JPH0796558B2 - Modified polypeptide - Google Patents

Description

TECHNICAL FIELD The present invention relates to human granulocyte colony stimulating factor (hG-CSF).
Chemical modification obtained by chemically modifying at least one amino group in a molecule of a polypeptide having activity
It relates to hG-CSF polypeptide and a method for producing the same.

hG-CSF is a kind of polypeptide essential for proliferating / differentiating hematopoietic stem cells to form various blood cells, and has an effect of promoting the increase of mainly granulocytes, especially neutrophils.
Although neutrophils play an important role in defense against infection of the body, they have a short life span and must be constantly supplied by the constant proliferation and differentiation of progenitor cells. The treatments for proliferative tumors that have been widely performed in recent years have the serious side effect of simultaneously inhibiting the proliferation of neutrophil progenitor cells and thus reducing the protective function against infection in cancer-bearing patients. hG-CSF is expected to reduce this side effect by promoting the increase of neutrophils and, at the same time, have the effect of preventing and treating infectious diseases. Furthermore, hG-CSF has an activity of differentiating leukemia cell lines in vitro, and thus may be a therapeutic drug for leukemia. Chemically modified hG of the present invention
-CSF polypeptide is a hG-better than known hG-CSF.
It has CSF activity and is expected to be used as a drug.

2. Description of the Related Art Genes of proteinaceous factors involved in proliferation and differentiation of blood cells have been isolated one after another by the recombinant DNA technology which has been rapidly developed in recent years. Those factors are produced by genetic engineering techniques using microorganisms and animal cells.

hG-CSF was obtained by Nagata et al. from the human squamous cell carcinoma cell line CHU-II.
cDNA was isolated, its nucleotide sequence was determined, and its expression in COS cells was reported [Nagata et al .: Nature 319,
415 (1986)]. In addition, Souza et al. Isolated cDNA from human bladder cancer cell line 5637 and determined its nucleotide sequence,
Expression in Escherichia coli has been reported [Souza et al .: Science 232, 61 (1986)].

The hG-CSF thus obtained, when administered in vivo, requires continuous administration to maintain its effect, and there is a report that the effect disappears promptly when the administration is stopped [Journal of Experimental Medicine (J. Exp.Med.) 165, 941-948 (1984)]. This is hG
-It is considered that CSF has low persistence in blood.

In addition, asparaginase [Inada, Y., et al.
Trends in Biotechnolog
y), 4, 68-73 (1986)], arginase [Savoca, K.
V., et al., Biochemica et Biophysica Acta, 578, 47-53,
(1979)], batroxobin [Nishimura, H., et al., Life Science, 33 , 1467-1473,
(1983)] and other enzymes are chemically modified with polyethylene glycol to improve blood persistence and antigenicity.

Generally, when a protein is used as a medicine, it is often supplied as a freeze-drying agent. However, it may be affected by external factors such as temperature, humidity, oxygen, and ultraviolet rays during or after freeze-drying, and may undergo physical or chemical changes such as association, polymerization, or oxidation, resulting in a decrease in activity. . Attempts have been made to stabilize freeze-drying with some additives (for hG-CSF, see, for example, JP-A-63-146).
826, JP-A-63-146829, etc.), but there is a demand for a more stabilized preparation for practical use.

Problems to be Solved by the Invention When using hG-CSF as a drug, hG-CSF administered in vivo is highly stable in blood while maintaining its activity, and has high durability, Further, it is desirable that its antigenicity is attenuated. In addition, when hG-CSF is used as a medicine, it is usually supplied as a freeze-dried preparation, so it is useful to improve the stability during and after freeze-drying. However, hG-CSF having such properties and its production method have not been developed.

Means for Solving the Problems The present inventor has chemically modified at least one amino group in a molecule of a polypeptide having hG-CSF activity to give a higher blood level than unmodified hG-CSF. The present invention has been completed based on the finding that it is possible to improve the durability of the composition and that the stability can be improved during and after freeze-drying.

The present invention will be described in detail below.

The present invention, hG-CSF polypeptide of the formula with at least one amino group in the molecule R ₁ having activity _{- (OCH 2 CH 2 n X} -R 2 - (I) { wherein R ₁ is an alkyl group or Alkanoyl group, n is a positive integer which can be changed arbitrarily, X is O, NH or S, R ₂ is [In the formula, R ₃ represents OH, Cl, O- (CH ₂ CH ₂ O) n-R ₁ (in the formula, R ₁ and n have the same meanings as described above), Y does not exist, or Z- (CH ₂ ) pCO (in the formula, Z represents O, S or NH, and p represents a positive integer which can be changed arbitrarily) or (CO) m- (CH ₂ ) _l CO (in the formula, m Represents 0 or 1, and 1 represents a positive integer which can be changed optionally), and a modified polypeptide.

The polypeptide having hG-CSF activity used in the present invention is a polypeptide having an amino acid sequence shown in Table 1 or at least one polypeptide in the amino acid sequence shown in Table 1 as represented by Table 2. Any of the above-mentioned polypeptides in which the amino acid of 5 is substituted with an amino acid of another species or a polypeptide in which 1 to 11 N-terminal amino acids are deleted and has hG-CSF activity may be used. Furthermore, the hG-CSF derivative described in JP-A-63-299 and JP-A-63-500636 can also be used.

Regarding the chemical modifying group used in the present invention, as the alkyl group and the alkanoyl group as the terminal oxygen protecting group represented by R ₁ , specifically, the alkyl group is a C _1-18 (methyl, ethyl, propyl, etc.) group. The alkanoyl group is C
_Examples include _{1 to 18} (formyl, acetyl, propionyl, etc.).

The positive integer represented by n is 500 or less. Especially 7
~ 230 is preferred.

The positive integer represented by 1 is 100 or less, preferably 0 to 6. p is a positive integer represented by 1 to 18, preferably 1 to 6. The molecular weight of the chemically modifying group of the present invention is 30,000 or less, preferably in the range of 300 to 10,000.

The chemically modified hG-CSF of the present invention includes, for example, hG-CSF and (Wherein R ₁ , n, X and R ₃ have the same meanings as described above), condensation with a halide or hG-CSF and R _1- (OCH ₂ CH ₂ ) _{n
-X- (CO) m (CH 2} ) l -COOH (III) (R 1, n, X, m, l
Is the same as the above) and condensation with carboxylic acid or hG-CSF It is produced by condensation with a carboxylic acid represented by the formula (wherein R ₁ , n, X, R ₃ , Z and p are as defined above).

The halide of formula (II) is R _1- (OCH ₂ CH ₂ ) n-XH (R
₁ , n, X have the same meaning as described above) and cyanuric chloride are condensed (for example, Matsushrma, A., et al., Chemistry Letters (Chemistry, Letters), 773-776, (198
0), Abuchowski, A., et al., Journal of Biological Chemistry (J. Biol. Chem.), 252, (12).
3578-3581 (1977)]. Since this halide is reactive, it can be directly reacted with a polypeptide having hG-CSF activity.

Carboxylic acid of formula _{(III), R 1 - (OCH 2 CH} 2) n -XH
(R ₁ , n and X have the same meanings as above) and a carboxyl group is introduced by dehydration condensation of the carboxyl group of alkanedicarboxylic acid, a carboxyl group is introduced by a halogenated monocarboxylic acid, or a hydroxyl group at the terminal is introduced. Oxidize
Obtained by converting to a carboxyl group. Since this carboxylic acid has no reactivity, it needs to be activated before use. Examples of the carboxylic acid activation method include N-
Hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, a method of forming an active ester with p-nitrophenol, isobutyl chloroformate, a method of forming a mixed acid anhydride with ethyl chloroformate, Alternatively, a method of reacting with a halogenating agent such as thionyl chloride to form an acid halide [see “Peptide Synthesis” (Nobuo Izumiya et al., Published by Maruzen)] and the like can be mentioned.

The above-mentioned carboxylic acid of formula (IV) can be converted to the chloride of formula (II) and HZ-
_{(CH 2) p-CO 2} H (Z, p has the same meaning as defined above) obtained by condensing. The carboxylic acid of the formula (IV) has the formula (II
It is used after activation in the same manner as the carboxylic acid of I).

These halides or carboxylic acid activators are
Add 2 to 100 times the amount (molar ratio) of amino groups present in the molecule of the polypeptide having hG-CSF activity, preferably at 4 to 37 ° C, preferably 4 to 10 ° C, pH 7 to 10 for 1 hour to 2 days. Is the target chemically modified hG by reacting for 1 to 24 hours.
-Can produce CSF.

In the chemically modified hG-CSF or the chemically modified hG-CSF derivative, the reaction product with the halide of the formula II is the type II, the reaction product with the carboxylic acid of the formula III is the type III, and the reaction product with the carboxylic acid of the formula IV is Type IV, respectively.

The degree of chemical modification is monitored by quantifying the amount of free amino group reduction with trinitrobenzenesulfonic acid or by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to track the change in mobility of chemically modified hG-CSF. Confirmed by

The chemically modified hG-CSF and the chemically modified hG-CSF derivative are provided for pharmaceutical use as a solution or lyophilization agent dissolved in water or an appropriate buffer.

The conditions for freeze-drying are not particularly limited, but usually, frozen at -50 ° C or lower for 1 to 5 hours, shelf temperature -20 ° C to 0 ° C, vacuum degree 50 to
It is dried at 150 mTorr for 24 to 48 hours, then dried at a shelf temperature of 10 to 30 ° C and a vacuum degree of 50 to 100 mTorr for 16 to 24 hours to obtain a freeze-dried product.

The solution is aseptically passed as it is and used as an injection. The lyophilization agent is dissolved in water or an appropriate buffer and then aseptically used as an injection.

The hG-CSF used in the present invention may contain: various usual pharmaceutical carriers, excipients, diluents, stabilizers or anti-adsorption agents.

The dose of this derivative is determined according to the target disease and the medical condition of the patient, but is usually 0.1 per adult.
~ 500 μg, preferably 0.5-200 μg of chemically modified hG-CSF
And 1 formulation per week containing chemically modified hG-CSF derivative
~ 7 doses.

One molecule to three molecules of polyethylene glycol derivative are bound to the chemically modified hG-CSF and the chemically modified hG-CSF derivative.
Therefore, the modified hG-CSF and the modified hG-CSF derivative are used as a mixture of the monomolecular conjugate to the trimolecular conjugate, or the monomolecular conjugate to the trimolecular conjugate are separately used.

The amount of protein in the present invention is measured by the experimental method described below.

Experimental method 1. Method of OH Lowry [Lowry, OH, et al., Journal of Biological Chemistry (J. Bio
Chem.), 193, 265 (1951)].

Experimental method 2. Remli's method [UK Laemmli: Nature 227,
680 (1970)] and perform SDS-polyacrylamide gel electrophoresis, and then measure with a chromatoscanner (CS-930 Shimadzu).

The G-CSF activity in the present invention was measured as follows. Bone marrow cells were aseptically removed from the femur of 8-12 week old C3H / He male mice (Shizuoka Experimental Animal Cooperative), and 10% of fetal bovine serum (FBS) was added to α-Minimum Essential Med.
It was suspended in ium (Flow Laboratories, hereinafter abbreviated as α-MEM medium). 1.5 ml of this cell suspension (approximately 5 x 10 ⁷ cells) was added to Nylon woll (Wako Pure Chemical Industries, Nylon Fiber 14
6-04231) in a packed column (0.3g) and 5% CO ₂
The reaction was carried out at 37 ° C for 90 minutes in an incubator. Then, the α-MEM medium preheated to 37 ° C. was passed through the column to obtain the eluted nylon-wool non-adhesive bone marrow cells.
The cells were washed once with α-MEM medium and adjusted to a predetermined concentration.

Then, the method of Okabe et al. [Okabe T. et al., Cancer
Research (Cancer Research) 44, 4503-4506 ( 1986) ]
The bone marrow hematopoietic stem cell colony forming ability was measured according to. That is, 0.2 ml of bone marrow cells (2 × 10 ⁶ cells / ml) prepared by the above method was mixed with a mixed solution of 0.2 ml of α-MEM medium, 0.4 ml of FBS and 0.2 ml of each sample diluted in two steps. 0.6% agar kept at 42 ℃ (Difco, Agar purified #
0560-01) Equal amount (1.0 ml) of the solution was mixed, and 0.5 ml thereof was added to 24
It was seeded in a hole multi-dish (Nunc, # 143982) (5 × 10 ⁴ cells / well, n = 3). The cells were cultured at 37 ° C for 7 days in a 5% CO ₂ incubator, and the number of colonies consisting of 40 or more cells was counted with a microscope (Olympus, x40).
After counting the colonies, carefully pick them out on a slide glass, fix them with an acetone / formalin mixture for 30 seconds, and then use Kubota.
Et al. [Kubota K., et al., Experimental
Hematology (Exp. Hematology) 8 , 339-344 (198
Double staining with esterase was performed in (0)] to identify each colony.

The titer of each sample was calculated as follows from the counting result in the two-step dilution of the colony formation test. The activity which gave half the maximum value of the colony formation of intact G-CSF used as a standard was defined as 50 units, and this was multiplied by 20 in order to convert it into the dilution ratio of each sample and the activity per ml. Was used as the titer (unit). The specific activity was expressed as a titer (unit / mg) per unit protein (mg).

Examples, reference examples and experimental examples are shown below.

Example 1. Includes 186 μg / ml hG-CSF having the amino acid sequence of Table 1.
To 3 ml of 0.1 M borate buffer (pH 10), 56 mg of the chlorinated compound obtained in Reference Example 1 described below was added, and the mixture was reacted at 4 ° C. for 24 hours while stirring.

After removing unreacted chloride by ultrafiltration (molecular weight cutoff of 30,000), reversed phase HP with a linear gradient of 0-70% acetonitrile using YMC-PackAM-312ODS (Kurita industry)
LC was performed. The chemically modified hG-CSF polypeptide was eluted in the elution fraction of about 50% acetonitrile (yield 30 μg, yield 5%). The resulting chemically modified hG-CSF polypeptide has h
It was confirmed by SDS-polyacrylamide gel electrophoresis that one chlorinated molecule was bound to one G-CSF molecule. The purity was 90% or more.

Example 2. Containing 570 μg / ml of hG-CSF having the amino acid sequence of Table 1.
To 50 ml of 50 mM phosphate buffer (pH 7.2), 240 mg of the active ester obtained in Reference Example 2 described below was added, and the mixture was mixed with stirring 4
The reaction was carried out at 0 ° C for 6 hours.

10 mM Tris-HCl buffer-0.7 M ammonium sulfate (pH 8.
0) After adding 50 ml, Butyl-Toyopearl 650 filled with 10 mM Tris / hydrochloric acid-0.35 M ammonium sulfate (pH 8.0)
It was passed through M (manufactured by Tosoh Corporation) (2.2 cm × 26 cm) at a flow rate of 100 ml / hr. Then, after washing with 100 ml of 10 mM Tris / hydrochloric acid-0.35 M ammonium sulfate (pH 8.0) at a flow rate of 100 ml / hr,
A total of 400 ml of 0.35 M ammonium sulfate to 0 M ammonium sulfate was added to a 10 mM Tris / hydrochloric acid (pH 8.0) system in a linear gradient, and elution was performed at a flow rate of 100 ml / hr. The target substance was eluted at 50 mM to 130 mM ammonium sulfate. Elution fraction 130
ml was ultrafiltered [molecular weight fraction 10,000: membrane YM10 (manufactured by Amicon)] and concentrated to 7 ml. Then, the concentrate was filled with 10 mM phosphate buffer-physiological saline (pH7.2) (PBS), Sephacryl S-200 (Pharmacia) (2.8 cm × 70).
cm) at a flow rate of 120 ml / hr. Then, at the same flow rate, PB
I passed S.

The chemically modified hG-CSF polypeptide is 150m after the polyethylene glycol derivative trimolecular conjugate (Tri body) begins to flow in PBS.
Eluted from l to 160 ml (Yield 2 mg Yield 7
%). Then, the polyethylene glycol derivative bimolecular conjugate (Di form) was eluted from 165 ml per 185 ml (amount 1.5 mg, yield 5%). Next, a polyethylene glycol derivative monomolecular conjugate (Mono form) was eluted from 190 ml per 210 ml (amount 4.5 mg, yield 16%). In the obtained polypeptide, one molecule of polyethylene glycol derivative carboxylic acid is bound to one molecule of hG-CSF in Mono body, two molecules are bound in Di body, and three molecules are bound in Tri body. It was confirmed by SDS-polyacrylamide gel electrophoresis. The purity of each was 90% or more.

Example 3. hG-CSF derivative (570 μg / ml) obtained in Reference Example 3 described later
54 mg of the active ester obtained in Reference Example 2 described later was added to 10 ml of a 0.1 M borate buffer solution (pH 9) containing, and the mixture was reacted at 4 ° C. for 10 hours.

After removing the unreacted active ester and its degradation products using the ultra-permeation membrane YM30 (manufactured by Amicon), 10
The internal solution was replaced with mM Tris / HCl buffer (pH 8).
DEAE-Toyopearl 650M (Tosoh) filled with 10 mM Tris / HCl buffer (pH 8.0) (1.7 cm x 4.4 cm)
The column was passed through at a flow rate of 10 ml / hr. Then, 20 ml of 10 mM Tris-HCl buffer (pH 8) is passed through the column at a flow rate of 5 ml / hr for washing, and then 1
0M with a linear gradient to a buffer system of 0 mM Tris / HCl (pH 8)
NaCl to 0.4 M NaCl was added over a total volume of 100 ml, and elution was performed at a flow rate of 5 ml / hr. The chemically modified hG-CSF polypeptide is N
It was eluted with aCl of 100 to 120 mM (yield 0.85 mg, 15% yield).
It was confirmed by SDS-polyacrylamide gel electrophoresis that the obtained polypeptide was bound to one molecule of the hG-CSF derivative and one molecule of the carboxylic acid of the polyethylene glycol derivative. The purity was 90% or more.

Example 4 Including the hG-CSF derivative (570 μg / ml) obtained in Reference Example 3
300 mg of the active ester obtained in Reference Example 2 was added to 50 ml of 50 mM phosphate buffer (pH 7.2), and the mixture was reacted at 4 ° C. for 6 hours.

10 mM Tris-HCl buffer-0.7 M ammonium sulfate (pH 8.
0) After adding 50 ml, Butyl-Toyopearl 6 filled with 10 mM Tris-HCl buffer-0.35 M ammonium sulfate (pH 8.0)
The solution was passed through 50M (manufactured by Tosoh Corporation) (2.2 cm x 26 cm) at a flow rate of 100 ml / hr. Then, 100 ml of 10 mM Tris-hydrochloric acid buffer-0.35 M ammonium sulfate (pH 8.0) was passed through the column at a flow rate of 100 ml / hr to wash, and then 0.3 mM of 10 mM Tris-hydrochloric acid buffer (pH 8.0) with a linear gradient.
Total amount of 5m ammonium sulfate to 0m ammonium sulfate 400m
It was added over 1 and elution was performed at a flow rate of 100 ml / hr. The target product was eluted at 50 mM to 150 mM ammonium sulfate. The eluted fraction (150 ml) was subjected to an ultrafiltration (molecular weight fraction 10,000: YM10 (manufactured by Amicon)) and concentrated to 10 ml. Then concentrate the PB
Sephacryl S-200 filled with S (made by Pharmacia)
It was passed through (2.8 cm × 70 cm) at a flow rate of 120 ml / hr. Next, PBS was passed through at the same flow rate. The chemically modified hG-CSF polypeptide is a polyethylene glycol derivative trimolecular conjugate (Tr
The i-form) was eluted from 150 ml to 160 ml after starting the flow of PBS (amount 1.5 mg, yield 5%). Next, polyethylene glycol derivative bimolecular conjugate (Di body) was added from 165 ml to 185
It was eluted per ml (3 mg, 11% yield). Next, polyethylene glycol derivative monomolecular conjugate (Mono
Body was eluted from 190 ml per 210 ml (yield 4 mg
Yield 14%). The obtained polypeptide is hG-CS in Mono form.
SDS-poacrylamide gel electrophoresis confirmed that one molecule of carboxylic acid of polyethylene glycol derivative is bound to one molecule of F, two molecules are bound in Di body, and three molecules are bound in Tri body. Was done. The purity of each was 90% or more.

Example 5 Contains the hG-CSF derivative (300 μg / ml) obtained in Reference Example 3
To 100 ml of 50 mM phosphate buffer (pH 7.2), 800 mg of the active ester obtained in Reference Example 4 was added and reacted at 4 ° C for 24 hours.

10 mM Tris-HCl buffer-0.7 M ammonium sulfate (pH 8.
0) After adding 100 ml, it was passed through butyl-Toyopearl 650M (manufactured by Tosoh Corp. 2.2 cm × 26 cm) filled with 10 mM Tris-hydrochloric acid buffer-0.35 M ammonium sulfate (pH 8.0) at a flow rate of 100 ml / hr. . Next, 100 ml of 10 mM Tris-hydrochloric acid buffer-0.35 M ammonium sulfate (pH 8.0) was passed through the column at a flow rate of 100 ml / hr to wash, and then a 10 mM Tris-hydrochloric acid buffer (pH 8.0) system was linearly gradient from 0.35 M ammonium sulfate to 0 M. Ammonium sulfate was added over a total volume of 400 ml, and elution was performed at a flow rate of 100 ml / hr. The target product was eluted with ammonium sulfate from 0 mM to 250 mM. Elute 250 ml of elution fraction with ultrafiltration [molecular weight fraction 10,000: YM10
(Manufactured by Amicon)] and concentrated to 10 ml. Then, PB
Sephacryl S-200 filled with S (Made by Farasia)
(5.6 cm × 40 cm) was passed through at a flow rate of 160 ml / hr. Next, PBS was passed through at the same flow rate. The chemically modified hG-CSF polypeptide is a polyethylene glycol derivative trimolecular conjugate (Tr
The i-form) was eluted from 360 ml per 400 ml after starting the flow of PBS (2.1 mg yield, 7% yield). Next, the polyethylene glycol derivative bimolecular conjugate (Di body) is 420 ml to 450 ml.
Was eluted around (about 1.5 mg yield 5%). Then
Polyethylene glycol derivative monomolecular conjugate (Mono body)
Was eluted per 500 to 530 ml (yield 1.5 mg, 5% yield). The obtained polypeptide is hG-CSF in Mono form.
It was confirmed by SDS-polyacrylamide gel electrophoresis that one molecule of the carboxylic acid of the polyethylene glycol derivative was bound to one molecule, two molecules were bound in the Di body, and three molecules were bound in the Tri body. It was The purity of each was 90% or more.

Example 6. Production of chemically modified hG-CSF freeze-dried preparation and its storage stability In the same manner as in Example 2, hG-CSF was reacted with an active ester, and unreacted with an ultrapermeabilized membrane. The active ester and its degradation products were removed. Using the same membrane, the internal solution was exchanged with 50 mM phosphate buffer (pH 7.2) containing 1 M sodium chloride. The resulting solution containing 200 μg / ml of modified hG-CSF was freeze-dried to produce a freeze-drying agent. As a control, a mixed solution of hG-CSF and polyethylene glycol was freeze-dried in the same manner to produce a freeze-dried agent. After each lyophilization agent was left at 65 ° C., it was dissolved over time, and the residual activity of hG-CSF was measured according to the above-mentioned activity measuring method. The results are shown in Table 3.

The residual activity (%) is a relative ratio to the initial activity before freeze-drying and is defined by the following formula.

Freeze-drying conditions: Both unmodified hG-CSF solution and modified hG-CSF solution are subjected to the following conditions. Place the hG-CSF solution in a glass vial and freeze at -50 ° C or below for 2 hours. Next, shelf temperature -20
Dry for 24 hours under vacuum at 100 Torr. Then, it is dried at 20 ° C. and a vacuum degree of 80 mTorr for 24 hours to obtain a freeze-dried product.

Example 7. Production of freeze-dried preparation of chemically modified hG-CSF derivative and its storage stability In the same manner as in Example 3, the hG-CSF derivative was reacted with the active ester, and an ultrapermeabilization membrane was used. Unreacted active ester and its decomposition products were removed. Using the same membrane, the internal solution was exchanged with 50 mM phosphorus buffer solution (pH 7.2) containing 1 M sodium chloride. The resulting solution containing 200 μg / ml of the modified hG-CSF derivative was freeze-dried to produce a freeze-drying agent. As a control, the hG-CSF derivative and the polyethylene glycol-added solution were freeze-dried in the same manner to produce a freeze-drying agent.
The freeze-drying agent was left at 37 ° C. for 7 days, and the residual activity of hG-CSF in the solution was measured over time according to the above-mentioned activity measuring method. The results are shown in Table 4. The residual activity (%) and freeze-drying conditions in the table are the same as in Example 5.

Reference Example 1. 2,4-bis (O-methoxypolyethylene glycol)-
Preparation of 6-chloro-S-triazine 10 g was dissolved in 100 ml of anhydrous toluene containing anhydrous sodium carbonate to dissolve 20 g of monomethoxypolyethylene glycol (manufactured by NOF Corporation) having an average molecular weight of 4000 and heated at 110 ° C for 30 minutes, followed by chlorination. 500 mg of cyanur was added and heated at 110 ° C for 24 hours. The reaction residue was removed, 300 ml of petroleum ether was added to cause precipitation, and the precipitate was washed several times with petroleum ether to obtain 2,4-bis (O-methoxypolyethylene glycol) -6-chloro-S-triazine. Was obtained (yield 50%).

Reference Example 2. Monomethoxypolyethylene glycol succinyl-N-
Synthesis of hydroxysuccinimide ester Sufficiently dehydrated monomethoxypolyethylene glycol with an average molecular weight of 5000 (manufactured by Union Carbide) 20 g, succinic anhydride 2
g was added to 50 ml of anhydrous toluene, heated to 150 ° C. and refluxed for 5 hours. Toluene was distilled off under reduced pressure, and methylene chloride was added to the residue.
After addition of 100 ml and complete solubilization, 400 ml of anhydrous ethyl ether was added to cause precipitation. This precipitate was recrystallized with methylene chloride: ethyl ether system (1: 3 volume ratio),
Succinylated monomethoxy polyethylene glycol 10g
(Yield about 50%) was obtained. 3.3 g of this succinyl compound and N-
Hydroxysuccinimide 100mg anhydrous methylene chloride 5ml
The solution was solubilized in, and 200 mg of dicyclohexylcarbodiimide (DCC) was added under ice cooling, and the mixture was stirred at room temperature for 20 hours. The resulting dicyclohexylurea (DCU) was separated, and ethyl ether was added to the solution to cause precipitation. The obtained precipitate was recrystallized from methylene chloride: ethyl ether to give monomethoxypolyethylene glycol-succinyl-N-
2.5 g (yield 72%) of hydroxysuccinimide ester was obtained.

Reference Example 3. The first threonine of hG-CSF having the amino acid sequence shown in Table 1 is alanine, the third leucine is threonine, the fourth glycine is tyrosine, and the fifth proline is arginine. HG-CSF derivatives in which the 17th cystine was replaced with serine were obtained as follows.

Plasmid containing DNA encoding the above hG-CSF derivative
E. coli W3110 strA strain harboring pCfBD28 (Escherichia c
oli ECfBD28 FERM BP-1479) is dissolved in LG medium [10 g of bactotryptone, 5 g of yeast extract, 5 g of NaCl, and 1 g of glucose in water 1 and adjusted to pH 7.0 with NaOH. ], And cultivated at 37 ℃ for 18 hours, and 5 ml of this culture solution was added with 25 μg / ml tryptophan
/ ml MCG medium containing ampicillin (Na ₂ HPO ₄ 0.6%, KH ₂ PO
₄ 0.3%, NaCl 0.5%, casamino acid 0.5%, MgSO ₄ 1 mM, vitamin B ₁ 4 μg / ml, pH 7.2) 100 ml, inoculated and incubated at 30 ° C for 4-8 hours, then tryptophan inducer 3β-indoleacrylic acid (3β-indoleacrylic acid, hereinafter abbreviated as IAA) was added at 10 μg / ml, and the culture was continued for 2 to 12 hours. The culture solution was centrifuged at 8,000 rpm for 10 minutes to collect the cells, and 30 mM N
It was washed with aCl, 30 mM Tris / HCl buffer (pH 7.5). The washed cells were suspended in 30 ml of the above buffer solution and ultrasonically disrupted at 0 ° C (SONIFIER CELL DISRU, BRANSON SONIC POWER COMPANY).
PTOR 200, OUTPUT CONTROL2, 10 minutes). this
Centrifugation was performed at 9,000 rpm for 30 minutes to obtain a cell residue. The Marston et manner from cell residue [FAOMarston et al Bio Tekunorojii (BIO / TECHNOLOGY) 2, 800 (1984) ] hG-CSF derivative extraction and purification, solubilization and play the by.

Reference Example 4. 2,4-bis (O-methoxypolyethylene glycol)-
Production of N-hydroxysuccinimide ester (IV b) of 6- (3-carpoxypropylamino) -s-triazine (IV a) 500 mg of the chloride obtained in Reference Example 1 was dissolved in 9 ml of anhydrous tetrahydrofuran. On the other hand, the above solution was added to a solution prepared by dissolving 10 mg of γ-aminobutyric acid and 28 μl of triethylamine in 1 ml of anhydrous dimethylamide, and then stirred at room temperature for 16 hours. Then, after drying under reduced pressure, 30 ml of methylene chloride and 15 ml of 10 mM phosphate buffer (pH 10) were added and distributed. The upper layer was adjusted to pH 1 with 2N HCl, 30 ml of methylene chloride was added, and the mixture was partitioned again. The lower layer was fractionated, dried over anhydrous sodium sulfate, separated, and concentrated under reduced pressure to obtain 15 mg (yield 30%) of the carboxylic acid (IV a). Then 150 mg of dried carboxylic acid (IV a) and N-
3 mg of hydroxysuccinimide was solubilized in 1 ml of anhydrous methylene chloride, and dicyclohexylcarbodiimide (DC
C) After adding 6 mg, the mixture was stirred at room temperature for 12 hours. The resulting dicyclohexylurea (DCU) was separated and ethyl ether was added to the solution to cause precipitation. Then, the precipitate was separated and dried under reduced pressure to obtain the desired ester (IV b) 100 mg (yield 6
7%).

Experimental Example 1. Specific activity of chemically modified hG-CSF derivative and mouse leukemia cell N
Proliferation promoting activity of FS60 In the same manner as in Example 3, the hG-CSF derivative was reacted with the active ester, and the unreacted active ester and its decomposition product were removed using an ultrapermeabilization membrane. Using the same membrane, the internal solution was exchanged for PBS, and the chemically modified hG-CSF in the resulting residual solution was
G-CSF activity and growth-promoting activity of the derivatives on NFS60 cells [KL Holmes, et al., Proceeding of the
National Academy of Science (Proc.Nat
L.Acad.Sci.USA) 82, measured 6687 (1985)]. The results are shown in Table 5.

The chemically modified hG-CSF derivative clearly retained CSF activity on mouse bone marrow stem cells. Furthermore, it also had a growth promoting activity on NFS60 cells showing G-CSF-dependent growth.

Experimental Example 2. Leukocyte (granulocyte) increasing effect The chemically modified hG-CSF derivative used in Experimental Example 1 was subcutaneously administered to C3H / He mice (male, n = 3) once or once a day for 6 days, Thereafter, blood was collected over time to count white blood cells (WBC) in peripheral blood. The results are shown in Table 6 (single dose) and Table 7 (daily dose).

After a single administration, the number of white blood cells increased at a peak of 8 hours after administration, but after that, the level of the hG-CSF derivative decreased to almost normal level at 48 hours, whereas the chemically modified hG-CSF derivative showed a decrease.
The CSF derivative had a significantly high sustained leukocytosis effect even at 48 hours.

With daily administration, especially in the low dose group, chemically modified hG
The -CSF derivative clearly showed a significant leukocytosis effect compared to the hG-CSF derivative.

Experimental Example 3. Changes in blood concentration The chemically modified hG-CSF derivative used in Experimental Example 1 was subcutaneously administered to C3H / He mice (male, n = 3) once or 6 times daily, and blood was collected over time. Then, the G-CSF concentration in plasma was measured. The results are shown in Table 8 (single dose) and Table 9 (daily dose). In some experiments, a single intravenous dose (10
The experiment shown in the table) was also performed.

In the case of a single administration (subcutaneous), the blood concentration of hG-CSF derivative peaked at 1 hour and decreased rapidly thereafter.
With the chemically modified hG-CSF derivative, the blood concentration gradually increased over 5 to 7 hours and remained relatively high even after 24 hours (Table 8). On the other hand, in the experiment of daily administration, the blood concentration after 1 hour was higher in the hG-CSF derivative in all cases, but it was low in 24 hours and could not be detected after the 3rd day. On the other hand, the chemically modified hG-CSF derivative was detectable even after 24 hours, and was higher than the hG-CSF derivative.

On the other hand, in the case of intravenous administration, as shown in Table 10, the chemically modified hG-CSF derivative showed a clearly higher blood concentration.

Experimental Example 4. Specific activity of chemically modified hG-CSF and chemically modified hG-CSF derivative and proliferation promoting activity of mouse leukemia cell NFS60 (1) Chemically modified hG-CSF (type III) obtained in Example 2
Was used to measure the specific activity and proliferation promoting activity in the same manner as in Experimental Example 1. The results are shown in Table 11.

(2) The chemically modified hG-CSF derivative (I
II type) and the chemically modified hG-CSF derivative (IV type) obtained in Example 5 were used to measure specific activity and growth promoting activity in the same manner as in Experimental Example 1. The results are shown in Table 12.

Experimental Example 5 Leukocyte (granulocyte) increase effect (1) Chemically modified hG-CSF (type III) obtained in Example 2
BALB / C mice (2.5 μg / mouse) (3 males, 4 in control group)
Subcutaneously, and blood was collected over time to calculate the number of white blood cells (WBC) in peripheral blood. The results are shown in Table 13.

(2) The chemically modified hG-CSF derivative (I
The type II) and the chemically modified hG-CSF derivative (type IV) obtained in Example 5 were used at 2.5 μg / animal, and the number of white blood cells (WBC) was counted in the same manner as in the above (1). The results are shown in Table 14.

EFFECTS OF THE INVENTION The chemically modified hG-CSF and the chemically modified hG-CSF derivative of the present invention have an increased effect of increasing peripheral leukocytes (granulocytes), have improved stability and persistence in blood, and are frozen. Since it has improved stability as a dry preparation, it can be advantageously used as an investigational drug.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-57-192435 (JP, A) JP-A-62-115280 (JP, A) JP-A-63-60938 (JP, A) JP-A-63- 146828 (JP, A) Japanese Patent Sho 47-24679 (JP, B1)

Claims (4)

[Claims] 1. A modified polypeptide having a group represented by the formula (1) bonded to at least one amino group in the molecule of the polypeptide having human granulocyte colony stimulating factor activity. Formula R ₁ — (OCH ₂ CH ₂ ) _n —X—R ₂ — (I) In the formula, R ₁ is an alkyl group or an alkanoyl group, n is a positive integer that can be changed arbitrarily, X is O, NH or S, R ₂ is [In the formula, R ₃ represents OH, Cl, O- (OCH ₂ CH ₂ ) _n- R ₁ (in the formula, R ₁ and n have the same meanings as described above), and Y does not exist or Z -(CH ₂ ) _p CO (where Z is O, S or N
Represents H, p represents represents) a positive integer which can vary arbitrarily] or _{(CO) m - (CH 2} ) in _l CO (wherein, m is 0 or
1, l represents a positive integer that can be changed arbitrarily). 2. A freeze-dried preparation containing the modified polypeptide according to claim 1. 3. A leukocyte proliferation promoter containing the modified polypeptide according to claim 1. 4. A novel carboxylic acid represented by the following formula (In the formula, R ₁ , n and X are as defined above, and Z is O
Or represents S and p represents a positive integer which can be changed arbitrarily)