JPH0615477B2 - Anti-infective agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an infection control agent comprising a human colony stimulating factor (hereinafter abbreviated as CSF) as an active ingredient.

CSF is known to act on granulocyte stem cells of human or animal bone marrow cells to induce mitotic proliferation and differentiation into monocyte macrophages and neutrophils (eg Me.
tcalf.D. Etc. Experimental Haematology 1 185 (1
973)).

Regarding human CSF, CS derived from normal human tissue has been used so far.
Many reports have been made on F and CSF derived from human tumor cells (eg, Stanley et al. Fed. Proc. 35 2272 (197).
5), Burgess et al. Blood 49 573 (1977), Shah et al. Blood
50 811 (1977), Fojo et al. Biochem, 17 3109 (197)
8), Okabe et al. Cancer Res 38 3910 (1978), Asano
Etc Blood 49 845 (1977), Golde etc Blood 57
1321 (1981), Wu et al. Biol. Chem 254 6226 (197
9), Dipersio et al. Blood 56 717 (1980), etc. ).

However, these human CSFs are not completely purified, and therefore the usefulness or efficacy of human CSF as a drug remains unclear.

In recent years, the remarkable development of chemotherapeutic agents in the field of infectious diseases has broad spectrum, and has promoted the emergence of antibacterial agents having strong bactericidal activity, while conventional bacteria producing strong poisonous substances with specific pathogens. Cause a crossing phenomenon of causative bacteria such as Gram-negative bacteria that compose normal human flora in humans, and clinically cause new problems such as opportunistic infections. In addition, fungi such as Candida have recently become a clinically important problem because of the growth of molds such as Candida in place of the bacteria reduced by the use of powerful antibiotics. It has very strong side effects and its use is severely restricted. For the treatment of these infectious diseases, in addition to the effects of conventional chemotherapeutic agents, it is desired to use a drug that activates the defense function of the host, but no effective drug has been found to date.

In the early stage of bacterial infection, it is considered that the phagocytic bactericidal action of leukocytes has the strongest effect on the defense function of the host, and therefore the host's infection defense ability can be improved by enhancing the proliferation and differentiation / maturation of neutrophils and macrophages. If it can be enhanced, it may be an effective therapeutic drug for infection.

Therefore, studies on mass production of pure human CSF have been under way, but none have been successful to date. In such a situation, the present inventors established a cell line having extremely high CSF-producing ability and good proliferating ability from tumor cells of patients with oral floor cancer (CNCM Accession No. “1-315”). ),
For the first time, we have succeeded in isolating pure human CSF exhibiting human neutrophil colony-promoting activity from the culture supernatant of this cell line (Japanese Patent Application No. 59-153273).

The present inventors have investigated the anti-infective effect of this human CSF using an infected animal model, and found that human CSF was found to be in vitro.
The present invention was found to be effective as a therapeutic agent for infectious diseases because it has a remarkable neutrophil maturation ability in vo and therefore has an effect of preventing infection, and thus completed the present invention.

The present invention relates to the provision of a novel infection control agent.

That is, the present invention is an infection control agent containing human CSF as an active ingredient.

Human CSF, which is the active ingredient of the present invention, can be obtained according to the method disclosed in the above-mentioned patent application.
Obtained by culturing a hybridoma obtained by cell fusion of a SF-producing cell and a malignant tumor cell having a self-proliferating property that is distinguishable from the cell, by culturing in the presence or absence of mitogen, or human It can also be obtained by preparing a recombinant DNA using a gene encoding CSF and expressing it in an appropriate host cell (eg, Escherichia coli, yeast, Chinese hamster ovary cell, etc.).

All human CSFs obtained by these methods are included in the present invention.

The obtained human CSF-containing solution may be further purified and concentrated by known means, if necessary, and then frozen or stored, or may be stored after removing water by means such as freeze-drying and vacuum-drying. If desired, human CSF may be dissolved in an appropriate buffer solution, and then sterile filtered with a Millipore filter or the like to prepare an injection.

Furthermore, the infection control agent of the present invention may contain a pharmaceutical carrier or excipient necessary for taking a form as a pharmaceutical preparation suitable for human or veterinary medicine, and further, a stabilizer and an adsorption inhibitor.

The dose of human CSF contained in the anti-infective agent of the present invention, the number of administrations can be determined in consideration of the medical condition of the target disease patient, but usually 0.1 to 500 μg per adult, preferably 5 to 100 μg of human. 1 to 1 weekly preparations with CSF
It can be administered 7 times. However, the present invention
Is not limited by the content of.

Examples of the target infectious bacteria for which the anti-infective agent containing human CSF of the present invention as an active ingredient can be used include various bacteria and are not particularly limited. Among them, Staphylococcus spp., Streptococcus spp. Gram-positive cocci, Gram-negative facultative anaerobes such as Escherichia, Serratia, Klebsiella and other Haemophilus, Gram-negative aerobes such as Pseudomonas and Alcaligenes, and Gram-negative anaerobes such as Bacteroides A high infection control effect can be obtained against sexually transmitted bacteria and single infections with fungi such as Candida and Aspergillus or combined infections with several fungi.

<Reference Example 1> Assay method The colony formation promoting activity by human CSF (hereinafter abbreviated as CSA) was measured by the following method.

Horse serum 0.4 ml, test sample 0.1 ml, C3H / He (female)
0.1 ml of mouse bone marrow cell suspension (0.5 to 1 × 10 ⁵ nucleated cells), modified McCoy's 5A culture medium containing 0.75% agar.
After mixing 4 ml and putting it in a plastic dish for tissue culture with a diameter of 35 mm to solidify, it was cultured for 5 days at 37 ° C., 5% carbon dioxide / 95% air, 100% humidity, and the number of colonies formed (A colony consisting of 50 or more cells was defined as one colony), and the activity for forming one colony was defined as 1 unit (Unit) to determine CSA.

<Reference Example 2> Isolation of human CSF Human CSF-producing cells (CNCM Accession No. "I-31
5 ") were collected from two 150 cm ² culture flasks in which they were completely densely grown, and the cells were suspended in 500 ml of F-10 culture medium containing 10% fetal bovine serum.
Transfer to a 0 cm ² glass roller bottle (Belco),
0.5r. p. m. The rotation culture was performed at the speed of. When the cells were completely and densely grown on the inner wall of the roller bottle, the culture solution was exchanged with serum-free RPMI1640, and the cells were cultured for 4 days, and then the culture supernatant was collected and 10% of fetal bovine serum was collected.
F-10 containing is added and culture is continued. After culturing for 3 days, the serum-free RPMI1640 was changed again, and after 4 days, the culture supernatant was collected. By repeating the same procedure below, 500 ml of serum-free culture supernatant can be obtained from 1 bottle each week, and this method allows the cells to be maintained for a considerably long time and the culture supernatant can be recovered. Met.

The obtained culture supernatant 5 was treated as 1 batch, and 0.01
% After adding Tween 20, Hollow Fiber DC-4 and A
After being concentrated about 1000 times by the ultrafiltration method using micon PM-10 (manufactured by Amicon), it was purified in the following order. Ultrogel with a diameter of 4.6 cm and a length of 90 cm
AcA 54 column (manufactured by LKB) using 0.15 MN
5 ml of the above concentrated culture supernatant was subjected to gel filtration at a flow rate of about 50 ml / hour using 0.01 M Tris-HCl buffer (pH 7.4) containing aCl and 0.01% Tween 20 (manufactured by Hanai Chemical Co., Ltd.). The column was previously bovine serum albumin (molecular weight: 67,000), ovalbumin (molecular weight: 45000), cytochrome C (molecular weight: 12400).
Was calibrated at. . After the completion of gel filtration, 0.1 ml was collected from each fraction, diluted 10-fold, and the fractions showing activity were examined by the above-mentioned CSA measurement method. As a result, it was first found that the Ve = 400 to 700 ml fraction showed macrophage-dominated CSA, and the Ve = 800 to 1200 ml fraction showed neutrophil-dominated CSA.
The latter fractions were collected and concentrated to about 5 ml by an ultrafilter using PM-10 (manufactured by Amicon).

After adding 0.1% trifluoroacetic acid aqueous solution containing 30% of n-propanol (manufactured by Tokyo Kasei Co., Ltd. for amino acid sequence determination) to the above concentrated fraction and leaving it in ice for about 15 minutes,
15,000 r. p. m. The precipitate was removed by centrifugation for 10 minutes. Then, μ Bondapak C equilibrated with the above aqueous solution containing n-propanol and trifluoroacetic acid
18 columns (Waters, semi-preparative, 8mm x 30)
cm), followed by sequential elution with a 0.1% trifluoroacetic acid aqueous solution containing 30 to 60% linear concentration gradient of n-propanol. High performance liquid chromatograph is Hitachi 685-50
The detection was performed using a Hitachi 638-41 type detector (both manufactured by Hitachi, Ltd.), and absorptions at 220 nm and 280 nm were simultaneously measured. After elution, 10 μl of each fraction was collected and diluted 100-fold, and the fraction showing activity was examined. As a result, neutrophil-dominated CSA was observed in the peak eluted with 40% of n-propanol, so this peak was collected and re-chromatographed again under the same conditions to perform C chromatography in the same manner as above.
When SA was examined, neutrophil-dominated CSA was observed at the peak at the position of n-propanol 40%.
This peak was collected (4 fractions = 4 ml) and freeze-dried.

The lyophilized powder was dissolved in 200 μl of 0.1% trifluoroacetic acid aqueous solution containing 40% of n-propanol,
TSK-G3000SW column (Toyo Soda Co., Ltd., 2.5
mm × 60 cm) and subjected to high performance liquid chromatography. Elution is performed with the same aqueous solution at a flow rate of 0.4 ml / min.
A fraction collector FRAC-100 (Pharmacia) was used to collect 0.4 ml each. As a result of examining CSA for each of the collected fractions in the same manner as described above, neutrophil-dominated CSA was found in the fraction having a retention time of 37 to 38 minutes (equivalent to a molecular weight of about 20,000). Collect the fractions,
Furthermore, μBondapak C18 column for analysis (4.6 mm × 30c
m), the main peak was recovered and freeze-dried.

The human CSF thus obtained had the following physicochemical properties.

I) Molecular weight: about 19,000 ± 1,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

II) Isoelectric points: Three isoelectric points (A,
At least one of B and C).

III) Ultraviolet absorption: maximum absorption at 280 nm, 25
It has a minimum value at 0 nm.

IV) The amino acid sequence from the N-terminal to the 21st residue is as follows.

V) Amino acid composition: It has the amino acid composition shown in Table-II in the specification.

Reference Example 3 Isoelectric Point Measurement of CSF of the Present Invention Flat Bed Type Isoelectric Focusing Device FBE-30
Polyacrylamide gel (T = 5%, C) containing Pharmalyte (manufactured by Pharmacia) having a pH of 4 to 6.5 using 00 (manufactured by Pharmacia) and 4M urea.
= 3%, 115 mm x 230 mm), and electrophoresed at 30 W constant power (maximum voltage 2000 V) for 2 hours, then fixed with 30% methanol / 10% trichloroacetic acid / 35% sulfosalicylic acid, and then Coomassie Brilliant Blue R
-250 staining was performed. Low pI as isoelectric point marker
Kit pH 2.5-6.5 (Pharmacia) was used.

When separation was examined between pH 4 and 6.5, pI = 5.73, 6.
Three bands, 03 and 6.37, were recognized. Of these, pI ＝
Two bands of 5.73 and 6.03 were the main components. When isoelectric points were measured in the same manner in the absence of urea, three bands with pI = 5.52, 5.80 and 6.13 were recognized.

As a result of repeating the isoelectric point measurement 10 times, the values are as shown in Table-I, and the isoelectric points A, B, and C were different from each other by about 0.3.

<Reference Example 4> Measurement of molecular weight of CSF of the present invention It was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis device is GE-2 / 4 (Pharmacia), gel is T = 15%,
C = 2.6% polyacrylamide slab gel (70 m
m × 70 mm × 3 mm) and concentrated gel (T = 3%, C
= 20%) was used. The sample is 0.64 M2-
After boiling in a solution prepared by adding sodium dodecylsulfate to mercaptoethanol to a concentration of 2% for 3 minutes and then adding urea to a concentration of 4M, 2 μg of the product was used. After running at 120 V for 3 hours, the gel was taken out, and methanol: acetic acid: water (4: 1:
After fixing with 5), a band was detected by silver staining (Silver Stain: manufactured by Piolad). Bovine serum albumin (BSA, molecular weight 67,000) as a molecular weight marker,
Ovalbumin (OVA, 45,000), Cytochrome C
(Cyt.C, 12,000), insulin (Ins. A chain: 3,300,
B chain: 2,400) was similarly treated and used. As a result, a single band with a molecular weight of about 18,000 was observed.

It was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis device is PROTEAN ^™ 16cm (manufactured by Bio-Rad), gel is T =
15%, C = 2.6% polyacrylamide slab gel (140 mm × 160 mm × 1.5 mm) and concentrated gel (T = 3
%, C = 20%) was used. Sample is 0.64 M in advance
2 = 4 µg of a solution obtained by boiling and denaturing a solution of sodium dodecyl sulfate added to mercaptoethanol at a concentration of 2% for 3 minutes. After running for 4 hours at a constant current of 30 mA, the gel was taken out and the band was detected by staining with 0.25% Coomassie Primitive Blue R250 (manufactured by Sigma). Phosphorylase B (Phosphorylass B, molecular weight 92,500), bovine serum albumin (BSA, 67,000) ovalbumin (OVA, 45,000), carbonic anhydrase (Ca) as molecular weight markers
rbonic Anhydrase, 31,000), Soybean Trypsin Inhibitor, 21,50
0) and lysozyme (Lysozyme, 14,400) were similarly treated and used. As a result, a single band with a molecular weight of about 19,000 was observed.

From the above results and above, it is considered that the molecular weight of CSF of the present invention is 19,000 ± 1,000 as measured by SDS-PAGE. <Reference Example 5> An amino acid composition sample of human CSF of the present invention was hydrolyzed by a conventional method, and the amino acid composition of the protein portion was analyzed by a special amino acid analysis method using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.). analyzed. The results are shown in Table-II. The hydrolysis conditions are as follows.

6N HCl, 110 ° C, 24 hours, 4N methanesulfonic acid + 0.2% 3- (2-aminoethyl) indole in vacuum, 110 ° C, 24 hours, 48 hours, 7
2 hours in vacuum The sample was dissolved in a solution containing 40% n-propanol and 0.1% trifluoroacetic acid (1.5 ml), and then dissolved in water.
After taking 1 ml and drying with dry nitrogen gas, or the reagent of (1) was added and the tube was sealed in a vacuum and subjected to hydrolysis.

In the table, the measured value is 24, 48, 72 with the value of 24 hours.
It is an average of four time values in total. However, Thr, Ser, 1 / 2C
ys, Met, Val, Ile and Trp were calculated by the following method.
(Biochemistry Laboratory, Protein Chemistry II (Tokyo Kagaku Dojin)
(Published)) ・ Thr, Ser, 1 / 2Cys, and Met change over time at 24, 48, and 72 hours and are extrapolated to zero hours.

・ Val and Ile are 72-hour values.

・ Trp is the average of 24, 48 and 72 hours.

The number of amino acid residues in the table is a predicted value calculated assuming 33 Leu. In general, amino acids that require correction as described above are either partially or substantially destroyed during hydrolysis, or are less susceptible to hydrolysis.
Since o has a low coloring rate, etc., the measured values (nmol) of those amino acids, and therefore the number of residues calculated from them tend to show lower values than they actually are (for example reference).

<Example> Infection prevention effect of human CSF 1. Protective effect against Pseudomonas aeruginosa infection 8 to 9 week-old (body weight 35.3 ± 1.38 g) ICR mice (male) Endoxan (Shionogi, trade name) 200
After intraperitoneal administration of mg / kg, it was divided into 3 groups, and the solvent (1% propanol, 5% (W / V) mouse serum albumin) containing human CSF (25000 μ / mouse or 50,000 μ / mouse) was divided into 2 groups, And another one group only solvent,
Each 24 hours, 0.1 ml was subcutaneously administered 4 times.
Pseudomonas was given to each group 3 hours after the 4th administration.
Aeruginosa (Pseudomonas aeruginosa) GNB-139
(3.9 × 10 ⁵ CFU / mouse) was subcutaneously administered to infect. Human CSF 21 hours after infection
A solvent containing (25,000 μ / mouse or 50,000 μ / mouse) or only the solvent was subcutaneously administered to the corresponding group.

The effect of preventing infection was examined by the number of surviving mice up to 10 days after infection (Table-III).

(Preparation of Bacterial Solution) Pseudomonas aeruginosa was used overnight at 37 ° C. using a heart infusion agar plate (Difco, trade name).
GNB-139 is cultured by shaking. The culture solution was prepared by suspending it in physiological saline.

2. Anti-protective effect against Candida infection 8-week-old (body weight 40.5 ± 1.60 g) ICR mouse (male)
Endoxan (Shionogi: trade name) 200 mg /
After intraperitoneal administration of kg, human CSF was divided into 2 groups and 1 group
(50000u / mouse) containing solvent (1% propanol,
(10% (W / V) allogeneic mouse serum) was subcutaneously administered to another group 4 times by 0.1 ml of the solvent alone every 24 hours. 4 hours after the 4th administration, Candida albicans U-50-1 (leukemia patient isolate, Tohoku University Bacteriology Department) 5.6 × 10 ⁵
CFU / mouse was administered intravenously to infect. After infection 1
The infection control effect was examined by the number of surviving mice up to day 0 (Table-IV).

(Preparation of bacterial solution) Sabouraud liquid medium (2% dextrose (manufactured by Junsei Kagaku) 10% tryptocase peptone (manufactured by BBL: trade name), 5% yeast extract (manufactured by Difco: trade name), pH5.
6) with Candida albicans U-5 at 37 ° C. overnight
Incubate 0-1 with shaking. The culture solution was washed twice with physiological saline and then suspended in physiological saline to prepare.

As shown in Tables III and IV, it was confirmed that the human CSF of the present invention has a remarkable anti-infective effect.

The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1 The human CSF-containing fraction obtained by the method of Reference Example 2 was subjected to aseptic treatment, and a frozen product frozen at -20 ° C was used to prepare an injection.

Example 2 Human CSF obtained by the method of Reference Example 2 was aseptically filled into a 10 ml vial in an amount of 5 ml, and the freeze-dried product was freeze-dried at −20 ° C. and capped with a rubber stopper to give an injection.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Masahiko Matsumoto 3-41-8 Takada, Toshima-ku, Tokyo Inside Chugai Pharmaceutical Co., Ltd. (56) References JP-A-58-59924 (JP, A) JP-A-SHO 59-210027 (JP, A) JP-A-61-227526 (JP, A)

Claims (4)

[Claims] 1. A human G-CS having the following physicochemical properties.
Anti-infective agent containing F as an active ingredient. [Physical and chemical properties] (i) Molecular weight: sodium dodecyl sulfate-polyacrylamide gel electrophoresis 19000 ± 100
0. (ii) Isoelectric points: Three isoelectric points (A,
At least one of B and C). (iii) Ultraviolet absorption: maximum absorption at 280 nm, 2
It has a minimum value at 50 nm. 2. The infection control agent according to claim 1, wherein the isoelectric point pl of human G-CSF is A in Table I of claim 1. 3. The infection control agent according to claim 1, wherein the isoelectric point pl of human G-CSF is B in Table-I of claim 1. 4. The infection control agent according to claim 1, wherein the isoelectric point pl of human G-CSF is C in Table-I of claim 1.