CA1091175A - Polyether antibiotic from streptomyces hygrosiopicus - Google Patents
Polyether antibiotic from streptomyces hygrosiopicusInfo
- Publication number
- CA1091175A CA1091175A CA269,005A CA269005A CA1091175A CA 1091175 A CA1091175 A CA 1091175A CA 269005 A CA269005 A CA 269005A CA 1091175 A CA1091175 A CA 1091175A
- Authority
- CA
- Canada
- Prior art keywords
- antibiotic
- follows
- absorption spectrum
- chloroform
- soluble
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Abstract of the Disclosure This invention provides a method for producing the new Antibiotic T-42082 by cultivating an Antibiotic T-42082-producing strain of the genus Streptomycos (ATCC 31080) and permitting said strain to elaborate and accumulate Antibiotic T-42082 in the resultant broth and harvesting the anti-biotic from said broth. The new antibiotic inhibits the growth of any gram-positive bacteria, acid-fast bacteria and certain fungi, and may be used for human treatment and for the treatment of coccidiosis in domestic fowls and animals.
Description
i`(l~ll'7~i The present invention relates to a novel Antiobiotic T-42082 and a method for the production thereof.
In search of new antibiotics, microorganisms were isolated from a number of soil samples and separated and screened for their metabolites. As a result, it was found, for instance, that certain microorganisms produced a new antiobiotic, that said microorganisms belonged to the genus Streptomyces and that by cultivating such a microorganism in an appropriate culture medium it was possible to cause said microorganism to accumulate in the broth said antibiotic which was found to be active against gram-positive bacteria, acid-fast bacteria and certain fungi, such as Candida albicans and Piricularia oryzae. The antiobiotic was isolated and, based on its physical, chemical and biological characteristics, it was confirmed that said antibiotic was a new polyether-type antiobiotic. The antibiotic was named Antibiotic T-42082.
In the method of the present invention there is employed an Anti-biotic T-42082-producing strain of the genus StrePtomyces. As an example thereof, there may be mentioned Strain T-42082 which was isolated from soil samples collected in a mountainous area of Ito City, Shizuoka Prefecture, Japan in April, 1973.
According to the invention, therefore, there is provided a process for preparing Antibiotic T-42082 which comprises aerobically cultivating an Antibiotic T-42082 producing microorganisms of Streptomyces hvgroscopicus in a liquid nutrient medium until substantial antibiotic activity accumulates in the resultant medium and then recovering the Antibiotic T-42082 from said medium, either as such or in the form of a pharmaceutically acceptable salt thereof.
The invention also provides the new Antibiotic T-42082 and its pharmaceutically acceptable salts, said Antibiotic T-42082 having the follow-ing properties:
(l) Melting point: 120-122C.
In search of new antibiotics, microorganisms were isolated from a number of soil samples and separated and screened for their metabolites. As a result, it was found, for instance, that certain microorganisms produced a new antiobiotic, that said microorganisms belonged to the genus Streptomyces and that by cultivating such a microorganism in an appropriate culture medium it was possible to cause said microorganism to accumulate in the broth said antibiotic which was found to be active against gram-positive bacteria, acid-fast bacteria and certain fungi, such as Candida albicans and Piricularia oryzae. The antiobiotic was isolated and, based on its physical, chemical and biological characteristics, it was confirmed that said antibiotic was a new polyether-type antiobiotic. The antibiotic was named Antibiotic T-42082.
In the method of the present invention there is employed an Anti-biotic T-42082-producing strain of the genus StrePtomyces. As an example thereof, there may be mentioned Strain T-42082 which was isolated from soil samples collected in a mountainous area of Ito City, Shizuoka Prefecture, Japan in April, 1973.
According to the invention, therefore, there is provided a process for preparing Antibiotic T-42082 which comprises aerobically cultivating an Antibiotic T-42082 producing microorganisms of Streptomyces hvgroscopicus in a liquid nutrient medium until substantial antibiotic activity accumulates in the resultant medium and then recovering the Antibiotic T-42082 from said medium, either as such or in the form of a pharmaceutically acceptable salt thereof.
The invention also provides the new Antibiotic T-42082 and its pharmaceutically acceptable salts, said Antibiotic T-42082 having the follow-ing properties:
(l) Melting point: 120-122C.
(2) Elemental analysis: Found ~ C, 62.73; H, 9.23; 0, 26.01;
C, 62.74; H, 9.36; 0, 27.03 t%)
C, 62.74; H, 9.36; 0, 27.03 t%)
(3) Molecular weight, 859 (by osmometry):
C~
.
lV91~
C~
.
lV91~
(4) Ultraviolet absorption spectrum; No characteristic absorptions;
(5) Infrared absorption spectrum: The dominant absorptions ~wave-numbers) measured in KBr disc method being as follows: 2940, 1700, 1462, 1383, 1085, 972 cm 1;
~ 6) Thin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10% aqueous H2S04;
Rf values as follows:
Solvent system Rf Ethyl acetate-benzene ~1:1) 0.52 Ethyl acetate 0.73 Benzene-acetone (9:1) 0.18 Benzene-acetone (1:1) 0.88 Chloroform-methanol (19:1) 0.87 Chloroform-ethyl acetate (2:3) 0.62 Plate: silica gel plate ~Kieselgel 60, Merck, Germany) C7~ Color reactîons:
Color reagent Sulfuric acid Positive ~brown) An~line-phthalic acid Positive ~indigo blue) ~anillin-sulfuric acid Positive (violet) Molisch Negative Dragendorff Positive ~light orange) Barton Negative Molybdenic acid Negative Benzidine Negative Ninhydrine Negative Alkaline potassium permanganate Negative C8~ Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride, sparingly soluble in cyclohexane; and very sparingly soluble in water and petroleum ether;
~09117$
when prepared by the above defined process or by an obvious chemical equiva-lent thereof.
The following are some characteristics of the strain as deposited in various culture collections.
a) Morphological characteristics From a well-branched vegetative mycelium extends an aerial mycelium measuring about 1 ~, monopodially branched. At the ends of the side branches thus formed are chains of closed spiral spores. The spore is elipsoidal to cylindrical C0.7 - 1.0 ~ x 0.9 - 1.4 ~), the spore configuration being verrucose Cwarty). No evidence of other special organs such as spherical sporangium, flagella, sclerotium, etc. are observed.
b) Cultural characteristics The characteristics displayed by the strain on various media are set forth in Table 1. Unless otherwise specified, the observations were made after 2 weeks' incubation at 28C.
- 2a -1(J'i~11`7~
~ r~
P '~ 3 1 `~ n 3 ~
~ _ ~ ~ ~., I ~ a ~ j !f ~, !, ' ' ~ f : `
c) Physiological ch~racteristics l, Temperature range for growth +
++
24 +++
28 +++
+++
34 +++
37 +++
+
- : No growth + : Doubtful growth : + : Growth ++ : Good growth +++ : ~uxuriant growth 2. ~iquefaction of gelatin: Positive 3. Hydrolysis of starch: Positive 4. Peptonization of skim milk: Positive Coagulation of skim milk : Negative 5. Production of melanoid pigment Tyrosine-agar : Negative P.eptone-yeaSt extract-iron-agar : Negative
~ 6) Thin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10% aqueous H2S04;
Rf values as follows:
Solvent system Rf Ethyl acetate-benzene ~1:1) 0.52 Ethyl acetate 0.73 Benzene-acetone (9:1) 0.18 Benzene-acetone (1:1) 0.88 Chloroform-methanol (19:1) 0.87 Chloroform-ethyl acetate (2:3) 0.62 Plate: silica gel plate ~Kieselgel 60, Merck, Germany) C7~ Color reactîons:
Color reagent Sulfuric acid Positive ~brown) An~line-phthalic acid Positive ~indigo blue) ~anillin-sulfuric acid Positive (violet) Molisch Negative Dragendorff Positive ~light orange) Barton Negative Molybdenic acid Negative Benzidine Negative Ninhydrine Negative Alkaline potassium permanganate Negative C8~ Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride, sparingly soluble in cyclohexane; and very sparingly soluble in water and petroleum ether;
~09117$
when prepared by the above defined process or by an obvious chemical equiva-lent thereof.
The following are some characteristics of the strain as deposited in various culture collections.
a) Morphological characteristics From a well-branched vegetative mycelium extends an aerial mycelium measuring about 1 ~, monopodially branched. At the ends of the side branches thus formed are chains of closed spiral spores. The spore is elipsoidal to cylindrical C0.7 - 1.0 ~ x 0.9 - 1.4 ~), the spore configuration being verrucose Cwarty). No evidence of other special organs such as spherical sporangium, flagella, sclerotium, etc. are observed.
b) Cultural characteristics The characteristics displayed by the strain on various media are set forth in Table 1. Unless otherwise specified, the observations were made after 2 weeks' incubation at 28C.
- 2a -1(J'i~11`7~
~ r~
P '~ 3 1 `~ n 3 ~
~ _ ~ ~ ~., I ~ a ~ j !f ~, !, ' ' ~ f : `
c) Physiological ch~racteristics l, Temperature range for growth +
++
24 +++
28 +++
+++
34 +++
37 +++
+
- : No growth + : Doubtful growth : + : Growth ++ : Good growth +++ : ~uxuriant growth 2. ~iquefaction of gelatin: Positive 3. Hydrolysis of starch: Positive 4. Peptonization of skim milk: Positive Coagulation of skim milk : Negative 5. Production of melanoid pigment Tyrosine-agar : Negative P.eptone-yeaSt extract-iron-agar : Negative
6. Assimilation of carbon sources (Pridham & Gottlieb agar) See Table 2.
. . . . ~ ., .
. ': ' .
" 1091~7~
Table 2 i-Inositol +
D-mannitol +
D-xylose +
L-arabinose +
D-glucose +
D-fructose +
Rhamnose +
Sucrose +
Raffinose +
Control (not added) - ; no growth; +; growth The above characteristics indicate clearly that strain T 'l2082 belongs to the genus ~treptom~ces. Comparison of the above characteristics with the descriptions of many Strepto-m~ces species in S.A, Waksman, ~he Actinomycetes, Vol.2(1961), R. HUtter, Systematik der Streptomyceten (1967~, International Streptomyces Project (ISP) and other literature shows that the present strain has much in common with Streptom,yces h~groscopicus Waksman and Henrici (1948) so far as the characteristics important for the taxonomic identification of species are concerned. ~he inventors have relegated the strain to the species Streptom~ces h~roscopicus and named it StrePtom~ces h~groscopicus ~-42082.
Streptom~ces h~roscopicus ~-42082, which is exploited according tothe present invention has been deposited at the Institute of Microbiological ~echnology of the Agency of Industrial ~echnology, the Institute for Fermentation, Osaka .~
.
- 1(J9117$
and American Type Culture Collection under the serial or accession number of ~RM-P-2691, IF0-13609 and ATCC 31080, respectively.
~ he new strain was submitted to the American ~ype Culture Collection in 12301 Parklawn Drive, Rockville Maryland 20852 on August 6, 1976 and identified as strePt m~ces ~y~roscopicus ~-42082 and assigned the ATCC number 31080. ~he permanency of the deposit and the ready accesi-bility to the deposit by the public are afforded inthe event the patent is granted. Access to the culture is available during the pendency of the application under Rule 14 and 35 U.S.C, 112. All restrictions on the avail-ability of the culture deposited to the public will be irrevocably removed upon the granting of the patent, Although the T-42082 strain has been described above, it is well known that characteristics of actinomycetes are not constant but are readily varied by spontaneous or artificial mutation and it should be understood that the strains of microorganisms employable according to the present invention include all the strains which belong to the genus Streptom~ces and which are able to elaborate Antibiotic ~-42082.
lhe cultivation according to the present invention is carried out by growing any of said strains in a medium containing nutrients which may be utilized by the particular strain. As to medium ingredients, the carbon source may for example be glucose, starch, glycerin, dextrin, sucrose, millet jelly, molasses or/and so forth. As the nitrogen , . . . .
. ' ' , ~O~il7S
source, there may be utilized meat extract, dried yeast, yeast extract, soybean flour, corn steep liquor, wheat embryos, cotton seed meal, ammonium sulfate, ammonium nitr~te and so forth. If necessary, there may be added inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphates, etc. as well as organic and inorganic materials which would assist in the growth of the particular microorganism or in the elaboration of Antibiotic ~-42082.
~ he cultivation may be carried out in generally the same manner as in the production of antibiotics in general, submerged aerobic culture in a fluid medium being particularly desirable. ~he cultivation is carried out aerobically at an appropriate pH level which is within the range of pH 5 to 9, preferably in the range of pH 6 to 8, and at a temperature in the range of 15 to 40C, preferably 23 to 34C, although in many instances the cultivation is prefer-ably carried out in the neighborhood of 28C. ~he incuba-tion time normally ranges from 2 to 12 days, preferably 3 to 7 days.
After completion of cultivation, Antibiotic ~-42082 is separated and harvested from the broth by conventional procedures which are normally employed for the harvest of microbial metabolites from culture broths, either alone or in a suitable combination ~hus, by taking advantage of its solubility in neutral lipids, the desired antibiotic may be separated by extraction with various organic solvents, phasic transfer, recrystallization, chromatography on various . ~ -lO91i`75 adsorbents and so ~orth.
~ he present antibiotic produced by the cultural methodthus described hereinbefore occurs in both the liquid phase of the broth and the grown cells. ~herefore, the following procedures, for example, may be exploited with advantage.
Thus, with the addition of a water-miscible solvent such as methanol, ethanol, acetone or the like, the broth is filtered and, after the solvent has been distilled off from the filtrate, the aqueous solution is adjusted to pH 4-9.
~he solution is then extracted with a solvent hardly miscible with water, such as ethyl acetate, butyl acetate, chloroform or the like. ~hen, following removal of the solvent by distillation, the residue is crystallized from a solvent system such as methanol-water, ethanol-water, acetone-water or the like, or with a solvent hardly miscible with water, such as ethyl ether, chloroform or carbon tetrachloride, Alternatively, the broth is filtered together with a filter aid and the filtrate is extracted with ethyl acetate, chloroform or the like. The cells are also extracted with methanol, ethanol, acetone or the like. ~he filtrate and cell extract are pooled and, after the solvent has been distilled off, the residue is crystallized from a solvent such as that described. Where the broth is rich in impuri-ties, the broth may be passed through a styrenic adsorbent resin or chromatographed on silica gel, alumina or other carrier material. ~hen, the eluate free from the contami-nants is concentrated under reduced pressure and the residue 109~17S
is crystallized from said solvent.
In the above procedure, crystals of Antibiotic ~-42082 can be obtained in good yield. Antibiotic T-42082 may be isolated as crystals of its salts, e.g sodium, potassium, lithium, ammonium and other salts.
The following are the physical and chemical properties of Antibiotic T-42082 and of its sodium salt, which are obtainable according to the procedures set forth herein-after in Example 1.
~I) The free form of Antibiotic T-42082 (1) Color and appearance: Colorless needles (2) Melting point: 120-122C
(3) Elemental analysis: ~ound 0 C, 62.73; H, 9.23; 0,26.01;
~ C, 62.74; H, 9.36; 0, 27.03(%) (4) Molecular weight: 859 (by osmometry) (5) Ultraviolet absorption spectrum: No characteristic absorptions (6) Infrared absorption spectrum: The spectrum (KBr) is given in ~ig l. ~he dominant absorptions (wave-numbers) are as follows: 2940, 1700, 1462, 1383, 1085, 972 cm 1
. . . . ~ ., .
. ': ' .
" 1091~7~
Table 2 i-Inositol +
D-mannitol +
D-xylose +
L-arabinose +
D-glucose +
D-fructose +
Rhamnose +
Sucrose +
Raffinose +
Control (not added) - ; no growth; +; growth The above characteristics indicate clearly that strain T 'l2082 belongs to the genus ~treptom~ces. Comparison of the above characteristics with the descriptions of many Strepto-m~ces species in S.A, Waksman, ~he Actinomycetes, Vol.2(1961), R. HUtter, Systematik der Streptomyceten (1967~, International Streptomyces Project (ISP) and other literature shows that the present strain has much in common with Streptom,yces h~groscopicus Waksman and Henrici (1948) so far as the characteristics important for the taxonomic identification of species are concerned. ~he inventors have relegated the strain to the species Streptom~ces h~roscopicus and named it StrePtom~ces h~groscopicus ~-42082.
Streptom~ces h~roscopicus ~-42082, which is exploited according tothe present invention has been deposited at the Institute of Microbiological ~echnology of the Agency of Industrial ~echnology, the Institute for Fermentation, Osaka .~
.
- 1(J9117$
and American Type Culture Collection under the serial or accession number of ~RM-P-2691, IF0-13609 and ATCC 31080, respectively.
~ he new strain was submitted to the American ~ype Culture Collection in 12301 Parklawn Drive, Rockville Maryland 20852 on August 6, 1976 and identified as strePt m~ces ~y~roscopicus ~-42082 and assigned the ATCC number 31080. ~he permanency of the deposit and the ready accesi-bility to the deposit by the public are afforded inthe event the patent is granted. Access to the culture is available during the pendency of the application under Rule 14 and 35 U.S.C, 112. All restrictions on the avail-ability of the culture deposited to the public will be irrevocably removed upon the granting of the patent, Although the T-42082 strain has been described above, it is well known that characteristics of actinomycetes are not constant but are readily varied by spontaneous or artificial mutation and it should be understood that the strains of microorganisms employable according to the present invention include all the strains which belong to the genus Streptom~ces and which are able to elaborate Antibiotic ~-42082.
lhe cultivation according to the present invention is carried out by growing any of said strains in a medium containing nutrients which may be utilized by the particular strain. As to medium ingredients, the carbon source may for example be glucose, starch, glycerin, dextrin, sucrose, millet jelly, molasses or/and so forth. As the nitrogen , . . . .
. ' ' , ~O~il7S
source, there may be utilized meat extract, dried yeast, yeast extract, soybean flour, corn steep liquor, wheat embryos, cotton seed meal, ammonium sulfate, ammonium nitr~te and so forth. If necessary, there may be added inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphates, etc. as well as organic and inorganic materials which would assist in the growth of the particular microorganism or in the elaboration of Antibiotic ~-42082.
~ he cultivation may be carried out in generally the same manner as in the production of antibiotics in general, submerged aerobic culture in a fluid medium being particularly desirable. ~he cultivation is carried out aerobically at an appropriate pH level which is within the range of pH 5 to 9, preferably in the range of pH 6 to 8, and at a temperature in the range of 15 to 40C, preferably 23 to 34C, although in many instances the cultivation is prefer-ably carried out in the neighborhood of 28C. ~he incuba-tion time normally ranges from 2 to 12 days, preferably 3 to 7 days.
After completion of cultivation, Antibiotic ~-42082 is separated and harvested from the broth by conventional procedures which are normally employed for the harvest of microbial metabolites from culture broths, either alone or in a suitable combination ~hus, by taking advantage of its solubility in neutral lipids, the desired antibiotic may be separated by extraction with various organic solvents, phasic transfer, recrystallization, chromatography on various . ~ -lO91i`75 adsorbents and so ~orth.
~ he present antibiotic produced by the cultural methodthus described hereinbefore occurs in both the liquid phase of the broth and the grown cells. ~herefore, the following procedures, for example, may be exploited with advantage.
Thus, with the addition of a water-miscible solvent such as methanol, ethanol, acetone or the like, the broth is filtered and, after the solvent has been distilled off from the filtrate, the aqueous solution is adjusted to pH 4-9.
~he solution is then extracted with a solvent hardly miscible with water, such as ethyl acetate, butyl acetate, chloroform or the like. ~hen, following removal of the solvent by distillation, the residue is crystallized from a solvent system such as methanol-water, ethanol-water, acetone-water or the like, or with a solvent hardly miscible with water, such as ethyl ether, chloroform or carbon tetrachloride, Alternatively, the broth is filtered together with a filter aid and the filtrate is extracted with ethyl acetate, chloroform or the like. The cells are also extracted with methanol, ethanol, acetone or the like. ~he filtrate and cell extract are pooled and, after the solvent has been distilled off, the residue is crystallized from a solvent such as that described. Where the broth is rich in impuri-ties, the broth may be passed through a styrenic adsorbent resin or chromatographed on silica gel, alumina or other carrier material. ~hen, the eluate free from the contami-nants is concentrated under reduced pressure and the residue 109~17S
is crystallized from said solvent.
In the above procedure, crystals of Antibiotic ~-42082 can be obtained in good yield. Antibiotic T-42082 may be isolated as crystals of its salts, e.g sodium, potassium, lithium, ammonium and other salts.
The following are the physical and chemical properties of Antibiotic T-42082 and of its sodium salt, which are obtainable according to the procedures set forth herein-after in Example 1.
~I) The free form of Antibiotic T-42082 (1) Color and appearance: Colorless needles (2) Melting point: 120-122C
(3) Elemental analysis: ~ound 0 C, 62.73; H, 9.23; 0,26.01;
~ C, 62.74; H, 9.36; 0, 27.03(%) (4) Molecular weight: 859 (by osmometry) (5) Ultraviolet absorption spectrum: No characteristic absorptions (6) Infrared absorption spectrum: The spectrum (KBr) is given in ~ig l. ~he dominant absorptions (wave-numbers) are as follows: 2940, 1700, 1462, 1383, 1085, 972 cm 1
(7) ~hin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10 %
aqueous H2S04. The Rf values are given in ~able 3.
. . ' " "
' , lO9il75 ~able ~
Solvent system Rf ___ Free form Sodium salt .
Ethyl acetate-benzene (1:1) 0 52 0.51 Ethyl acetate 0.73 0.73 Benzene-acetone (9:1) 0.18 0.18 Benzene-acetone (1:1) 0 88 0.89 Chloroform-methanol(19:1) 0.87 0.87 Chloroform-ethyl acetate~2:3) 0.62 0.62 Plate: silica gel plate (Kieselgel 60, ~lerck, Germany)
aqueous H2S04. The Rf values are given in ~able 3.
. . ' " "
' , lO9il75 ~able ~
Solvent system Rf ___ Free form Sodium salt .
Ethyl acetate-benzene (1:1) 0 52 0.51 Ethyl acetate 0.73 0.73 Benzene-acetone (9:1) 0.18 0.18 Benzene-acetone (1:1) 0 88 0.89 Chloroform-methanol(19:1) 0.87 0.87 Chloroform-ethyl acetate~2:3) 0.62 0.62 Plate: silica gel plate (Kieselgel 60, ~lerck, Germany)
(8) Color reactions: Refer to ~able 4.
~able 4 Color rea~ent ~ree form Sodium salt Sulfuric acid Positive(brown) Positive (brown) Aniline-phthalic acid Positive(indigo Positive(indigo blue) blue) Vanillin-sulfuric acid Positive (violet) Positive(violet) Molisch Negative Negative Dragendorff Positive(light Positive(light orange) orange) Barton ~egative Negative Molybdenic acid Negative Negative Benzidine Negative Negative Ninhydrin Negative ~egative Alkaline potassium ~egative ~egative permanganate
~able 4 Color rea~ent ~ree form Sodium salt Sulfuric acid Positive(brown) Positive (brown) Aniline-phthalic acid Positive(indigo Positive(indigo blue) blue) Vanillin-sulfuric acid Positive (violet) Positive(violet) Molisch Negative Negative Dragendorff Positive(light Positive(light orange) orange) Barton ~egative Negative Molybdenic acid Negative Negative Benzidine Negative Negative Ninhydrin Negative ~egative Alkaline potassium ~egative ~egative permanganate
(9) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride;
1091~7~
sparingly soluble in cyclohexane; and very hardly soluble in water and petroleum ether, ~ he sodium salt of Antibiotic ~-42082 (1) Color and appearance: Colorless needles (2) Melting point: 180-182C(with brownish discoloration) (3) Elemental analysis: Found ~ C, 62,05, H, 8.99;
Na, 2.08; ~ C, 62 27; H, 9.01; ~a, 2.79(%) (4) Moleculax weight: 871, 878(by osmometry) (5) Optical rotation (a)D5-4.5+0.5 (in chloroform, c=l.O) (6) Ultraviolet absorption spectrum: No characteristic absorption at and over 210m~.
(7) Infrared absorption spectrum: ~he spectrum(KBr) is given in Fig.2. ~he dominant absorptions (wave-numbers) are as follows: 2935, 1610, 1461, 1382, 1080, 972 cm 1 (8) NMR: ~he nuclear magnetic resonance spectrum is reproduced in Fig.3 which attests to the presence of 3 methoxy groups.
(9) Thin-layer chromatography: Refer to Table 3.
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride;
1091~7~
sparingly soluble in cyclohexane; and very hardly soluble in water and petroleum ether, ~ he sodium salt of Antibiotic ~-42082 (1) Color and appearance: Colorless needles (2) Melting point: 180-182C(with brownish discoloration) (3) Elemental analysis: Found ~ C, 62,05, H, 8.99;
Na, 2.08; ~ C, 62 27; H, 9.01; ~a, 2.79(%) (4) Moleculax weight: 871, 878(by osmometry) (5) Optical rotation (a)D5-4.5+0.5 (in chloroform, c=l.O) (6) Ultraviolet absorption spectrum: No characteristic absorption at and over 210m~.
(7) Infrared absorption spectrum: ~he spectrum(KBr) is given in Fig.2. ~he dominant absorptions (wave-numbers) are as follows: 2935, 1610, 1461, 1382, 1080, 972 cm 1 (8) NMR: ~he nuclear magnetic resonance spectrum is reproduced in Fig.3 which attests to the presence of 3 methoxy groups.
(9) Thin-layer chromatography: Refer to Table 3.
(10) Color reactions: Refer to Table 4.
(11) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetra-chloride; sparingly soluble in cyclohexane; and very hardly soluble in water and petroleum ether.
The biological characteristics of Antibiotic ~-42082 sodium salt are given in ~able 5.
.. . .
. .
' . . ~ .
~091~7S
Table Minimal inhibitory Assay organismconcentration(mcg/m~) . .
Bacillus subtilis PCI 219 0.31 Bacillus subtilis A~CC 6633 0~31 Bacillus cereus IF0 3466 < 0.1 Bacillus megaterium IF0 121080.2 - 0.31 Bacillus pumilus IF0 3813 0.2 Staphylococcus aureus 209P 0.2 Escherichia coli I~0 3044 > 100 Proteus vulgaris I~0 3045 ~ 100 Pseudomonas aeruginosa I~0 3080> 100 Mycobacterium sp. A~CC 607 10 Mycobacterium smegmatis I~0 3083 0.4 Candida albicans IF0 0583 10 Aspergillus niger I~0 4066 > 50 Penicillium chrysogenum I~0 4626 > 50 Piricularia oryzae P-18 10 The acute oral toxicity (LD50) of Antibiotic T-42082 sodium salt in mice is about 2000 mg/kg, with the ~D50 value in mice by the intraperitoneal route being 125-250 mg/kg.
~ he above physical and chemical properties suggest that Antibiotic ~-42082 is a polyether antibiotic compound containing three methoxy groups.
When the physical and chemical properties of Anti-biotic ~-42082 are compared with those of known polyether antibiotics, it is found that Antibiotic X-537A ~The Journal of American Chemical Society 7~, 5295 (1951)~, Salinomycin
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetra-chloride; sparingly soluble in cyclohexane; and very hardly soluble in water and petroleum ether.
The biological characteristics of Antibiotic ~-42082 sodium salt are given in ~able 5.
.. . .
. .
' . . ~ .
~091~7S
Table Minimal inhibitory Assay organismconcentration(mcg/m~) . .
Bacillus subtilis PCI 219 0.31 Bacillus subtilis A~CC 6633 0~31 Bacillus cereus IF0 3466 < 0.1 Bacillus megaterium IF0 121080.2 - 0.31 Bacillus pumilus IF0 3813 0.2 Staphylococcus aureus 209P 0.2 Escherichia coli I~0 3044 > 100 Proteus vulgaris I~0 3045 ~ 100 Pseudomonas aeruginosa I~0 3080> 100 Mycobacterium sp. A~CC 607 10 Mycobacterium smegmatis I~0 3083 0.4 Candida albicans IF0 0583 10 Aspergillus niger I~0 4066 > 50 Penicillium chrysogenum I~0 4626 > 50 Piricularia oryzae P-18 10 The acute oral toxicity (LD50) of Antibiotic T-42082 sodium salt in mice is about 2000 mg/kg, with the ~D50 value in mice by the intraperitoneal route being 125-250 mg/kg.
~ he above physical and chemical properties suggest that Antibiotic ~-42082 is a polyether antibiotic compound containing three methoxy groups.
When the physical and chemical properties of Anti-biotic ~-42082 are compared with those of known polyether antibiotics, it is found that Antibiotic X-537A ~The Journal of American Chemical Society 7~, 5295 (1951)~, Salinomycin
- 12 --` lV91175 (Japanese Patent Application Laid Open No.25392/1972), Antibiotic K-178(Zeitschrift f~r Allgemeine Mikrobiologie 4 236(1964)~, Allgemeine Mikrobiologie 4, 269(1964)~, Dianemycin (The Journal of Antibiotics 22, 161(1969)), Antibiotic A-130A (Japanese Patent Publication No.4558/1973), etc. have characteristic ultraviolet absorption spectra, whereas Antibiotic ~-42082 has no such characteristic absorption spectrum. Antibiotic X-206 ~Chemical Communica-tions (1971), 927~ has no methoxy group. Monensin ~he Journal of American Chemical Society 89, 5737(1967)), Nigericin ~Biochemical and Biophysical Research Communica-tions 33, 29(1968)~ and Grisorixin ~Chemical Communications, 1421(1970)~ contain one methoxy group each. Antibiotic A-28695 A(Japanese Patent Application Laid Open ~o.68795/
1973) and Antibiotic A-218(Japanese Patent Application Laid Open No.80793/1973) each have four methoxy groups, and Antibiotic A-2C4A~he Journal of American Chemical Society 95, 3399(1973)) and Antibiotic K-41 (Japanese Patent Appli-cation Laid Open No.14692/1974) contain five methoxy groups.
Therefore, the aforementioned antibiotics are clearly distinct from Antibiotic ~-42082 which has three methoxy groups. As an antibiotic having three methoxy groups like Antibiotic ~-42082, there may be mentioned Antibiotic A-28695 B(Japanese Patent Application Laid Open ~o.68795/I973) but in view of their differences in melting point, optical rotation, infrared absorption spectrum and other properties, these antibiotics are considered to be dissimilar substances.
Based on the above findings, A~tibiotic ~-42082 according -` 10~:~17S
to the present invention is considered to be a novel compound.
Figure 1 is an infrared absorption spectrum (KBr) of the free form of Antibiotic T-42082.
Figure 2 is an infrared absorption spectrum (KBr) of Antibiotic T-42082 sodium salt; and Figure 3 is a nuclear magnetic resonance spectrum of Antibiotic T-42082 sodium salt.
As described hereinbefore, Antibiotic T-42082 according to the present invention inhibits growth of gram-positive bacteria, acid-fast bacteria and certain fungi.
Therefore, the present antibiotic is of value in the treatment of infections with such microorganisms. For example, it can be utilized as a cream or ointment for topical application containing 0.3 to 1.0 % of the same in the prevention or treatment of fester of a wound caused by Staphylococcus aureus.
For the purpose of the prevention or treatment of intestine infection by Staphylococcus aureus, it can also be administered as tablets, capsules, etc. at a normal dose level of 20 to 200 mg/kg daily or as injections at a daily dose of 1 to 20 mg/kg per adult human. Since the present antibiotic inhibits growth of Piricularia oryzae, it may also be employed as an agricultural aid.
In addition, Antibiotic T-42082 is useful for the pre-vention and treatment of coccidiosis.
Coccidiosis is an infectious disease caused by para-sitic protozoa in domesticated fowls and animals, manifestations of which include diarrhea and nutritional disturbances.
~09~1`75 htrl poultry, ducks, turkeys, quails, rabbits, goats, sheep and cattle, for instance, often succumb to the disease and sustain serious damages, Various drugs have been employed for the prevention or treatment of coccidiosis, but they have such drawbacks as inadequate potency, serious side effects or/and liability for the emergence of drug-resistance protozoas.
Antibiotic ~-42082, of this invention, has been found to display positive effects in the prevention and treatment of coccidiosis, overcoming the drawbacks of the anticoccidial drugs thus far available.
~ he anticoccidial drug is produced by processing Anti-biotic T-42082, in the absence or presence of a solid or liquid diluent, into powders, dusts granules, tablets, liquids, capsules and so on, or by adding to feed, drinking water or the like with or without previous dispersion in a diluent. The said diluent may be any diluent which per se is physiologically harmless, although it is preferably a substance that, by itself, may be a feedstuff or a feed ingredient.
As a solid diluent, there may be mentioned barley flour, wheat flour, rye flour, corn flour, soybean flour, soybean cake, rapeseed cake, wheat bran, rice bran, extracted rice bran, sweet potato flour, potato flour, soybean curd residue, starch, lactose, sucrose, glucose, fructose, yeast, spent yeast, fish meal, talc, acid clay, clay and so forth.
As examples of said liquid diluent, there may be mentioned water, physiological saline, organic solvents which are lU~il75 physiologically harmless and so forth In addition to said diluent, there may also be com-pounded appropriate auxiliary agents such as an emulsifier, dispersing agent, suspending agent, wetting agent, thickener, gelling agent, solubilizer and so forth in appropriate proportions The resultant composition may be further supplemented with a preservative, fungicide, antibiotic, enzyme preparation, lactobacillus preparation and so forth, Moreover, pyrimethamine, sulfa drugs, vitamin preparations, etc may be further incorporated.
The proper dosage of the anticoccidial drug varies with the species and breed of domesticated fowl or animal, its age, the route of dosi~g, symptoms and other factors ~or the prevention of coccidiosis in poultry, for example, the present drug is desirably given in such amounts that the poultry will receive about 4.0 to 12 5 mg/kg/day of Antibiotic ~-42082. For the treatment of the same disease, the poultry preferably takes about 10 to 30 mg of the anti-biotic per kg body weight daily, ~o achieve this dose level, the drug may be added to the feed in such a propor-tion that the concentration of Antibiotic T-42082 will be somewhere between about 50 and 300 ppm.
The toxicity of Antibiotic ~-42082 in animals is low.
For example, the acute toxicity of Antibiotic ~-42082 sodium salt in mice (ID50) is about 2000 mg/kg by the oral route, or 125 to 250 mg/kg by the intraperitoneal route. ~he oral toxicity (LD50) in poultry is 1100 mg (423-2860 mg)/kg.
An outstanding characteristic of the anticoccidial 10~11`7~
drug according to this invention is that it is by far safer than any known anticoccidial drug of the polyether type.
The experimental data shown are in the Experimental Description which follows Example 1 are illustrative of the superior effectiveness of the anticoccidial drug.
Example 1 A culture medium prepared by adding 0.5 % of precip-itated calcium carbonate to an aqueous solution (pH 7.0) containing 2 % of glucose, 3 % of soluble starchJ 1 % of raw soybean flour, 1 % of corn steep liquor, 0.5 % of peptone and 0.3 % of sodium chloride was inoculated with Strepto myces hygroscopicus T-42082 (FERM P 2691, IFO 13609, ATCC
31080). The inoculated medium was incubated under shaking at 28C for 48 hours. One liter of the resultant culture fluid was transferred to a 100-liter tank containing 30 -~
liters of a culture medium prepared by adding 0.5 % of precipitated calcium carbonate to an aqueous solution ~pH 7.0~ containing 5 % of dextrin, 3 % of raw soybean flour and 0.1 % of peptone and aerobic stirred culture was carried out at 28C with sparging at the rate of 30 liters/min. for 140 hours. -To the resultant broth was added an equal volume of acetone and after 30 minutes' stirring, the cells were separated from the broth. The cells were resuspended in acetone, the volume of which was equal to that of the broth, and following 30 minutes stirring, the filtrate was separated and pooled with the filtrate previously obtained. The lO9i~:`75 filtrate was concentrated under reduced pressure until the acetone was distilled off and, then, extracted with ethyl acetate, followed by drying over anhydrous sodium sulfate.
~he ethyl acetate solution was then passed through a column of 400 m~ activated carbon, which was washed with ethyl acetate. ~he active fractions were pooled and the solvent was distilled off. The oily residue was dissolved in benzene and the solution was passed through a column of 500 m~ silica gel. ~lution was carried out with benzene, benzene-ethyl acetate (9:1), benzene-ethyl acetate (8:2), benzene-ethyl acetate (1:1) and ethyl acetate in the order mentioned. ~he active fractions were pooled and the solvent was distilled off, whereupon 12.5 g crude powders of Anti-biotic ~-42082 were obtained. ~he powders were dissolved in 80 /0 acetone water and, after being adjusted to pH 9.0 with lN-sodium hydroxide, the solution was concentrated under reduced pressure to remove the acetone. By the above -procedure were obtained crystals of Antibiotic T-42082 sodium salt. ~he crystals were collected and recrystallized from acetone water. By this procedure were obtained 4 g colorless needles of Antibiotic ~-42082 sodium salt. ~he recrystallization mother fluid was adjusted to pH 5.0 with lN-hydrochloric acid and concentrated under reduced pressure to remove the acetone. The resultant crude crystals were collected and recrystallized from acetone water. ~he pro-cedure described above provided 2.5 g colorless needles of free Antibiotic T-42082.
1(~91175 EXPERIMEN~AL
The basal diet composition used in the ~ollowing experiment is a nutritionally balanced feed and it i5 known that chicks show quite good growth on it. By way of example, the chicks grown on this diet in a breeding battery at room temperature, 26+1C. (40C. within the battery) on an ad libitum basis for both feed and drinking water will have body weights in excess of 90 grams on the 9th day after hatching. ~he test of anticoccidial effect was performed with 3 or 5 healthy chicks having even body weights in a stainless steel cage at 26+1C, under 24 hour lighting, with free access to feed and water and under conditions precluding microbial infections.
The term "healthy control!! as used hereinafter means "uninfected, untreated control". All parts are by weight.
EXPERIMENTA~ 1 ~ rom the caecal contents of a chick, fresh oocysts were collected on the 8th day after infection with Eimeria tenella. After sporulation, the oocysts were artificially threshed by the method of Doran and Vettering (Journal of Protozoology 14, 657-662(1967)) to obtain the sporozoides.
On the other hand, using Eagle ~DEM(Gibco, U.S.A.), BK cells were allowed to multiply well on a glass slip sealed in a Rayton tube. Then, 3x104/0.3~ of the sporozoides previously prepared, 0.2 m~ of a test sample (Antibiotic ~-42082 as dissolved in methanol to a concentration of 1 mg/m~ and, then, diluted 10-fold with the medium to 10 5 mcg/m~) and 1 5 m~ of medium were injected. Cultivation was carried 109117~
out at 41C for 3 da~s, ~he sporozoides which had entered the BK cells and the grown and multiplied schizonts were stained and counted under a microscope, The results are shown in Table No.l, _able No.l Level of addition of Grown schizonts sample, mcg/m~(-log) 1 2-O x x 4 ++ _ +++ ++ _ +++
+++ +++
Inoculated control +++ +++
Untreated control Scoring scheme:
he exfoliation and denaturation of cells (cytotoxicity):
x The exfoliation of cells from the glass surface as well as a denaturation of cells are clearly in evidence.
2. Inhibition of growth of schizonts: - - +++
- : when the specimen (24x9mm) was examined under a microscops ~x400(40xlO)), no grown schizont at all in two rows.
+ : About 5 schizonts in two rows under the microscope.
+ : About one schizonts per field, ++ : About 5 schizonts per field.
+++ : About 10 schizonts per field.
i~)9il~75 ''.
EXPERIMEN~A~ 2 ~est materials and testing procedure:
(1) Test chicks : White ~eghorn, male, 10 days old at the start of the experiment.
(2) Coccidium : Eimeria tenel1a; The inoculum size per bird: 5xlO sporulated oocysts/0.2 -0.5 m~.
~ est compound: Antibiotic ~-42082 (4) Levels of addition of the test drug: ~he test compound was added to portions of a compound mash for newly hatched chicks, ~eed EP-l-A(composition hereinafter in Example 1), which contained no anticoccidial agent, at the varying levels of 30, 60, 90 and 120 ppm.
(5) ~esting procedure: The test chicks were bred in quarters protected against coccidial infections and after it had been confirmed that they had no other disease, either, but were healthy, each chick was weighed. ~hey were classed into groups of three birds in such a manner that the body weight distributions of the groups would be substantially identical. ~wo of the groups were used as "infected control" and "healthy control", respectively.
After the grouping, each test group of chicks were placed on the diet containing the test drug, while the "infected control" and "healthy control" birds were kept on the drugfree diet.
After 24 hours, excepting the birds in the healthy control group, all the birds were orally inoculated with , . .
.
, 10~117S
imeria tenella at a rate of 5x104 sporulated oocysts per bird.
(6) Scoring scheme: ~ill the end of the test period, each chick was weighed every morning before feeding and, at the same time, the drops of blood (h~emorrhage) in its droppings were counted, ~he chicks were also investi-gated for deaths. Eight days following the oocyst inoculation, all the test chicks were autopsied and the apparent pathological changes of the caeca and the caecal contents were microscopically examined to evaluate the effectiveness of the drug.
(7) ~otes to the description in the table.
(a) Relative weight gain =
Percent weight gain for test group x 100 Percent weight gain for healthy control group (b) Pathological change of the caecum:
~ he ~est Procedures for Coccidiosis in Poultry (See Kiyoshi ~sunoda and Toshio Ishii. ~he Research Society of Poultry Diseases (1971), p.20) ~he results of post-mortem examinations were scored according to the following scheme.
he caecum is completely normal.
(+) : ~he caecum retains its normal shape. The contents are slightly fluid, with a tinge of yellow, The mucous membrane of the caecum is slightly swollen and whitish.
10911~75 (+~he caecum is substantially normal in shape. The ; entire mucous membrane is swollen No haemorrhage is noted in the contents The viscous fluid shows a yellowish discoloration. Within the mucous membrane, a few white dot-like necrotic lesions and blood patches are in evidence.
(+++) : ~he atrophy and deformation of the caecum are obvious. The caecum extends slightly longer than the rectum. ~he contents include nothing normal and, in many cases, the caecum is full of blood clots or grayish white cheesy denaturation products ~he caecal wall is considerably thickened and brittle, sometimes left with dot-like patches of blood. The lesion extends to the base of the caecum but not to the rectum.
(++++): ~he atrophy and deformation of the caecum are pro-nounced. Generally the caecum looks like a sausage, as long as the rectum or shorter. The lesion extends to about one-third or one-quater o~ the rectum. Other findings are similar to those described in (3).
In the above evaluation, where one side of the caecum had been affected more seriously than the other side, the findings on the more seriously affected side were recorded.
- .: - ' ' ' ' , ' ~
lO9il75 . .. . _ __ .
~rl X ~ C~ ~ ~ ~ ~ O ~ C~ ~
0,bD~ ~; O O' O' ~ ~:' .D O; ~D ~
0 _ _ __ _ _ __ __ _ .
o.~ + _ K~ _ ___ __ _ _ h h + _ _ _ _ _ o ~0~ + ~ ~ ~ ~ ~ ~q h ~ ~ ~ _ . _ -- h ¢ ~ ~ o ~ ~ O
O ~ Ll\ \ ~ \ ~ ~ ~ ~ r(~ ~ h 1~1 _ _ . _ _ ~ .,_1 h O o o O O ~1 O o O O
C~ __ _ ~ , o.~ c~ O o o o ~ o o o o o a~ h 0 ~0 ~
~J~o ~ ~) O O O O O O O O O O 0 o o _ _ -- s~
~i ~ ~ O O O O O O ~ O O O a) ~
a)~:i . _ _ _ _ h ~ h~ O O O O O O O O O O ~ 0 ,D0~ ~) O O O O ~ cs~ r-l' O O O I~
~ O h ~u~ O O O O ~ O ~ O O O 'hl ~
O h ~ 0 ._ ~1 _ bD
~ .q, ~ O O O O O ~D N~ O O O ~ ~
~' ~ ,~
~ ~U ~ O
/ ~ /~ ~ ~0 ~0 ~o ~ ~ ~ p o ,, ~/~ H ~, h h h h h h EXPERIMEN~A~ ~
Heretofore, clopidol, amplolium, decoquinate, etc, have been employed as anticoccidial drugs and, more recently, lobenidine has been introduced However, continued administ-ration of the same anticoccidial drug induced the emergence of drug-resistant strains. ~he following experiment was carried out with a wild strain resistant to the conventional anticoccidial drugs.
~ he oocysts, predominantly of Eimeria tenella, obtained from a wild strain were used to inoculate chicks at a rate of lOx105 sporulated oocysts per bird. Other test materials are the same as those used in Experimental 2. ~he battery test procedure was repeated.
.
iU9~5 l ~4 O .~ J. J. it 01 ~ u\o ,0 ,W"~ ~ co ~; ~ D o C~ a) ;~; r;
~ D .~
.~ ___ .
0 l O O O O ~ O O
'~4 _ O O_ O O ~ 00 ~' ~'o + .~ .~ O O O O O O O
0 ~ J O r-J O O O O O O O
~1 ~ + ___ a) 0 ~ +
~ ~, ++ O .~ ~ N~ O O O ~ ~U
\ 4 h-. _ _ ~ 0 h ~ ~ ~ ~ ~ ~ K~ ~
~'h \ N~ ~ ~ K~ N~ N~ ~ ~ ~i i 4~ U~ o~ O O O O O O O O O
o ~ C' O O O O O O O O O
o' o ~ ~ O O O O O O O O O
~i ~; ~ U\ O O O O O O O O ~
.~ ~ O rt~Lr\
C' O ,~ J' ~ O O O ~ ~-E~ ~ .~ . . .
o." ~9 o ~ o o h ~g O O ,~ O O O ,~
~ L~ O .~ O N~ O O O O' O' O h ~ H
~;~ J O O O' ~`J' O O O
_ ._ _ . .
~ ^ U~ O O
~o,,~ ~D ~ O O O O O
c~ h _ .~ ~ .~ ~.~ ~.~
0 " oo .~ C) ~0 ~ h h ~ O ~ ~ ~ c) ~
~ ~ ~ ,~io ,a~O
~ ._ .H
-~9~1'75 The same test materials and testing procedure as those described in Experimental 2 were employed.
Notes to the description in the table:
(a) Average lesion index (degree) ~ he testing procedure used for coccidiosis in poultry was in conformity with the method of J. Johnson and W. M.
Reid (Experimental Parasitology 28, 33(1970)).
The results of post-mortem examinations performed on the 6th to 7th day following the infection were recorded according to the following scheme, - : The small intestine was completely normal (no patho-- logical change at all).
+ : ~ed small dots of blood appeared on the serous membrane in the middle of the small intestine.
Though the small intestine shows neither swelling nor thickening, it sometimes contains an orange-colored viscous fluid.
++ : A number of red small patches of blood appear on the mucous membrane of the small intestine. It is also full of orange-colored viscous fluid. The swelling of the small intestine is slight, the intestinal wall being slightly thickened.
+++ : The intestinal wall is swollen and thickened, The surface of the mucous membrane become loose and the intestine is full of blood clots and viscous fluids ++++ : The small intestine is swollen all over and contains a great many blood clots. Moreover, owing to the lO9il~5 digestion of red blood cells, the small intestine assumes a peculiar shade and gives off a rank odor, The intestinal wall is exceedingly swollen. Dead -chicks are included in this category.
(b) OPG: Oocysts per gram of feces ~ he mark " - " in the OPG column means that the value is less than log 3 (not measurable).
Table No.4 Infective Test material: Relative Average OPG
oocysts Antibiotic weight patho- (log) 4T-42082 gain logical - 1 --- ~
(lOxlO / index 4 5 1 6 ( bird)(ppm) (degree) _ _ days Eimeria125 108.3 _ _ _ _ /
acervulina 62.5 98.4 ++ _ 5. 5.4 /
_ . .
O 90.2 ++++6.6 6. 6.4 /
.... _ , . , Eimeria125 97.4 + / / 4.8 4.2 maxima62.5 94.9 + ~ ~ 5. 5.5 0__ 78.8 ++ / ~ 5.2 EXPERIMENTAL ~
~he test materials ? testing procedure, and scheme of evaluation are similar to those used in Experimental 2 and 4, 10~3~175 Table No,5 Infectlve Test material: Relative Average I OPG
oocystsAntibiotic weight pathologica _ (lo 3) (lOxlO4/bird) ~-42082 gain i7dex 4 5 lays . . .,.
Eimeria 120 100.3 _ _ _ _ acervulina90 100.4 _ _ _ _ _ 60 _ 92.6 ++__ 5.1 6.0 4,4 _ _ 0 90.2 ++++ _ 6.5 6.9 6.6 Example : ~en parts of Antibiotic ~-42082, previously milled to sizes not exceeding 149 ~, was mixed with 90 parts of dry extracted soybean flour to prepare a 10 k powder of Anti-biotic ~-42082. l.Ox103 parts of this powder was compounded with l.Ox106 parts of Feed KP-l-A (composition hereinafter) (for young broilers) to obtain an avian anticoccidial diet for broilers ~he concentration of Antibiotic T-42082:
100 ppm).
Formula of KP-l-A
(for young broilers) . .
Ingredient Compounding ratio, weight %
Yellow corn 55.
Wheat bran 5.
Soybean cake 18.0 Fish meal 8.0 Fish soluble 3.0 Alfalfa meal 3.
Tallow complex 5.7 Calcium carbonate 0.9 ~ribasic calcium phosphate o.7 Sodium chloride 0.25 A-Food E beads * o.o5 B-Feed S * 0,1 Neo-Mine feed C~ o.o5 Vitamin B12 - T * o.o5 Mixture of active agent and extracted soybean flour 0.2 .. ~ --- _ .
otal 100.0 : ~rade names (Distributor: ~akeda Chemical Industries, ~td.(Japan)) ExamPle~_ ?
25 parts of Antibiotic ~-42082, previously milled to sizes not exceeding 149 ~ in diameter, was blended well with 75 parts of dry extracted soybean flour to prepare a 10911 ~5 powder containing 25 % of Antibiotic ~-42082. 4.0xlO
parts of this powder was compounded with l.Ox106 parts of ~eed KP-l-A to obtain an anticoccidial diet for poultry ~he concentration of Antibiotic ~-42082: 100 ppm).
Example 0.5x103 parts of the 10 % powder obtained in Example 1 was compounded with l.Ox106 parts of Feed KP-l-A to prepare an anticoccidial diet for poultry ~he concentration of Antibiotic ~-42082: 50 ppm) Example 4 10 parts of Antibiotic T-42082, previously milled to sizes not exceeding 149 ~, was blended with 2 parts of hydroxypropyl-cellulose and 88 parts of lactose. ~he mixture was kneaded with 20 parts of water and granulated in a beater-granulator. ~he wet granules thus obtained were dried at 35C overnight, whereby 10 k granules 150 to 1,000 in diameter are obtained. l.Ox103 parts of this granular product was compounded with l.Ox106 parts of ~eed KP-l-B
(composition hereinafter)(for grown broilers) to prepare an anticoccidial diet for broilers ~he concentration of Antibiotic ~-42082: 100 ppm~.
, lO~il`7S
Formula of KP-l-B
(for grown broilers) . .
Ingredient _ --' Y
Yellow corn 58,0 Wheat bran 5.o Soybean cake 14,0 Fish meal 6,8 Fish soluble 3.o Alfalfa meal 3.o Tallow complex 8.0 Calcium carbonate 0,9 Tribasic calcium phosphate 0,68 Sodium chloride 0.25 A-Feed E beads o~o5 B-Feed S 0.1 Mixture of active agent 0,1 with lactose, etc, 0,12 Total 100.0 Example ~
25 parts of Antibiotic 'r-42082, previously milled to sizes not exceeding 149 11, were blended with 2 parts of hydroxypropyl-cellulose and 88 parts of lactose. The mixture was kneaded with 20 parts of water and granulated in a beater-granulator. The resultant wet granules were dried at 55C overnight to obtain 25 % dry granules 150 to 1,000 11 in diameter. 0,4x103 parts of the granules were .'" --' -. ' ' ' ' ' " ' , '' :- ' .
" iO9~1`7S
compounded with l,Ox106 parts of Feed KP-l-B to prepare an anticoccidial diet for poultry (~he concentration of Anti-biotic ~-42082 : 100 ppm).
Example 6 2.0x103 parts of the 10 % granules obtained in Example 4 were compounded with 1,Ox106 parts of ~eed KP-l-B to prepare an anticoccidial diet for poultry (~he concentration of Antibiotic ~-42082: 200 ppm), Example 7 1.2x103 parts of the 25 % granules obtained in Example 5 were compounded with l.Ox106 parts of Feed KP-l-B to prepare an anticoccidial diet for poultry (The concentration of Antibiotic ~-42082: 300 ppm).
By a procedure similar to that described in ~xample 4, the 10 /0 granules obtained in Example 4 were compounded with feed in various ratios to prepare diets containing Anti-biotic T-42082 in various concentrations.
Amount of the Amount of Concentration of 10 % granules ~eed KP-l-B Antibiotic obtained in ~-42082 Ex 4 ~parts) (parts) (ppm) 0.6 x 103 1.0 x 1o6 60 0.9 x 103 1~0 x 106 go 1.2 ~ 103 1.0 x 1o6 120 1.8 x 103 1.0 x 1o6 180 2.4 x 103 1.0 x 1o6 240 .
1973) and Antibiotic A-218(Japanese Patent Application Laid Open No.80793/1973) each have four methoxy groups, and Antibiotic A-2C4A~he Journal of American Chemical Society 95, 3399(1973)) and Antibiotic K-41 (Japanese Patent Appli-cation Laid Open No.14692/1974) contain five methoxy groups.
Therefore, the aforementioned antibiotics are clearly distinct from Antibiotic ~-42082 which has three methoxy groups. As an antibiotic having three methoxy groups like Antibiotic ~-42082, there may be mentioned Antibiotic A-28695 B(Japanese Patent Application Laid Open ~o.68795/I973) but in view of their differences in melting point, optical rotation, infrared absorption spectrum and other properties, these antibiotics are considered to be dissimilar substances.
Based on the above findings, A~tibiotic ~-42082 according -` 10~:~17S
to the present invention is considered to be a novel compound.
Figure 1 is an infrared absorption spectrum (KBr) of the free form of Antibiotic T-42082.
Figure 2 is an infrared absorption spectrum (KBr) of Antibiotic T-42082 sodium salt; and Figure 3 is a nuclear magnetic resonance spectrum of Antibiotic T-42082 sodium salt.
As described hereinbefore, Antibiotic T-42082 according to the present invention inhibits growth of gram-positive bacteria, acid-fast bacteria and certain fungi.
Therefore, the present antibiotic is of value in the treatment of infections with such microorganisms. For example, it can be utilized as a cream or ointment for topical application containing 0.3 to 1.0 % of the same in the prevention or treatment of fester of a wound caused by Staphylococcus aureus.
For the purpose of the prevention or treatment of intestine infection by Staphylococcus aureus, it can also be administered as tablets, capsules, etc. at a normal dose level of 20 to 200 mg/kg daily or as injections at a daily dose of 1 to 20 mg/kg per adult human. Since the present antibiotic inhibits growth of Piricularia oryzae, it may also be employed as an agricultural aid.
In addition, Antibiotic T-42082 is useful for the pre-vention and treatment of coccidiosis.
Coccidiosis is an infectious disease caused by para-sitic protozoa in domesticated fowls and animals, manifestations of which include diarrhea and nutritional disturbances.
~09~1`75 htrl poultry, ducks, turkeys, quails, rabbits, goats, sheep and cattle, for instance, often succumb to the disease and sustain serious damages, Various drugs have been employed for the prevention or treatment of coccidiosis, but they have such drawbacks as inadequate potency, serious side effects or/and liability for the emergence of drug-resistance protozoas.
Antibiotic ~-42082, of this invention, has been found to display positive effects in the prevention and treatment of coccidiosis, overcoming the drawbacks of the anticoccidial drugs thus far available.
~ he anticoccidial drug is produced by processing Anti-biotic T-42082, in the absence or presence of a solid or liquid diluent, into powders, dusts granules, tablets, liquids, capsules and so on, or by adding to feed, drinking water or the like with or without previous dispersion in a diluent. The said diluent may be any diluent which per se is physiologically harmless, although it is preferably a substance that, by itself, may be a feedstuff or a feed ingredient.
As a solid diluent, there may be mentioned barley flour, wheat flour, rye flour, corn flour, soybean flour, soybean cake, rapeseed cake, wheat bran, rice bran, extracted rice bran, sweet potato flour, potato flour, soybean curd residue, starch, lactose, sucrose, glucose, fructose, yeast, spent yeast, fish meal, talc, acid clay, clay and so forth.
As examples of said liquid diluent, there may be mentioned water, physiological saline, organic solvents which are lU~il75 physiologically harmless and so forth In addition to said diluent, there may also be com-pounded appropriate auxiliary agents such as an emulsifier, dispersing agent, suspending agent, wetting agent, thickener, gelling agent, solubilizer and so forth in appropriate proportions The resultant composition may be further supplemented with a preservative, fungicide, antibiotic, enzyme preparation, lactobacillus preparation and so forth, Moreover, pyrimethamine, sulfa drugs, vitamin preparations, etc may be further incorporated.
The proper dosage of the anticoccidial drug varies with the species and breed of domesticated fowl or animal, its age, the route of dosi~g, symptoms and other factors ~or the prevention of coccidiosis in poultry, for example, the present drug is desirably given in such amounts that the poultry will receive about 4.0 to 12 5 mg/kg/day of Antibiotic ~-42082. For the treatment of the same disease, the poultry preferably takes about 10 to 30 mg of the anti-biotic per kg body weight daily, ~o achieve this dose level, the drug may be added to the feed in such a propor-tion that the concentration of Antibiotic T-42082 will be somewhere between about 50 and 300 ppm.
The toxicity of Antibiotic ~-42082 in animals is low.
For example, the acute toxicity of Antibiotic ~-42082 sodium salt in mice (ID50) is about 2000 mg/kg by the oral route, or 125 to 250 mg/kg by the intraperitoneal route. ~he oral toxicity (LD50) in poultry is 1100 mg (423-2860 mg)/kg.
An outstanding characteristic of the anticoccidial 10~11`7~
drug according to this invention is that it is by far safer than any known anticoccidial drug of the polyether type.
The experimental data shown are in the Experimental Description which follows Example 1 are illustrative of the superior effectiveness of the anticoccidial drug.
Example 1 A culture medium prepared by adding 0.5 % of precip-itated calcium carbonate to an aqueous solution (pH 7.0) containing 2 % of glucose, 3 % of soluble starchJ 1 % of raw soybean flour, 1 % of corn steep liquor, 0.5 % of peptone and 0.3 % of sodium chloride was inoculated with Strepto myces hygroscopicus T-42082 (FERM P 2691, IFO 13609, ATCC
31080). The inoculated medium was incubated under shaking at 28C for 48 hours. One liter of the resultant culture fluid was transferred to a 100-liter tank containing 30 -~
liters of a culture medium prepared by adding 0.5 % of precipitated calcium carbonate to an aqueous solution ~pH 7.0~ containing 5 % of dextrin, 3 % of raw soybean flour and 0.1 % of peptone and aerobic stirred culture was carried out at 28C with sparging at the rate of 30 liters/min. for 140 hours. -To the resultant broth was added an equal volume of acetone and after 30 minutes' stirring, the cells were separated from the broth. The cells were resuspended in acetone, the volume of which was equal to that of the broth, and following 30 minutes stirring, the filtrate was separated and pooled with the filtrate previously obtained. The lO9i~:`75 filtrate was concentrated under reduced pressure until the acetone was distilled off and, then, extracted with ethyl acetate, followed by drying over anhydrous sodium sulfate.
~he ethyl acetate solution was then passed through a column of 400 m~ activated carbon, which was washed with ethyl acetate. ~he active fractions were pooled and the solvent was distilled off. The oily residue was dissolved in benzene and the solution was passed through a column of 500 m~ silica gel. ~lution was carried out with benzene, benzene-ethyl acetate (9:1), benzene-ethyl acetate (8:2), benzene-ethyl acetate (1:1) and ethyl acetate in the order mentioned. ~he active fractions were pooled and the solvent was distilled off, whereupon 12.5 g crude powders of Anti-biotic ~-42082 were obtained. ~he powders were dissolved in 80 /0 acetone water and, after being adjusted to pH 9.0 with lN-sodium hydroxide, the solution was concentrated under reduced pressure to remove the acetone. By the above -procedure were obtained crystals of Antibiotic T-42082 sodium salt. ~he crystals were collected and recrystallized from acetone water. By this procedure were obtained 4 g colorless needles of Antibiotic ~-42082 sodium salt. ~he recrystallization mother fluid was adjusted to pH 5.0 with lN-hydrochloric acid and concentrated under reduced pressure to remove the acetone. The resultant crude crystals were collected and recrystallized from acetone water. ~he pro-cedure described above provided 2.5 g colorless needles of free Antibiotic T-42082.
1(~91175 EXPERIMEN~AL
The basal diet composition used in the ~ollowing experiment is a nutritionally balanced feed and it i5 known that chicks show quite good growth on it. By way of example, the chicks grown on this diet in a breeding battery at room temperature, 26+1C. (40C. within the battery) on an ad libitum basis for both feed and drinking water will have body weights in excess of 90 grams on the 9th day after hatching. ~he test of anticoccidial effect was performed with 3 or 5 healthy chicks having even body weights in a stainless steel cage at 26+1C, under 24 hour lighting, with free access to feed and water and under conditions precluding microbial infections.
The term "healthy control!! as used hereinafter means "uninfected, untreated control". All parts are by weight.
EXPERIMENTA~ 1 ~ rom the caecal contents of a chick, fresh oocysts were collected on the 8th day after infection with Eimeria tenella. After sporulation, the oocysts were artificially threshed by the method of Doran and Vettering (Journal of Protozoology 14, 657-662(1967)) to obtain the sporozoides.
On the other hand, using Eagle ~DEM(Gibco, U.S.A.), BK cells were allowed to multiply well on a glass slip sealed in a Rayton tube. Then, 3x104/0.3~ of the sporozoides previously prepared, 0.2 m~ of a test sample (Antibiotic ~-42082 as dissolved in methanol to a concentration of 1 mg/m~ and, then, diluted 10-fold with the medium to 10 5 mcg/m~) and 1 5 m~ of medium were injected. Cultivation was carried 109117~
out at 41C for 3 da~s, ~he sporozoides which had entered the BK cells and the grown and multiplied schizonts were stained and counted under a microscope, The results are shown in Table No.l, _able No.l Level of addition of Grown schizonts sample, mcg/m~(-log) 1 2-O x x 4 ++ _ +++ ++ _ +++
+++ +++
Inoculated control +++ +++
Untreated control Scoring scheme:
he exfoliation and denaturation of cells (cytotoxicity):
x The exfoliation of cells from the glass surface as well as a denaturation of cells are clearly in evidence.
2. Inhibition of growth of schizonts: - - +++
- : when the specimen (24x9mm) was examined under a microscops ~x400(40xlO)), no grown schizont at all in two rows.
+ : About 5 schizonts in two rows under the microscope.
+ : About one schizonts per field, ++ : About 5 schizonts per field.
+++ : About 10 schizonts per field.
i~)9il~75 ''.
EXPERIMEN~A~ 2 ~est materials and testing procedure:
(1) Test chicks : White ~eghorn, male, 10 days old at the start of the experiment.
(2) Coccidium : Eimeria tenel1a; The inoculum size per bird: 5xlO sporulated oocysts/0.2 -0.5 m~.
~ est compound: Antibiotic ~-42082 (4) Levels of addition of the test drug: ~he test compound was added to portions of a compound mash for newly hatched chicks, ~eed EP-l-A(composition hereinafter in Example 1), which contained no anticoccidial agent, at the varying levels of 30, 60, 90 and 120 ppm.
(5) ~esting procedure: The test chicks were bred in quarters protected against coccidial infections and after it had been confirmed that they had no other disease, either, but were healthy, each chick was weighed. ~hey were classed into groups of three birds in such a manner that the body weight distributions of the groups would be substantially identical. ~wo of the groups were used as "infected control" and "healthy control", respectively.
After the grouping, each test group of chicks were placed on the diet containing the test drug, while the "infected control" and "healthy control" birds were kept on the drugfree diet.
After 24 hours, excepting the birds in the healthy control group, all the birds were orally inoculated with , . .
.
, 10~117S
imeria tenella at a rate of 5x104 sporulated oocysts per bird.
(6) Scoring scheme: ~ill the end of the test period, each chick was weighed every morning before feeding and, at the same time, the drops of blood (h~emorrhage) in its droppings were counted, ~he chicks were also investi-gated for deaths. Eight days following the oocyst inoculation, all the test chicks were autopsied and the apparent pathological changes of the caeca and the caecal contents were microscopically examined to evaluate the effectiveness of the drug.
(7) ~otes to the description in the table.
(a) Relative weight gain =
Percent weight gain for test group x 100 Percent weight gain for healthy control group (b) Pathological change of the caecum:
~ he ~est Procedures for Coccidiosis in Poultry (See Kiyoshi ~sunoda and Toshio Ishii. ~he Research Society of Poultry Diseases (1971), p.20) ~he results of post-mortem examinations were scored according to the following scheme.
he caecum is completely normal.
(+) : ~he caecum retains its normal shape. The contents are slightly fluid, with a tinge of yellow, The mucous membrane of the caecum is slightly swollen and whitish.
10911~75 (+~he caecum is substantially normal in shape. The ; entire mucous membrane is swollen No haemorrhage is noted in the contents The viscous fluid shows a yellowish discoloration. Within the mucous membrane, a few white dot-like necrotic lesions and blood patches are in evidence.
(+++) : ~he atrophy and deformation of the caecum are obvious. The caecum extends slightly longer than the rectum. ~he contents include nothing normal and, in many cases, the caecum is full of blood clots or grayish white cheesy denaturation products ~he caecal wall is considerably thickened and brittle, sometimes left with dot-like patches of blood. The lesion extends to the base of the caecum but not to the rectum.
(++++): ~he atrophy and deformation of the caecum are pro-nounced. Generally the caecum looks like a sausage, as long as the rectum or shorter. The lesion extends to about one-third or one-quater o~ the rectum. Other findings are similar to those described in (3).
In the above evaluation, where one side of the caecum had been affected more seriously than the other side, the findings on the more seriously affected side were recorded.
- .: - ' ' ' ' , ' ~
lO9il75 . .. . _ __ .
~rl X ~ C~ ~ ~ ~ ~ O ~ C~ ~
0,bD~ ~; O O' O' ~ ~:' .D O; ~D ~
0 _ _ __ _ _ __ __ _ .
o.~ + _ K~ _ ___ __ _ _ h h + _ _ _ _ _ o ~0~ + ~ ~ ~ ~ ~ ~q h ~ ~ ~ _ . _ -- h ¢ ~ ~ o ~ ~ O
O ~ Ll\ \ ~ \ ~ ~ ~ ~ r(~ ~ h 1~1 _ _ . _ _ ~ .,_1 h O o o O O ~1 O o O O
C~ __ _ ~ , o.~ c~ O o o o ~ o o o o o a~ h 0 ~0 ~
~J~o ~ ~) O O O O O O O O O O 0 o o _ _ -- s~
~i ~ ~ O O O O O O ~ O O O a) ~
a)~:i . _ _ _ _ h ~ h~ O O O O O O O O O O ~ 0 ,D0~ ~) O O O O ~ cs~ r-l' O O O I~
~ O h ~u~ O O O O ~ O ~ O O O 'hl ~
O h ~ 0 ._ ~1 _ bD
~ .q, ~ O O O O O ~D N~ O O O ~ ~
~' ~ ,~
~ ~U ~ O
/ ~ /~ ~ ~0 ~0 ~o ~ ~ ~ p o ,, ~/~ H ~, h h h h h h EXPERIMEN~A~ ~
Heretofore, clopidol, amplolium, decoquinate, etc, have been employed as anticoccidial drugs and, more recently, lobenidine has been introduced However, continued administ-ration of the same anticoccidial drug induced the emergence of drug-resistant strains. ~he following experiment was carried out with a wild strain resistant to the conventional anticoccidial drugs.
~ he oocysts, predominantly of Eimeria tenella, obtained from a wild strain were used to inoculate chicks at a rate of lOx105 sporulated oocysts per bird. Other test materials are the same as those used in Experimental 2. ~he battery test procedure was repeated.
.
iU9~5 l ~4 O .~ J. J. it 01 ~ u\o ,0 ,W"~ ~ co ~; ~ D o C~ a) ;~; r;
~ D .~
.~ ___ .
0 l O O O O ~ O O
'~4 _ O O_ O O ~ 00 ~' ~'o + .~ .~ O O O O O O O
0 ~ J O r-J O O O O O O O
~1 ~ + ___ a) 0 ~ +
~ ~, ++ O .~ ~ N~ O O O ~ ~U
\ 4 h-. _ _ ~ 0 h ~ ~ ~ ~ ~ ~ K~ ~
~'h \ N~ ~ ~ K~ N~ N~ ~ ~ ~i i 4~ U~ o~ O O O O O O O O O
o ~ C' O O O O O O O O O
o' o ~ ~ O O O O O O O O O
~i ~; ~ U\ O O O O O O O O ~
.~ ~ O rt~Lr\
C' O ,~ J' ~ O O O ~ ~-E~ ~ .~ . . .
o." ~9 o ~ o o h ~g O O ,~ O O O ,~
~ L~ O .~ O N~ O O O O' O' O h ~ H
~;~ J O O O' ~`J' O O O
_ ._ _ . .
~ ^ U~ O O
~o,,~ ~D ~ O O O O O
c~ h _ .~ ~ .~ ~.~ ~.~
0 " oo .~ C) ~0 ~ h h ~ O ~ ~ ~ c) ~
~ ~ ~ ,~io ,a~O
~ ._ .H
-~9~1'75 The same test materials and testing procedure as those described in Experimental 2 were employed.
Notes to the description in the table:
(a) Average lesion index (degree) ~ he testing procedure used for coccidiosis in poultry was in conformity with the method of J. Johnson and W. M.
Reid (Experimental Parasitology 28, 33(1970)).
The results of post-mortem examinations performed on the 6th to 7th day following the infection were recorded according to the following scheme, - : The small intestine was completely normal (no patho-- logical change at all).
+ : ~ed small dots of blood appeared on the serous membrane in the middle of the small intestine.
Though the small intestine shows neither swelling nor thickening, it sometimes contains an orange-colored viscous fluid.
++ : A number of red small patches of blood appear on the mucous membrane of the small intestine. It is also full of orange-colored viscous fluid. The swelling of the small intestine is slight, the intestinal wall being slightly thickened.
+++ : The intestinal wall is swollen and thickened, The surface of the mucous membrane become loose and the intestine is full of blood clots and viscous fluids ++++ : The small intestine is swollen all over and contains a great many blood clots. Moreover, owing to the lO9il~5 digestion of red blood cells, the small intestine assumes a peculiar shade and gives off a rank odor, The intestinal wall is exceedingly swollen. Dead -chicks are included in this category.
(b) OPG: Oocysts per gram of feces ~ he mark " - " in the OPG column means that the value is less than log 3 (not measurable).
Table No.4 Infective Test material: Relative Average OPG
oocysts Antibiotic weight patho- (log) 4T-42082 gain logical - 1 --- ~
(lOxlO / index 4 5 1 6 ( bird)(ppm) (degree) _ _ days Eimeria125 108.3 _ _ _ _ /
acervulina 62.5 98.4 ++ _ 5. 5.4 /
_ . .
O 90.2 ++++6.6 6. 6.4 /
.... _ , . , Eimeria125 97.4 + / / 4.8 4.2 maxima62.5 94.9 + ~ ~ 5. 5.5 0__ 78.8 ++ / ~ 5.2 EXPERIMENTAL ~
~he test materials ? testing procedure, and scheme of evaluation are similar to those used in Experimental 2 and 4, 10~3~175 Table No,5 Infectlve Test material: Relative Average I OPG
oocystsAntibiotic weight pathologica _ (lo 3) (lOxlO4/bird) ~-42082 gain i7dex 4 5 lays . . .,.
Eimeria 120 100.3 _ _ _ _ acervulina90 100.4 _ _ _ _ _ 60 _ 92.6 ++__ 5.1 6.0 4,4 _ _ 0 90.2 ++++ _ 6.5 6.9 6.6 Example : ~en parts of Antibiotic ~-42082, previously milled to sizes not exceeding 149 ~, was mixed with 90 parts of dry extracted soybean flour to prepare a 10 k powder of Anti-biotic ~-42082. l.Ox103 parts of this powder was compounded with l.Ox106 parts of Feed KP-l-A (composition hereinafter) (for young broilers) to obtain an avian anticoccidial diet for broilers ~he concentration of Antibiotic T-42082:
100 ppm).
Formula of KP-l-A
(for young broilers) . .
Ingredient Compounding ratio, weight %
Yellow corn 55.
Wheat bran 5.
Soybean cake 18.0 Fish meal 8.0 Fish soluble 3.0 Alfalfa meal 3.
Tallow complex 5.7 Calcium carbonate 0.9 ~ribasic calcium phosphate o.7 Sodium chloride 0.25 A-Food E beads * o.o5 B-Feed S * 0,1 Neo-Mine feed C~ o.o5 Vitamin B12 - T * o.o5 Mixture of active agent and extracted soybean flour 0.2 .. ~ --- _ .
otal 100.0 : ~rade names (Distributor: ~akeda Chemical Industries, ~td.(Japan)) ExamPle~_ ?
25 parts of Antibiotic ~-42082, previously milled to sizes not exceeding 149 ~ in diameter, was blended well with 75 parts of dry extracted soybean flour to prepare a 10911 ~5 powder containing 25 % of Antibiotic ~-42082. 4.0xlO
parts of this powder was compounded with l.Ox106 parts of ~eed KP-l-A to obtain an anticoccidial diet for poultry ~he concentration of Antibiotic ~-42082: 100 ppm).
Example 0.5x103 parts of the 10 % powder obtained in Example 1 was compounded with l.Ox106 parts of Feed KP-l-A to prepare an anticoccidial diet for poultry ~he concentration of Antibiotic ~-42082: 50 ppm) Example 4 10 parts of Antibiotic T-42082, previously milled to sizes not exceeding 149 ~, was blended with 2 parts of hydroxypropyl-cellulose and 88 parts of lactose. ~he mixture was kneaded with 20 parts of water and granulated in a beater-granulator. ~he wet granules thus obtained were dried at 35C overnight, whereby 10 k granules 150 to 1,000 in diameter are obtained. l.Ox103 parts of this granular product was compounded with l.Ox106 parts of ~eed KP-l-B
(composition hereinafter)(for grown broilers) to prepare an anticoccidial diet for broilers ~he concentration of Antibiotic ~-42082: 100 ppm~.
, lO~il`7S
Formula of KP-l-B
(for grown broilers) . .
Ingredient _ --' Y
Yellow corn 58,0 Wheat bran 5.o Soybean cake 14,0 Fish meal 6,8 Fish soluble 3.o Alfalfa meal 3.o Tallow complex 8.0 Calcium carbonate 0,9 Tribasic calcium phosphate 0,68 Sodium chloride 0.25 A-Feed E beads o~o5 B-Feed S 0.1 Mixture of active agent 0,1 with lactose, etc, 0,12 Total 100.0 Example ~
25 parts of Antibiotic 'r-42082, previously milled to sizes not exceeding 149 11, were blended with 2 parts of hydroxypropyl-cellulose and 88 parts of lactose. The mixture was kneaded with 20 parts of water and granulated in a beater-granulator. The resultant wet granules were dried at 55C overnight to obtain 25 % dry granules 150 to 1,000 11 in diameter. 0,4x103 parts of the granules were .'" --' -. ' ' ' ' ' " ' , '' :- ' .
" iO9~1`7S
compounded with l,Ox106 parts of Feed KP-l-B to prepare an anticoccidial diet for poultry (~he concentration of Anti-biotic ~-42082 : 100 ppm).
Example 6 2.0x103 parts of the 10 % granules obtained in Example 4 were compounded with 1,Ox106 parts of ~eed KP-l-B to prepare an anticoccidial diet for poultry (~he concentration of Antibiotic ~-42082: 200 ppm), Example 7 1.2x103 parts of the 25 % granules obtained in Example 5 were compounded with l.Ox106 parts of Feed KP-l-B to prepare an anticoccidial diet for poultry (The concentration of Antibiotic ~-42082: 300 ppm).
By a procedure similar to that described in ~xample 4, the 10 /0 granules obtained in Example 4 were compounded with feed in various ratios to prepare diets containing Anti-biotic T-42082 in various concentrations.
Amount of the Amount of Concentration of 10 % granules ~eed KP-l-B Antibiotic obtained in ~-42082 Ex 4 ~parts) (parts) (ppm) 0.6 x 103 1.0 x 1o6 60 0.9 x 103 1~0 x 106 go 1.2 ~ 103 1.0 x 1o6 120 1.8 x 103 1.0 x 1o6 180 2.4 x 103 1.0 x 1o6 240 .
Claims (6)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing Antibiotic T-42082 which comprises aero-bically cultivating an antibiotic T-42082 producing microorganisms of Streptomyces hygroscopicus in a liquid nutrient medium until substantial antibiotic activity accumulates in the resultant medium and then recovering the Antibiotic T-42082 from said medium, either as such or in the form of a pharmaceutically acceptable salt thereof.
2. Antibiotic T-42082 and its pharmaceutically acceptable salts, said Antibiotic T-42082 having the following properties:
(1) Melting point: 120-122°C, (2) Elemental analysis: Found ? C,62.73; H, 9.23; 0, 26.01;
? C, 62.74; H, 9.36; 0, 27.03(%) (3) Molecular weight: 859 (by osmometry);
(4) Ultraviolet absorption spectrum; No characteristic absorptions, (5) Infrared absorption spectrum: The dominant absorptions (wave-numbers) measured in KBr disc method being as follows: 2940, 1700, 1462, 1383, 1085, 972 cm-1;
(6) Thin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10% aqueous H2S04;
Rf values s follows:
Plate: silica gel plate (Kieselgel 60, Merck, Germany) (7) Color reactions:
(brown) (8) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride; sparingly soluble in cyclohexane; and very sparingly soluble in water and petroleum ether;
when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
(1) Melting point: 120-122°C, (2) Elemental analysis: Found ? C,62.73; H, 9.23; 0, 26.01;
? C, 62.74; H, 9.36; 0, 27.03(%) (3) Molecular weight: 859 (by osmometry);
(4) Ultraviolet absorption spectrum; No characteristic absorptions, (5) Infrared absorption spectrum: The dominant absorptions (wave-numbers) measured in KBr disc method being as follows: 2940, 1700, 1462, 1383, 1085, 972 cm-1;
(6) Thin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10% aqueous H2S04;
Rf values s follows:
Plate: silica gel plate (Kieselgel 60, Merck, Germany) (7) Color reactions:
(brown) (8) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride; sparingly soluble in cyclohexane; and very sparingly soluble in water and petroleum ether;
when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
3. A process according to claim 1 wherein the Antibiotic T-42082 is recovered as such and then converted into its sodium salt.
4. A process according to claim 1 wherein the Antibiotic T-42082 is recovered as its sodium salt.
5. The sodium salt of the Antibiotic T-42082 having the following properties:
(1) Melting point: 180-182°C (with brownish discoloration) (2) Elemental analysis: Found ? C, 62.05; H, 8.99; Na, 2.08;
? C, 62.27; H, 9.01; Na, 2.79(%) (3) Molecular weight: 871, 878 (by osmometry) (4) Optical rotation [.alpha.]? -4.5?0,5° (in chloroform, c=1.0) (5) Ultraviolet absorption spectrum: No characteristic absorp-tion at and over 210 mµ, (6) Infrared absorption spectrum: The dominant absorptions (wave-numbers) measured in KBr disc method being as follows: 2935, 1610, 1461, 1382, 1080, 972 Cm-1 (7) Thin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10 % aqueous H2S04;
Rf values as follows:
(9) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride; sparingly soluble in cyclohexane; and very sparingly soluble in water and petroleum ether when prepared by the process of claim 3 or 4 or by an obvious chemical equivalent thereof.
(1) Melting point: 180-182°C (with brownish discoloration) (2) Elemental analysis: Found ? C, 62.05; H, 8.99; Na, 2.08;
? C, 62.27; H, 9.01; Na, 2.79(%) (3) Molecular weight: 871, 878 (by osmometry) (4) Optical rotation [.alpha.]? -4.5?0,5° (in chloroform, c=1.0) (5) Ultraviolet absorption spectrum: No characteristic absorp-tion at and over 210 mµ, (6) Infrared absorption spectrum: The dominant absorptions (wave-numbers) measured in KBr disc method being as follows: 2935, 1610, 1461, 1382, 1080, 972 Cm-1 (7) Thin-layer chromatography: silica gel, ascending method, detected as a brown spot by a spray of 10 % aqueous H2S04;
Rf values as follows:
(9) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl ether, chloroform, benzene and carbon tetrachloride; sparingly soluble in cyclohexane; and very sparingly soluble in water and petroleum ether when prepared by the process of claim 3 or 4 or by an obvious chemical equivalent thereof.
6. A method according to claim 1, 3 or 4 wherein the Antibiotic T-42082 producing microorganisms of Streptomyces hygroscopicus is Streptomyces hygroscopicus T-42082 (ATCC 31080).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA269,005A CA1091175A (en) | 1976-12-31 | 1976-12-31 | Polyether antibiotic from streptomyces hygrosiopicus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA269,005A CA1091175A (en) | 1976-12-31 | 1976-12-31 | Polyether antibiotic from streptomyces hygrosiopicus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1091175A true CA1091175A (en) | 1980-12-09 |
Family
ID=4107633
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA269,005A Expired CA1091175A (en) | 1976-12-31 | 1976-12-31 | Polyether antibiotic from streptomyces hygrosiopicus |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1091175A (en) |
-
1976
- 1976-12-31 CA CA269,005A patent/CA1091175A/en not_active Expired
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1041448A (en) | Antibiotic a-28086 complex from streptomyces aureofaciens | |
| JPS584720B2 (en) | Anticoccidial substance and method for producing the same | |
| DE2952520A1 (en) | ANTIBIOTIC TM-531 | |
| US4075323A (en) | Coccidiocidal combinations | |
| US4069316A (en) | Method for producing antibiotic T-42082 and antibiotic T-42082 | |
| HU179463B (en) | Process for preparing deoxy-narasin antibiotic complex | |
| US3949070A (en) | New antibiotic substance, its preparation and its use as growth promoting agents | |
| US5317030A (en) | Method and compositions for helmintic, arthropod ectoparasitic and acaridal infections with novel agents | |
| US4226879A (en) | Antibiotic composition | |
| CA1091175A (en) | Polyether antibiotic from streptomyces hygrosiopicus | |
| EP0071970B1 (en) | Novel antibiotic 76-11, process for the production thereof, anticoccidiosis agent and domestic animals growth accelerator comprising the same as an effective ingredient | |
| US4670260A (en) | Antibiotic for animal feeds | |
| US3991183A (en) | Antibiotic produced by a species of micromonospora | |
| US3927211A (en) | Antibiotic MYC 8003 and process for producing same | |
| CA1046965A (en) | Naphthyridinomycin antibiotics from streptomyces | |
| US5017561A (en) | Antibotic 6270B and its use as an anticoccidiosis agent and a feed additive | |
| US4232006A (en) | Antibiotic W-10 complex, antibiotic 20561 and antibiotic 20562 as antifungal agents | |
| US3966913A (en) | Antibiotics No. K-73 and method for producing the same | |
| US5198464A (en) | Method and compositions for helmintic, arthropod ectoparasitic and acaridal infections with novel agents | |
| US3655876A (en) | Antibiotic cp-21 635 | |
| US4137224A (en) | Process for the preparation of antibiotic W-10 complex and for the isolation of antibiotic 20561 and antibiotic 20562 therefrom | |
| US4204039A (en) | Process for producing deoxynarasin antibiotics | |
| US5034224A (en) | Method and composition for treating protozoal infections | |
| CA1240278A (en) | Antibiotic 6270, process for its production and its use as an anticoccidiosis agent and a feed additive | |
| US3923981A (en) | Antibiotic MYC 8003 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |