Antibiotic TKR2999, process for the preparation thereof and microbe

The antibiotic TKR2999 having the physicochemical properties described below and its pharmacologically acceptable salt: PA1 (1) FAB-MS m/z 971 [M+H)].sup.+, (2) the molecular formula: C.sub.44 H.sub.78 N.sub.10 O.sub.14, and high-resolution FAB-MS m/z 971.5776 [M+H].sup.+, (3) the ultraviolet absorption spectrum in methanol has an end absorption, (4) the infrared absorption spectrum by KBr method shows the major absorption wave numbers at 3320, 2920, 1680, 1540, 1210, 1140, 840, 800, and 720 cm.sup.-1, (5) aspartic acid, threonine, serine, glycine, alanine, .beta.-alanine, and ornithine are detected by the amino acid analysis using ninhydrin reaction, and (6) the solubility is that it is soluble in methanol, and practically insoluble in hexane, chloroform, and water.

TECHNICAL FIELD
 The present invention relates to the antibiotic TKR2999, which is useful as
 a therapeutic agent for fungal infection diseases, a method for its
 production, and microorganisms producing it.
 BACKGROUND ART
 Fungi are known to cause a variety of infectious diseases in man, animals,
 and plants. In man, for instance, they cause superficial mycosis affecting
 the skin, oral cavity, etc. and systemic mycosis affecting the viscera,
 brain, etc. They cause similar infections in pet and domestic animals as
 well. Furthermore, fungi inflict various hazardous effects on plants such
 as orchard trees and vegetables.
 As the principal pathogenic fungi causing systemic mycosis in man, those of
 the genera Candida, Cryptococcus, and Aspergillus, among others, are
 known. As to superficial mycosis, Candida species affecting the skin, oral
 cavity, and vagina and trichophytons infecting the skin of the extremities
 are regarded as the major pathogenic fungi. Besides those fungi, many
 other fungi exist in the environment and are suspected to contaminate the
 animal and vegetable kingdoms.
 As antimycotics of use for the prevention and treatment of such fungal
 infections and contaminations, only a very few are known. As therapeutic
 drugs for systemic mycosis in man and animals, for instance, amphotericin
 B, flucytosine, miconazole, and fluconazole can be mentioned. However,
 those compounds are not fully satisfactory in potency, toxic potential, or
 antifungal spectrum, thus being not impeccable as therapeutic drugs.
 In view of the above-mentioned prior art, the present invention has for its
 object to provide a novel antibiotic which is of value as a therapeutic
 agent for fungal infections.
 In the search for a novel antibiotic, the inventors of the present
 invention isolated a large number of microorganisms from the natural
 kingdom, isolated the antibiotics they produced, and scrutinized their
 biological properties. As a result, they discovered that the culture broth
 of a strain of microorganism of the Fungi Imperfecti contained an
 antibiotic having antifungal activity against pathogenic fungi inclusive
 of Candida albicans, C. kefyr, Cryptococcus neoformans, and Aspergillus
 fumigatus. Accordingly the inventors isolated this antibiotic and studied
 its physicochemical properties. As a result, they discovered that it was a
 novel substance having distinct physicochemical characteristics, which had
 been described in no literatures yet, and named it TKR2999. The present
 invention intends to provide the above antibiotic TKR2999 and a method of
 production thereof.
 The present inventions are described in detail as follows.
 DISCLOSURE OF THE INVENTION
 The above antibiotic TKR2999 has the physicochemical properties of (1),
 (2), (3), (4), and (5) described below:
 (1) the mass spectrumby FAB-MS method gives an ion peak of [M+H].sup.+ at
 m/z 971;
 (2) the molecular formula is represented by C.sub.44 H.sub.78 N.sub.10
 O.sub.14, and its mass spectrum by high-resolution FAB-MS gives m/z
 971.5776 as [M+H].sup.+ ;
 (3) the ultraviolet absorption spectrum in methanol has an end absorption;
 (4) the infrared absorption spectrum by KBr method shows the major
 absorption wave numbers at 3320, 2920, 1680, 1540, 1210, 1140, 840, 800,
 and 720 cm.sup.-1 ;
 (5) aspartic acid, threonine, serine, glycine, alanine, .beta.-alanine, and
 ornithine are detected by the amino acid analysis using ninhydrin
 reaction; and
 (6) the solubility is that it is soluble in methanol, and practically
 insoluble in hexane, chloroform, and water.
 The antibiotic TKR2999 mentioned above shows the .sup.1 H-NMR spectrum
 shown in FIG. 3 and the .sup.13 C-NMR spectrum shown in FIG. 4, and is
 characterized in the reversed-phase high-performance liquid
 chromatography, in which it is eluted at the position indicated in FIG. 5.
 The above-mentioned antibiotic TKR2999 can be produced by growing a strain
 of microorganism belonging to the class Fungi Imperfecti and capable of
 producing said TKR2999 in a culture medium, and by isolating the substance
 from the culture broth of the above strain.
 The strain of microorganism used in the present invention is not limited
 and can be used only provided it is capable of producing said TKR2999. An
 example of the strain that is used for production of the said compound is
 the fungi TKR2999 (hereinafter referred to as the TKR2999-strain)
 belonging to the class of Fungi Imperfecti.
 The above-mentioned TKR2999-strain is a novel strain not heretofore
 described in a literature, and was isolated and characterized for the
 first time by the inventors of the present invention. The strain has the
 property to produce TKR2999 with advantage. The mycological
 characteristics of this TKR2999-strain are now described in detail as
 follows.
 The colors of colonies of said TKR2999-strain on various media are shown in
 Table 1. The description of colors in the table is based on those
 prescribed in Japanese Industrial Standard (JIS) Z 8102 (1985). The
 results of observation on days 14 of culture at 25.degree. C. after
 inoculation are shown.
 TABLE 1
 Diameter Surface
 of colony Color of color of Appearance
 Medium (mm) colony colony of colony
 Malt 29 grayish sepia velvety
 extract yellow-red 10YR3/2
 agar 5YR5/2
 Potato 37 dark gray little dark velvety
 dextrose N3 yellow
 agar 2.5Y3/2
 Sabouraud 28 dark yellowish little dark velvety
 agar gray yellow
 5Y4/1 2.5Y3/2
 YpSs agar 26 light dark grayish velvety
 yellowish gray blue-green
 5YR7/1 2.5BG3/2
 The above TKR2999-strain grows slowly on malt extract agar, potato dextrose
 agar, and Sabouraud agar etc. The colony has velvety surface and rises in
 the center with dense hard mycelia. Conidia of the TKR2999-strain are
 unicellular, cylindrical with smooth surface, and the both edges are
 round. Their size is 3-8.times.1.5-2.5 .mu.m. Conidia are formed well on
 the above medium, but the conidia formation style in view of the
 conidiophore is uncharacterized.
 Among the mycological characters of the TKR2999-strain, its physiological
 characteristics are as follows.
 Temperature range for growth: the temperature range for growth is 10 to
 30.degree. C. and the optimum range of temperature for growth is around
 25.degree. C.
 The pH range for growth: the pH range for growth is pH 3 to 9 and the
 optimum range of pH for growth is pH 5.
 The above mycological characters are compared with the descriptions of
 species of the class Fungi Imperfecti described in "A Manual of Soil
 Fungi" authored by Joseph C. Gilman (Constable and company Ltd.) (1959)
 etc. The species of the TKR2999-strain can not be identified because its
 conidia formation style is unidentified.
 However, no report was available on a strain of microorganism having the
 ability to product TKR2999 among strains of the class Fungi Imperfecti.
 Therefore, the inventors of the present invention regarded it as a novel
 strain and named it Fungi strain TKR2999 of the Fungi Imperfecti. The
 strain was deposited under the Budapest Treaty with the National Institute
 of Bioscience and Human Technology (Address, 1-3, Higashi 1-chome,
 Tsukuba-shi, Tbaraki, Japan (Zip code 305-0046)) under the accession
 number of FERM BP-6524 (original date of deposit: Nov. 21, 1997; date of
 request for transfer to international deposit: Sep. 24, 1998).
 The present invention can be carried into practice not only with the
 above-mentioned TKR2999-strain but also with any spontaneous or artificial
 mutant of the TKR2999-strain or any other strain of microorganism
 belonging to the class Fungi Imperfecti and capable of producing TKR2999.
 In accordance with the present invention, TKR2999 is produced by
 cultivating a TKR2999-producing strain described above in a nutrient
 medium. Nutrients to be used for the medium include various carbon sources
 such as glucose, fructose, saccharose, starch, dextrin, glycerol,
 molasses, malt syrup, oils and fats, and organic acids.
 Nutrients to be used for the medium include nitrogen sources, organic and
 inoragnic materials such as soybean meal, cotton seed meal, corn steep
 liquor, casein, peptone, yeast extract, meat extract, wheat germs, urea,
 amino acids, ammonium salts, etc. Salts as nutrients are various inorganic
 salts such as salts of sodium, potassium, calcium, magnesium, etc. and
 salts of phosphoric acid. Those materials can be used independently or in
 a suitable combination.
 Where necessary, the nutrient medium may be supplemented with heavy metal
 salts such as iron salts, copper salts, zinc salts, cobalt salts, etc.,
 vitamins such as biotin, vitamin B1, etc., and other organic and inorganic
 substances which would assist in growth of the microorganism and promote
 production of TKR2999.
 In addition to the above components, an antifoamer and/or a surfactant, for
 example silicone oil, polyalkylene glycol ethers, etc., can be added to
 the nutrient medium.
 In cultivating a strain of microorganism capable of producing TKR2999 in
 said nutrient medium, a variety of cultural methods which are generally
 used in the production of antibiotics by means of microorganisms can be
 employed. However, a liquid culture method, particularly a method by shake
 culture or submerged aerobic culture, is preferred.
 The cultivation is preferably carried out at 15 to 25.degree. C. The pH of
 the medium may range from pH 3 to 8 and is preferably around pH 5.
 Regarding the incubation time, generally a sufficient output of the
 substance can be expected by 6 to 15 days of culture.
 By means of the above cultivation, TKR2999 is contained both
 intracellularly and extracellularly and accumulated in the culture broth.
 In the present invention, the TKR2999 accumulated in the culture broth can
 be recovered and isolated from the broth by utilizing its physicochemical
 characteristics and, where necessary, by further purification.
 The above-mentioned recovery can be achieved by extracting the whole broth
 with a non-hydrophilic organic solvent such as ethyl acetate, butyl
 acetate, chloroform, butanol, methyl isobutyl ketone, or the like. As an
 alternative, it is possible to subject the broth to centrifugation or
 filtration to separate into the medium and cells and isolate the
 antibiotics from each of the medium and cells.
 For separation of TKR2999 from the medium not only the above-mentioned
 extraction method using a non-hydrophilic organic solvent but also the
 method which comprises contacting the medium with an adsorbent to let
 TKR2999 adsorbed on the adsorbent and desorbing or eluting them with a
 solvent can be employed.
 The adsorbent that can be used includes, for example, activated carbon,
 cellulose powder, and adsorbent resins. As the above-mentioned solvent, a
 variety of solvents can be selectively used according to the kind and
 properties of the adsorbent and either singly or in combination. Thus, an
 aqueous solution of one or more water-soluble organic solvents, such as
 aqueous acetone, aqueous alcohol, etc., can be employed. For separation of
 TKR2999 from the cells, the extraction technique using a hydrophilic
 organic solvent such as acetone can be employed.
 In the present invention, the crude extract of TKR2999 obtained from the
 culture broth as described above can be subjected to a procedure for
 purification when necessary. The purification can be carried out by
 conventional methods for separation and purification of hydrophobic
 antibiotics. Examples of the methods are column chromatographies or
 high-performance liquid chromatographies, using a column packed with a
 stationary phase such as silica gel, activated alumina, activated
 charcoal, adsorbent resin, etc. The eluent that can be used for silica gel
 column chromatography includes chloroform, ethyl acetate, methanol,
 acetone, water, a mixture of thereof, etc.
 The resin for high-performance liquid chromatography includes
 chemically-derivatized silica gel, such as silica gel derivatives having
 octadecyl, octyl, or phenyl groups, and polystyrenic porous polymer gels,
 while the mobile phase that can be used includes aqueous solutions of
 water-soluble organic solvents, such as aqueous methanol, aqueous
 acetonitrile, etc.
 TKR2999 of the present invention can each be put to use as such or in the
 form of a pharmacologically acceptable salt in medicinal applications.
 There is no particular limitation on the type of pharmacologically
 acceptable salt. Thus, the salt includes salts of mineral acids such as
 hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,
 hydrofluoric acid, hydrobromic acid, etc., salts of organic acids 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, camphorsulfonic acid, etc., and salts of alkali metals or alkaline
 earth metals, such as sodium, potassium, calcium, etc.
 To administer TKR2999 or its pharmacologically acceptable salt, as a drug,
 they can be administered to animals inclusive of humans either as such or
 in the form of a pharmaceutical composition containing typically 0.1 to
 99.5%, preferably 0.5 to 90% thereof in a pharmaceutically acceptable, non
 toxic and inert carrier.
 The carrier mentioned above includes solid, semisolid or liquid diluents,
 fillers, other formulation auxiliaries, etc. and such carriers can be used
 alone or in combination.
 The above-mentioned pharmaceutical composition is preferably administered
 in unit dosage forms and can be administered orally, parenterally,
 topically (e.g. transdermally) or rectally. Of course, those
 pharmaceutical compositions should be administered in dosage forms suited
 for the respective route of administration.
 For administration of TKR2999, or its pharmacologically acceptable salt, as
 a drug, the dose as an antifungal agent is preferably selected with
 reference to patient factors such as age and body weight, route of
 administration, nature and severity of disease, etc. Usually in man,
 however, the daily dose of the active ingredient for an adult patient is
 10 to 2000 mg. While a daily dose lower than the above range may be
 sufficient in some cases, a dose higher than the range may be required in
 other cases. When a high dose is used, the daily dosage is preferably
 administered in several divided doses.
 The above oral administration can be made using solid, powdery, or liquid
 dosage forms such as bulc powders, powders, tablets, dragees, capsules,
 drops, subligual tablets, etc.
 For the above parenteral administration, liquid unit dosage forms for
 subcutaneous, intramuscular, or intravenous administration, typically
 solutions and suspensions, can be employed. These preparations can be
 manufactured by suspending or dissolving a predetermined amount of
 TKR2999, or a pharmacologically acceptable salt thereof, in a nontoxic
 liquid carrier suitable for injection, such as an aqueous medium or an
 oily medium, and sterilizing the resulting suspension or solution.
 The topical administration (e.g. transdermal administration) can be carried
 out using a variety of topical dosage forms such as liquids, creams,
 powders, pastes, gels, and ointments. These dosage forms can be
 manufactured by using a predetermined amount of TKR2999 or a
 pharmacologically acceptable salt thereof, in combination with one or more
 of the perfume, coloring agent, filler, surfactant, humectant, emollient,
 gelatinizer, carrier, preservative, stabilizer, etc., suitable for topical
 dosage formulations.
 The rectal administration can be made using, for example, suppositories
 each mixing a predetermined amount of TKR2999, or its pharmacologically
 acceptable salt of the present invention, with a low-melting solid base
 such as higher esters, e.g. myristyl palmitate, polyethylene glycol, cacao
 butter, or a mixture of them.

BEST MODE OF CARRYING OUT THE INVENTION
 A loopful of the TKR2999-strain (FERM BP-6524) from a slant culture was
 used to inoculate into a 500-ml Erlenmeyer flask containing 100 ml of
 liquid medium (Difco yeast nitrogen base 0.67% (w/v) and glucose 2.0%
 (w/v)) and incubated on a shaker at 25.degree. C. for 10 days to prepare a
 seed culture. This seed culture, 1.0 ml, was transferred to each of 26
 Erlenmeyer flasks of 500 ml capacity each containing 120 ml of the same
 liquid medium as above and incubated (under shaking at 220 rpm) at
 25.degree. C. for 12 days. The culture broth obtained was centrifuged and
 separated into the supernatant and the cells.
 The cells obtained were mixed with 1 L of methanol and subjected to
 sufficient mixing for extraction. The extract was concentrated under
 reduced pressure. The residue was mixed with 300 ml each of water and
 butanol, and subjected to sufficient mixing to perform extraction with
 butanol. The extract was concentrated under reduced pressure to recover
 362 mg of a residue. The residue was dissolved in 2 ml of methanol and
 subjected to high-performance liquid chromatography to provide an active
 fraction. The fraction was concentrated under reduced pressure to recover
 1.2 mg of purified TKR2999 as white powder. The high-performance liquid
 chromatography was carried out under the following conditions.
 Apparatus: LC-8A (Shimadzu)
 Column: YMC pack C18 (2.0 cm.times.25 cm) (YMC)
 Mobile phase: 60% (v/v) of acetonitrile/water containing 0.05%
 trifluoroacetic acid
 Physicochemical Properties
 Mass spectrometry was carried out by JMS-DX302 mass spectrometer (Jeol
 Ltd.). .sup.1 H-NMR spectrum (in deuterated dimethylsulfoxide with
 deuterated dimethylsulfoxide as reference) and .sup.13 C-NMR spectrum (in
 deuterated dimethylsulfoxide with deuterated dimethylsulfoxide as
 reference) were measured by JNM-A500 nuclear magnetic resonance
 spectrometer (Jeol Ltd.). Ultraviolet spectrophotometry (in methanol) was
 carried out by UV-250 self-recording spectrophotometer (Shimadzu), and
 infrared absorption spectrometry (KBr method) was by 270-30 infrared
 spectrophotometer (Hitachi). Physicochemical properties of the substance
 TKR2999 are desribed below.
 (1) Mass Spectrometry and Determination of the Molecular Formula
 The purified white powdery product available upon vacuum concentration of
 the active fraction in said high-performance liquid chromatography was
 found to be a substance with m/z 971 [M+H].sup.+ by measurement with
 FAB-MS. In addition, its high resolution FAB-MS was measured and an
 [M+H].sup.+ was observed at m/z 971.5776. This result gave the molecular
 formula of C.sub.44 H.sub.78 N.sub.10 O.sub.14 (calculated, 971.5699) to
 TKR2999.
 (2) Ultraviolet Absorption Spectrum
 The UV absorption in methanol of the purified white powdery product
 available upon vacuum concentration of the active fraction in the
 high-performance liquid chromatography was found to be end absorption.
 The UV absorption spectrum is shown in FIG. 1.
 (3) Infrared Absorption Spectrum
 The IR spectrophotometric characterization (KBr method) of the purified
 white powdery product available upon vacuum concentration of the active
 fraction in the high-performance liquid chromatography was as follows. IR
 (KBr) (cm.sup.-1): 3320, 2920, 1680, 1540, 1210, 1140, 840, 800, 720.
 The IR absorption spectrum is shown in FIG. 2.
 (4) .sup.1 H-NMR and .sup.13 C-NMR Spectra
 The purified white powdery product available upon vacuum concentration of
 the active fraction in the high-performance liquid chromatography was used
 to measure .sup.1 H NMR spectrum and .sup.13 C NMR spectrum.
 The .sup.1 H-NMR spectrum and .sup.13 C-NMR spectrum of this product are
 presented in FIG. 3 and FIG. 4, respectively.
 (5) Amino Acid Analysis
 The purified white powdery product available upon vacuum concentration of
 the active fraction in the high-performance liquid chromatography was
 hydrolysed in 6 N HCl at 110.degree. C. for 24 hours. The decomposed
 product was analyzed by the amino acid analyzer, and aspartic acid,
 threonine, serine, glycine, and alanine were detected. Additionally
 .beta.-alanine and ornithine were detected.
 (6) As for the solubility of the this substance in various solvents, it was
 soluble in methanol, but practically insoluble in hexane, chloroform and
 water.
 Based on the above analyses, the purified white powdery product available
 upon vacuum concentration of the active fraction in the high-performance
 liquid chromatography was identified to be TKR2999.
 The above TKR2999 was analyzed by reversed-phase partition high-performance
 liquid chromatography (HPLC) using LC-10A high-performance liquid
 chromatography (Shimadzu). This HPLC analysis was carried out under the
 following conditions.
 Column: CAPCELL PAK C.sub.18 (6 mm.times.150 mm) (Shiseido)
 Mobile phase: 50% (v/v) acetonitrile/water containing 0.05% trifluoroacetic
 acid
 Column temperature: 40.degree. C.
 Detection UV wavelength: 220 nm
 As a result, the above TKR2999 was eluted at the position indicated in FIG.
 5.
 Biological Characteristics
 The TKR2999 obtained was tested for the antimicrobial spectrum to various
 microorganisms. Using the liquid medium dilution method for the
 measurement, the concentration causing substantially complete inhibition
 of micorbial growth was determined as the minimal inhibitory concentration
 (.mu.g/ml). The results are shown in Table 2. The minimal concentration
 causing partial inhibition of fungal growth was determined as the
 sub-inhibitory concentration (.mu.g/ml) and are shown in parentheses in
 the table. In the table, YNBG stands for a medium comprising 0.67% of
 yeast nitrogen base (Difco) and 1.0% of glucose, and BHI does for a medium
 comprising 0.5% of brain heart infusion bouillon (Nissui).
 TABLE 2
 Minimal inhibitory
 concentration
 Test strain Medium (.mu.g/ml)
 Candida albicans TIMM0136 YNBG 6.25
 Candida kefyr TIMM0301 YNBG 12.5
 (6.25)
 Cryptococcus neoformans TIMM0354 YNBG 6.25
 (3.13)
 Aspergillus fumigatus TIMM1776 BHI 0.78
 It is apparent from Table 2 that TKR2999, the antibiotic according to the
 present invention, is active against pathogenic fungi such as Candida
 albicans, Candida kefyr, Cryptococcus neoformans, Aspergillus fumigatus,
 etc. Intraperitoneal administration of the TKR2999 obtained above at a
 dose of 50 mg/kg to ICR mice caused no toxic signs.
 INDUSTRIAL APPLICABILITY
 The present invention provides the antibiotic TKR2999 which are of use in
 clinical medicine, for example in the therapy of fungal infectious
 diseases, and a method for production of the substance.