Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-08-02
2001-12-25
Tate, Christopher R. (Department: 1651)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C435S254100, C435S071300, C514S183000, C514S192000, C514S200000, C260S66500B, C260S66500B, C930S010000
Reexamination Certificate
active
06333305
ABSTRACT:
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]
+
at m/z 971;
(2) the molecular formula is represented by C
44
H
78
N
10
O
14
, and its mass spectrum by high-resolution FAB-MS gives m/z 971.5776 as [M+H]
+
;
(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
−1
;
(5) aspartic acid, threonine, serine, glycine, alanine, &bgr;-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
1
H-NMR spectrum shown in FIG.
3
and the
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° 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 3-8×1.5-2.5 &mgr;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° C. and the optimum range of temperature for growth is around 25° 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 a
Awazu Naoyuki
Kato Ikunoshin
Onishi Yoshimi
Takesako Kazutoh
Ueno Mitsuhiro
Ozga Brett
Sughrue & Mion, PLLC
Takara Shuzo Co. Ltd.
Tate Christopher R.
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