JPH08119984A - Actinomyces blonging to genus streptomyces and tgf-beta inhibitor derived from the same actinomyces - Google Patents

Abstract

PURPOSE: To obtain the subject new actinomyces belonging to the genus Streptomyces, having TGF-β-inhibiting activity and capable of producing TGF-β inhibiting active substance for treating diabetic nephropathy, acute glomerulonephritis, cirrhosis of the liver, pulmonary fibrosis, etc., caused by TGF-β. CONSTITUTION: This actinomyces comprises actinomyces Streptomyces sp. NKK-908 (FERM P-14410) belonging to the genus Streptomyces, having gray- based aerial mycelium color, forming key-like or spiral spore chain in sporophore of aerial mycelium and having smooth surface of spore and capable of assimilating L-arabinose, D xylose, D-fructose, L-rhamnose, sucrose and raffinose on Pridham-Gottlieb agar culture medium and having TGF-β inhibiting substance-producing ability. The actinomyces is cultured in a culture medium and then, a substance produced by the actinomyces is separated and collected from a cultured products to provide the objective TGF-β-inhibiting substance useful as a medicine for treating diabetic nephropathy, acute glomerulonephritis, cirrhosis of the liver, pulmonary fibrosis, etc., proliferative vitreous body retinopathy, etc., to which TGF-β is related.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to actinomycetes belonging to the genus Streptomyces and having a TGF-β inhibitory substance-producing ability, and a TGF-β inhibitory activity obtained from a culture of the actinomycete. It relates to a method for producing a substance. Furthermore, the present invention relates to a TGF-β inhibitor containing the obtained substance as an active ingredient. TGF-β inhibitors are diseases related to TGF-β, such as diabetic nephropathy,
It is useful as a therapeutic drug for glomerulonephritis, liver sclerosis, pulmonary fibrosis, proliferative vitreoretinopathy and the like.

[0002]

2. Description of the Related Art TGF-β (Transforming Growth Fact
or-β) is a kind of peptide hormone in vivo, enhances cell adhesion and controls cell growth and differentiation through regulation of extracellular matrix such as collagen, fibronectin and decorin. The action of TGF-β on cell growth and differentiation acts both stimulatory and inhibitory depending on the type of cell. The action of TGF-β in vivo is mainly to promote wound healing and angiogenesis. But,
On the other hand, TGF-β is a disease associated with fibrosis of various organs,
For example, it is also a cause of diabetic nephropathy, glomerulonephritis, liver sclerosis, pulmonary fibrosis, and proliferative vitreoretinopathy. Therefore, by inhibiting TGF-β, the above diseases can be treated.

So far, as TGF-β inhibitors, anti-TGF-β antibody (Nature; 1990, vol.346, p371-374) and decorin (Nature; 1990, vol.
vol.346, p281-284) and TGF-β receptor (Cel
l; 1992, vol.69, p1067-1070) is known. Of these, decorin is a type of proteoglycan (protein to which mucopolysaccharide is bound) whose production is promoted by TGF-β and whose molecular weight is approximately 120 kilodaltons (kD). Decorin can bind to TGF-β, and TGF-
It has the effect of inactivating β.

[0004]

The TGF-β inhibitors in the above-mentioned prior art are all derived from animals and animal cells and are high molecular weight proteins, so that they are complicated to handle and lack stability.

Furthermore, it takes time and money to extract and purify a minute amount of protein from an animal body, and it is economically difficult to put it into practical use. Also, when using animal cells,
It is economically disadvantageous because serum is used as the medium and the culture time is long.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a TGF-β inhibitor other than a high molecular weight protein. More specifically, the present invention provides actinomycetes capable of growing in a simple medium and producing a TGF-β inhibitory active substance, and at the same time an inexpensive and efficient TGF-β inhibitory action from a culture in which this microorganism is cultured. It is intended to provide a method for obtaining a substance. As a result, various TGF-β inhibitors can be provided.

[0007]

The invention according to claim 1 belongs to the genus Streptomyces and has a basal bacterium color of gray and has a key-like or spiral long spore on an aerial spore spore. It forms chains and has a smooth surface of spores, assimilates L-arabinose, D-xylose, D-fructose, L-rhamnose, sucrose, and raffinose on a Pridham-Gotrieve agar medium, and is a TGF-β inhibitor. It is an actinomycete with a production ability.

In addition to the above, the invention according to claim 2 is that the actinomycetes belonging to the genus Streptomyces and having the ability to produce a TGF-β inhibitor is Streptomyces sp. NKK-
908 (Streptomyces sp. NKK-908).

The invention of claim 3 is a strain belonging to the genus Streptomyces and having the ability to produce a TGF-β inhibitor, particularly Streptomyces sp. A method for producing a TGF-β inhibitory active substance, which comprises culturing NKK-908 to produce and accumulate a TGF-β inhibitory active substance, and separating and collecting from the culture.

The invention according to claim 4 is the above-mentioned claim 3
A TGF-β inhibitor containing the TGF-β inhibitory active substance obtained by the method according to the item 1 as an active ingredient.

The details of the present invention will be described below. <Strains> NKK-908 strain used in the present invention is Shizuoka
Using the following medium from the soil of Futo beach, Minamiizu-cho, Japan
It was separated by. Specifically, separation medium
(% W / V): glycerin 0.1, glucose 0.1, L-
Arginine 0.03, K₂ HPO_Four0.03, NaCl 0.03, MgSO_Four ・ 7H
₂O 0.02, agar 1.5, Fe (SO_Four)₃ 0.001, CuSO_Four ・ 5H ₂O 0.
0001, ZnSO_Four・ 7H₂28 days at O 0.0001 (pH 6.4)
After continuous culture, colonies were transformed into the following agar medium (% W / V): yeast
Extract 0.1, meat extract 0.1, N.Z. amine A 0.2, gluco
Incubate at 1.0 ℃, agar 1.5 (pH 7.3) at 28 ℃ for 7 days,
The obtained colonies were designated as ISP (International Streptomyc
es Project) No. Separated in 2 media.

The NKK-908 strain has the following mycological properties. (A) Morphological properties The basal hyphae develop well on yeast / malt agar medium, oatmeal agar medium, starch / inorganic salt agar medium, glycerin / asparagine agar medium, etc., and fragmentation is not observed. Aerial aerial hyphae grow abundantly in the above-mentioned medium, the color tone is grayish, and fragmentation is not observed. When observed under a microscope, the spore stalk on the aerial mycelium had a key-like or incomplete spiral 1
Formation of zero or more spore chains is observed. The spores have a diameter of 0.7-1.0 μm and a length of 0.6-1.2 μm, and the surface is smooth with no motility or flagella. No sporangia, sclerotium, or zoospores were observed.

(B) Culture properties Yeast / malt agar medium (ISP No2), oatmeal agar medium (ISP No3), starch / inorganic salt agar medium (ISP No4), or glycerin / asparagine agar medium of NKK-908 strain ( The growth conditions on ISP No5) are as shown in Table 1.

[0014]

[Table 1]

Regarding the color description, see A. Kornerup and J.
H. Wanscher, "Methuen handbookof color" third ed
ition; 1978, Eyre Methuen, with reference to the method, the color code name and its code are also shown. Observation is 3
It was performed after culturing at 0 ° C. for 7 to 14 days.

In Table 1, along with the NKK-908 strain, Streptomyces gardneri IFO
12865 ^T ) and Streptomyces narbonensis (Str
The characteristics of eptomyces narbonensis IFO 12801 ^T ) on various media are also shown for reference.

(C) Physiological properties (1) Growth temperature range: yeast / malt agar medium (ISP No.
In 2), it was grown at 11 to 35 ° C. The optimum growth temperature was 23 to 31 ° C. (2) Formation of melanin-like pigment: not observed (3) Utilization of carbon source: Carbon assimilation was carried out using Pridham-Gotlieve agar medium (ISP No9). The results are shown below. Use: L-arabinose, D-xylose, D-fructose, L-rhamnose, sucrose, raffinose Not use: Inositol, D-mannitol

(D) Chemotaxonomic properties (1) Cell wall composition Becker et al. (B. Becker, et. Al., Appl. Microbio
l.; 1964, vol.12, p421-423), and the composition of cell wall components was analyzed. The presence of arabinose and galactose was estimated by using the sulfuric acid hydrolyzate of all bacterial cells. The results are shown in Table 2.

[0019]

[Table 2]

(2) Kind of bacterial cell lipid The quinone system in the bacterial cell is menaquinone, and the number of isoprene units and hydrogen saturation of its side chain were MK-9 (H8) and MK-9 (H6).

(3) Base composition of DNA The GC content of intracellular DNA was determined with reference to the bacterial identification method described in Eiko Yabuuchi et al. And a new taxonomy; 1987. The results are shown in Table 3.

[0022]

[Table 3]

(4) DNA-DNA homology with closely related bacterial species DNA-DNA hybridization test by photobiotin labeling method using microplate (Takayuki Ezaki et al., Journal of Japanese Bacteriology; 1990, vol 45, p851) The results are shown in Table 4.

[0024]

[Table 4]

Although the mycelial properties of the NKK-908 strain have been described in detail above, in summary, diaminopimelic acid in the cell wall is LL type, forms basal hyphae, and has a long spore chain on the spore stalk of aerial mycelia. Is characterized by forming. As for various nutritional properties, the basal hyphae have a grayish tone and the aerial mycelia also have a grayish tone. The bacterium does not produce melanin pigment.

From the above results, it is appropriate to consider that this strain belongs to the genus Streptomyces in actinomycetes. Nono
mura's reference (H. Nonomura, J. Ferment. Technol .; 197
4, vol 52, p78), the species was searched, but there was no description of the bacterial species that completely matched the properties of this cell.

Then, a search was conducted by focusing on only the color tone and assimilation property of the surface of the village, and it was found that Streptomyces gardneri
(Streptomyces gardneri IFO 12865 ^T ) and Streptomyces narbonensis I
FO 12801 ^T ), so these two strains were selected as reference strains, and the culture properties, the GC content of intracellular DNA,
A DNA-DNA hybridization test was performed for comparative examination. As a result, as shown in the table, this strain differed from the reference strain in color tone of the colony and the like. Although the GC content showed a close value, DN
A homology test showed low homology with the reference strain. Therefore, it is considered appropriate to identify the NKK-908 strain as a new strain.

From the above results, this strain belongs to the genus Streptomyces in actinomycetes, and is a new strain different from Streptomyces gardneri and Streptomyces narbonesis. The strain NKK-908 was deposited under the deposit number FERM P-14 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.
It has been deposited as 410.

The NKK-908 strain of the present invention, as seen in other strains of the genus Streptomyces, is likely to change its properties, and for example, irradiation treatment with X-rays, ultraviolet rays, radiation, etc., such as nitrogen mustard, Azeserine, nitrous acid, 2
-Aminopurine, mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine, and easily mutated by commonly used bacterial species mutation treatment methods such as phage contact, transformation, transduction, conjugation, etc. Any of the artificial strains and the strains capable of producing the TGF-β inhibitory active substance can be used in the method of the present invention.

<Culturing Method> In order to obtain a TGF-β inhibitory active substance which is an active ingredient of the TGF-β inhibitor of the present invention, first, the NKK-908 strain is inoculated into a nutrient-containing medium and aerobically cultured. To do. As the nutrient source, those known as the nutrient source for actinomycetes can be used. For example, as the carbon source, commercially available glucose, glycerin, maltose, starch, saccharose, molasses, dextrin or the like can be used alone or as a mixture. As the nitrogen source, commercially available corn steep liquor, soybean meal, meat extract, yeast extract, cottonseed flour, peptone,
Wheat germ, fish meal, inorganic ammonium salts, sodium nitrate and the like can be used alone or as a mixture. As the inorganic salt, commercially available sodium chloride, magnesium sulfate, calcium chloride, zinc sulfate, copper sulfate, manganese sulfate, cobalt chloride, iron sulfate, potassium chloride and the like can be used. In addition, if necessary, a trace amount of a metal salt such as cobalt or molybdenum can be added. Hereinafter, one example of a preferable medium for producing the target TGF-β inhibitory active substance will be described.

Culture medium 1% Glucose 0.5% Cotton seed oil 1.5% Corn steep liquor 1% Soybean meal 0.2% CaCO ₃ 100 μl Trace salt solution ^* (pH 7.0) *: Trace salt solution composition (Unit: g /
l) NaOH 15 EDTA Na 60 MgSO ₄ .7H ₂ O 20 CaCl ₂ .6H ₂ O 5 ZnSO ₄ .7H ₂ O 2 MnSO ₄ .4H ₂ O 2 CuSO ₄ .5H ₂ O 0.5 CoCl ₂ .6H ₂ O 0.05 FeSO ₄ .7H ₂ O 10 (dissolved in 1.75 ml of sulfuric acid in advance) H ₃ BO ₄ 0.031 Na ₂ MoO ₄ .2H ₂ O 0.002 KCl 75

The culture method may be the same as the method for producing a general microbial metabolite, and liquid culture is particularly preferable. In the case of liquid culture, any of static culture, stirring culture, shaking culture, aeration culture and the like can be performed, but shaking culture or deep aeration stirring culture is particularly preferable. The culture temperature is 25 ° C or higher on the low temperature side, preferably 27 ° C or higher, and 37 ° C on the high temperature side.
The following is preferably 30 ° C. or less. PH of medium is 6 ~
8 is suitable, but pH 6.5-7.5 is preferable. The culture time is 24 hours to 10 days, preferably 3 to 7 days.

<Separation / Purification Method> In order to collect the desired TGF-β inhibitory active substance from the culture, a separation means usually used for collecting from the culture of the microorganism is appropriately used. The TGF-β inhibitory active substance, which is an active ingredient of the medicine of the present invention, is accumulated in the culture medium and the cells, but it is preferable to collect it from the culture medium in consideration of the efficiency of separation and purification. The culture solution obtained above is separated from the cells by centrifugation or filtration. For example, 2000r
Centrifuge at pm for 20-30 minutes. The TGF-β inhibitory activity of the obtained culture supernatant is measured using the TGF-β inhibitory activity test described below.

As shown in Example 1 <ii>, the culture supernatant has TGF-β inhibitory activity. This culture supernatant and its freeze-dried product were used as the present active substance A, and the following STEP
Isolate and purify.

STEP1 First, the culture supernatant is extracted with an organic solvent to obtain an aqueous layer.
As the organic solvent, for example, hexane, ethyl acetate, chloroform can be used. As an extraction method, for example, the culture supernatant is extracted twice with an equal volume of hexane, the aqueous layer is further extracted twice with an equal volume of ethyl acetate, and the obtained aqueous layer is extracted twice with chloroform. Is preferred.

The aqueous layer after solvent extraction is freeze-dried and dissolved in an appropriate amount of water. An appropriate amount means 1/20 to 1 / of the culture supernatant
The amount is 2, preferably about 1/10. This lysate is fractionated by ion exchange chromatography. For example, DIAION WA- which is one of the synthetic resin ion exchangers.
30 (trade name, manufactured by Mitsubishi Kasei Co., Ltd., inner diameter 1 cm x 25 cm)
After using the column and supplying the above-mentioned solution, the column is washed with about 3 volumes of water to remove non-adsorbed substances and eluted with 1M-NH ₄ Cl. Each eluted fraction was subjected to TGF-
The β inhibitory activity is measured.

As illustrated in Example 1, the TG of the eluted fraction
The F-β inhibitory activity shows one peak. The active fractions are collected, and the lyophilized product as it is or as the active substance B,
Proceed to STEP 2 below.

STEP 2 The fraction of the present active substance B obtained in STEP 1 is precipitated with an organic solvent such as ethanol, and the resulting precipitate is collected by centrifugation. For example, 40 ml of TGF-β inhibitory activity fraction
80 ml of ethanol was added to the mixture to cause precipitation, and 50
It is preferable to centrifuge at 15000 rpm using a ml centrifuge tube. The obtained precipitate is dissolved with water and the insoluble matter is removed by filtration. Then, this solution is subjected to high performance liquid chromatography (HPLC) using a cellulose ion exchanger column. Next, elution is performed with the same buffer solution in which the concentration gradient of NaCl is gradually increased. For example, 20mM HEPES
(N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid) buffer solution equilibrated with TSKgel DEAE
-2SW (trade name, manufactured by Tosoh Corporation, inner diameter 2.15 cm x 25)
cm) column, and apply the above-mentioned lysis solution to 0M-1M NaC
Elution with a concentration gradient of 1 is preferred. Each of the eluted fractions is subjected to TGF-β inhibitory activity measurement described below. The active fraction or its lyophilized product is referred to as the present active substance C.

As shown in Example 1, the active substance C
Is Mv1lu, which is a TGF-β inhibitory activity test described below.
In the cell test system, its TGF-β inhibitory activity is divided into three peaks. Fractions of the three peaks are each eluted with the following concentration range of NaCl. That is, 0.35
0.5 M (this active substance C-I), 0.5 to 0.6 M (this active substance C-II), 0.6 to 0.9 M (this active substance C-I)
III). Of these, it was the fraction of the active substance C-III that showed TGF-β inhibitory activity in the second TGF-β inhibitory activity test described below, and this fraction was analyzed by the following ST.
Proceed to EP3.

STEP3 Fractions of the active substance C-III were collected and freeze-dried for several meters.
After concentrating to l, it is subjected to gel filtration HPLC. For example,
20 mM HEP on TSKgel G3000PX _XL column (trade name, manufactured by Tosoh Corporation, inner diameter 0.78 x 30 cm)
When eluted with ES buffer, TGF-β inhibitory activity can be confirmed in the fraction around molecular weight 2000. Collect this fraction,
As it is or by freeze-drying, the active substance D is obtained.

As shown in Example 1, the active substance D has a molecular weight of about 2000. The inhibitory activity of the present active substance D on TGF-β is stable in the presence of a reducing agent (eg, treated with 1 M dithiothreitol overnight) and is inactivated by heat, acid or alkali. For example, incubating at 100 ° C for 10 minutes, treating in 1N HCl at 22 ° C for 3 hours, and in the presence of 10% ammonia at 22 ° C for 3 hours.
When treated with time, the inhibitory activity of sample D on TGF-β is lost.

<Method for measuring TGF-β inhibitory activity> TGF-
The β inhibitory activity can be measured by the following two test systems.

The first test system was mink lung epithelial cells (Mv
This is a test system using 1lu cells). For Mv1lu cells, for example, ATCC No. CCL64 can be used. TGF-β has the effect of suppressing the proliferation of Mv1lu cells. Therefore, if a substance having a TGF-β inhibitory activity is added, Mv1l is present even in the presence of TGF-β.
The growth of u cells is not inhibited. Since cells can be stained with crystal violet, the absorbance at 415 nm of crystal violet reflects the number of cells. Therefore, the effect of the present active substance can be confirmed by the fact that when Mv1lu cells are grown in the presence of TGF-β, the absorbance at 415 nm is larger than that in the drug-free group.

The second test system is rat skeletal muscle myoblasts.
This is a test system using (L6 cells). For L6 cells, for example
For example, ATCC No. Can use CRL1458
it can. TGF-β was found to induce extracellular matrix in L6 cells.
It has the effect of accelerating the synthesis of X. The deco mentioned above
Phosphorus has a sugar chain structure of chondroitin sulfate
Therefore, with TGF-β³⁵S-SO_Four ^2-To L6 cell culture system
Add it to Decorin ³⁵S] -SO_Four ^2-Is captured
You. Use the culture supernatant for polyacrylamide gel electrophoresis.
If you do an autoradio guffy, the position of Decorin,
That is, a band appears at a position with a molecular weight of about 120 kD. Obedience
Therefore, the effect of this active substance is that L6 cells are present in the presence of TGF-β.
Molecular weight detected from the supernatant of the cell culture as described above
Band of about 120 kD becomes smaller or disappears
By doing so, it is possible to confirm. The control experiment
As for, TGF-β was added without adding the culture supernatant
The (+) or non-added (-) system is similarly measured. cell
Since there is also endogenous TGF-β in the
In the test (-) as well, the amount produced by the action of TGF-β
A thin band around 100 kD is observed.

Example 1 As shown in Table 1, Example substances A to D obtained from the culture of the strain of the present invention show TGF-β inhibitory activity in either or both of the above two test systems. Had.

<Toxicity> The toxicity of the present active substance can be evaluated by intraperitoneal administration of 500 mg / kg to ICR male mice aged 4 to 5 weeks. As shown in Example 2, with the substance of Example prepared in Example 1, no death was observed in any of the administration groups.

No cytotoxicity was observed in the substances of these Examples in the TGF-β inhibitory activity test by the above two test systems.

<Usage (including formulation), capacity> T of the present invention
The GF-β inhibitor contains the present active substance as an active ingredient. The administration route is not particularly limited, and oral administration or parenteral administration (for example, intramuscular injection, intravenous injection, subcutaneous injection, rectal administration,
Mucosal absorption, transdermal administration, eye drop). Also,
Regarding the dosage form (form), a pharmaceutically acceptable auxiliary agent is mixed to prepare an injection, tablet, capsule, granule, powder, pill, fine granule, rectal administration, suppository, eye drop. It can be a spray, a coating, or a local tissue administration agent. Examples of pharmaceutically acceptable adjuvants include diluents, excipients, binders, disintegrants, lubricants, buffers, preservatives, solubilizers, preservatives, flavoring agents, soothing agents, and stabilizing agents. Examples include an agent and a colorant, which can be appropriately combined and used by a conventional method.

Examples of the diluent include sterile water and sterile physiological saline. Examples of excipients include lactose, sucrose, glucose, sorbitol, corn starch, crystalline cellulose. Examples of binders include cellulose derivatives, gum arabic, gelatin, polyvinyl alcohol, polyvinyl ether. Examples of disintegrants include carboxymethyl cellulose calcium. Examples of lubricants include talc, magnesium stearate, polyethylene glycol. Examples of buffers include boric acid, boric acid-sodium sulfite,
Sodium dihydrogen phosphate-sodium hydrogen phosphate and the like can be mentioned. Examples of preservatives include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like. Examples of solubilizers include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, and maglogol. Examples of preservatives include glycerin. Examples of the flavoring agent include cocoa powder, peppermint oil, and cinnamon powder. Examples of soothing agents include benzyl alcohol. Examples of stabilizers include sodium sulfite, sodium metasulfite, and ether. Examples of colorants include tar dyes, iron sesquioxide, titanium oxide, β-carotene, and chlorophyll.

Example 3 shows a formulation example of the active substance using the substance of Example prepared in Example 1.

The dose of the TGF-β inhibitor of the present invention varies depending on age, body weight, sex, symptoms, administration method, administration period, etc. 0.5 to 5000 mg, 0.5 to 10 for dwarfs
It is 00 mg, and this may be administered in 1 to 5 divided doses per day.

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

[0053]

【Example】

Example 1: Collection of the present active substance, TGF-β inhibitory activity test <i> Cultivation of strain 100 ml of the culture medium described above was placed in a 500 ml flask, and high temperature high pressure steam sterilization was performed at 121 ° C for 15 minutes. It was Actinomyces NK cultured in agar medium in this flask
The K-908 strain was inoculated and cultured at a constant temperature of 28 ° C. for 5 days on a rotary shaker (rotation speed: 120 rpm).

<Ii> Separation / Purification and TGF-β Inhibitory Activity of Each Fraction FIG. 3 shows a scheme of a method for collecting the active substance from the culture solution.

<Ii-1> Separation of culture supernatant The culture solution obtained in the above <i> was dispensed into a 50 ml centrifuge tube and centrifuged at 2000 rpm for 30 minutes to remove bacterial cells.

This culture supernatant is shown in FIG. 1 as follows <iii-1>
The results obtained by adding 10 μl each to the TGF-β inhibitory activity test system of No. 2 are shown. As shown in FIG. 1, the culture supernatant of the NKK-908 strain inhibited the growth inhibitory effect of TGF-β on Mv1lu cells, and cell proliferation equivalent to that of the TGF-β-free control was confirmed. This culture supernatant and its dried product were used as the active substance A.
And

FIG. 2 shows the culture supernatant of the following <iii-2>
The results obtained by adding 10 μl each to the TGF-β inhibitory activity test system of No. 2 are shown. As shown in FIG. 2, the culture supernatant of the NKK-908 strain suppressed decorin production of L6 cells promoted by TGF-β.

<Ii-2> STEP 1: Solvent extraction and ion exchange chromatography 300 ml of the culture supernatant obtained in <ii-1> above was subjected to solvent extraction to obtain an aqueous layer. Hexane is extracted twice with solvent,
Perform ethyl acetate twice and chloroform twice, and perform 3 times each.
00 ml of organic solvent was used.

The obtained aqueous layer was freeze-dried by a conventional method. 30 ml of water was added to this lyophilized product to give a 10-fold concentrated solution. A DIAION WA-30 (trade name, manufactured by Mitsubishi Kasei Kogyo KK, inner diameter 1 cm × 25 cm) column was equilibrated with water, and the 10-fold concentrated solution was provided. After washing with about 100 ml of water to remove non-adsorbed substances, the mixture was eluted with 1M-NH ₄ Cl. The size of the fraction was 5 ml. The elution pattern was detected by the absorbance at 280 nm.

FIG. 4 shows the results obtained by adding 10 μl of the fractions obtained from the above column to the TGF-β inhibitory activity test system of <iii-1> below. As shown in FIG. 4, TGF-β
The inhibitory activity was eluted as one peak. 40 ml of this active fraction (fraction Nos. 26 to 32) and this lyophilized product were designated as the present active substance B.

<Ii-3> STEP 2: Ethanol precipitation and DEA
E-HPLC To 40 ml of the active substance B fraction was added a double volume of ethanol (80 ml) to give a white precipitate. The suspension was placed in a 50 ml centrifuge tube and centrifuged at 15000 rpm to collect the precipitate. Discard the supernatant and discard the precipitate
After dissolving with ml of water, it was filtered with a 0.2 μm filter.

20 mM HEPES (N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid) buffer (p
H7.2) TSKgel DEAE-2S equilibrated with H7.2)
The above solution was applied to a W (trade name, manufactured by Tosoh Corporation, inner diameter 2.15 cm × 25 cm) column. After flowing with a 20 mM HEPES buffer (pH 7.2) for 20 minutes at a flow rate of 5 ml / min, elution was performed with the same buffer in which the concentration gradient of NaCl was linearly increased from 0 to 1 M over 80 minutes. Fraction size is 5m
l.

FIG. 5 shows the results obtained by adding 10 μl of the fractions obtained from the above column to the Mv1lu cell test system of <iii-1> below. As shown in FIG. 5, the TGF-β inhibitory activity of the fraction showing the activity (this active substance C) was divided into three peaks, and each fraction was eluted with NaCl in the following concentration range. That is, 0.35-0.5M (this active substance C-
I), 0.5-0.6M (this active substance C-II), 0.6
-0.9 M (this active substance C-III).

FIG. 6 shows active substances C-I, C-II and C-II.
10 μl each of I was added to the test system of <iii-2> L6 cells described below to measure the TGF-β inhibitory activity. As shown in FIG. 6, among the active substances C, C-II
Only I was found to have an action of suppressing decorin production of L6 cells promoted by TGF-β.

<Ii-4> STEP 3: Gel filtration-HPLC 20 ml of the fraction of the active substance C-III was freeze-dried by a conventional method and dissolved in 1 ml of water. This concentrated solution is referred to as TSKg
el G3000PX _XL column (trade name, manufactured by Tosoh Corporation,
Subjected to gel filtration HPLC using an inner diameter of 0.78 x 30 cm). Elution was performed with a 20 mM HEPES buffer (pH 7.2) at a flow rate of 1 ml / min and a fraction size of 1 ml.

FIG. 7 shows the results obtained by adding 10 μl each of the fractions obtained from the above column to the TGF-β inhibitory activity test system of <iii-1> below. As shown in FIG. 7, TGF-β
The inhibitory activity was eluted as one peak in the fraction around molecular weight 2000. This active fraction or its lyophilized product was designated as the present active substance D.

<Iii> Test method for pharmacological activity <iii-1> Test system using mink lung epithelial cells (Mv1lu cells) Mv1lu cells (ATCC No. C) suspended in a medium.
CL64) in a 96-well microplate with 30 cells
50 μl was dispensed so that it became 00 / well. The medium contains DMEM containing 5% fetal bovine serum (FBS).
(Dulbecco's Modified Eagle Media, product name, GIBCO
BRL Catalog No. 12800-017) was used. The above cells were cultured under the conditions of 37 ° C., 5% CO ₂ /95% air for 2 hours, and then dissolved in the same medium to 1 ng / ml of TGF-β (TGF-β +) or only the medium ( TGF
-Β-free control: TGF-β-) was added at 50 μl / well each. Here, a solution of the test substance in the medium (compound added) or medium (compound-free control: Cont in the figure)
10 μl / well was added to each), and the cells were cultured under the same conditions as above for 4 days.

After culturing, the cells were stained with crystal violet as follows. First, discard the culture supernatant,
A 20% methanol solution of 0.2% crystal violet was added at 100 μl / well. After 20 minutes, the staining solution was discarded, washed twice with water, and 100 μl of 0.1N NaH ₂ PO ₄ / methanol (1: 1) solution was added to each well. After standing for 1 hour, the absorbance at 415 nm was measured with a microplate detector (manufactured by Tosoh Corporation).

<Iii-2> L6 suspended in a medium similar to the medium used in the test system <iii-1> using rat skeletal muscle myoblasts (L6 cells)
100 μl of cells (ATCC No. CRL1458) were dispensed into a 96-well microplate so that the cell number was 5 × 10 ⁵ / well. After culturing for 3 hours under the culture condition of <iii-1>, the medium was changed to a serum-free medium. RPMI-1640 (Sanko Junyaku Co., Ltd., Catalog No. SS610-0101) was used as the serum-free medium. TG
F-β was dissolved in SO ₄ ^2- -free RPMI-1640 medium (manufactured by Nikken Biomedical Research Institute) at 100 ng / ml, and 10 μl was added to each well. As a control without addition of TGF-β, SO ₄ ^2- -free RPMI-1640 was used.
Only the medium was added. After adding 10 μl / well of a test substance medium solution (compound addition) or medium (compound-free control: indicated as Control in the figure) to each, [ ³⁵ S] -Na ₂ SO ₄ was further added at 20 μCi / Wells were added, and the cells were cultured for 16 hours under the same culture conditions as above.

After culturing, 10 μl of a protease inhibitor mixture (1% leupeptin, 1% antipain, 10% trypsin inhibitor) was added to each well.
Collect the culture supernatant in a microtube, 1200 rpm
It was centrifuged for 3 minutes. 90 μl of this supernatant and sample buffer (125 μM Tris (trihydroxymethylaminomethane) -HCl, 4% SDS (sodium dodecyl sulfate), 10 mM β-mercaptoethanol,
30 μl of 25% glycerol, pH 6.8) was mixed and subjected to SDS-PAGE (polyacrylamide gel electrophoresis) with a gradient of 4 to 20%.

After electrophoresis, autoradiography was performed to
Incorporation of ³⁵ S] -SO ₄ ^2- was detected by the appearance of a band at a molecular weight of approximately 120 kD. A control experiment (TGF-
β-), a molecular weight of 100 derived from endogenous TGF-β
A thin band around kD was observed.

The results of Example 1 are summarized in Table 5 below.
become that way.

[0073]

[Table 5]

Example 2: Toxicity test This active substance was intraperitoneally administered to 4 to 5 week-old male ICR mice (Charles River) at 500 mg / kg.
Ten mice were used for each compound. as a result,
With the substance of Example prepared in Example 1, no deaths were observed in any of the administration groups.

Further, no cytotoxicity was observed in these Example substances in the pharmacological tests using the above-mentioned two test systems using cultured cells.

Example 3: Formulation Example The following is an example of a TGF-β inhibitor containing the substances of Examples A to D obtained in Example 1 above as an active ingredient.

Formulation Example 1 Example 1 The present active substance A (lyophilized product) 20 g Lactose 100 g Corn starch 36 g Microcrystalline cellulose 30 g Carboxymethyl cellulose calcium 10 g Magnesium stearate 4 g The ingredients of the above composition were uniformly mixed to give the present active substance. Each tablet contained 200 mg.

Formulation Example 2 Example 1 Active substance B (lyophilized product) 20 g Lactose 315 g Corn starch 125 g Microcrystalline cellulose 25 g The components of the above composition were uniformly mixed and then granulated to give granules.

Formulation Example 3 Example 1 The active substance C-III (lyophilized product) 20 g Lactose 100 g Microcrystalline cellulose 70 g Magnesium stearate 10 g The above components were uniformly mixed and then granulated. This was filled into a gelatin capsule so as to contain 200 mg of this active substance per capsule to obtain a capsule.

Formulation Example 4 Example 1 5 g of the active substance D (freeze-dried product) was added in a total amount of 100
It was dissolved in distilled water for injection to 0 ml. This one
Each ml was sealed in an ampoule to give an injection containing 5 mg of the active substance.

[0081]

Industrial Applicability As described above, the TGF-β inhibitor of the present invention has a safe and excellent TGF-β inhibitory action.
Therefore, the TG of the present invention containing these as active ingredients
The F-β inhibitor is useful for treating diseases in which TGF-β, which is easy to handle and inexpensive, is involved in the onset and progression of disease states, such as diabetic nephropathy.

The TGF-β inhibitor of the present invention is a low-molecular substance derived from Streptomyces sp. Therefore, it is easier to handle than high molecular weight proteins, and since it is obtained from a culture of actinomycetes, it can be manufactured at low cost in a short time and is economically advantageous.

[Brief description of drawings]

FIG. 1 shows that the inhibitory effect of the culture supernatant of NKK-908 strain on TGF-β was measured by an Mv1lu cell test system at 415n.
It is the figure shown by the light absorbency of m.

FIG. 2 is a diagram showing an autoradiograph when the inhibitory effect of the culture supernatant of the NKK-908 strain on TGF-β is measured by the L6 cell test system.

FIG. 3 is a diagram showing a scheme of a method for collecting a TGF-β inhibitory active substance of the present invention from a culture solution.

FIG. 4 shows the TGF-β inhibitory effect of each elution fraction when the culture supernatant of NKK-908 strain was subjected to WA-30 open column chromatography after extraction with an organic solvent, Mv1l.
It is the figure shown by the light absorbency of 415 nm measured by the u cell test system.

FIG. 5 is a graph showing a fraction in which a TGF-β inhibitory activity was observed in WA-30 open column chromatography was further added.
TGF of each elution fraction when subjected to EAE-HPLC
-Β inhibitory effect was measured by Mv1lu cell test system at 415n
It is the figure shown by the light absorbency of m.

FIG. 6 is an autoradiograph showing three fractions showing a TGF-β inhibitory effect in the DEA-HPLC in the Mv1lu cell test system when the fractions were subjected to the test system using L6 cells.

FIG. 7 shows the TGF-β inhibitory effect of each elution fraction when the fraction showing TGF-β inhibitory activity in the test system using L6 cells in DEAE-HPLC was further subjected to gel filtration-HPLC. It is the figure shown by the light absorbency of 415 nm measured by the test system.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 35/74 ACS 7431-4C ACV ADN G 7431-4C (C12N 1/20 C12R 1: 465) ( (72) Inventor Haruhito Yokota 1-2-1, Marunouchi, Chiyoda-ku, Tokyo Nihon Kokan Co., Ltd. (72) Inventor Takeshi Yago 1-chome, Marunouchi, Chiyoda-ku, Tokyo No. 2 Nihon Steel Pipe Co., Ltd. (72) Inventor Yasuko Yao 1-2-1, Marunouchi, Chiyoda-ku, Tokyo Nihon Steel Pipe Co., Ltd.

Claims (4)

[Claims] 1. A bacterium belonging to the genus Streptomyces, which has a gray color of a basal bacterium type, forms a long key-like or spiral spore chain on an aerial spore stalk, and has a smooth spore surface,
L-arabinose, D-xylose, D-fructose, L-rhamnose, sucrose, raffinose were assimilated on a Pridham-Gotrieve agar medium, and TG
An actinomycete capable of producing an F-β inhibitor. 2. A TG belonging to the genus Streptomyces
The actinomycete having the ability to produce an F-β inhibitor is Streptomyces sp. NKK-908 (Streptomyces sp. NKK-90
The actinomycetes according to claim 1, which is 8). 3. A TGF-, which comprises culturing the strain according to claim 1 or 2 to produce and accumulate a TGF-β inhibitory active substance, and separating and collecting from the culture. Method for producing β-inhibiting active substance. 4. A TGF-β inhibitor containing a TGF-β inhibitory active substance obtained by the method according to claim 3 as an active ingredient.