JP2002371009A - Method for extending duration of pharmacological effect of granulocyte colony stimulating factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for extending the pharmacological effect duration of granulocyte colony stimulating factor (G-CSF), and a low-viscosity water-soluble composition having an extended pharmacological effect duration of G-CSF. To provide. SOLUTION: The present invention provides a method for extending the pharmacological effect duration of G-CSF, characterized by including a surfactant in a G-CSF-containing water-soluble composition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for extending the pharmacological effect duration of granulocyte colony stimulating factor (hereinafter abbreviated as G-CSF), and a water-soluble method for extending the pharmacological effect duration of G-CSF. Composition.

[0002]

BACKGROUND OF THE INVENTION Various attempts have been made to reduce the number of administrations by maintaining the blood level of a therapeutically effective amount of a protein in a therapeutically effective range for a longer period of time and extending the duration of its pharmacological effect. For example, the administration cycle of drugs in general cancer chemotherapy is as short as about 3 to 4 days, but G-CSF in cancer chemotherapy for neutropenia.
The dosing cycle is generally as long as one day. Various attempts have been made to formulate a continuous preparation of a protein requiring such frequent administration.

For example, Japanese Patent Application Laid-Open No. 2-213 discloses a sustained-release preparation of a hyaluronic acid-containing physiologically active peptide utilizing diffusion delay caused by increasing the viscosity of a drug solution. Several other hyaluronic acid-containing sustained release preparations have been disclosed (JP-A-5-97694, JP-A-2-231429, etc.), but a high concentration of hyaluronic acid is necessary to obtain sufficient durability. As a result, the drug solution has a high viscosity, and has problems such as viscosity control at the time of use, air bubbles at the time of administration, and limitation of the thickness of the injection needle.

[0004] Further, a system for obtaining a sustained-release effect by gelling at a site of administration using a temperature-sensitive polymer is also known. As a base for this, for example, US Pat. No. 4,188,373 and US Pat. (Pluronics), International Publication WO99 / 18142, poly (lactide-glycolide) copolymer [poly (lactide-co-gly)
coli) and a polyethylene glycol polymer (polyethylene glycol polymer). However, in vivo gelled preparations using these bases must be used with the temperature of the preparation controlled below the gelling temperature so that the viscosity of the drug solution during administration does not become too high to make administration with an injection needle impossible. Must. This problem is the same in the process of formulation, and sufficient temperature control is required to prevent gelation during production. In addition, there is a concern that tissues at the administration site may be damaged by physical stress due to gelation of the preparation at the administration site. Furthermore, in order to gel at the administration site, these polymers must be contained at a high concentration, and there is a concern about toxicity to the living body.

Further, sustained-release microcapsule injections based on poorly water-soluble polymers, and emollient-type sustained-release preparations using a molded matrix are also known. For example, G-CSF
About Japanese Patent Publication No. Hei 7-25689, G-CSF microcapsule sustained-release preparations based on in vivo histocompatibility polymer substances, and JP-A-6-321803 based on biodegradable polymers Disclosed is a shaped-in, recessed G-CSF sustained release formulation, respectively. However, with respect to sustained-release preparations using these poorly water-soluble polymers, formulation techniques have been an issue. For example, in general, an organic solvent must be used for dissolving the base in the formulation, so it is extremely difficult to formulate a protein or the like which has a strong tendency to be inactivated or denatured by the organic solvent, and the solvent in the formulation is extremely difficult. Consideration must also be given to the residue of waste. In addition, in the art, the duration of the effect is generally set to one week or more, which is always consistent with the currently practiced cycle of, for example, twice-weekly cancer chemotherapy. Not something.
Surfactants are known to exhibit a protein stabilizing effect by appropriately setting the amount of addition.For example, JP-A-2000-504696 discloses a formulation of a combination of growth hormone and pluronic polyol, and G- Regarding CSF, Patent 2577742 discloses a stable preparation comprising natural G-CSF and various surfactants such as polyoxyethylene sorbitan monofatty acid ester.However, regarding the sustained pharmacological effect of these preparations or preparations, unknown.

[0006]

The object of the present invention is to provide a G-CS
Providing a way to extend the pharmacological effect duration of F;
Another object of the present invention is to provide a low-viscosity water-soluble composition having a prolonged pharmacological effect duration of G-CSF.

[0007]

A practical and simple method for maintaining the blood level of a therapeutically effective amount of G-CSF in a therapeutically effective range for a long period of time and extending the pharmacological effect duration of G-CSF. In order to avoid the problems of viscosity control and temperature control in viscosity preparations and gelling preparations in the body, the viscosity is low enough not to impede the normal administration operation at 25 to 37 ° C, and the preparation is simple. Compatible with chemotherapy administration cycles
The aim of G-CSF formulation with a duration of about 2 days to 1 week was investigated. As a result, the presence of a surfactant in the G-CSF-containing water-soluble composition allows
It has been found that the pharmacological effect duration of F can be extended. Further, a water-soluble composition containing G-CSF and polyoxyethylene polyoxypropylene glycols and having a viscosity of 0.5 Pa second or less in a temperature range of 25 to 37 ° C.,
It has been found that G-CSF has a prolonged pharmacological effect duration and can be administered with a sufficiently thin injection needle. That is, it has been found that a water-soluble composition having a sufficiently long duration effect can be obtained even at a low concentration of polyoxyethylene polyoxypropylene glycol which does not gel at around body temperature. As a result, the problem of temperature control of the in-body gelation preparation was solved, and the amount of the polymer added could be significantly reduced.

That is, the present invention relates to the following. (1) A method for extending the pharmacological effect duration of G-CSF, wherein a surfactant is present in the G-CSF-containing water-soluble composition. (2) The pharmacological effect of G-CSF, wherein a surfactant is present in the G-CSF-containing water-soluble composition so that the viscosity is 0.5 Pa · s or less in a temperature range of 25 to 37 ° C. How to extend time. (3) The surfactant has the formula (I)

Embedded image (Wherein a, b and c each represent a positive integer). The method according to the above (1) or (2), which is a polyoxyethylene polyoxypropylene glycol represented by the formula: (4) The molecular weight of the polyoxypropylene portion of the polyoxyethylene polyoxypropylene glycols is 1500 to
The method according to the above (3), wherein the number of polyoxyethylene units is in the range of 4000 to 30 to 80% of the total units. (5) The method according to (3) above, wherein the polyoxyethylene polyoxypropylene glycol is selected from the group consisting of poloxamer 188, poloxamer 237, poloxamer 238, poloxamer 338, poloxamer 403 and poloxamer 407. (6) The method according to the above (3), wherein the polyoxyethylene polyoxypropylene glycol is poloxamer 407. (7) The method according to the above (6), wherein the content of poloxamer 407 in the G-CSF-containing water-soluble composition is 1 to 10% by weight. (8) The method according to any one of (1) to (7) above, wherein G-CSF is a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a mixture thereof. (9) G-CSF and at least one formula (I)

Embedded image (Wherein a, b and c each represent a positive integer) containing a polyoxyethylene polyoxypropylene glycol represented by the formula: and having a viscosity of 0.5 Pa · s or less in a temperature range of 25 to 37 ° C. Composition. (10) The molecular weight of the polyoxypropylene portion of the polyoxyethylene polyoxypropylene glycols is 1500
The water-soluble composition according to the above (9), wherein the number of polyoxyethylene units is from 30 to 80% of the total units. (11) The water-soluble composition according to the above (9), wherein the polyoxyethylene polyoxypropylene glycol is selected from the group consisting of poloxamer 188, poloxamer 237, poloxamer 238, poloxamer 338, poloxamer 403 and poloxamer 407. (12) The method according to the above (9), wherein the polyoxyethylene polyoxypropylene glycol is poloxamer 407. (13) Poloxamer 407 in G-CSF-containing water-soluble composition
The water-soluble composition according to the above (9), wherein the content of the compound is 1 to 10% by weight. (14) the amino acid sequence of G-CSF is SEQ ID NO: 1 and / or
Or the water-soluble composition according to any one of the above (9) to (13).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The G-CSF used in the present invention may be any polypeptide having G-CSF activity included in the definition of G-CSF, such as an extract from a natural product, A product obtained by using recombinant DNA technology (for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 3) or a part of the amino acid sequence of natural G-CSF is replaced with another amino acid And modified amino acids such as derivatives or those in which a part thereof is deleted or added. Examples of the amino acid variants include, for example, preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a mixture thereof (JP-A-63-267292).

[0010] The surfactant used in the present invention includes:
Polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene polyoxypropylene glycols and polyoxyethylene sorbitan monolaurate; glycerin fatty acid esters such as glyceryl monostearate; polyethylene glycol fatty acid esters such as polyoxyl stearate; Polyethylene glycol monoethers such as ethylene cetyl ether, polyoxyethylene hydrogenated castor oils such as polyoxyethylene glycerin trioxystearic acid, sucrose fatty acid esters and the like, and polyoxyethylene polyoxypropylene glycols are preferable. Among them, polyoxyethylene polyoxypropylene glycols represented by the formula (I) are preferable, and further, the molecular weight of the polyoxypropylene portion is 1500 to 400. Polyoxyethylene polyoxypropylene glycols having a range of 0 and having a number of polyoxyethylene units of 30 to 80% of the total units are preferred. Poloxamer 18 is particularly preferred.
8, Poloxamer 237, Poloxamer 238, Poloxamer 3
38, poloxamer 403, poloxamer 407 and the like. The amount or content of these surfactants is such that the finally obtained water-soluble composition has a temperature of 25 to 37 ° C.
The amount is preferably in a low viscosity state of 0.5 Pa · s or less, and as the content in the water-soluble composition of the present invention, for example, in poloxamer 188 and poloxamer 238, preferably 1 to 40% by weight, more preferably Is in the range of 5 to 25% by weight, and in the case of poloxamer 237, poloxamer 338, poloxamer 403 and poloxamer 407, it is preferably in the range of 0.5 to 15% by weight, more preferably 1 to 10% by weight.

G-CSF contained in the water-soluble composition of the present invention
The amount of is not particularly limited as long as its effect is recognized, but is preferably 0.01 to 10 mg per 1 mL of the composition, and more preferably 0.05 to 1 mg per 1 mL of the composition.

Further, the water-soluble composition of the present invention can be provided as a preparation by filling it in a preparation container. In the case of formulation, it is desirable to be in a unit dosage form suitable for injection. Examples of the form include liquids filled in ampules, vials, syringes, and the like. Further, the water-soluble composition of the present invention can be freeze-dried and used as a freeze-dried preparation. The lyophilization conditions are not particularly limited, but are usually frozen at -50 ° C or lower for 1 to 5 hours, and the shelf temperature is -20 ° C or higher.
Dry at 0 ° C and a vacuum of 1-10 Pa for 24-48 hours, and then
A freeze-dried preparation can be obtained by drying at 0 ° C. to 30 ° C. and a degree of vacuum of 1 to 10 Pa for 16 to 24 hours, and sealing the obtained freeze-dried product with a rubber stopper or an aluminum cap. In the case of preparing the freeze-dried preparation, as an excipient, for example, sugars such as lactose, glucose, maltose, mannitol, trehalose or sugar alcohols, glycine, cysteine, threonine, tryptophan, histidine, amino acids such as arginine , Polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol,
It is also possible to use a water-soluble polymer such as dextran. Further, the composition of the present invention can be provided by adding a polyoxyethylene polyoxypropylene glycol to a solution used for re-dissolving a freeze-dried preparation containing G-CSF.

The water-soluble composition of the present invention contains commonly used tonicity agents, pH regulators, soothing agents, buffers, preservatives, thickeners, antioxidants, antiadsorption agents and the like. You can also. Examples of the tonicity agent include inorganic salts such as sodium chloride, glucose, fructose, mannitol, xylitol, glycerin, propylene glycol, benzyl alcohol and the like. Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, lactic acid, sodium hydroxide, potassium hydroxide and the like. Examples of the soothing agent include inositol, chlorobutanol, propylene glycol, benzyl alcohol and the like. Examples of the buffer include acids such as phosphoric acid, citric acid, acetic acid, and lactic acid and salts thereof. Examples of the preservative include benzoic acid, ethanol, tetrasodium edetate, citric acid, glycerin, chlorobutanol, phenol, propylene glycol, benzyl alcohol and the like. Examples of the thickener include glycerin, gelatin, hydroxypropylcellulose, polyethylene glycols and the like. Antioxidants include, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, sodium bisulfite, sodium sulfite, sodium pyrosulfite,
Cysteine hydrochloride, citric acid, sodium edetate and the like can be mentioned. Examples of the adsorption inhibitor include polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monostearate, sorbitan monolaurate,
Examples include sorbitan fatty acid esters such as sorbitan monopalmitate, and polyethylene glycol monoethers such as polyoxyethylene lauryl ether.

The dose and frequency of administration of the water-soluble composition of the present invention are determined according to the administration form, the age and weight of the patient, the disease to be treated, the condition of the patient, and the like. 0.001 to 10 mg, preferably 0.01 to 1 mg, of the polypeptide having CSF activity per week
Administer 1-4 times. Although the method of administration may vary depending on the purpose of treatment, subcutaneous injection or intramuscular injection is used.
The water-soluble composition of the present invention has a viscosity low enough to be usable for injection, and the viscosity of the finally obtained water-soluble composition is preferably in a temperature range of 25 to 37 ° C,
It is 0.5 Pa · s or less, more preferably 0.3 Pa · s or less, and most preferably 0.1 Pa · s or less. Hereinafter, examples, comparative examples, and test examples of the present invention will be described, but the present invention is not limited thereto.

[0015]

Example 1 Measurement of G-CSF Concentration in Rat Plasma The compositions 1 to 11 obtained in Examples 3 to 10, Comparative Examples 1, and Reference Examples 1 and 2 were used for 7-week-old Crj: CD (SD) IGS. It was subcutaneously administered to the back of a rat (Charles River Japan). The dose was 0.1 mg / kg as G-CSF. After administration, about 150 μL of venous blood was collected from the tail vein with time using a heparin-treated capillary tube. Plasma was separated by centrifugation and stored frozen at -80 ° C until measurement. The G-CSF concentration in the obtained plasma was measured by ELISA. Composition 1
The results of 3, 9, and 11 are shown in FIG. 1, the results of compositions 4, 5, and 9 are shown in FIG. 2, and the results of compositions 6 to 9 are shown in FIG.

FIGS. 1 to 3 show the sustained effect of the plasma G-CSF concentration by the presence of a surfactant in the G-CSF-containing water-soluble composition. In FIG. 1 poloxamer 40
G-CSF concentration in plasma was sustained in accordance with the amount of 7 added. On the other hand, no clear difference was observed in the change in plasma concentration between the gelled preparation of composition 11 and composition 1, and even when gelation was not performed in vivo as in composition 1, gelation of composition 11 The sustained effect of plasma G-CSF concentration was comparable to that of the preparation. In FIG. 2, the poloxamer 188 (composition 4) and the poloxamer 238 (composition 5)
It was confirmed that the addition of about 0% by weight had the effect of maintaining the plasma G-CSF concentration. In FIG. 3, poloxamer 338 (composition 6) and poloxamer 403 (composition 7)
Also, it was confirmed that the addition of about 5% by weight of poloxamer 237 (composition 8) had a sustained effect on the plasma G-CSF concentration. Example 2 Measurement of Rat Leukocyte Count The compositions 1, 9 and 11 obtained in Example 3, Comparative Example 1 and Reference Example 2 were applied to the back of a 7-week-old Crj: CD (SD) IGS rat (Charles River Japan Co., Ltd.). Was administered subcutaneously. The dose was 0.1 mg / kg of G-CSF. Using a heparin-treated capillary tube, 70 μL was collected from blood collected from the tail vein using an Eppendorf pipette, and 31
It was mixed with 2 μL of an aqueous solution of disodium mg / mL EDTA to prepare a sample for leukocyte measurement. The white blood cell count is a Sysmex multi-item automatic blood cell counter (manufacturer: Toa Medical Electronics Co., Ltd., model: K-20
00). FIG. 4 shows the results.

FIG. 4 shows that the method of the present invention extends the time during which the white blood cell count is maintained at a high level. In addition, in the comparison between Composition 1 and Composition 11, which did not show a clear difference in the persistence of plasma G-CSF concentration in Example 1, almost the same long-lasting effect of the white blood cell count was observed. It was shown that the sustained plasma G-CSF concentration prolonged the time during which the white blood cell count was maintained at a high level. Example 3 G-CSF amino acid variant mixture at a concentration of 1 mg / mL [SEQ ID NO: 1
About 6: 1 mixture of a polypeptide having an amino acid sequence represented by the following formula: and a polypeptide having an amino acid sequence represented by SEQ ID NO: 2 (JP-A-63-267292). L phosphate buffer, pH 7) and 5.6 wt% poloxamer 407 [Pluronic F-127 (SIGMA
Company)] aqueous solution (50 mmol / L phosphate buffer, pH 7) in 1: 9
(Composition 1).

Example 4 An aqueous solution of a modified amino acid of G-CSF having a concentration of 1 mg / mL (50 mmol / L)
Phosphate buffer, pH 7) and 1.1 wt% poloxamer 407
[Pluronic F-127 (SIGMA)] aqueous solution (50 mmol / L)
Phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (Composition 2).

Example 5 An aqueous solution of a modified amino acid of G-CSF having a concentration of 1 mg / mL (50 mmol / L)
Poloxamer 407 in phosphate buffer, pH 7) and 0.56% by weight
[Pluronic F-127 (SIGMA)] aqueous solution (50 mmol / L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (composition 3).

Example 6 A 1-mg / mL G-CSF amino acid-modified solution (50 mmol / L phosphate buffer, pH 7) and 22.2% by weight poloxamer 188
[Pluronic F-68 (SIGMA)] aqueous solution (50 mmol / L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (composition 4).

Example 7 G-CSF amino acid variant solution (50 mmol / L phosphate buffer, pH 7) at a concentration of 1 mg / mL and 22.2% by weight of poloxamer 238
[Pluronic F-88 (Asahi Denka Kogyo)] aqueous solution (50 mmol /
L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (Composition 5).

Example 8 G-CSF amino acid modified solution (concentration: 1 mg / mL, 50 mmol / L phosphate buffer, pH 7) and 5.6% by weight of poloxamer 338 [Pluronic F-108 (Asahi Denka Kogyo)] An aqueous solution (50 mmol / L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (composition 6).

Example 9 A solution of a modified G-CSF amino acid solution (50 mmol / L phosphate buffer, pH 7) at a concentration of 1 mg / mL and a 5.6% by weight aqueous solution of poloxamer 403 [Pluronic F-123 (BASF)] (50 mmol / L phosphate buffer, pH 7) were mixed at a volume ratio of 1: 9 (composition 7).

Example 10 An aqueous solution of a modified amino acid of G-CSF having a concentration of 1 mg / mL (50 mmol / L)
Poloxamer 237 with phosphate buffer, pH 7) and 5.6% by weight
[Pluronic F-87 (BASF)] aqueous solution (50 mmol / L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (composition 8).

Comparative Example 1 An aqueous solution of a modified amino acid of G-CSF having a concentration of about 1 mg / mL (50 mmol / mL)
L phosphate buffer (pH 7) and 50 mmol / L phosphate buffer (pH
7) were mixed in a 1: 9 volume ratio (Composition 9).

Reference Example 1 An aqueous solution of a modified amino acid of G-CSF having a concentration of 1 mg / mL (50 mmol / L phosphate buffer, pH 7) and 0.11% by weight of poloxamer 407
[Pluronic F-127 (SIGMA)] aqueous solution (50 mmol / L)
Phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (Composition 10). Reference Example 2 An aqueous solution of a modified G-CSF amino acid having a concentration of 1 mg / mL (50 mmol / L)
Poloxamer 407 in phosphate buffer, pH 7) and 22.2% by weight
[Pluronic F-127 (SIGMA)] aqueous solution (50 mmol / L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (composition 11). Reference Example 3 Modified aqueous solution of G-CSF amino acid at a concentration of 1 mg / mL (50 mmol / L
Poloxamer 338 with phosphate buffer, pH 7) and 22.2% by weight
[Pluronic F-108 (Asahi Denka Kogyo)] aqueous solution (50 mmol
/ L phosphate buffer, pH 7) was mixed at a volume ratio of 1: 9 (Composition 12).

Test Example 1 Viscosity Measurement About 1 mL of each of Compositions 1 to 8, 11 and 12 was sampled, and the viscosities at 25 ° C. and 37 ° C. were measured using a digital viscometer (DVR-E, Tokyo Keiki). . The results are shown in Table 1.

[Table 1] Table 1 shows that the present water-soluble composition has low viscosity at 25 ° C and 37 ° C. Further, gelation was observed at 25 ° C. and 37 ° C. in composition 11, and at 37 ° C. in composition 12.

[0028]

According to the present invention, a simple and practical G-CS
Methods are provided for extending the duration of the pharmacological effect of F. Further, there is provided a low-viscosity water-soluble composition having an extended duration of pharmacological effect of G-CSF, which is easy to prepare and formulate.

[Sequence List] SEQUENCE LISTING <110> KYOWA HAKKO KOGYO CO., LTD. <120> Methods for long-lasting pharmacological effect of granulocyte colony-stimulating factor <130> H14-0694D1 <140> <141> <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 175 <212> PRT <213> Artificial Sequence <223> Peptide substitution on 5 amino acids of hG-CSF. <400> 1 Met Ala Pro Thr Tyr Arg Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu 1 5 10 15 Lys Ser Leu Glu GlnVal Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu 20 25 30 Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 35 40 45 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 50 55 60 CysPro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 80 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 85 90 95 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 175 <210> 2 <211> 174 <212 > PRT <213> Artificial Sequence <223> Peptide substitution on 5 amino acids of hG-CSF. <400> 2 Ala Pro Thr Tyr Arg Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gl n Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 174 <210> 3 <211> 174 <212> PRT <213> Homo Sapience <400> 3 Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 5 10 15 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 20 25 30 Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 35 40 45 Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 50 55 60 Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 65 70 75 80 Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 85 90 95 Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 100 105 110 Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125 Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140 Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 145 150 155 160 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 174

[Brief description of the drawings]

FIG. 1 shows plasma G-CSF concentrations over time after a single subcutaneous administration of Compositions 1-3, 9, and 11 to rats. The horizontal axis represents elapsed time (hours), and the vertical axis represents plasma G-CSF.
Indicates the concentration (ng / mL).

[Explanation of symbols]

 -◇-: Composition 1-□-: Composition 2-△-: Composition 3-◆-: Composition 9-■-: Composition 11

FIG. 2 shows plasma G-CSF concentrations over time after a single subcutaneous administration of Compositions 4-5 and 9 to rats.
The horizontal axis represents elapsed time (hours), and the vertical axis represents plasma G-CSF concentration (n
g / mL).

[Explanation of symbols]

 -△-: Composition 4-□-: Composition 5-◆-: Composition 9

FIG. 3 shows plasma G-CSF concentrations over time after a single subcutaneous administration of Compositions 6 to 9 to rats. The horizontal axis represents elapsed time (hours), and the vertical axis represents plasma G-CSF concentration (ng / mL).

[Explanation of symbols]

 -◇-: Composition 6-□-: Composition 7-△-: Composition 8-◆-: Composition 9

FIG. 4 shows the time course of white blood cell count after a single subcutaneous administration of Compositions 1, 9 and 11 to rats. The horizontal axis represents elapsed time (hours), and the vertical axis represents white blood cell count (10 ² / μL).

[Explanation of symbols]

 -◇-: Composition 1-◆-: Composition 9-■-: Composition 11

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mayumi Mukai 1188 Shimotsukari, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture Inside the Pharmaceutical Research Laboratory, Kyowa Hakko Kogyo Co., Ltd. (72) Eiji Hayakawa 1-6-1, Otemachi, Chiyoda-ku, Tokyo F-term (Reference) 4K076 AA12 BB11 CC27 EE23 FF11 FF17 FF70 4C084 AA02 AA03 AA14 BA01 BA08 BA22 BA23 CA18 CA53 CA56 CA59 DA19 MA05 MA17 MA66 NA05 ZB262

Claims (14)

[Claims] 1. A method for extending the duration of the pharmacological effect of G-CSF, comprising adding a surfactant to a water-soluble composition containing granulocyte colony-stimulating factor (G-CSF). 2. The G-CSF-containing water-soluble composition has a viscosity of 25 to 37.
A method for extending the pharmacological effect duration of G-CSF, wherein a surfactant is present at a temperature of 0.5 Pa · sec or less in a temperature range of ° C. 3. The method according to claim 1, wherein the surfactant is of the formula (I) 3. The method according to claim 1, wherein the polyoxyethylene polyoxypropylene glycol is represented by the formula: wherein a, b and c each represent a positive integer. 4. 4. The molecular weight of the polyoxypropylene portion of the polyoxyethylene polyoxypropylene glycols is
The method of claim 3 wherein the number of polyoxyethylene units ranges from 1500 to 4000 and 30 to 80% of the total units. 5. The polyoxyethylene polyoxypropylene glycols are poloxamer 188, poloxamer 237,
Poloxamer 238, Poloxamer 338, Poloxamer 403
The method according to claim 3, which is selected from the group consisting of poloxamer 407 and poloxamer 407. 6. The method according to claim 3, wherein the polyoxyethylene polyoxypropylene glycol is poloxamer 407. 7. The method according to claim 6, wherein the content of poloxamer 407 in the G-CSF-containing water-soluble composition is 1 to 10% by weight. 8. G-CSF is SEQ ID NO: 1 or SEQ ID NO: 2
The method according to any one of claims 1 to 7, which is a polypeptide having an amino acid sequence represented by: or a mixture thereof. 9. A granulocyte colony-stimulating factor (G-CSF) and at least one of the formulas (I) (Wherein a, b and c each represent a positive integer) containing a polyoxyethylene polyoxypropylene glycol represented by the formula: and having a viscosity of 0.5 Pa · s or less in a temperature range of 25 to 37 ° C. Composition. 10. The method according to claim 9, wherein the molecular weight of the polyoxypropylene portion of the polyoxyethylene polyoxypropylene glycols is in the range of 1500 to 4000, and the number of polyoxyethylene units is 30 to 80% of the total units. Water-soluble composition. 11. Polyoxethylene polyoxypropylene glycols are poloxamer 188, poloxamer 23
7, Poloxamer 238, Poloxamer 338, Poloxamer 4
The water-soluble composition according to claim 9, which is any one selected from the group consisting of 03 and poloxamer 407. 12. The method according to claim 9, wherein the polyoxyethylene polyoxypropylene glycol is poloxamer 407. 13. The water-soluble composition according to claim 9, wherein the content of poloxamer 407 in the water-soluble composition containing G-CSF is 1 to 10% by weight. 14. The water-soluble composition according to claim 9, wherein the amino acid sequence of G-CSF is SEQ ID NO: 1 and / or 2.