JPH09249680A - Antibiotic tkr842, its production and microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel antibiotic substance which has excellent antifungal activity and is useful as a clinical medicine, for example, a therapeutic agent for fungus infection by culturing a strain in Chalara capable of producing an antibiotic substance: TKR842 and isolating the substance from the culture mixture. SOLUTION: This novel antibiotic substance: TKR842 (salt) has a m/z peak of 454 [M+H]<+> in the mass spectrum according to the FAB mass spectrometry; 24 of carbon atom number and 1 of nitrogen atom number; 212nm and 289nm of major peak wavelengths in the ultraviolet absorption spectrometry in methanol, their E<1> 1cm are 603 and 106, respectively; soluble in chloroform and methanol, slightly soluble in hexane and water, and is useful as a therapeutic agent for fungus infections. This antibiotic substance is obtained by culturing a strain in Chalara, capable of producing an antibiotic substance, TKR842, for example, Chalara sp. TKR 842 strain (FERM P-15474) and isolating the substance from the culture mixture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibiotic TKR842 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 (As
pergillus ) is known, and in superficial mycosis,
Candida, which infects the skin, oral cavity, vagina, etc., and Trichophyton, which infects the skin of limbs, 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. where found that in culture of a microorganism belonging to Carrara (Chalara) genus, antibiotic exists which exhibits antimicrobial activity against Cryptococcus neoformans, pathogenic fungi such as Aspergillus fumigatus. Then we isolated the antibiotic,
As a result of examining its physicochemical properties, it was confirmed that it is a novel substance having unique physicochemical properties and not described in the literature, and this antibiotic was named TKR842. The present invention provides the above antibiotic TKR842 and a method for producing the same.

The above antibiotic TKR842 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 454 [M + H] ⁺ (2) The number of carbon atoms is 24 and the number of nitrogen atoms is 1. (3) The main absorption wavelengths of the ultraviolet absorption spectrum in methanol are 212 nm and 289 nm. E ^1% _{1 cm} is 603 and 106 respectively. (4) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 3380 _cm ^-1 , 2920 cm ^-1 , 1660.
^{cm -1, 1570cm -1, 1460cm -1} , 1400c
m ^-1 , 1250 cm ^-1 , 1130 cm ^-1 , 1040 cm
^-1 (5) Soluble in chloroform and methanol, sparingly soluble in hexane and water

[0007] The above TKR842 also ¹ shown in FIG. 3
It has an H-NMR spectrum and a ¹³ C-NMR spectrum shown in FIG. 4, and has a characteristic of being eluted at the position shown in FIG. 5 in reversed phase partition high performance liquid chromatography.

[0008] The TKR842 is a Carara ( Chala).
It can be produced by culturing a strain which belongs to the genus ra ) and produces the above TKR842, and then isolating it from the culture of the above strain. The above-mentioned strain used in the present invention belongs to the genus Caralara and has the above-mentioned T
There is no particular limitation as long as it produces KR842, and for example, Carrara sp.
Examples thereof include KR842 strain (hereinafter referred to as "TKR842 strain") and the like.

The above-mentioned TKR842 strain is a new strain not described in the literature, which was first isolated and identified by the present inventors from a sample collected in Shiga Prefecture.
It has the property of advantageously producing 842. Hereinafter, the mycological properties of the TKR842 strain will be described in detail.

The color tone of the above-mentioned TKR842 strain in colonies (hereinafter also referred to as "community") in various media is as shown in Table 1. The colors in the table are based on the Japanese Industrial Standard JI.
Based on the color name by SZ 8102 (1985),
This is shown by the result of observing after 14 days of culturing at 25 ° C. after inoculating the medium.

[0011]

[Table 1]

The above-mentioned TKR842 strain grows slowly on malt extract agar medium, potato dextrose agar medium, Sabouraud agar medium, etc., and the surface of the colony is velvety and the center is dense and hard mycelium rises. TK
The conidia stalk of the R842 strain has a long cylindrical shape that is upright from the hyphae, the lower part is thick and the tip is thin to form a phialide.
Conidia are unicellular or long linked. The surface is cylindrical to barrel-shaped, and both ends are round or cut. or,
Its size is 6 to 10 × 2.0 to 3.0 μm.
Also, ascospores and chlamydospores are not formed in the above medium.

Among the mycological properties of the above TKR842 strain,
The physiological properties are as shown below. Growth temperature range: The growth temperature range is 10 to 30 ° C, and the optimum growth temperature is around 25 ° C. Growth pH range: The pH range in which growth is possible is pH 4 to pH 9, and the optimum growth pH is pH 5 to 8.

Bacterial species having the above-mentioned mycological properties were selected from Joseph C. Gilman.
Written by A Manual of Soil Fanjay (A
manual of soil fungi, Constable and Company Limited (Const
available and company ltd) (195
The above TKR842 strain can be identified as a strain belonging to the genus Carrara by searching the strains of the genus Carrara described in the literature such as 9 years).

However, regarding strains belonging to the genus Carrara, which have the ability to produce TKR842,
No report has been made so far, and the present inventors named it a new strain, and named it Carara sp. TKR842 strain (Chalara sp. TKR842), and designated C
halara sp. It is displayed as TKR842 and is deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (address :; 1-3-1, Higashi 1-3, Tsukuba, Ibaraki, Japan (postal code 305)) with deposit number FERM P-15474 (deposit date; 1996). February 23).

In the present invention, in addition to the above-mentioned TKR842 strain, a natural or artificial mutant strain of the TKR842 strain, other fungal species belonging to the genus Carrara, and the like having the ability to produce TKR842 are used. You can

In the present invention, TKR842 is produced by inoculating a nutrient source-containing medium with the above-mentioned TKR842-producing strain and culturing. Among the above nutrient sources, examples of the carbon source include glucose, fructose, sucrose, starch, dextrin, glycerin,
Examples include 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 ; Organic substances, inorganic substances and the like that promote the production of TKR842 can be added as appropriate. 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 the strain producing TKR842 is cultivated in the above nutrient-source-containing medium, a method generally used when 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 culture is preferably performed at 15 to 25 ° C,
The pH of the medium is usually 3-8, but is preferably around pH 5. The culture period is usually 6 to 15 days, and a sufficient production amount can be obtained.

By the above-mentioned culture method, TKR842
Is contained in the culture solution and cells and accumulated in the culture. In the present invention, TKR8 accumulated in the culture
42 can be obtained by separating it from the culture by utilizing the physicochemical properties of these antifungal substances and then further purifying it if necessary. 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. Further, after the culture is separated into a culture solution and bacterial cells by filtration or centrifugation, the culture solution,
It can also be separated from each of the cells.

In order to separate TKR842 from the culture medium, the method of extraction with the non-hydrophilic organic solvent can be adopted. Further, the culture medium is contacted with an adsorbent carrier to remove TKR842 in the culture medium. A method of adsorbing onto a carrier and then eluting with a solvent can be adopted. Examples of the carrier include activated carbon, powdered cellulose, adsorptive resin, and the like. As the solvent, the type of carrier,
One kind or a combination of two or more kinds may be appropriately used depending on the properties and the like, and examples thereof include those in which an aqueous solution of a water-soluble organic solvent such as hydrous acetone and hydrous alcohols is appropriately combined. TKR from the above cells
In order to separate 842, a method of extracting with a hydrophilic organic solvent such as acetone can be adopted.

In the present invention, the crude extract of TKR842 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, a column chromatography method using a carrier such as silica gel, activated alumina, activated carbon, an adsorptive resin, 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 TKR842 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 TKR842 of the present invention or a pharmacologically acceptable salt thereof is administered as a medicine, the TKR842 of the present invention or a pharmacologically acceptable salt thereof is
As a pharmaceutical composition containing, for example, 0.1 to 99.5%, preferably 0.5 to 90% as it is or in a pharmaceutically acceptable non-toxic and inert carrier, it can be applied to animals including humans. Is administered. Examples of the carrier include solid,
Examples include 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. When TKR842 of the present invention or a pharmacologically acceptable salt thereof is administered as a medicine, the dose as an antifungal agent depends on the patient's condition such as age and body weight, administration route, nature and degree of disease, etc. Although it is desirable to adjust the amount after taking into consideration, in general, for humans, the daily dose of the active ingredient of the present invention for an adult is 1
It is in the range of 0 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 obtained by suspending a fixed amount of TKR842 of the present invention or a pharmacologically acceptable salt thereof in a non-toxic liquid carrier suitable for injection purpose such as an aqueous or oily medium or It is prepared by dissolving and then sterilizing the suspension or solution.

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 TKR842 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 TKR842 of the present invention or a pharmacologically acceptable salt thereof is 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 a slope culture of the TKR842 strain (FERM P-15474) was treated with 100 ml of liquid medium (Difco yeast nitrogen base 0.67% (W / V), glucose 2.0% ( W / V)) was put into a 500 ml Erlenmeyer flask and shaken at 25 ° C. for 7 days to obtain a seed culture solution. 1.0 ml of this seed culture solution was added to the above liquid medium 1
Inoculate 36 500 ml Erlenmeyer flasks containing 40 ml and shake culture at 25 ° C for 13 days (shaking 220 rp).
m). The culture broth thus obtained was centrifuged to separate into a supernatant and cells. To the obtained supernatant, 2 L of ethyl acetate was added, mixed well and extracted with ethyl acetate. The extract was concentrated under reduced pressure to obtain 468 mg of a residue.

Further, the obtained cells were 800 m of methanol
After adding 1 and thoroughly mixing and performing an extraction operation, concentration under reduced pressure was performed. Water and ethyl acetate (300 ml each) were added to the obtained residue, mixed well and extracted with ethyl acetate.
The extract was concentrated under reduced pressure to give a residue of 198 mg.
I got The supernatant and the ethyl acetate extract of the bacterial cells were combined and extracted with methanol to obtain 549 mg of a residue.

This was dissolved in a small amount of methanol and subjected to high performance liquid chromatography to obtain an active fraction. By concentrating this fraction under reduced pressure, 238 mg of a residue was obtained.
The conditions for high performance liquid chromatography were as follows. Device: LC6A (manufactured by Shimadzu Corporation) Column: YMCpackC ₁₈ (2.0 cm × 25 cm)
(Manufactured by WMC) Mobile phase: 25 to 10 containing 0.05% trifluoroacetic acid
0% (V / V) methanol

This was dissolved in a small amount of methanol and applied to a Sephadex LH-20 (Pharmacia) column (150 ml) equilibrated with methanol and eluted with methanol to obtain an active fraction. This fraction was concentrated under reduced pressure to obtain 98 mg of residue. This was dissolved in a small amount of methanol and subjected to high performance liquid chromatography to obtain an active fraction. This fraction was concentrated under reduced pressure to obtain 11.4 mg of a purified product of TKR842 as a white powder. The conditions for high performance liquid chromatography were as follows. Device: LC6A (manufactured by Shimadzu Corporation) Column: YMCpackC ₁₈ (2.0 cm × 25 cm)
(Manufactured by WMC) Mobile phase: 51% (V containing 0.05% trifluoroacetic acid)
/ V) acetonitrile / water

For physicochemical properties , a JMS-DX302 type mass spectrometer (made by JEOL Ltd.) was used for mass spectrometry. ¹ H-NMR spectrum (in deuterated chloroform, standard substance: tetramethylsilane) and ¹³
C-NMR spectrum (in deuterated chloroform: standard substance:
For the measurement of deuterated chloroform), a JNM-A500 nuclear magnetic resonance apparatus (made by JEOL Ltd.) was used. A UV-250 type self-recording spectrophotometer (manufactured by Shimadzu Corporation) was used for ultraviolet absorption spectrum analysis (in methanol). A 270-30 type infrared spectrophotometer (manufactured by Hitachi, Ltd.) was used for infrared absorption spectrum analysis (KBr method). Below is TKR84
Describe the physicochemical properties of the two substances.

(1) Mass spectrometry A purified white powder obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure was
FAB-MS measurement by mass spectrometer, m / z 454
It was found to be [M + H] ⁺ .

(2) Carbon number, nitrogen number A white powder purified product obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure is
¹ H-NMR spectrum measurement, ¹³ C-NMR spectrum measurement and its analysis show that it has 24 carbon atoms and 1 nitrogen atom.
It turned out to be. The ¹ H-NMR spectrum is shown in FIG. 3 and the ¹³ C-NMR spectrum is shown in FIG.

(3) Ultraviolet absorption spectrum A white powder purified product obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure,
The ultraviolet absorption spectrum in methanol was as follows. UV (nm) (E ^1% _{1 cm} ): 212 (603), 289
(106). The 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 infrared spectrum measurement results by the KBr method were as follows. IR (KBr) (cm ^-1 ): 3380, 2920, 16
60, 1570, 1460, 1400, 1250, 11
30, 1040. The infrared absorption spectrum is shown in FIG.

The solubility of this substance in various solvents was soluble in chloroform and methanol, and sparingly soluble in hexane and water. From the above analysis results, it was revealed that the white powder purified product obtained by subjecting to high performance liquid chromatography and concentrating the obtained active fraction under reduced pressure was TKR842.

The above TKR842 was subjected to analysis by reversed-phase partition high performance liquid chromatography (HPLC) using an LC-10A type high performance liquid chromatography device (manufactured by Shimadzu Corporation). 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 TKR842 was eluted at the position shown in FIG.

Biological properties The obtained TKR842 was used to examine 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 the table, YNBG is YNBG medium containing yeast nitrogen base (manufactured by Difco) 0.67% and glucose 1.0%, and BHI is brain heart infusion broth (manufactured by Nissui Pharmaceutical Co., Ltd.) 0.5. % Of BHI medium is represented respectively.

[0043]

[Table 2]

From the results of Table 2, the antifungal substance T of the present invention is shown.
KR842 was found to have antibacterial activity against pathogenic fungi such as Cryptococcus neoformans and Aspergillus fumigatus.

Each of the obtained TKR842 was intraperitoneally administered to ICR mice at 100 mg / kg, but no toxicity was observed.

[0046]

INDUSTRIAL APPLICABILITY The present invention can provide an antifungal substance TKR842 which is useful as a clinical drug such as a therapeutic agent for fungal diseases and a method for producing the same.

[Brief description of drawings]

FIG. 1 is a diagram showing an ultraviolet absorption spectrum of an antifungal substance TKR842. The vertical axis represents wavelength (nm).

FIG. 2 is a diagram showing an infrared absorption spectrum of an antifungal substance TKR842. The horizontal axis represents the wave number (cm ^-1 ).

FIG. 3 is a diagram showing a ¹ H-NMR spectrum of an antifungal substance TKR842. Horizontal axis shows chemical shift value (ppm)
Is shown.

FIG. 4 is a diagram showing a ¹³ C-NMR spectrum of an antifungal substance TKR842. Horizontal axis shows chemical shift value (ppm)
Is shown.

FIG. 5 is a view showing elution positions in HPLC of an antifungal substance TKR842. The vertical axis represents relative absorption intensity, and the horizontal axis represents retention time (minutes).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12R 1: 645) (C12P 1/02 C12R 1: 645) (72) Inventor Junko Yamamoto 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 TKR842 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 454 [M + H] ⁺ (2) The number of carbon atoms is 24 and the number of nitrogen atoms is 1. (3) The main absorption wavelengths of the ultraviolet absorption spectrum in methanol are 212 nm and 289 nm. E ^1% _{1 cm} is 603 and 106 respectively. (4) The main absorption wave numbers of the infrared absorption spectrum by the KBr method are 3380 _cm ^-1 , 2920 cm ^-1 , 1660.
^{cm -1, 1570cm -1, 1460cm -1} , 1400c
m ^-1 , 1250 cm ^-1 , 1130 cm ^-1 , 1040 cm
^-1 (5) Soluble in chloroform and methanol, sparingly soluble in hexane and water 2. A Carrara (Chalara) a strain belonging to the genus, culturing the strains producing antibiotics TKR842, then antibiotics TKR842 for and isolating the desired product from the culture of the strain Manufacturing method. 3. A Carrara (Chalara) belonging to the genus microorganisms characterized in that it produces an antibiotic TKR842.