JPH05105693A - Ws4418 substance, its production and use - Google Patents

Abstract

PURPOSE:To provide the subject new substance having an excellent collagenase- inhibiting activity, and useful for preventing and treating the destruction of bone in chronic articular rheumatism, osteoarthritis, etc., as an inhibiting agent agains collagenase originated from human fibroblasts. CONSTITUTION:WS4418 substance having the following physical properties. The color and state: acidic white powder; melting point: 152-154 deg.C; FAB-MS: FAB-MS m/z 362 (M+H); <+>HR-FAB-MS: HR-FAB-MS m/z 362, 1933; practically measured value (X) in an elemental analysis: C 47.49, H 7.62, N 10.71; solubility: easily soluble in water or methanol; slightly soluble in acetone; insoluble in chloroform and ethyl acetate; coloring reaction: positive to a ferric chloride reaction and an iodine vapor reaction, negative to a ninhydrin reaction. The substance is obtained by culturing a WS4418 substance-producing bacterium of the genus Kitasatoporia [e.g. new Kitasatoporiaowasenea No.4418 strain (FERM P-12106)] preferably at 10-36 deg.C underaerating and stirring and subsequently recovering the substance from the culture product.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a WS4418 substance, a process for producing the same, and a use thereof. The WS4418 substance is a novel substance which has not been heretofore known and has been isolated and collected from a culture of the genus Kitazatosporia. It exhibits an excellent collagenase inhibitory action, is useful as a human fibroblast-derived collagenase inhibitor, and is useful as a drug, for example, chronically. It can be used as a preventive or therapeutic drug for bone destruction in rheumatoid arthritis, osteoarthritis and the like.

[0002]

2. Description of the Related Art Rheumatoid arthritis, osteoarthritis, etc.
It is painful and causes bone destruction, making it one of the most difficult diseases to treat. However, safe and highly effective therapeutic agents that can be administered for a long time are:
The reality is that it has not been developed yet.

[0003]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the state of the art as described above, and pays particular attention to the aspect of bone destruction in rheumatoid arthritis, osteoarthritis and the like, and prevents and And / or for the purpose of developing new substances that can be used for therapy.

[0004]

Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and as a result of examination from various aspects, a collagenase inhibitor has been focused as one of the preventive and therapeutic agents for the bone destruction. did.

Therefore, for the purpose of developing a human-derived fibroblast collagenase inhibitor which is highly effective and can withstand long-term administration, attention was paid to fermentation products of microorganisms. As a result of searching various microorganisms, the microorganism No. isolated from the soil sample near Owase City, Mie Prefecture
It was discovered that strain 4418 produced and accumulated the target substance in the culture medium. Furthermore, when the physicochemical properties of this substance were studied in detail, it was confirmed that it was a novel substance that was previously unknown, and this substance was newly named WS4418 substance, and as a result of further research, Not only was the industrial process established, but the collagenase inhibitory action was also confirmed. The 4418 strain has also been identified as a new bacterial species which has been unknown so far, and the present invention has been finally completed based on these new findings.

The WS4418 substance according to the present invention has the physicochemical properties shown in Tables 3 to 4 below.

[0007]

[Table 3]

[0008]

[Table 4]

The WS4418 substance according to the present invention is, for example, a microorganism No. 1 newly isolated by the present inventors from a soil sample near Owase City, Mie Prefecture. Produced by 4418 strains.

Among the studies on the morphology, culture properties, physiological properties, and other mycological properties of this strain, the medium and method were shirring and Gottlieb (refer to each reference shown in Tables 5, 6, and 7 below). 1), Waxman (also reference 2). Each culture was carried out at 30 ° C. for 14 days and then observed. The morphology was observed with an optical and scanning electron microscope after culturing on oatmeal agar and starch / yeast extract agar. The growth temperature was in the pH growth range, and the salt tolerance was yeast extract / malt extract agar. Color names are quoted from "Methuen Handbook of Colors" (reference 3). Cell wall type (references 4 and 5),
Whole cell sugar (reference 6), phospholipid component (reference 7), and menaquinone (reference 8) were examined according to the method described in each reference.

[0011]

[Table 5]

[0012]

[Table 6]

[0013]

[Table 7]

The microorganism No. 1 according to the present invention is described below. 441
The mycological properties of 8 strains are shown.

[0015]

[(1) Morphological properties] Basal hypha of strain 4418 develops well and branches irregularly. Aerial aerial hyphae extending from basal hyphae form long spore chains. The morphology of aerial hyphae and spore chains is straight or wavy (reference 1). The mature spore chain is
Link 20 or more spores. The shape of the spores was cylindrical, the size was 0.5-0.7 × 1.0-2.3 μm, the surface was smooth, and the sclerotium, sporangium, and zoospores were not observed.

[0016]

[(2) Culture properties and physiological properties] The culture characteristics of the 4418 strain are shown in Table 1 shown in Tables 8 and 9 below, the physiological properties are shown in Table 2 shown in Table 10 below, and the carbon source availability is shown in Table 11 below. Third indicated by
Shown in the table.

[0017]

[Table 8]

[0018]

[Table 9]

[0019]

[Table 10]

[0020]

[Table 11]

As is apparent from the above results, the formation of aerial mycelia was poor or poor in many of the tested media, and when the aerial mycelia were settled, the color tone was white. In the early stage of growth, the surface of growth may be wet. The color of the basal hyphae is yellow orange to reddish brown. Yeast extract / malt extract agar, tyrosine agar, and starch / yeast extract agar produced a small amount of reddish brown soluble pigment. These dyes are not pH sensitive. No production of melanoid pigment was observed. Carbon source availability was generally poor, with glucose, glycerin and galactose among the tested.

[0022]

[(3) Chemotaxonomic properties] The whole cell decomposition product of this strain contained mesodiaminopimelic acid and a small amount of LL-diaminopimelic acid. Galactose is contained as a sugar in the decomposed product of whole cells. It contains phosphatidylethanolamine as a characteristic phospholipid. MK-9 (H ₆ ) and MK-9 (H ₈ ) were detected as major menaquinones (Table 4 shown in Table 12 below).

[0023]

[Table 12]

[0024]

[(4) Identification] The above morphological characteristics and the results of the chemical analysis show that the strain is Kitasatospori.
(Oria ) genus (references 9, 1)
0). Therefore, considering the description of known species of the same genus (references 11 to 20), the characteristics of this strain are Kitazatosporia
Kitasatosporia setae
It was clearly different from other species. So, with this strain
K. A detailed comparative control experiment was carried out with setae IFO 14216 and the results in Table 5 shown in Table 13 below were obtained.

[0025]

[Table 13]

As is clear from these results, many differences were found between the two strains in terms of culture and physiological properties. Then, these differences are considered to be sufficient to separate both strains from each other. 4418 strain is Ki
We conclude that it is a new species of the genus Tasatosporia, and named it Kitasatosporia owasenea due to the soil collection site of the separation source.
sp. nov. ) Was named. This new variety, No. Four
The 418 strain has been deposited at the Institute of Microbial Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (accession number FERM P-12106,
Date of deposit: March 14, 1991).

It should be understood that the production of this substance is not limited to the use of the particular microorganisms described herein merely for purposes of illustration. This invention includes artificial mutants and natural mutants that can be obtained from the microorganisms described by X-ray irradiation, ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine and other mutation treatments. It also includes the use of all mutants capable of producing this substance.

The substance according to the present invention is a substance-producing bacterium belonging to the genus Kitazatosporia (for example, Kitasatospo).
ria owasenea sp. nov. No.
4418) can be produced by inoculating a nutrient medium containing a carbon and nitrogen source capable of assimilating and culturing under aerobic conditions (eg, shaking culture, aeration and agitation culture).

As the carbon source, glucose, sucrose, starch, modified starch, fructose, glycerin and other carbohydrates are preferably used.

As the nitrogen source, oatmeal, yeast extract, peptone, gluten meal, cottonseed meal, cottonseed oil meal, soybean meal, corn steep liquor, dried yeast, wheat germ,
Peanut flour, chicken bone meat meal and the like are preferably used, but inorganic and organic nitrogen compounds such as ammonium salts (for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.) urea and amino acids can also be advantageously used.

It is advantageous to use these carbon sources and nitrogen sources in combination, but it is not necessary to use pure ones. This is because an impure substance may be advantageous in some cases because it may contain a growth factor or a trace element.

If necessary, the following inorganic salts may be added to the medium: sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, sodium iodide, iodine. Potassium chloride, magnesium salt, copper salt, cobalt salt, etc.

In particular, if the medium strongly foams, liquid paraffin, animal oil, vegetable oil, mineral oil, silicone or the like may be added when necessary.

For large-scale industrial production of the target substance, it is preferable to carry out aeration-agitation culture as in the case of other fermentation products. For small-volume production, shake culture using a flask is suitable.

When the culture is carried out in a large tank, in order to prevent the growth delay of the bacteria in the production process of this substance,
First, after inoculating and culturing the production bacteria in a relatively small amount of medium,
The culture is then preferably transferred to a large production tank for production culture. In this case, the composition of the medium used for the pre-culture and the composition of the medium used for the production culture may be the same or may be different if necessary.

The culture is preferably carried out under aeration and stirring conditions,
For example, known methods such as stirring with a propeller or other machine, rotation or shaking of a fermenter, pumping, blowing of air are appropriately used. Use sterilized air for ventilation.

The culturing temperature may be appropriately changed within the range in which the substance-producing bacterium produces the substance, but it is usually 1 to 40 ° C, preferably 10 to 36 ° C. The culturing time varies depending on the culturing conditions and the culturing amount, but is usually about 1 day to 1 week.

After completion of fermentation, the desired substance of interest is recovered from the culture. That is, bacterial cells are directly extracted with water and / or an organic solvent, or disrupted mechanically or by a known means such as ultrasonic waves, and then extracted with water and / or an organic solvent, followed by a conventional method. Recover and purify according to. In the case of a culture solution, it may be directly recovered and purified by a conventional method.

The recovery and purification methods include, for example, water, an organic solvent, solvent extraction with a mixed solvent thereof: chromatography; a conventional method such as recrystallization from a single solvent or a mixed solvent, alone or in combination. it can.

The recovery and purification of this substance is carried out by appropriately utilizing the known method as described above, but may be carried out as follows, for example. First the culture was filtered and the filtrate was filtered over Dowex 1x2.
Adsorbed on a resin such as (Dow Chemical Co.), eluted with saline, and adsorbed the eluate on a neutral adsorption resin [for example, Diaion HP-20 (manufactured by Mitsubishi Kasei Co.)],
Elute with aqueous methanol, concentrate the eluate to dryness, and perform silica gel column chromatography to obtain an aqueous isopropanol eluate. The active fractions therein were subjected to reverse-phase silica gel chromatography and the aqueous methanol eluate containing the active substance was concentrated and then adsorbed on, for example, Diaion HP-20 SS (manufactured by Mitsubishi Kasei) and eluted with aqueous methanol. Then, the eluate is concentrated and freeze-dried to obtain the target substance powder.

The pharmaceutical composition according to the present invention is WS4418.
A substance and / or salt thereof as an active ingredient is added to an inorganic or organic carrier that is commonly used therein, and is formulated into a solid, semisolid or liquid form for oral administration as well as parenteral administration such as external preparations. Turn into.

The preparations for oral administration include tablets, pills, granules, soft / extended capsules, powders, fine granules, powders,
Examples thereof include emulsions, suspensions, syrups, pellets and elixirs. Formulations for parenteral administration include injections, drip infusions, infusions, ointments, lotions, tonics, sprays, suspensions, oils, emulsions, suppositories and the like. The active ingredient of the present invention may be formulated according to any other method such as surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents. Other commonly used adjuvants should be used as appropriate.

The dose of the pharmaceutical composition according to the present invention varies depending on its type, type of disease to be treated or prevented, administration method, age of patient, symptom of patient, treatment time, etc.
In the case of intravenous administration, the active ingredient (this substance) is 0.01 to 1000 mg / kg, preferably 0.1 per adult per day.
1 to 100 mg / kg, and in the case of intramuscular administration, 0.01 to 1000 mg / kg, preferably 0.1 to 1
00 mg / kg was administered, and 0.5
~ 2000 mg / kg, preferably 1-1000 mg / kg
It is administered within the range of kg.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[0045]

Example 1

[0046]

[(1) No. Cultivation of strain 4418] Pinedex #
3 (trade name, acidic soluble starch) 1%, glucose 0.5
%, Yeast extract 0.5%, malt extract 0.5%, polypeptone 0.5%, 0.1% K ₂ HPO ₄ was added to a liquid seed medium (pH adjusted to 6.0 with NaOH) to give 500
160 ml of each was dispensed into 25 ml Erlenmeyer flasks and sterilized at 125 ° C. for 30 minutes. To this, Kita
satosporia owaseneasp. no
v. No. One platinum loop of 4418 (FERM P-12106) slant was inoculated and cultivated on a rotary shaker (220 rpm, 5.1 cm stroke) at 30 ° C. for 4 days to obtain a seed culture solution.

Separately, soluble starch 5%, peanut powder 1%,
Soybean powder 1%, corn steep liquor 1%, CaCO
₃ Sterile medium containing 0.2% (pH with NaOH
(Adjusted to 6.9) into a 200 l jar fermenter and inoculated with 120 l of the above-mentioned seed culture solution, and aerated at 120 l / min, rotation speed of 200 rotations / min.
Aeration-agitation culture was carried out for a day.

[0048]

[(2) Separation and Purification of WS4418 Substance] The culture broths obtained by the above culture method were combined (450 l) and filtered using Radiolite (trade name, filter aid). Filtrate (4
001) to Dowex 1x2 (trade name, US Dow
Chemical Co. ) (Cl type, 34 l), the column was washed with water (90 l) and then eluted with 0.2 M NaCl (120 l). The eluate was adsorbed on a column of Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd., 6 l), and the column was mixed with water (18 l) and
After washing with% aqueous methanol, it was eluted with 50% aqueous methanol (18 l). The eluate was concentrated under reduced pressure, dried, and subjected to silica gel column chromatography (Silica).
gel 60, manufactured by Merck & Co., Inc., 41). After washing the column with isopropyl alcohol (41), 90% aqueous isopropyl alcohol (81), the active substance was eluted from the column with 85% aqueous isopropyl alcohol (121).

The active fractions were combined and concentrated until the volume of the aqueous solution was 300 ml. The solution was applied to a reversed phase silica gel column ODS 60A (trade name, Yamamura Chemical Laboratory) (Type I-40 / 64, 41), the column was washed with water (12 1), and 5% aqueous methanol (1
The active substance was eluted from the column with 6 l). The eluate was concentrated to obtain 80 ml of an aqueous solution, the pH was adjusted to 7.0, and the solution was passed through a column of Diaion HP-20 SS (manufactured by Mitsubishi Kasei Co., Ltd., 300 ml). The column is filled with water (1.
2 l), and 5% aqueous methanol (400 m
1) was used to elute the active substance, and the eluate was concentrated and lyophilized to give purified WS441.
682.6 mg of white powder of 8 substances was obtained.

[0050]

[(3) Physicochemical properties of WS4418 substance] WS441
The physicochemical properties of the 8 substances have been clarified in Tables 3 and 4, and further amino acid analysis was performed to obtain the following results. That is, WS4418 substance (1 mg)
It was hydrolyzed with N HCl (1 ml) at 110 ° C. for 20 hours. The reaction mixture was evaporated to dryness, and the resulting hydrolyzate was analyzed with an amino acid automatic analyzer (Hitachi 835 A).
automatic Amino-Acid Analysis
Ser) gave the following results: Ser (1) and Val (1). Furthermore, based on the above-mentioned physicochemical properties, the chemical structure of the WS4418 substance was presumed to be as shown in Chemical Formula 1 below by utilizing the structural analysis method for natural products.

[0051]

[Chemical 1]

[0052]

Example 2 Collagenase Inhibition Assay

[0053]

[(1) Preparation of human dermal fibroblasts] The cells were obtained from a normal healthy human skin graft, which contained 10% fetal bovine serum and penicillin (50 U / m 2).
l), grown in a Dulbecco's modified minimum essential medium (DMEM) supplemented with streptomycin (50 U / ml) at 37 ° C. in a humid atmosphere (5% CO ₂ -95% air). Therefore, the cells were subcultured by trypsin treatment and used for 5 to 15 passages.

[0054]

[(2) Induction of collagenase] Fibroblasts (1.5 x 1
0 ⁶ ) were seeded in a 75 cm ² plastic flask and cultured. After 3 days, remove the medium and add 20 ml of cells.
After washing with DMEM for 3 times, bovine serum albumin (3
mg / ml) and interleukin 1β (Genzym
e company, USA, 100 U / ml) added DMEM (1
The cells were cultured in 1.25 ml for 3 days. Collect the culture, 15
After centrifugation at 00 rpm for 5 minutes to remove cell fragments, the cells were stored at -20 ° C until use. For use, 1 ml of the culture supernatant was added to trypsin (Sigma, 10
Soybean trypsin inhibitor (Sigma, 400 μg / ml) after activation with 200 μl
200 μl was added.

[0055]

[(3) Assay] All reactions were performed in a U-shaped microtiter plate. 0.4M NaCl and 10m
0.1 M Tris-HCl containing M CaCl ₂
25 μl of inhibitor was serially diluted with buffer (pH 7.5). 50 μl of FITC (fluorescein isothiocyanate) -collagen (1 mg / ml manufactured by Collagen Technology Workshop) and 25 μl of collagenase (trypsin-activated fibroblast supernatant) diluted 10-fold were added at 37 ° C. Incubated for 2 hours. After the incubation, 5 μl of 80 mMo-
5 μl of phenanthroline was added and then incubated for 1 hour. To the reaction mixture was added 70 μl ethanol and 30 μl Tris-HCl buffer, stirred and centrifuged at 3000 rpm for 10 minutes. FITC removed by collagenase in the supernatant
Intensity from excitation (excitation 485 nm, emission 538
nm) was measured and the% inhibition of the test sample was calculated and the results in Table 14 below were obtained.

[0056]

[Table 14]

[0057]

[Example 3: Production of injection] (1) WS4418 substance produced in Example 1 5g (2) Salt 9g (3) Sodium hydrogencarbonate 1g

All the components (1) to (3) were added to distilled water 1000
After dissolving in ml, 1 ml was dispensed into ampoules to manufacture 1000 injections.

[0059]

INDUSTRIAL APPLICABILITY The present invention provides a WS4418 substance, which is a previously unknown novel pharmacologically active substance, which exhibits an excellent collagenase inhibitory action,
For example, it is useful for preventing / treating diseases caused by collagenase as a preventive / therapeutic agent for bone destruction in rheumatoid arthritis, osteoarthritis and the like.

Further, according to the present invention, not only has an industrial method for producing the above-mentioned substance utilizing a microorganism been established, but since the above-mentioned substance-producing bacterium is a novel species which has not been known so far, it is possible to improve the microbiological research. In addition, in terms of discovering new physiologically active substances, the present invention is highly expected in the technical field of biotechnology.

[Brief description of drawings]

FIG. 1 is a drawing showing a ¹ H nuclear magnetic resonance spectrum of WS4418 substance.

FIG. 2 is a drawing showing a ¹³ C nuclear magnetic resonance spectrum of WS4418 substance.

Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12P 1/02 B 2114-4B // (C12N 1/20 C12R 1:01) (C12P 1/02 C12R 1: 01) (72) Inventor Masanori Okamoto 3-22-6 Namiki, Tsukuba City, Ibaraki Prefecture (72) Inventor Masakuni Okuhara 2-14-10 Umezono, Tsukuba City, Ibaraki Prefecture

Claims (4)

[Claims] 1. A WS4418 substance having the physical properties shown in Table 1 below. [Table 1] 2. Kitasatos
A method for producing a WS4418 substance, which comprises culturing a WS4418 substance-producing bacterium belonging to the genus Poria) to produce the WS4418 substance, and collecting the WS4418 substance. 3. A collagenase inhibitor, which comprises the WS4418 substance as an active ingredient. 4. A new bacterial species Kitazatosporia owassenea having the mycological properties shown in Table 2 below.
osporia owasenea). [Table 2]