JPH07103158B2 - Colony stimulating factor-gelatin conjugate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] The present invention relates to a water-soluble CSF-gelatin conjugate comprising colony stimulating factor (CSF) and gelatin, a method for producing the same, and a water-soluble CSF-gelatin conjugate ( Hereinafter, a tumor cell growth inhibitor using CSF-gelatin conjugate) and CSF-
A leukopenia therapeutic agent using a gelatin conjugate.

[Conventional technology]

CSF is a substance that acts on mammalian bone marrow leukocyte progenitor cells and promotes the differentiation and proliferation of these cells into granulocytes or macrophages. Ichikawa, Y. et al; National Ac
ademy of science 56, p488, 1966; Metcalf, D .; Expe
rimental Hematology 1 vol. p185, 1973].

Various different forms of activators are known in CSF, including granulocyte / CSF (G-CSF), macrophage / CSF (MC
This includes SF), granulocyte / macrophage CSF (GM-CSF) and multifunctional CSF (multiCSF or IL-3). Due to the function of CSF to differentiate and proliferate into these leukocytes, CSF is expected to be applied to the administration of anticancer agents and the treatment of leukopenia associated with irradiation. Further, it has been reported that CSF has a function of activating mature macrophages and inducing them into antitumor macrophages [Weinberg, JB et al .; Journal of Immunolog.
y, 121, p72, 1978, Ralph, P. and Nakoing, I.; Cellul
ar Immunology, 105, p270, 1978]. CSF is a macrophage activator for cancer treatment, viruses and bacteria,
It is expected to be applied to the prevention and treatment of infectious diseases caused by parasites.

However, since CSF has a small macrophage activating action and is susceptible to degradation in vivo and is unstable, it has an enhanced macrophage activating action and an increased in vivo stability for the above-mentioned therapeutic applications. Etc. must be improved.

[Problems to be Solved by the Invention]

The present invention improves the property of CSF that it has a small macrophage activating effect and is easily degraded in vivo, and provides a CSF administration system in which macrophage activating action is enhanced and stability in vivo is increased. It is the one we are trying to provide.

[Means for Solving the Problems]

As a result of intensive studies to solve the above problems, the present inventors have found that CSF and gelatin are condensing agents (water-soluble carbodiimide).
It was found that the CSF-gelatin conjugate obtained by conjugation in the presence of E. coli has extremely advantageous characteristics and completed the present invention.

Therefore, the present invention provides a water-soluble colony-stimulating factor-gelatin conjugate comprising a colony-stimulating factor and gelatin; a water-soluble colony-stimulating factor characterized by binding the colony-stimulating factor and gelatin in the presence of a water-soluble carbodiimide-
A method for producing a gelatin conjugate; and a tumor cell growth inhibitor and a leukopenia therapeutic agent using the aforementioned water-soluble colony stimulating factor-gelatin conjugate.

[Specific description]

The CSF-gelatin conjugate of the present invention can be obtained, for example, by the following method.

After adding CSF to a buffer in which gelatin is dissolved, water-soluble carbodiimide is added and reacted at 4 ° C. for 5 to 30 hours. Unreacted CSF due to dialysis or gel filtration after reaction
And gelatin are removed, and then sterile
An F-gelatin conjugate is obtained.

The CSF used in the present invention includes M-CSF, GM-CSF, G-CSF and mul.
ti CSF, or a mixture thereof.
As a method for producing CSF, a cell culture method, an extraction method from biological components (urine, ascites, etc.), and a gene recombination method have been reported. For the purpose of the present invention, CSF produced by any method should be used. You can In particular, M-CSF obtained by purifying from human urine described in Japanese Patent Application No. 61-258163, M-CSF such as M-CSF obtained by highly purifying from cell culture supernatant described in Japanese Patent Application No. 62-291776. CSF
Is preferably used as high-purity CSF containing substantially no impurities.

Any gelatin may be used as the gelatin used in the present invention, and among them, those produced by heating and extracting crude collagen obtained by treating animal bones or skins with acid or alkali with water are preferable.

The mixing ratio of gelatin and CSF is not particularly limited, but 10 ² to 10 ⁹ CSF unit (1 ng to 10 mg) per 1 mg of gelatin
Is preferred.

1-Ethyl-3-as a condensing agent water-soluble carbodiimide
(3-Dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3- (2-morpholinyl-4
-Ethyl) carbodiimidometh-p-toluenesulfonate, 1-benzyl-3- (3-dimethylaminopropyl) carbodiimide and the like are used, and 1-ethyl-3-
(3-Dimethylaminopropyl) carbodiimide hydrochloride is particularly preferably used. The pH during the binding reaction is preferably 7 or less.

The CSF-gelatin conjugate of the present invention is water-soluble and can be dissolved in a diluent such as a buffer, saline, or an injection solvent and used for assay and treatment as it is, but it is lyophilized and dissolved in a diluent before use. May be.

The CSF-gelatin conjugate of the present invention has the following excellent properties as compared with the original CSF.

(1) The macrophage activating action using the tumor cell growth inhibitory action as an index is significantly higher than that of the original CSF.

(2) Macrophages, which are highly stable and activated by the CSF-gelatin conjugate, maintain the tumor cell growth inhibitory action for a long period of time.

(3) The effect of promoting recovery of the reduced number of white blood cells in the body is high.

〔The invention's effect〕

Since the macrophage activated by the CSF-gelatin conjugate of the present invention has a tumor cell growth inhibitory action, it can be used as a tumor cell growth inhibitor (a carcinostatic agent and a leukopenia therapeutic agent), as well as a preventive and therapeutic agent for infectious diseases. Expected to be used as a medicine.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1. Gelatin [alkaline type, pI4.9, Nitta Gelatin Co., Ltd.]
Made in PBS (phosphate buffered saline, pH)
4.7] 6.9 × 10 ⁵ units of HK cell-derived M-CSF (hereinafter abbreviated as HK-CSF) or human urine-derived M-CSF (hereinafter abbreviated as Hu-CSF) were placed in 10 ml and stirred at 4 ° C. for 10 minutes . After that, water-soluble carbodiimide [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride], (Nacalai Tesque, Inc.)
10 mg) was added, and the mixture was further reacted at 4 ° C. for 19 hours. Then, the reaction solution was dialyzed and sterilized to obtain a CSF-gelatin conjugate. As a result of performing the same reaction using ¹²⁵ I-CSF,
The recovery rate of CSF is 42.5% for both HK-CSF and Hu-CSF.
The CSF content in the F-gelatin conjugate was 68940 units (u) / 1 mg gelatin.

Example 2. P815 masto mouse tumor cells of macrophages activated with CSF (HK-CSF) -gelatin conjugate prepared in Example 1.
The growth inhibition rate against cytoma was measured according to the following method. Thioglycollate-induced mouse peritoneal macrophages 5 × 10 ⁵ cells / ml and CSF dispersed in RPMI-1640 medium containing 10% FCS (hereinafter abbreviated as RPMI-FCS) in a 24-well plate.
A sample such as (HK-CSF) -gelatin conjugate or gelatin was added and incubated for 2 days or 3 days at 37 ° C. in a 5% CO ₂ -95% air atmosphere (macrophage activation). After washing the macrophages with RPMI-1640 medium and completely removing the sample, the target tumor cells P815 mastocytoma 2.5 × 10 ⁴ cells / ml RPMI-FCS were added to the wells.
It was cultured at 7 ° C for 48 hours. Live cells of P815 mastocytoma were measured, and the cell growth inhibition rate (%) was calculated from the following formula.

The results are shown in Table 1.

As described above, the CSF-gelatin conjugate of the present invention has the same C
It showed a much higher tumor cell growth inhibitory effect than SF or gelatin.

Moreover, macrophage activation did not occur at all just by mixing gelatin and HK-CSF.

Example 3. Mouse tumor cells R1 fibrosa of macrophages activated with the CSF (HK-CSF) -gelatin conjugate prepared in Example 1.
Growth inhibition rate against rcoma is targeted to target tumor cells R1 fibrosar
The measurement was performed in the same manner as in Example 2 except that coma was used. The results are shown in Table 2.

Example 4. Mouse tumor cells P815 masto of macrophages activated with CSF (Hu-CSF) -gelatin conjugate prepared in Example 1.
The growth inhibition rate against cytoma or R1 fibrosarcoma was measured according to the following method. Thioglycolate-induced mouse peritoneal macrophages 5 × 10 ⁵ cells / ml dispersed in RPMI-FCS and a sample such as CSF (Hu-CSF) -gelatin conjugate or gelatin were added to a 24-well plate at 37 ° C. for 2 days. 5% CO ₂ -95% were incubated in an air atmosphere (macrophage activation). Then, macrophages were washed with RPMI-1640 medium to completely remove the sample, and then the target tumor cells P815 mastocytoma or R1 fibrosarcom were added to the same well.
a 2.5 × 10 ⁴ cells / ml RPMI-FCS was added and cultured at 37 ° C. for 48 hours. The cell growth inhibition rate was calculated as in Example 2. The results are shown in Table 3.

Example 5. Mouse tumor cell P815 mastocytoma growth inhibition rate of macrophages activated with various concentrations of CSF (HK-CSF) -gelatin conjugate prepared in Example 1 was activated with various concentrations of original HK-CSF. It is shown in FIG. 1 in comparison with the tumor cell growth inhibition rate of macrophages.

The CSF (HK-CSF) -gelatin conjugate showed a similar cell growth inhibitory rate at a concentration of about 1 / 2,000 that of the original HK-CSF.

Example 6. The relationship between the macrophage activation time by the CSF (HK-CSF) -gelatin conjugate or the original HK-CSF prepared in Example 1 and the growth inhibition rate of mouse tumor cell P815 mastocytoma by the activated macrophages is shown in FIG. Show. Macrophages were activated in a short time by the CSF (HK-CSF) -gelatin conjugate and the activity duration was extended.

Example 7. Balb / C mice (8 weeks old, female) were intravenously inoculated with 150 mg / kg (3.75 mg / mouse) of 5-fluorouracil (manufactured by Sigma) to prepare bone marrow-suppressed mice. 4 hours after administration of 5-fluorouracil, 5 days (5 times) CSF (Hu-CSF) -gelatin conjugate 5.3 × 10 ³ u / mouse / time, or original CSF (Hu-C)
SF) 2 × 10 ⁵ u / mouse / dose was intraperitoneally administered. After the end of administration, blood was collected from the orbital venous plexus and the number of white blood cells was quantified over time. (Measurement of white blood cells: Red blood cells in blood were crushed with Turk's solution, and the number of nuclei was counted.) The results are shown in FIG.

As shown in FIG. 3, the recovery of the white blood cell count of the CSF (Hu-CSF) -gelatin conjugate or the original CSF (Hu-CSF) -administered mouse was recovered as compared with the mouse not administered with the CSF (Hu-CSF) -gelatin conjugate. Earlier, the CSF (Hu-CSF) -gelatin conjugate was
At a concentration 50 times lower than that of F), similar recovery was shown.

Reference Example 1. Method for preparing mouse HK cell-derived CSF Mouse fibroblast-derived HK-CSF (3 × 10 ⁸ cells / roller bottle) was incubated at 37 ° C. in DM-160 medium containing 1 mM lithium chloride and 0.1% sodium hydrogen carbonate, Culture supernatant obtained by culturing for 3 days [containing CSF titer of 3500u per ml. CSF titer in vit
ro Mouse bone marrow culture method (Shikita, M. et al .; Journal of Cellul
ar Physiollogy, Vol.109, p161-169, 1981). The titer of CSF was defined as 1 unit (u) for the activity to form one colony], the pH was adjusted to 4.5 with 10% phosphoric acid, the precipitate formed was removed, and the supernatant was subjected to DEAE-cellulose chromatography, phenyl sepharose. Chromatography, Sephacryl S-300 and Superrose 12 column chromatography were used to purify the product.
000 ± 1,000, specific activity 1.8 × 10 ⁸ u / mg protein [For protein quantification, use BCA Protein Assay Reagent (Pierce Chemical
Company) (Smith, PK et al .; Analytical Biochemistry 1
50, p76-85, 1985), and M-CSF forming only macrophage colonies was obtained by the in vitro mouse bone marrow culture method.

Reference example 2. Method for producing CSF derived from human urine Adsorbed on 20 kg of granular hydrous silicic acid (trade name "White Carbon" manufactured by Tokuyama Soda Co., Ltd.) of 10,000 l of adult male urine, the protein was extracted with 1% ammonia water, and then ammonium sulfate was added. 80
It was added so as to be% saturated and precipitated. The precipitate was dissolved in water and then heat-treated at pH 4.5 at 60 ° C for 10 hours. The deposited precipitate was removed by centrifugation, and the supernatant was desalted to obtain a CSF stock solution. Stock solution is DEAE-cellulose chromatography, phenyl sepharose chromatography, Sephacryl S
-300 Chromatography, Superrose 12 Chromatography, Microbonder C18 column chromatography, SDS-PAGE analysis of molecular weight 82.0
M-CSF having a physical property value of 00 ± 6,000 and a specific activity of 1.3 × 10 ⁸ u / mg protein and forming only macrophage colonies was obtained by the in vitro mouse bone marrow culture method.

[Brief description of drawings]

FIG. 1 shows the in vitro cytostatic activity of macrophages activated with various concentrations of CSF (HK-CSF) -gelatin conjugate. In the figure,-●-is the original HK-CSF,-○-is the CSF (HK-CS
F) -Gelatin conjugate,-□-is the result for original HK-CSF + gelatin. FIG. 2 shows the in vitro cytostatic activity of macrophages activated with CSF (HK-CSF) -gelatin conjugate for various times. In the figure,-●-is the original HK-CSF (1000μ),-○-is the CS
Results are for F (HK-CSF) -gelatin conjugate (586u). Figure 3 shows 5-fluorouracil-induced myelosuppressed mice.
CSF (Hu-CSF) -gelatin conjugate 5.2 × 10 ³ u / mouse / time or original CSF (Hu-CSF) 2 × 10 ⁵ u / mouse / time, white blood cell count change after 5 days (5 times) administration Shows the result of comparison with the CSF-untreated mouse. -● -in the figure is the original CSF (Hu-CSF),-
◯ -indicates the results for the CSF (Hu-CSF) -gelatin conjugate administration group, and -Δ-indicates the results for the CSF non-administration group.

Claims (5)

[Claims] 1. A water-soluble colony stimulating factor-gelatin conjugate comprising a colony stimulating factor and gelatin. 2. The colony stimulating factor is macrophage
The water-soluble colony stimulating factor-gelatin conjugate according to claim 1, which is a colony stimulating factor. 3. The method for producing a water-soluble colony stimulating factor-gelatin conjugate according to claim 1, wherein the colony stimulating factor and gelatin are bound in the presence of a water-soluble carbodiimide. 4. A tumor cell growth inhibitor which uses the water-soluble colony stimulating factor-gelatin conjugate according to claim 1. 5. A therapeutic agent for leukopenia, which comprises the water-soluble colony stimulating factor-gelatin conjugate according to claim 1.