44 44 44 4 4 4 t44 44 te~ Table 1 (cont'd) Activity Amount Amount After After Stabilizing agent (parts by s storage at storage at weight) 4 0 C for 370C for 6 months 1 month human serum albumin 2,000 polyoxyethylene 100 99 96 sorbitan monolaurate mannitol 2,000 human serum albumin 2,000 hydroxypropyl cellulose 500 98 92 dextran 40,000) 2,000 polyoxyethylene 100 sorbitan monolaurate 98 96 sorbitol 2,000 polyoxyethylated 100 hardened castor oil 94 92 dextran 40,000) 2,000 not added 74 58 Example 2 To 10 gg 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 (containing 5 100 mM sodium chloride; pH, 7.4) to make a pharmaceutical preparation containing 10 pg of G-CSF per ml. The preparation was aseptically charged into a sulfa-treated glass vial and sealed to make a G-CSF solution. The time-dependent change in the activity of G-CSF in this solution was measured by the same method as used in Example 1 and the results are shown in Table 2.

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444 4 I 4 4I 44 I 44 44 -19- Table 2 Activity Amount Amunt After After After Stabilizing agent (parts storage storage storage by at 4 0 C at 4 0 C at RT weight) for 7 for 2 for 1 days months month mannitol 5,000 91 87 82 hyaluronic acid 2,000 93 87 dextran 40,000) 2,000 96 95 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 globulin 1,000 98 94 acid-treated gelatin 2,000 97 96 87 alkali-treated gelatin 500 99 95 92 collagen 2,000 99 94 88 polyethylene glycol 10,000 94 89 4,000) hydroxypropyl cellulose 2,000 98 95 92 sodium carboxymethyl 2,000 92 91 cellulose hydroxyethyl cellulose 4,000 92 94 polyvinyl alcohol 4,000 97 93 50,000) polyvinylpyrrolidone 4,000 95 95 92 50,000) sorbitan monolaurate 400 97 96 polyoxyethylene 400 100 96 94 sorbitan monolaurate 4 I -20- L 4 14 I 4 44'.

4 #4 4* 4 4444 44 44 4 4*4 4 44 o 4 4 444 444 44 4 444 4 0 ~4 4 44 44 4 4 Ii *444 o 44 44 *4.4 4 44441 *4 Table 2 (cont'd) Activity(% Amount After After After Stabilizing agent (parts storage storage storage by at 4 0 C at 4 0 C at RT weight) for 7 for 2 for 1 days months month polyoxyethvlene 409 79 sorbitan monostearate 409 79 polyoxyethylene polyoxypropylene 400 100 94 93 glycol ether polyoxyethylated 400 99 98 9C hardened castor oil sodium lauryl sulfate 2,000 97 93 87 lecithin 2,000 97 94 human serum albumin 2,000 mannitol 2,000 100 99 97 cysteine 100 human serum albumin 2,000 polyoxyethylene 109 79 sorbitan monolaurate 109 79 mannitol 2,000 human serum albumin 1,000 hydroxypropyl cellulose 500 99 97 dextran 40,000) 2,000 sorbitan monopalmitate 96 96 93 sorbitol 2,000 hardened castor 92 92 dextran 40,000) 2,000 not added -72 61 47 -21- Example 3 To 10 pg of G-CSF, one of the stabilizing agents 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 gg of G-CSF per ml. One milliliter of the preparation was charged into a sulfa-treated silicone-coated glass vial and left at 4 0 C. The effectiveness of each stabilizing agent in preventing G-CSF adsorption was evaluated by measuring the residual activity of 2 G-CSF in the .solution after 0.5, 2 and 24 hours. The measurement was conducted by method using reverse-phase high-performance liquid chromatography. The results are 01. 5 shown in Table 3.

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4 1 71 0 0 ,0 000 000 6 a a tea atd~ *h 0 0I 1O It -22- Table 3 Amount Residual activity Stabilizing agent (parts by initial 0.5 h 2 h 24 h weight) monnitol 5,000 100 93 90 91 hyaluronic acid 2,000 100 97 92 92 dextran 40,000) 2,000 100 98 95 96 glycerin 10,000 100 94 91 heparin 2,000 100 92 90 glucuronic acid 5,000 100 96 90 91 ketoglycolic acid 5,000 100 92 88 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 acid-treated gelatin 2,000 100 99 97 97 collagen 2,000 100 100 98 99 polyethylene glycol (m.w.polyethylene glycol4,000) 10,000 100 100 100 99 4,000) hydroxypropyl cellulose 2,000 100 100 100 99 sodium carboxymethyl cellulose 2,000 1 00 98 96 hydroxyethyl cellulose 4,000 100 96 93 92 polyvinyl alcohol (m.w.poly l a l 4,000 100 99 100 98 50,000) polyvinylpyrrolidone 50,000) 4,000 1 0 0 98 98 96 50,000) sorbitan monocaprylate 400 100 100 100 98 polyoxyethylene sorbitan monostearate 400 00 00 98 00 polyoxyethylated hardened castor oil 400 100 99 101 99 Cl tC I LI -23- LI LILI LI~ LI LI~4 LI LI LILI LI LI LI LI LI I LILI LI LI p., LI LILI LI LI LI LILILI LI

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LI LIII Table 3 (cont'd) Amount Residual activity() Stabilizing agent (parts by initial 0.5 h 2h 2 weight) sodium lauryl sulfate 2,000 100 100 99 97 lecithin 2,000 100 99 100( 98 human serum albumin 2,000 mannitol 2,000 100 100 100 101 cysteine 100 human serum albumin 2,000 polyoxyethylene1010 10 98 9 sorbitan monolaurate1010 10 98 9 mannitol 2,000 human serum albumin 1,000 hydroxypropyl cellulose 500 100 101 99 100 dextran 40,000) 2,000 sorbitan 100 100 99 99 sorbitol 2,00 0 hardened castor 100 100 98 97 dextran 40,000) 2,000 not added 7 100 91 72~ 73

Claims

1. A stable granulocyte colony stimulating factor (G-CSF) containing pharmaceutical preparation that contains, in addition to a highly purified granulocyte colony stimulating factor having human granulocyte stimulating factor activity characterized by a molecular weight in the range of 18,000 to 20,000 daltons on SDS PAGE 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 as hereinbefore defined. €1 tI Ir t t ALI 7 I A *e 0 0* A r AtA tlA -24- f Tihe--s en-o are-a Ifll4w 'I 1. A stable granulocyte colony stimulating fact containing pharmaceutical preparation that ntains, in addition to the granulocyte colon imulating factor present as the effectivej redient, at least one substance selected from th eroup consisting of a pharmaceutically accepta surfactant, saccharide, protein and high- lnfppilar wjight componnd,

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.

A stable granulocyte colony stimulating factor con- taining 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 glycerin 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 4 i 4 6C 6 I. 1. IU r/0 H r/- 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. 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 S" sulfate and salts thereof, heparin, inulin, chitin and derivatives thereof, chitosan and derivatives thereof, dextrin, dextran with an average molecular weight of 5,000 S150,000, and alginic acid and salts thereof.

J 1

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. I-- -26-

7. A stable granulocyte colony stimulating factor con- taining 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 a 4 a the granulocyte colony stimulating factor, l

9. A stable granulocyte colony stimulating factor con- taining 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 i cellulose, hydroxyethyl cellulose, polyethylene glycol with a molecular weight of 300 6,000, polyvinyl alcohol with a 6 molecular weight of 20,000 100,000, and polyvinylpyrroli- done with a molecular weight of 20,000 100,000. A---eees s--f--pr-GdG4inRg a -abie granulocyte colony stimulating factor containing pharmaceutical preparati n that contains, in addition to the granulocyte ony stimulating factor present as the effec e ingredient, at least one substance selected fro he group consisting of a pharmaceutically acceptab esurfactant, saccharide, protein and high-molecular ight compound.

P|L PHILLIPS MONDE AND FITZPATRICK SC AI SEIYAKU KABUSHIKI KAISHA fr4 4 B r~nu Sf 26a A process for producing a stable granulocyte colony stimulating factor (G-CSF) containing pharmaceutical preparation that contains, in addition to a highly purified granulocyte colony stimulating factor having human granulocyte stimulating factor activity characterized by a molecular weight in the range of 18,000 to 20,000 daltons on SDS PAGE 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 as hereinbefore defined.

11. A stable granulocyte i ony stimulating factor containing pharmaceutical preparation as claimed in claim 1 substantially as hereinbefore described with reference to any one of the examples. o*

12. A process as claimed in claim 10 substantially as o° hereinbefore described with reference to any one of the examples. DATED: 5 September 1990 PHILLIPS ORMONDE FITZPATRICK Attorneys for: A CHUGAI SEIYAKU KABUSHIKI KAISHA EJD 'i 11 Z IJ