JPH0925286A - Antibiotic substance tkr2554 and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibiotic substance useful as an agent for the treatment of mycotic infection. SOLUTION: This is an antibiotic substance TKR2554 or its pharmacologically allowable salt having the following physical and chemical properties: (1) mass spectrum by FAB-MS method shows a peak at m/z 436[M+H]<+> ; (2) it contains 22 carbon atoms and one nitrogen atom; (3) the main absorption wavelengths (nm) of ultraviolet absorption spectrum in methanol are 231 and 271sh and the E<1> 1cm are 413 and 297; (4) the main absorption wavenumbers of infrared absorption spectrum by Kbr method are 2920cm<-1> , 2840cm<-1> , 1750cm<-1> , 1700cm<-1> , 1630cm<-1> , 1510cm<-1> , 1410cm<-1> , and 1080cm<-1> ; and (5) it is soluble in methanol and scarcely soluble in chloroform, water and hexane; etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibiotic TKR2554 useful as a therapeutic agent for fungal infections, a method for producing the same, and a microorganism producing the same.

[0002]

2. Description of the Related Art Fungi are known to infect humans, animals, plants and the like to cause various diseases. For example,
It causes superficial mycosis in human skin, oral cavity, etc., systemic mycosis in viscera, brain, etc., and similar infectious diseases in animals such as pets and livestock. Further, it causes various diseases on plants such as fruit trees and vegetables. Of these, infected with human, as caused major fungi causing systemic mycosis, Candida (Candida), Cryptococcus (Cryptococcus), Aspergillus (A
spergillus ) and the like, and in superficial mycosis, candida that infects the skin, oral cavity, vagina and the like, and ringworm which infects the skin of limbs and the like are considered to be the main ones. In addition to these, various fungi exist in the living environment and are considered to be the cause of contamination of plants and animals.

[0003]

At present, very few antifungal agents are known which can be used for the purpose of treating and protecting against such infectious diseases and contamination by fungi. Absent. Among these, as therapeutic agents for systemic infectious diseases of animals including human beings in particular, amphotericin B, flucytosine, miconazole, fluconazole and the like can be mentioned. But these things
There are problems in efficacy, toxicity, antibacterial spectrum, etc., and it is not sufficient as a therapeutic agent.

In view of the above-mentioned present situation, an object of the present invention is to provide a novel antibiotic useful as a therapeutic agent for fungal infections.

[0005]

[Means for Solving the Problems] In order to search for novel antibiotics, the present inventors isolated a large number of microorganisms from nature, isolated the produced antibiotics, and investigated their biological properties. By the way, Aspergills
It has been found that in a culture of a microorganism belonging to the genus ( us ), an antibiotic having an antibacterial activity against pathogenic fungi such as Candida albicans and Cryptococcus neoformans is present. Then, the present inventors isolated this antibiotic and examined its physicochemical properties. As a result, it was confirmed that it was a novel substance with unique physicochemical properties that had not been described in the literature, and this antibiotic was identified as TKR2554. I named it.
The present invention provides the above antibiotic TKR2554 and a method for producing the same.

The above antibiotic TKR2554 has the following physicochemical properties (1), (2), (3), (4) and (5). (1) The mass spectrum by FAB-MS method is m / z
It has a peak of 436 [M + H] ⁺ (2) The number of carbon atoms is 22, and the number of nitrogen is 1. (3) The main absorption wavelength (nm) of the ultraviolet absorption spectrum in methanol is 231 and 271 sh. Yes,
Their E ^1% _{1 cm} are 413 and 297. (4) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 2920 cm ^-1 , 2840 cm ^-1 and 1750.
^{cm -1, 1700cm -1, 1630cm -1} , 1510c
m ^-1 , 1410 cm ^-1 , 1080 cm ^-1 (5) Soluble in methanol, sparingly soluble in chloroform, water and hexane

The above antibiotic TKR2554 was also analyzed by ¹ H-NMR spectrum shown in FIG. 3 and ¹³ C-N shown in FIG.
It has an MR spectrum and is characterized by being eluted at the position shown in FIG. 5 in reversed phase partition high performance liquid chromatography.

The above TKR2554 belongs to the genus Aspergillus , and the above TKR25
It can be produced by culturing a strain producing 54 and then isolating it from a culture of the above strain. The above-mentioned strain used in the present invention includes Aspergillus (A
spergillus ), and the above-mentioned TKR2554
As long as it produces not particularly limited, for example, Aspergillus sp. (Aspergillus s
p. ) TKR2554 strain (hereinafter referred to as "TKR2554 strain") and the like.

The above-mentioned TKR2554 strain is a novel strain not described in the literature, which was first isolated and identified by the present inventors, and has a characteristic of producing TKR2554 in an advantageous manner. Hereinafter, the mycological properties of the TKR2554 strain will be described in detail.

The color tone of the above-mentioned TKR2554 strain in colonies (hereinafter also referred to as "communities") in various media is as shown in Table 1. The colors in the table are based on Japanese Industrial Standard J
Based on the color name according to ISZ 8102 (1985), the medium was inoculated, cultured at 25 ° C., and observed 7 days later.

[0011]

[Table 1]

The above-mentioned TKR2554 strain grows rapidly on malt extract agar medium, Czapek agar medium, Sabouraud agar medium, etc., and the surface of the colony is powdery to velvety, and conidial head formation is very good. . TKR255
The conidia stalks of the 4 strains have a smooth surface of 60 to 220 × 4.0 to 7.0 μm, and stand up from the basal hypha layer. The summit has a diameter of 2
Spherical to subspherical diameters of 2-32 μm with 6.0-8.0 × 3.0-3.2 μm phialides occurring from the entire apex surface. Metre does not form. Conidia are stab-shaped to subspherical on the surface, and their size is 3.0-4.2μ.
m.

Among the mycological properties of the TKR2554 strain, physiological properties are as shown below. Growth temperature range: The growth temperature range is 15 to 37 ° C, and the optimum growth temperature is around 30 ° C. Growth pH range: The pH range in which growth is possible is pH 3 to pH 9, and the optimum growth pH is pH 5 to pH 6.

A bacterial strain having the above-mentioned mycological properties was prepared from Kenneth B. Raper,
Dorothy I. Fennel
Ennell), The Genus Aspergillus (T
he genus Aspergillus), The Williams & Wilkins Company (The
Williams & Wilkins compa
ny) (1965) and the like, and search for the Aspergillus spp.
The 2554 strain can be identified as a strain belonging to the genus Aspergillus.

However, no report has been made so far regarding strains belonging to the genus Aspergillus and having the ability to produce TKR2554. Therefore, the present inventors have designated this as a new strain and designated Aspergillus sp. TKR2554 ( Aspergillus s).
p. TKR2554) and Aspergill
us sp. Displayed as TKR2554 and deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, deposit number FERM P-14
Deposited as 831.

In the present invention, in addition to the above TKR2554 strain, a natural or artificial mutant strain of the TKR2554 strain,
Other species belonging to the genus Aspergillus, such as T
A microorganism capable of producing KR2554 can be used.

In the present invention, TKR2554 can be produced by inoculating a nutrient source-containing medium with the strain producing TKR2554 and culturing.
Among the above nutrient sources, examples of the carbon source include glucose, fructose, saccharose, starch, dextrin, glycerin, molasses, starch syrup, fats and oils, organic acids and the like.

Among the above nutrient sources, examples of the nitrogen source include soybean flour, cottonseed flour, corn steep liquor, casein, peptone, yeast extract, meat extract, germ, urea, amino acids, ammonium salts, and other organic nitrogen compounds. , Inorganic nitrogen compounds, and the like. Among the above nutrient sources, examples of salts include inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and phosphate salt. These may be used alone or in appropriate combination.

In the nutrient source-containing medium, if necessary, heavy metals such as iron salts, copper salts, zinc salts, and cobalt salts; vitamins such as biotin and vitamin B ₁ ; An organic substance, an inorganic substance or the like that promotes the production of TKR2554 can be appropriately added. In addition to the above nutrient sources, a defoaming agent such as silicone oil and polyalkylene glycol ether, a surfactant, and the like can be added to the above nutrient source-containing medium, if necessary.

When culturing the above TKR2554-producing strain in the above nutrient-source-containing medium, a method generally used for culturing an antibiotic to produce an antibiotic can be adopted. A culturing method, especially a shaking or deep aeration stirring culturing method can be preferably used. The above culture is preferably carried out at 15 to 30 ° C. The pH of the medium is usually 3 to 8, but preferably about pH 5. The culture period is usually 3 to 15 days, and a sufficient production amount can be obtained.

By the above-mentioned culture method, TKR2554
Is contained in the culture solution and cells and accumulated in the culture. In the present invention, TKR2 accumulated in the culture
554 was isolated from the culture using the physicochemical properties of these antifungal substances, and then further purified, if necessary,
Can be obtained. The above separation can be carried out by extracting the entire culture with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, chloroform, butanol, methyl isobutyl ketone. Alternatively, the culture may be separated into a culture solution and cells by filtration or centrifugation, and then separated from the culture solution and cells.

In order to separate TKR2554 from the above culture solution, the method of extraction with the above-mentioned non-hydrophilic organic solvent can be adopted, and the culture solution can be brought into contact with an adsorptive carrier,
It is also possible to employ a method in which TKR2554 in the culture solution is adsorbed on a carrier and then eluted with a solvent. Examples of the carrier include activated carbon, powdered cellulose, adsorptive resin, and the like. As the above-mentioned solvent, one kind or a combination of two or more kinds may be appropriately used depending on the kind and properties of the carrier, and for example, an aqueous solution of a water-soluble organic solvent such as hydrous acetone and hydrous alcohols may be appropriately combined. The thing etc. can be mentioned. T from the above fungus body
In order to separate KR2554, a method of extracting with a hydrophilic organic solvent such as acetone can be adopted.

In the present invention, the crude extract of TKR2554 thus separated from the culture can be subjected to a step of further purification, if necessary. The above-mentioned purification can be carried out by a method usually used for separation and purification of fat-soluble antibiotics, and as such a method, for example, column chromatography using a carrier such as silica gel, activated alumina, activated carbon, adsorptive resin, etc. Graphography,
A high performance liquid chromatography method and the like can be mentioned. When a column chromatography method using silica gel as a carrier is adopted, examples of the elution solvent include chloroform, ethyl acetate, methanol, acetone, water and the like, and these can be used in combination of two or more kinds. .

When the above high performance liquid chromatography method is adopted, the carrier is, for example, an octadecyl group,
Chemically bonded silica gel having octyl group, phenyl group and the like bonded thereto; polystyrene type porous polymer gel and the like can be mentioned, and as the mobile phase, for example, an aqueous solution of a water-soluble organic solvent such as hydrous methanol or hydrous acetonitrile is used. can do.

The TKR2554 of the present invention can be used in medicine as it is or as a pharmacologically acceptable salt thereof. The salt is not particularly limited as long as it is pharmacologically acceptable, and examples thereof include salts of mineral acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid and hydrobromic acid;
Organic acid salts such as formic acid, acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid and camphorsulfonic acid A salt of an alkali metal such as sodium, potassium or calcium, or an alkaline earth metal, or the like.

When the TKR2554 of the present invention or a pharmacologically acceptable salt thereof is administered as a medicine, the TKR2554 of the present invention or a pharmacologically acceptable salt thereof is used as it is or pharmaceutically. It is administered to animals, including humans, as a pharmaceutical composition containing, for example, 0.1 to 99.5%, preferably 0.5 to 90% in an acceptable non-toxic and inert carrier. Examples of the carrier include solid, semi-solid or liquid diluents, fillers and other auxiliaries for formulation, and these may be used alone or in combination.

The above-mentioned pharmaceutical composition is preferably administered in a dosage unit form, and can be administered orally, intratissueally, topically (such as transdermally) or rectally.
It goes without saying that the above-mentioned pharmaceutical composition is administered in a dosage form suitable for these administration methods. TKR2554 of the present invention,
Alternatively, when a pharmacologically acceptable salt thereof is administered as a drug, the dose as an antifungal agent is adjusted in consideration of the patient's condition such as age and weight, administration route, and nature and degree of disease. Normally, for humans,
As an active ingredient amount of the present invention for adults, per day,
It is in the range of 10 to 2000 mg. In some cases, a dose below the above range may be sufficient, while in other cases, a dose above the above range may be required. When administering a large amount, it is desirable to administer it in several divided doses a day.

The above oral administration can be carried out in solid, powder or liquid dosage units, for example powders, powders, tablets,
Sugar coating, capsules, drops, sublingual agents, and other dosage forms can be used. The above-mentioned parenteral administration can be carried out, for example, by using a liquid dosage unit form for subcutaneous, intramuscular or intravenous injection such as a solution or suspension. These are TKR2554 of the present invention, or
A certain amount of the pharmacologically acceptable salt is suspended or dissolved in a non-toxic liquid carrier suitable for injection purpose such as an aqueous or oily medium, and then the suspension or solution is sterilized. It is manufactured by

The above-mentioned local administration (transdermal administration, etc.) can be carried out, for example, by
It can be carried out by using a form of external preparation such as liquid, cream, powder, paste, gel, ointment and the like. These are a perfume, a colorant, a filler, a surfactant, a moisturizer, and an emollient which are suitable for the purpose of an external preparation, in which a certain amount of TKR2554 of the present invention or a pharmacologically acceptable salt thereof is used. It is produced by combining with one or more agents, gelling agents, carriers, preservatives, stabilizers and the like. For rectal administration, a fixed amount of TKR2554 of the present invention or a pharmacologically acceptable salt thereof may be used, for example, a higher melting point such as higher esters such as myristyl palmitate, polyethylene glycol, cocoa butter, or a mixture thereof. It can be carried out by using a suppository or the like mixed in the solid.

[0030]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 One platinum loop from the slope culture of the TKR2554 strain (FERM P-14831) was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of a liquid medium (Difco potato dextrose broth 2.4% (W / V)). Then, the mixture was shaken at 25 ° C. for 2 days to obtain a seed culture solution. This seed culture 1.0
13 ml was inoculated into 13 500 ml Erlenmeyer flasks containing 140 ml of the above liquid medium, and shake culture (shake 220 rpm) was performed at 25 ° C for 11 days. The culture broth thus obtained was centrifuged to separate into a supernatant and cells. The obtained supernatant was adsorbed on a Diaion HP20 (Mitsubishi Chemical Co.) column (1 L) equilibrated with water,
It was washed with 50% methanol. This was eluted with 3 L of methanol to obtain an active fraction.

Further, 1 L of methanol was added to the cells, and they were thoroughly mixed and extracted, and then concentrated under reduced pressure. 1 L of water was added to the obtained residue and mixed well. This was adsorbed on an HP20 column that had been washed with 50% methanol. After washing this column with 50% methanol, 3 L
Was eluted with methanol to obtain an active fraction. By collecting the culture supernatant and the active fractions derived from the bacterial cells and concentrating under reduced pressure,
Residue 537 mg was obtained.

50% (V / V) acetonitrile /
It was dissolved in 75 ml of methanol and subjected to preparative high performance liquid chromatography to obtain an active fraction. The conditions for high performance liquid chromatography were as follows. Device: Delta Prep 4000 system (Waters) Column: Soken Pack (8.0 cm x 50 cm) (Soken Kagaku) Mobile phase: 70% (V / V) acetonitrile / water

By concentrating this active fraction under reduced pressure,
229 mg of a crude product of TKR2554 was obtained. This was dissolved in 1.6 ml of methanol and subjected to high performance liquid chromatography to obtain an active fraction. By concentrating this fraction under reduced pressure, 108 mg of a purified product of TKR2554 was obtained as a white powder. The conditions for high performance liquid chromatography were as follows. Device: LC-8A (manufactured by Shimadzu Corporation) Column: YMCpack (2.0 cm x 25 cm) (manufactured by WMC) Mobile phase: 50% (V containing 0.05% trifluoroacetic acid)
/ V) acetonitrile / water

Physicochemical properties Next, in order to confirm that the obtained white powder is TKR2554, mass spectrometry using a JMS-DX302 type mass spectrometer (manufactured by JEOL Ltd.), JNM-A500 nuclear magnetic resonance apparatus (Made by JEOL Ltd.) ¹ H-NMR (standard substance: tetramethylsilane) in deuterated methanol, and ¹³ C-NMR (standard substance: deuterated methanol), UV-2
Ultraviolet absorption spectrum analysis using a 50 type self-recording spectrophotometer (manufactured by Shimadzu Corporation) (methanol solution: about 30 μg / m
1), the infrared absorption spectrum analysis (KBr method) using a 270-30 type infrared spectrophotometer (manufactured by Hitachi, Ltd.) was performed, and each physicochemical property was investigated.

(1) Mass spectrometry A white powder purified product obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure is
FAB-MS measurement by mass spectrometer, m / z 436
It was [M + H] ⁺ .

(2) Number of carbons and number of nitrogens Purified white powder obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure
¹ H-NMR spectrum measurement, ¹³ C-NMR spectrum measurement and its analysis show that the number of carbon atoms is 22 and the number of nitrogen atoms is 1
Met. The ¹ H-NMR spectrum in FIG. 3, ¹³ C
-The NMR spectrum is shown in FIG.

(3) Ultraviolet absorption spectrum The purified white powder obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure is as follows:
The main absorption wavelengths measured by the ultraviolet absorption spectrum measuring apparatus in methanol are as follows. UV (nm) (E ^1% _{1 cm} ): 231 (413), 27
1sh (297) Further, its ultraviolet absorption spectrum is shown in FIG.

(4) Infrared absorption spectrum A white powder purified product obtained by subjecting to high-performance liquid chromatography and concentrating the obtained active fraction under reduced pressure was obtained.
The main absorption wave numbers in the infrared absorption spectrum measurement by the KBr method are as follows. IR (KBr) (cm ^-1 ): 2920, 2840, 17
50, 1700, 1630, 1510, 1410, 10
80 The infrared absorption spectrum is shown in FIG.

All of them were soluble in methanol, but hardly soluble in chloroform, water and hexane. Based on the above analysis results, the white powder purified product obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure was TKR2554.

The above TKR2554 was subjected to analysis by reversed phase partition high performance liquid chromatography (HPLC) using a 600E high performance liquid chromatography apparatus (manufactured by Waters). The conditions for high performance liquid chromatography were as follows. Column: CAPCELL PAKC ₁₈ (6 mm x 150 m
m) (manufactured by Shiseido Co., Ltd.) Mobile phase: 50% (V containing 0.05% trifluoroacetic acid)
/ V) Acetonitrile / water Column temperature: 40 ° C. Detection UV wavelength: 220 nm As a result, it was revealed that the above TKR2554 was eluted at the position shown in FIG.

Biological properties The obtained TKR2554 was used to investigate the antibacterial spectrum against various microorganisms. The measurement was carried out by the liquid medium dilution method, and the concentration at which the growth of bacteria was almost completely inhibited was determined as the minimum growth inhibitory concentration (μg / ml). The results are shown in Table 2. In addition, the minimum concentration that partially inhibits the growth of bacteria was determined as a half-inhibitory concentration (μg / ml), and is also shown in parentheses in Table 2. In the table, YNBG is yeast nitrogen base (manufactured by Difco) 0.67%, glucose 1.0.
% Represents YNBG medium.

[0042]

[Table 2]

From the results of Table 2, the antifungal substance T of the present invention is shown.
KR2554 is Candida albicans, Candida
It was found to have antibacterial activity against pathogenic fungi such as kefir and cryptococcus neoformans.

Further, the obtained TKR2554 was ICR
50 mg / kg was intraperitoneally administered to strain mice, but no toxicity was observed.

[0045]

INDUSTRIAL APPLICABILITY The present invention can provide the antifungal substance TKR2554, which is useful as a clinical drug such as a therapeutic agent for fungal diseases, and a method for producing them.

[Brief description of drawings]

FIG. 1 is a diagram showing an ultraviolet absorption spectrum of an antifungal substance TKR2554. The horizontal axis represents wavelength (nm).

FIG. 2 is a diagram showing an infrared absorption spectrum of an antifungal substance TKR2554. The horizontal axis represents the wave number (cm ^-1 ).

FIG. 3 is a diagram showing a ¹ H-NMR spectrum of an antifungal substance TKR2554. The horizontal axis is the chemical shift value (pp
m).

FIG. 4 is a diagram showing a ¹³ C-NMR spectrum of an antifungal substance TKR2554. The horizontal axis is the chemical shift value (pp
m).

FIG. 5 is a view showing elution positions in HPLC of an antifungal substance TKR2554. The vertical axis represents relative ultraviolet absorption intensity, and the horizontal axis represents retention time (minutes).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication C12R 1:66) (C12P 1/02 C12R 1:66) (72) Inventor Hideharu Saito Otsu, Shiga Prefecture 3-4-1, Seta Ichi, Central Research Laboratory, Takara Shuzo Co., Ltd. (72) Inventor, Ikuno Kato 3-4-1, Seta 3-chome, Otsu, Shiga Prefecture

Claims (3)

[Claims] 1. An antibiotic TKR2554 or a pharmacologically acceptable salt thereof, which has the physicochemical properties of the following (1), (2), (3), (4) and (5). (1) The mass spectrum by FAB-MS method is m / z
It has a peak of 436 [M + H] ⁺ (2) The number of carbon atoms is 22, and the number of nitrogen is 1. (3) The main absorption wavelength (nm) of the ultraviolet absorption spectrum in methanol is 231 and 271 sh. Yes,
Their E ^1% _{1 cm} are 413 and 297. (4) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 2920 cm ^-1 , 2840 cm ^-1 and 1750.
^{cm -1, 1700cm -1, 1630cm -1} , 1510c
m ^-1 , 1410 cm ^-1 , 1080 cm ^-1 (5) Soluble in methanol, sparingly soluble in chloroform, water and hexane 2. Aspergillus
s ) belonging to the genus, which is an antibiotic TKR2554
The antibiotic TKR2, which comprises culturing a strain that produces TCR2, and then isolating the target product from the culture of the strain.
554 manufacturing method. 3. Aspergillus
s ) A microorganism belonging to the genus and producing the antibiotic TKR2554.