Note: Descriptions are shown in the official language in which they were submitted.
~039;~5
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18 This invention relates to the novel production
l9 by fermentation and isolation of a useful antibiotic
substance that heretofore has not been reported in the
21 prior art. More particularly, this invention relates to :
22 the preparation of antibiotic FR-02A by fermenting Str~p-
23 tomyces lactamdurans under controlled conditions, followed :~
24 by isolation of said hitherto undescribed antibiotic desig-
nated FR-02A.
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lQ3~215 ~ ;
1 The antibiotic FX-02A is obtained by growing
2 under controlled conditions the previously known micro-
3 organism, Streptomyces lactamdurans, in a fermentation
4 broth and extracting the whole broth with a water immisci-
ble polar organic solvent to obtain the antibiotic. The
6 fermentation may be carried out in media containing sus
7 pended nutrient matter or predominantly clear media wherein
8 the media is substantially free of suspended nutrient matter.
9 In the case wherein the fermentation is carried
10 out in media containing suspended nutrient matter the anti- -
11 biotic is found both in the solids comprising the mycelia
12 and suspended nutrient matter and in the broth. The anti-
13 biotic is isolated from the solids by separating the solids ``
14 from the fermentation broth by filtration, centrifugation or
other suitable means and extracting the cake comprising the
16 solids with an organic solvent, preferably a polar organic
17 solvent. The antibiotic remaining in the broth is isolated
18 from the broth, from which the solids have been previously
19 separated, by extraction with a water immiscible polar
organic solvent. It will be appreciated that upon delayed
21 harvesting the total level of solids in the fermentation
22 broth will be reduced and a smaller proportion of the anti-
23 biotic will be found in the solids recovered from the broth.
24 In media containing no suspended nutrient matter,
most of the FR-02A is found in the predominantly clear fer-
26 mentation broth. In such cases the whole broth is extracted
27 with a water immiscible polar organic solvent such as chloro-
2~ form to obtain the antibiotic. ~lternatively any solids,
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1 such as mycelia, may be separa-ted from the broth prior to
2 extractiny the broth with an immiscible polar organic
3 solvent. Furthermore, to obtain any residual FR~02A,
~ the myceliumseparated from the broth is extracted with a ~`
S suitable polar organic solvent to obtain the antibiotic FR-
6 02A.
7 A preferred method for obtaining the antibiotic
8 of this invention is by growing, under controlled condi-
9 tions, the previously known microorganism, Streptomyces
lactamdurans in a medium containing suspended nutrient mat-
11 ter or in a clear medium substantially free of suspended
12 nutrient matter and extracting the whole broth with a
13 water immiscible polar organic solvent. The extraction is
14 carried out by adjusting the pH o the broth to acid pH and
adding the solvent to the broth. After mixing, the solids
16 are separated from the broth. The broth is allowed to stand
17 until the solvent layer separates. The solvent layer is
18 drawn off, washed with water, dried with a suitable drying
19 agent and evaporated ln vacuo. The residue is washed with
20 a non-polar organic solvent such as petroleum ether or -;
21 hexane and air dried to yield the antibiotic FR-02A.
22 The further preferred method for obtaining the
23 antibiotic FR-02A is by growing, under controlled conditions~
24 the previously lcnown microorganism, Streptomyces lactam-
durans in a medium containing suspended nutrient matter.
26 The fermentation broth is filtered to recover the solids
27 comprising mycelium and suspended nutrient matter. After
28 blowing the filtered cake comprising the solids as dry as
29 possible the cake is stirred in a polar organic solvent and
..... . ~ . , .
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15523IB
1~3~3~1S
1 filtered again. After -the cake has been washed with addi-
2 tional solvent, the combined sol~ent extract and wash are
3 vacuum evaporated to leave an aqueous slurry. The pH is
4 made acidic. The aqueous slurry is washed with a non-polar
S organic solvent such as petroleum ether, ~hexane and the like -
6 until the washings are colorless. The aqueous slurry is
7 reextracted with a polar organic SOlvent. The solvent ex-
8 tract is dried, filtered, and evaporated ln vacuo to yield
9 the antibiotic FR 02A. For example, in accordance with the -
process of the invention, antibiotic FR-02A may be obtained
11 in from about 40-50 percent pure form.
12 Alternatively, the ermentation ma~ be carried ;
13 out in a medium substantially free of suspended nutrient
1~ matter. q`he fermentation broth is filtered to recover
the mycelium. The mycelium is extracted with a polar
16 organic solvent. The e~tract is dried and evaporated to
17 dryness in vacuo. The residue is shaken with a non-polar
,
18 organic solvent such as petroleum ether, hexane and the
19 like. The organic solvent is decanted after centrifugation
to sediment the residue. The residue is air-dried to obtain
21 antibiotic FR-02A.
22 A still further preferred method of obtaining
23 the antibiotic of this invention is by growing, under con-
24 trolled conditions, the microorganism, Streptomyces lac-
tamdurans, in a fermentation broth and extracting the
26 broth after separating the solids comprising mycelium or ~;
27 mycelium and suspended nutrient matter. The pH of the
28 fermentation broth is made acid and the solids comprising
, " ~,
~ . . .. . , . ... .- , . , .. ~ .; ~
15523IB
1 mycelium or mycelium and suspended nutrient matter is sep-
2 arated. The fermentation broth free of solids is mixed
3 with a water i~miscible polar organic solvent. ~fter
4 mixing, the layers are allowed to separate. The organic
layer is drawn off, dried with a suitable drying agent,
6 and evaporated to dryness in vacuo. The residue is washed
7 with a non-polar organic solvent such as petroleum ether
8 or hexane and air dried to obtain antibotic FR-02A.
9 In the processes described above wherein extrac-
tions are carried out with water immiscible polar organic
11 solvents, representative examples of said solvents include
12 alkyl esters of lower alkanoic acids such as methyl formate,
13 ethyl formate, methyl acetate, ethyl acetate/ n-butyl
1~ acetate, isobutyl acetate, ethyl propionate; a ketone such
as cyclohexanone; or a halogenated lower hydrocarbon such
16 as chloroform, methylene chloride, carbon tetrachloride,
17 ethylene dichloride, 1-chloro-2,2-dimethylpropane, tetra-
18 chloroethylene, or bromoform.
19 In the processes described above wherein extrac-
tions are carried out with polar organic solvents, repre-
21 sentative examples of said solvents include lower alkyl
22 esters of lower alkanoic acids such as methyl formate,
23 ethyl formate, methyl acetate, ethyl acetate, n-butyl
24 acetate, isobutyl acetate, ethyl propionate; a ketone such
as acetone, methyl ethyl ketone, or cyclohexanone; or a
26 halogenated lower hydrocarbon such as chloroform, methylene
27 chloride, carbon tetrachloride, ethylene dichloride, 1-
28 chloro-2,2-dimethylpropane, tetrachloroethylene, or bromo-
29 form.
~.
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~LQ3~5
1 In addition to the production of FR-02A, it has
2 been reported in the literature that S. ~actamduxans NRRL
3 3802 elaborates the antibiotic Cephamycin C (Antimicrobial
4 Agents and Chemotherapy, Sept. 1972, p. ~22-131, Vol. 2,
No. 3, "Cephamycins, a New Family of ~-Lactam Antibiotics"
6 and Belgian Patent 764,160). As a consequence of FR-02A
7 being highly soluble in water-immiscible organic solvents
8 whereas Cephamycin C is virtually insoluble in water
9 immiscible organic solvents, the two materials are readily
separated from each other. Accordingly, extraction of the
11 broth with a water immiscible solvent permits each anti- !
12 biotic to be obtained in a form free of contamination by
13 the other.
14 The antibiotic FR-02A isolated from the fermen-
tation broth is subjected to further purification by chroma-
16 tography through a molecular sieve followed by chromatog-
17 raphy over a surface active adsorbing agent. A suitable
18 molecular sieve is a cross-linked dextran such as the
19 molecular sieve known by the Pharmacia Fine Chemicals Inc.
trade name Sephadex LH-20. FR-02A may be eluted by a lower
21 alkanol such as me~hanol. A suitable surface active ad~
22 sorbing agent is a hydrophobic non-ionic macro porous co-
23 polymer of polystyrene cross-linked with divinylbenzene
24 known by the Rohm and Haas trade names Amberlite~XAD-l to
XAD-12. A preferred resin for purifying FR-02A is XAD-2.
26 Suitable solvents for eluting adsorbed FR-02A are aqueous
27 solutions of lower alkanols e.g. aqueous solutions of
28 methanol, ethanol, isopropanol, butanol and the like. The
~` .
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1~39~1S
1 preferred solvent for eluting F~-02A from XA~-2 resin is 50%
2 isopropanol-water.
3 The organism which produces FR-02A is a pre-
4 viously known strain of ~treptomyces lactamdurans
designated as MA-2908 in the culture collection of
6 Merck & Co., Inc., Rahway, New Jersey. It was isolated
7 from a soil sample and has been placed on permanent de-
8 posit without restrictions as to availability with the
9 culture collection of the Northern Utili2ation Research and
Development Division, Agricultural Research Service, U.S.
11 Department of Agriculture (formerly Northern Regional Re-
12 search Laboratories), Peoria, Illinois 61604 and is avail-
13 able to the public under culture No. NRRL 3802.
1~ Complete taxorlomy and morphology studies of
Streptomyces lactamdurans are reported in Belgian Patent
16 No. 764,160. Based on taxonomic studies Streptomyces
17 lactamdurans was identified as a new actinomycete. It
18 was found to belon~ to the genus Streptomyces and it
19 possesses many attributes of the known species Strepto-
myces fradiae. Biochemically the two are almost a perfect
21 match but morphologically there are important differences.
22 For example, the aerial mycelium of S. fradiae is seashell
23 pink as compared to the cream color of S. lactamdurans. On
24 the basis of this difference and other characteristics the
microorganism was assigned the species name Streptomyces
26 lactamdurans.
27 Streptomyces lactamdurans is simply illustrative
28 of the type of strain of microor~anism which can be used
:
15523IB
~L~?39~S
1 in the production of FR-02A and it should be understood
2 that the present invention is not limited to organisms
3 meeting these particular descriptions. This invention
4 includes the use of the other microorganisms, including
strains of actinomycetes either isolated from nature or
6 obtained by mutation as, for example, those obtained by
7 natural selection or those produced by mutating agents,
8 for example, X-ray irradiation, ultraviolet irradiation,
9 nitrogen mustards and the like which, under suitable con-
ditions will yield FR-02A.
11 FR-02A is produced during the aerobic fermenta-
12 tion of suitable aqueous nutrient media under controlled `
13 conditions via the inoculation with the organism Strepto-
14 myces lactamdurans. Aqueous media, such as those employed
for the production of other antibiotics are suitable for
16 producing the antibiotic FR-02A. Such media contain sources ; ;
17 of carbon, nitrogen and inorganic salts assimilable by the
18 microorganism. The choice of media is not critical and
19 the fermentation may be carried out in media containi~g
20 suspended nutrient matter or predominantly clear media ;
21 wherein the media is substantially free of suspended nu-
22 trient matter.
23 In general, carbohydrates such as sugars, for -
24 example dextrose, glucose, arabinose, maltose, raffinose,
:.~, , .
xylose, mannitol and the like and starches such as grains,
26 for example, oats, rye, corn starch, corn meal and the like
27 can be used either alone or in combination as sources of ~`
28 assimilable carbon i~ the nutrient medium. The exact
-8-
,
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.: . : . . . . : .. . . . .
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1 quanti-ty of the carbohydrate source or sources utilized
2 in the medium depends in part upon the other ingredients
3 of the medium bu-t, in general, the amount of carbohydrate
4 usually varies between about 1% and 6% by weight of the
5 medium. These carbon sources can be used individually, or
6 se~eral such carbon sources may be combined in the medium.
7 In general, many proteinaceous materials may be used as
8 nitrogen sources in the fermentation process. Suitable
9 nitrogen sources include, for example, nutrient broth,
yeast extract, yeast hydrolysates, primary yeast, soybean
11 meal, cottonseed flour, hydrolysates of casein, corn steep
12 liquor, distiller's solubles or tomato paste and the
13 lik~. The sources of nitrogenl either alone or in com-
14 bination, are used in amounts ranging from about 0.2%
to 6~ by weight of the aqueous medium.
16 Media described in the Examples are merely
17 illustrative of the wide variety of media which may be
18 employed, and are not intended to be limitative.
19 The fermentation is carried out at temperatures
ranging rom about 20C. to 37C.i however, for optimum
21 regults it is preferable to conduct the fermentation at
22 temperatures of from about 24C. to 32C. The pH of the
23 nutrient media sui~table for growing the Streptomyces
24 lactamdurans culture and producing the antibiotic FR-02A
should be in the range of from about 6.0 to 8Ø
26 A small scale fermentation of the antibiotic is
27 conveniently carried out by inoculating a suitable nu-
28 trient medium with the antibiotic-producing cuiture and,
_9_
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t~
15523IB
31.Q3~J~5 ; ~
1 after transfer to a production medium, permitting the fer-
2 mentation to proceed at a constant temperature of about
3 28C. on a shaker for several days. At the end of the ?
4 incubation period the mycelium and the suspended nutrient
5 matter can be recovered by centrifugation or filtration and
6 extracted with solvent, or the whole broth can be extracted
7 with chloroform or other water immiscible solvents or
8 alternatively the broth may be extracted after separating ;-~
9 the solids comprising mycelium or mycelium and suspended
10 nutrient matter.
11 The small scale fermentation is conducted in a
12 sterilized flask via a one, two, three or four stage seed
13 development. The nutrient medium for the seed stage may
1~ be any suitable combination of carbon and nitrogen sources.
15 The seed flask is shaken in a constant temperature chamber
16 at about 28C. for a period of from one to two days and
17 some of the resulting growth is used to inoculate either
18 a second stage seed or the production medium. Intermediate
. . .
19 stage seed flasks, when used, are developed in essentially
,",,.
20 the same manner; that is, part of the contents of the flask ;
21 are used to inoculate the production medium. The inocu-
22 lated flasks are shaken at a constant temperature for ~
23 several days and at the end of the incubation period the '!`'' . ~ ,. '' :;
24 antibiotic FR-02A is isolated as already described. ;`
For large scale work, it is preferable to con-
26 duct the fermentation in suitable tanks provided with an
27 agitator and a means of aerating the fermentation medium.
28 According to this method, the nutrient medium is made up ;
29 in the tank and sterilized by heating at temperatures of
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15523IB
1~P39~1S
1 up to about 120C. Upon cooling, the st rilized medium is
2 inoculated with a pre~iously grown seed of the producing
3 culture, and the fermentation is permitted to proceed for
4 a period of several days as, for example, rom two to
four days while agitating and/or aerating the nutrient
6 medium and maintaining the temperature at about 28C. The
7 yield of FR-02A is generally between 2Q mg. to 200 mg. per
8 liter of production broth as determined by bioassay.
9 Assay Procedure;
Assays were run by the disc-plate procedure using
11 3/8 inch filter paper discs. The assay plates were pre-
12 pared using Difco nutrient agar plus 2.0 g./l. Difco
13 yeast extract at 10 ml. per plate. An overnight ~rowth
14 of the assay organism, Vibrio percolans American Type
Culture Collection (ATCC 8461) in nutrient broth plus
16 0.2% yeast extract was diluted in sterile saline solution
17 to a suspension having 40% transmittance at a wave length
18 of 660 m~. This suspension WâS added at 20 ml./liter of
19 medium prior to pouring the plates.
The assay plates were heid at ~C. until used
21 (5 day maximum). Following the application o the anti-
22 biotic-saturated assay discs the plates were incubated
23 at 28C. for a period of froml6 to 24 hours. Zones of
24 inbibition were read as mm. diameter. They were used to
determine relative potencies or, when compared with a
26 purified reference standard, the potency in ~g./ml. -
27 Assays of FR-02A in fermentation broths, mycelia and
2~ suspended nutrient matter separated from fermentation
29 broths and in broths free of solids were performed after -
. ~ .
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1 extracting the FR-02A into a suitable solvent. Assays on
2 solutions containing FR-02A, 200 ~g./ml. using 3/8 inch
3 discs showed 16 mm. zones of inhibition. When such an
4 assay is performed in a quantitative ~ashion, from 50
5 to 100 ~g-. ~l. of antibiotic can be detected. ~ -
6 FR-02A shows activity against gram-negative and
7 gram-positive bacteria, coccidia and species of Mycoplasma.
8 In vitro, FR-02A is effective against E. acervulina, Bor-
9 detella, Streptococcus faecalis, Streptococcus faecum,
10 Streptococcus agalactiae, Streptococcus pyo~enes, Proteus -~
11 vulgaris, M. hyorhinis, M. synoviae, M. arthritidis, M.
12 ~allisepticum and species of Pasteurella. Activity was also ;~
. .. .
13 Eound against Vibrio percolans (ATCC 8461), Salmonella
14 ~allinarum (MB 1287), E. coli (MB 1418), Klebsiella pneu- ;
moniae (MB 1264), Pseudomonas stutzeri ~MB 1231) and (MB
16 2765), Bacillus subtilis (MB 964) and (MB 797), Staphylo- ~;
17 coccus aureus (MB 108), (MB 210) and (MB 703), and Pseudo- ; - ~ -
18 monas aeruginosa (MB 3210).
~... . ... .
19 FR-02A is useful both as an antibiotic and as a
growth promoting agent in animals.
21 When FR-02A is used as an antibiotic, the speci- i
22 ~ic means employed for administering it to the animal is not `
23 critical and any of the methods now used or available for
24 treating infected animals or animals susceptible to infec~
tion are satisfactory.
26 FR-02A can be used as an antibiotic, for example,
27 in the form of pharmaceutical preparations which contain -
28 it in admixture or conjunction with an organic or inor-
29 ganic, solid or liquid pharmaceutical excipient suitable
:::
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:: . . : ~ : - -
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~ 15523IB
~L~?3~
1 for enteral, parenteral or local administration. Suitable
2 excipients are substances that do not react with the anti-
3 biotic, for example, water, gelatin, lactose, starches,
4 stearyl alcohol, magnesium stearate, talcum, vegetable oils,
benzyl alcohols, gums, propyleneglycol, polyalkylenegly-
6 cols, white petroleum jelly, cholesterol or other known
7 medicinal excipients. The pharmaceutical preparations may
8 be, for example, tablets, dragees, ointments, creams or
9 capsules, or in liquid form solutions, suspensions or
emulsions. They may be sterilized and/or contain assistants,
}1 such as preserving, stabilizing, wetting or emulsifying
12 agents; solution promoters, salts for regulating the
13 osmotic pressure or buffers.
14 Where it is desired to administer the antibiotic
in dry, solid unit dosage form, capsules, boluses or tab-
16 lets containing the desired amount of antibiotic are em-
17 ployed. These dosage forms are prepared by intimately and
18 uniformly mixing the active ingredient with suitable
19 finely divided diluents, fillers, disintegrating agents
and/or binders such as starch, lactose, talc, magnesium
21 stearate, vegetable gums and the like. Such unit dosage
22 formulations may be varied widely with respect to their
23 total weight and content of FR-02A depending upon factors --
24 such as the type of host animal to be treated, the severity
and type of infection and the weight of the host. The
26 antibiotic may be administered on a daily basis at from
27 about 5 to 100 mg. per kilograms of body weight.
28 Included in this invention are the non-toxic,
29 pharmaceutically acceptable salts of FR-02A, for example,
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15523Is
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3~12~5
1 the alkali and alkaline earth metal salts such as those
2 derived from sodiuri~, potassium, ammonium and calclum or
3 salts with organic bases, for example, triethylamine,
4 N-ethylpiperidine, dibenzylethylenediamine.
5 In addition to its use as an antibiotic, FR-02A `
6 is useful as a feed additive to promote the growth of
7 animals quch as chickens, sheep and cattle. The use of ~
8 FR-02A shortens the time re~uired for bringing animals ; ~-
9 up to marketable weight. ^
lO When FR-02A is used as a growth promoter in ;~
ll animals, it can be administered as a component of the
12 feed of the animals or may be dissolved or suspended in
13 the drinking water.
14 When FR-02A is used as a component of animal
15 feed, it is first formulated as a feed supplement. In such ! .
16 feed supplements, FR-02A is present in relatively concen-
17 trated amounts intimately dispersed in an inert carrier
:. .
18 or diluent. The feed supplement can be added directly to
l9 the feed or made into a premix by an intermediate dilution
. . ,
or blending step. By inert carrier is meant one that will
21 not react with the antibiotic and one that may be admini-
22 stered safely to animals. Preferably, the carrier is
23 one that is, or may be, an ingredient of the animal ration.
24 Typical carriers or diluents suitable for such compositions
25 include, for example, distillers' dried grains, corn mealj ;
26 citrus meal, fermentation residues, ground oyster shells,
27 wheat shorts, molasses solubles, corn cob meal, edible
28 bean mill feed, soya grits, crushed limes~one and the like.
:`~ ' .
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1 The antibiotic is intimately dispersed throughout the
2 carrier by methods such as grinding, stirring, milling or
3 tumbling. Compositions containing from about 5 to 50%
4 by weight of the antibiotic are particularly suitable as
feed supplements.
6 Examples of typical feed supplements containing
7 FR-02A disperSed in a solid carrier are:
8 lbs.
9 (A) FR-02A . . . . ~ . . . . . . . . . . . 5
Wheat Standard Middling . . . . . . . 95
11 (B) FR-02A .............................. 50
12 Corn distiller's grains . . . . . . . 50
13 These and s.imilar feed supplements are prepared by uni-
14 formly mixing the antibiotic with the carrier.
The feed supplement can be added directly
16 to the feed or made into a premix by an intermediate
17 dilution or blending step with an orally ingestable
18 carrier. Compositions containing 0.03~ to 5% by weight
19 of the antibiotic are particularly suitable as premixes.
These premixes are prepared by uniformly mixing the
21 antibiotic with an orally ingestable carrier.
22 Such supplements or premixes are added to the -
23 animal feed in an amount to give the finished feed the
2~ concentration of FR-02A desired for growth promotion. In
chickens, FR-02A is fed at a final concentration of be-
26 tween 50 gm. to 300 gm. per ton of feed in order to achieve
27 the desired growth promoting result. In the case of swine,
28 including swine infected with M. hyorhinis, FR-02A may
29 be administered in the feed at similar levels.
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~L~39~1.S ~;
1 In the above discussion of this invention,
2 emphasis has been placed on solid compositions wherein
3 the FR-02A is mixed with an edible carrier in a feed
4 supplement, in a so-called premix or in the final feed-
stuff. This is the preferred m~thod of administering the
6 FR-02A. An alternate method is to dissolve or suspend the
7 FR-02A in the drinking water of the animals. The quantity
8 that may be suspended in the water without undue settling
9 is limited~ Emulsifiers or surface-active agents may be
employed for this latter purpose.
11 It will likewise be understood by those skilled
12 in this art that special feed supplement formulations and
13 inished animal eeds containing FR-02A may also include
14 vitamins, other antibiotics and growth-promoting agents
and other nutritional substances.
16 FR-02A is useful against poultry PPLO at a range
17 of 5 to 100 mg./kg. A preferred range for a single dose
18 is from 35 to 45 mg./kg. For reasons of convenience a
19 preferred method of administering the antibiotic in the
treatment of PPLO is to admix the FR-02A with the animal
21 eed. A preferred range for PPLO is from 0.0055~ to 0.02%
22 by weight of feed.
23 In the treatment of air sacculitis in broilers
24 the ED50 is 40 to 100 mg./kg. Accordingly, a useful dosage -~
of FR-02A may vary from 10 to 150 mg./kg.
26 A solution or suspension for subcutaneous injec~
27 tion for treatment of air sacculitis in broilers may be
28 prepared as follows:
.
:: : . ,,~ . ,
15523IB
1~39~5 ~
1 Subcutaneous Solution or Sus~ension Cont~inin~_20 mg._of
2 FR-02~ Am~oule:
. . . _
3 FR-02A 20 mg.
4 Diluent: Sterile water for injection 2 cc. `;
In the treatment of coccidiosis the preferred
6 method of administering FR-02A is in the feed at a level of
7 from about 0.05 to 2% by weight of feed.
8 It will be appreciated that the dosage to be
9 administered depends to a large extent upon the condition
and weight of the host; the parenteral route is preferred
11 for air sacculitis and the oral route is preferred for
12 PPLO and coccidiosis. The preferred route of admini-
13 strating FR-02A for growth promotion is by admixing in feed.
14 The examples which follow illustrate methods
by which the product of this invention may be obtained.
16 The claimed process is capable of wide variation and modi-
17 fication and, therefore, any minor departure therefrom or
18 extension thereof is considered as being within the skill
19 of the artisan and as falling within the scope of this
20 invention. -
' '
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~
103~ L5 : ~:
1 EXAMPLE 1
2 Production of Antibiotic YR-02A by Shake Flask Fermenta-
3 tion of Streptomyces lactamdurans
4 A lyophilized culture of Streptomyces lactamdur~
ans, designated MA-2908 is used as inoculum.
.. . ~ . , . :
6 The lyophilized culture is opened into a 250 ml.
7 three-baffled Erlenmeyer flask containing 40 ml. of First
8 Stage Seed Medium~
9 First Stage Seed Medium
Primary yeast 1~ -
11 in distilled water
12 pH adjusted to 7.0 with NaOH
13 The first stage seed flask, and the subsequent
14 second stage and production flasks, are incubated a-t 28C.
on a rotary shaker operating at 220 rpm.
16 After two days in first stage medium, one ml.
17 from the first stage seed flask is inoculated into 40 ml.
18 of Second Stage Seed Medium in a 250 ml. three-baffled
19 Erlenmeyer flask.
_econd Stage Seed Medium
21 Ardamine~YEP t99F) 1%
22 in distilled water
23 pH adjusted to 7.0 with NaOH
24 The second stage seed flask is incubated for one
day, then 1 ml. of the culture is inoculated into each of
26 ten non-baffled 250 ml. Erlenmeyer flasks containing 40 ml.
27 of Production Medium:
28 Production Medium
29 Primary Yeast 1 %
Distiller's Solubles 3 %
31 Glycine o 05%
32 L-phenylalaine 0.3 %
33 Cornstarch 2.0 %
34 Dimethylformamide 1.0 % (by volume)
Mobil~par-S defoamer 0.25% (by volume)
36 in distilled water
37 pH adjusted to 7.0 with NaOH
, ~ ~ -18-
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15523IB
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1 A sodium thiosulfate solution is prepared by
2 dissolving 12.5 g. Na2S2O3 5H2O in 100 ml. distilled
3 water. This solution is filter-sterilized, then 1 ml.
4 is added to each of the 10 production flasks.
5 The ten flasks are incubated for four days `~
6 at 28C., and harvested.
7 Isolation of FR-02A
~ Ths pH of the 400 ml. of broth obtained above
g is adjusted to 5 with hydrochloric acid, and two volumes
of chloroform are added to the broth. After thorough mixing
11 the broth is filtered through a Supercel pad. Sufficient
12 water is added to speed up the filtration. The filtrate is
13 allowed to stand until the chloroform layer separates. The
14 chloroform layer is drawn off, washed twice with 500 ml. of
15 water, dried over anhydrous magnesium sulfate and evapor- ~;
16 ated to dryness in vacuum. Five hundred ml. of petroleum
17 ether is added to the solid residue and the solid is col-
18 lected by filtration, washed with 50 ml. of petroleum ether
19 and air dried to yield 144 mg. of FR-02A. The FR-02A is ~ ;
converted to the ammonium salt by freeze drying a solution
21 of the FR-02A in 50 ml. water made basic to pH 10 with ;;
22 ammonium hydroxide. After freeze-drying, the product
23 weighs 140 mg. ;
24 Since the FR-02A is found in association with
25 the mycelia and suspended nutrient matter, the mycelia -
26 and suspended nutrient matter may first be filtered out
27 of the fermentation broth and the FR-02A extracted from the
28 filtered cake with a polar water miscible organic solvent
29 such as acetone. This alternate method of isolating FR-02A
is as follows:
-19-
- .: . . . -: ~ . . .
.
:. . . . . .
~ 15523IB
1~39Z15
. .
. ., . ~
1 Four hundred ml. of whole broth obtained from ~;
2 the above fermentation is filtered to recover the mycelia
3 and suspended nutrient matter. The filtered cake is then
4 stirred in 40 ml. of acetone for 30 minutes and filtered
again. The cake is washed with 15 ml. acetone. The com-
6 bined acetone extract and washings are vacuum evaporated
7 at 30C. to remove the acetone. The residue is taken up
8 in 15 ml. of water. The pH is ad~usted to 4.0 with hy-
9 drochloric acid. ;
An equal volume of hexane is added and stirred
11 10 minutes. After settling, the hexane is decanted and
12 discarded. TWo more extractions with equal volumes of
13 hexane are made, the last one being colorless.
14 The aqueous slurry is then extracted with an
15 equal volume of chloroform. A second extract with an -
16 equal volume is then made and combined with the first
17 extract. The aqueous phase is discarded.
18 The chloroform extract is dried over anhydrous
19 godium sulfate, filtered, and vacuum evaporated to yield
106 mg. of FR-02A.
21 The molecular weight of FR-02A is determined to
22 be about 1,000 by subjecting the material obtained from the
23 fexmentation process described above to chromatography on
24 Sephadex LH-20. The material obtained from this treatment
is rechromatographed on Amberlite XAD~2 to obtain an analy-
26 tically pure sample.
27 Chromatography on Sephadex LH-20 Gel
28 A 0.5 ml. solution of methanol containing 106 mg.
29 of FR-02A obtained from Example 1 above is charged to a 75
-2~-
: . :
~-~ 15523IB
~3~12~
1 ml. hed (1.25 cm. dia. x 56 cm. ht.) of Sephadex LH-20
2 gel in methanol (Pharmacia Fine Chemicals, Inc., Piscataway,
3 N.J.). The column is developed with methanol. The eluate
4 stream is monitored with a Meeco differential refractometer
and the record shows three mass peaks. Fractions are
6 bioassayed at a 1-10 dilution with 1/2" dia. paper discs ~
7 on agar-diffusion assay plates seeded with Vibrio percolans. ~ ;
8 Zones of inhibition are obtained corresponding to the third
g mass peak (KD = 0.3) detected. The fractions corresponding
10 to the third mass peak are combined and evaporated to yield
11 43 mg. of FR-02A.
12 The product when assayed in the above agar-
13 di~fusion bioassay using Vibrio percolans gives a 16 mm.
14 zone of inhibition at a concentration of 200 ~g./ml. The
15 U.V. absorption in pH 7.0 phosphate buffer was~
16 ~ max. 327 nm ElCm = 216
17 ~ max. 232 nm ElCm = 464
18 The purification with Sephadex LH-20 gel may be
19 omitted if the product obtained from Example 1 is dissolved
20 in water at a pH of 9 (pH adjusted with dilute sodium hy- Ç
21 droxide solution) and filtered to remove insoluble material
22 and the filtrate fr~eze-dried.
23 Chromatograph~ on Amberlite XAD-2 Resin
,;~
24 The 43 mg. of LH-20 product is further purified
25 by chromatography on a 150 ml. bed (1.25 cm. dia. x 112
26 cm. ht.) of Amberlite XAD-2 resin (Rohm & Haas Co., Phila.,
27 Pa.) in 50% isopropanol-water. The charge was acidified
28 to pH 2 to convert it to the free acid form before applying
29 it on the column. The column was monitored by a Meeco
-21-
.",
, .. . . . . .
15523I~ ~
iO39~15
1 differential refractometer and the record showed two mass
2 peaks. Agar diffusion bioassays using 1/4" dia. discs
3 and Vibrio percolans plates showed the mass peak centered
4 at KD3.5 to 4.0 to contain the antibiotic. Fractions cor-
responding to these KD values were combined and evaporated
6 to dryness under vacuum. 16.4 mg. of antibiotic FR-02A
7 was obtained. FR-02A is obtained as a pale yellow solid,
8 stable to normal handling, and analytically pure.
:` :
9 EXAMPLE 2
Production of Antibiotic FR-02A by Shake Flask Fermentation
11 of Streptomyces Lactamdurans
_
12 A lyophilized culture of Streptomyces lactamdur-
13 ans, designated MA-2908 is used as inoculum.
14 The lyophil is opened into a 250 mI. three-
baffled Erlenmeyer flask containing 40 ml. of Firs-t Stage
16 Seed Medium:
17 First Stage Seed Medium
. .
18 Primary yeast 1
19 in distilled water
pH adjusted to 7.0 with NaOH
21 The first stage seed flask, and the subsequent
22 second stage and production flasks, are incubated at 28C. ;
23 on a rotary shaker operating at 220 rpm.
24 After two days in first stage medium, one ml.
from the first stage seed flask is inoculated into 40 ml.
26 of Second Stage Seed Medium in a 250 ml. three-baffled
27 Erlenmeyer flask.
-22-
15523IB
~3~ 1S ; ~ :
1 Second Skage Seed Medium ~~
.~ . .. .
2 Ardamine~YEP (99F) 1% -
3 in distilled water
4 pH adjusted to 7.0 with NaOH
The second stage seed flask is incubated for
6 one day, 'hen 1 ml. of the culture is inoculated into each
7 of ten non-baffled 250 ml. Erlenmeyer flasks containing
8 40 ml. of Production Medium having the following composi-
9 tion:
Production Medium
; ~ :
11 Corn steep li~uor 2.8% by weight -~
12 cerelose 5.6% by weight
13 Proflo~ 2.8% by weight
14 glycerol 1.4% by volume
~Dimethylformamide 1.4~ by volume
16 in tap water
17 pH is adjusted to 7.3 with NaOH
18 One drop of P-2000 defoamer is`added to each
19 flask prior to autoclaving. ;~
A sodium thiosulfate solution is prepared by
21 dissolving 6.25 g. Na2S2O3 5112O in 100 ml. distilled
22 water. This solution is filter sterilized, then 0.5 ml.
23 is added to each of the production flasks after autoclaving.
24 The ten production flasks (totaling 400 ml.) are ~ ~`
then incubated for five days at 28C. and harvested.
26 The ten f1asks are pooled, the pH is lowered to
27 5.5 with hydrochloric acid, then 500 ml. of chloroform is
28 added, and the mixture is stirred for one-half hour at
29 room temperature. The mixture is then centrifuged to
30 separate the phases, and the aqueous phase is discarded. ; -
31 The chloroform layer is dried over anhydrous magnesium
32 sulfate and the solvent evaporated off in vacuo. The
:
-23-- ~
^. .
15523 IB
''
1~39~S ~: ~
.. . .
1 The residue is shaken with 30 ml. of hexane. The hexane is
2 decanted off after centrifuging down the residue. Thirty
3 ml. of water, made basic to pH 10 with ammonium hydroxide,
4 is added to the residue to yield a slightly cloudy solution.
For the purpose of bioassay the pH is then lowered to 8.3
6 with HCl, and diluted in pH 8.3 water for assay by disc -
7 test. The bioassay indicates that the yield in the fer~
8 mentation flask was 327 micrograms of FR-02A per ml. of
9 fermentation broth or 130 mg. total. ;~
EXAMPLE 3
11 Production of Antibiotic FR-02A by Shake Flask Fermentation
12 o Streptomyces Lactamdurans
-
13 A lyophilized culture of Streptomyces lactamdurans
14 designated MA-2908 is used as inoculum.
-15 The lyophil is opened into a 250 ml. three- ;
16 baffled Erlenmeyer flask containing 40 ml. of First Stage
17 Seed Medium:
18 First Stage Seed Medium
lq Primary yeast 1%
in distilled water
21 pH adjusted to 7.0 with NaOH
22 The first stage seed flask, and the subsequent
23 production flasks are incubated at 28C. on a rotary
24 shaker operating at 220 rpm.
25 After two days in the first stage seed medium, ;
26 one ml. from the first stage seed flask is inoculated
27 into each of 10 non-baffled 250 ml. Erlenmeyer flasks
28 containing 40 ml. of production medium substantially free
29 of suspended nutrient matter and having the following com-
position~
-24-
, : ' ,. .
. ~ ,, ,. . ,. : : ,,
!
'-- 15523IB , ~
~39~S
1 Production Medium
2 Nutrient Broth (Difco) 0O8
3 Yeast extract (Difco) 0,2%
4 Dextrose 1"0%
S in distilled water
6 One drop of P-2000 defoamer is added to each
7 flask prior to autoclaving.
8 The ten production flasks are then incubated for
9 five days at 28C. and harvested. `
Isolation of FR-02A
,, ~ . -
11 The ten production flasks (totaling 400 ml.) are
12 pooled, the pH is lowered to 5.5 with hydrochloric acid, and
13 the broth is filtered to remove mycelia. The clear filtrate ~!~
14 is then mixed with 500 ml. of chloroform and stirred for
1~ one-half hour. The mixture is then centrifuged to separate
16 the phases and the aqueous phase discarded. The chloroform ~;
17 layer is dried over anhydrous magnesium sulfate and eva-
18 porated to dryness in vacuo. The residue is washed with
19 30 ml. of hexane and air dried to obtain the antibiotic
FR-02A.
.
.
21 EXAMPLE 4
22 Production of Antibiotic FR-02A by Shake Flask Fermentation
23 of Streptomyce~ Lactamdurans
_. ~
24 A lyophilized culture of Streptomyces lactamdur- ~ -
25 ans designated MA-2908 is used as inoculum. ~
.
26 The lyophil is opened into a 250 ml. three-
27 baffled Erlenmeyer flask containing 40 ml. of First Stage
28 Seed Medium:
.
-25-
:~ ^
r~
15523IB
~L039~5 .~ ~
1 First Sta~e Seed Medium
2 Primary yeast 1
3 in distilled water
4 pH adjusted to 7.0 with NaOH
The first stage seed flask, and the subsequent ;
6 production flasks are incubated at 28C. on a rotary
7 shaker operating at 220 rpm. ~ ;
8 After two days in the first stage seed medium, ~ ;
9 one ml. from the first stage seed flask is inoculated
into each of 10 non-baffled 250 ml. Erlenmeyer flasks
11 containing 40 ml. of production medium substantially free
12 of suspended nutrient matter and having the following com-
13 pOsition:
14 Production Medium
:~ .
Nutrient Broth (Difco) 0.8
16 Yeast extract (Difco) 0.2~
17 Dextrose 1.0%
18 in distilled water
19 One drop of P-2000 defoamer is added to each
20 flask prior to autoclaving. -~;
21 - The ten production flasks are then incubated for
22 five days at 28C. and harvested. ~
23 Isolation of FR-02A ~ ;
24 The ten production flasks (totaling 400 ml.) are
pooled, the pH is lowered to 5.5 with hydrochloric acid,
26 and the mixture is filtered to recover the mycelia. The ~
27 mycelia is extracted with 500 ml. of chloroform. The chloro- - ;
28 form extract is dried over anhydrous magnesium sulfate and ~ -
29 evaporated to dryness ln vacuo. The residue is washed
with 30 ml. hexane and air dried to obtain the antibiotic
31 FR-02A.
., '
-26-
- . , , : , :
15523IB
~L~35~S
1 EXAMPLE 5
2 Production of Antibiotic FR-02A by Shake Flask Fermentation
3 of Streptomyces Lactamdurans
4 A lyophilized culture of Streptomyces lactamdur-
ans designated MA~2908 is used as inoculum.
6 The lyophil is opened into a 250 ml. three- ~`
7 baffled Erlenmeyer flask containing 40 ml. of Firs-t Stage
~, ,
8 Seed Medium:
9 First Sta~e Seed Medium
:. .
Primary yeast l~
ll in distilled water --
12 pH adjusted to 7.0 with NaOH ~;
13 The first stage seed flask, and the subsequent
14 production flasks are incubated at 28C. on a rotary ~;;
shaker operating at 220 rpm.
16 After two days in the first stage seed medium,
17 one ml. from the first stage seed flask is inoculated
18 into each of 10 non-baffled 250 ml. Erlenmeyer flasks
19 containing 40 ml. of Production Msdium substantially free
of suspended nutrient matter and having ~he following com-
21 position:
.
22 Production Medium
23 Nutrient Broth (Difco) 0.8%
24 Yeast extract (Difco) 0.2
Dextrose 1.0%
26 in distilled water
27 One drop of P-2000 defoamer is added to each
:
28 flask prior to autoclaving. `~
29 The ten production flasks are then incubated for
five days at 28C. and harvested.
.
': .
-27- ~
~ .
:.: ~. , .- . - . ; .
~ : . ~ . : . ~ .
~ 15523IB
~l~39;~S
1 Isolation of FR-02A
2 The ten production flasks (totaling 400 ml.) are
3 pooled, the pH is lowered to 5.5 with hydrochloric acid.
4 Five hundred ml. of chloroform is added, and the mix- ;
ture stirred for one-half hour at room temperature.
6 The mixture is then centrifuged to separate the phases
7 and the aqueous phase discarded. The chloroform layer
8 is dried over anhydrous magnesium sulfate and evaporated
9 to dryness in ~racuo. The residue is washed with 30 ml.
10 of h~xane and air dried to obtain the antibiotic FR-02A. ~ -
11 EX~MPLE 6
12 Production of Antibiotic FR-02A by Shake Flask Fermentation
13 of Streptomyces Lactamdurans
14 A lyophilized culture of Strepto~ ces lactamdur- ~ -
is ans, designated MA-2908 is used as inoculum.
16 The lyophil is opened into a 250 ml. three-
17 baffled Erlenmeyer flask containing 40 ml. of First Stage
18 Seed Medium:
19 First Stage Seed Medium
Primary yeast 1
21 in distilled water
22 pH adjusted to 7.0 with NaOH
23 The first stage seed flask, and the subsequent
24 second stage and production flasks, are incubated at 28C.
on a rotary shaker operating at 220 rpm.
26 After twa days in first stage medium, one ml.
27 from the first stage seed flask is inoculated into 4q ml.
28 of second stage medium in a 250 ml. three-baffled Er~nmeyer ;
29 flask.
-28-
. . ~: ,, '. :: :
15523IB
:`
~ ~ .
1 Second Staqe Seed Medium
2 Ardamine YEP (99F) 1
3 in distilled water
4 pH adjusted to 7.0 with NaOH
The second stage seed flask is incubated for
6 one day, then 1 ml. of the culture is inoculated into each
7 of ten non-baffled 250 ml. Erlenmeyer flasks containing
8 40 ml. of Production Medium having the following composi~
9 tion:
10 Production Medium
._ ,
11 Corn steep liquor 2.8~ by weight
12 cerelose 5.6% by weight
13 Proflo 2.8~ by weight
14 glycerol 1.4% by volume
15 Dimethylformamide 1.4~ by volume
16 in tap water
17pH is adjusted to 7.3 with NaOH
18One drop of P~2000 defoamer is added to each
19 flask prior to autoclaving.
A sodium thiosulfate solution is prepared by
21 dissolving 6.25 g. Na2S2O3 5H2O in 100 ml. distilled
22 water. This solution is filter sterilized, then 0.5 ml.
23 is added to each of the production flasks after autoclaving.
24 The ten production flasks (totaling 400 ml.) are
then incubated for five days at 28C. and harvested.
26 Isolation of FR-02A
27 The ten flasks are pooled, the pH is lowered to
28 5.5 with hydrochloric acid, and the mycelium and suspended
. .
29 nutrient matter is separated from the fermentation broth
by filtration. The filtrate is mixed with 500 ml. of
31 chloroform and stirred for one-half hour. The mixture is ;
32 then centrifuged to separate the phases and the aqueous
33 phase is discarded. The chloroform layer is dried over
.
-29-
: . . , . , : , , .. : . . ~
15523IB
2~S
1 anhydrous magnesium sulfate and the solvent evaporated off
2 in vacuo. The residue is washed with 30 ml. hexane and
3 air dried to obtain the antibiotic FR-02A.
4 Ph~sical Properties
Elemental analysis of FR-02A is as follows~
6 C 60.98
7 H 7.60%
8 N 2.60~
9 The empirical formula is c59H9o~96N2o2l- Thi ~,
is in general agreement with a molecular weight of about
11 1,168 obtained by mass spectrometry.
12 FR-02A as the ammonium salt is soluble in
13 alcohol and chloroform. It is moderately soluble in water
14 at pH 7.0 or higher. A U.V. spectrum of the ammonium salt
lS in water showed:
16 ~ max. 233 nm; Elcm = 320
17 ,~,max. 328 nm; ElCm = 180
18 For FR-02A, ~ = 19 x 10 .
19 The nuclear magnetic resonance spectrum of
antibiotic FR-02A was obtained at 100 ~Iz in CDC13 as
21 the solvent and tetramethylsilane (TMS) as the internal
22 standard. Representative features of the spectrum were
23 doublets at 1.21, 1.31, and 4.63 ~ and a siglet at 4.87
24 FIG. 1 shows the infrared absorption spectrum
of antibiotic FR-02A in a Nujol mull. FR-02A exhibits
26 characteristic absorption in the infr~red region of the
27 spectrum at the following wave lengths expressed in re-
28 ciprocal centimeters:
-30-
. .
,
,
15523IB
~L~3~5 ~: ~
1 Broad band at: 3400
2 Strong bands at: 1640, 1460, 1380, 1080, 1020 `
3 Prominent bands at: 1550, lS05, 1240, 1195, 940, ~ -
4 860, 720, 620
FR-02A was subjected to several analytical sys- -
6 tems and the results are set forth below~
7 1. LH-20 colu~.n in MeOH KD = 0.302
8 ~Charged NH4+salts)
9 2. XAD-2 column 50% iPrOH-H20 -
10 emergence ~Charged free acids) D.V. = 3.5 to 4.0
11 3. T.L.C. Silica gel ~;
12 CHCl3 - MeOH - Rf = 0.344
13 Conc. NH40H 80-20-1
14 4. Paper chromatography
15 isopropanol, phosphate buffer R = 0 9
16 pH 6.0 (0.01 M.), 70-30
17 The E'R-02A is a substantially pure material as ;
18 judged by its being a single spot material on TLC and a
19 single, gaussian shape profile detected by refractometer
monitoring of the LH-20 and XAD-2 analytical columns.
21 To further characterize FR-02A in vitro, ac-
22 tivity data for it was obtained using an antibiotic
23 spectrum profile. The test in,volves applications of a
24 droplet of antibiotic of approximately 0.015 ml. on the
surface of seeded complex agar plates. FR-02A was dissolved
26 in 10~ methanol which, by itself, did not produce zones of
27 inhibition. The results are reported in Table 1 in terms of
28 mm. of inhibition zones.
-31-
. , .:: :: . . : - , ,;. .
.. ...
15523IB
S
1TABLE 1
. . _
2Antibiotic Spectrum Profiles (ASP) of FR-02A
3_ in Agar Diffusion Tests
:
~ .
4Diameters (mm.) ~
5of ~nhihition Zones ~ ~ '
6 FR-02A
7 Organism, MB~ 1 mg./ml.
8 Bacillus sp. 633 18
9 Proteus vulgaris 1012 10
Pseudomonas aeruginosa 979 10H ~ -
11 Serratia marcescens 252 10H
12 Staph ~ us 108 11
13 Bacillus subtilis 964 19
14 Sarcina'''-'lutea 1101 28 '
Staphylococcus aureus 698~ 12H
16 Streptococcus faecalis 753 11
17 Alcallgenes faecalls 10 19
18 Brucella'~bronchiseptica 965 18
19 Sa'lmonel gall~rnarum 1-287 13
Vibrlo percolans 1272 21
21 Xanthomonas vesicatoria 815 13
22 Escherichia coli 1418 15
23 Ps. stutzeri 1231 10
24 Klebsiella pnsumoniae 1264 16
Aerobacter aerogenes 835 13
26 Erwinia atroseptica 1159 12
27 Corynebact. pseudodipth. 261 27
28 S. aureus 3032 12
29 S. aureus 2756 23 -'
~ e faecium 2820 20
31 Proteus v ~ulgaris'838 18
32 E. coli 60 10 '
33 S. aureus (res. Erythromycin) 1909 16
34 H = Hazy Zone
~Resistant to streptomycin, streptothricin,
36 neomycin, and viomycin. ;~
,~ ~
~", ' '' .
- .
-32-
15523Is
.~
~)39~15 ~:1 Antibiotic FR-02A ln vivo has a low toxicity and ~ ~
2 broad spectrum oE antibacterial activity when tested in ~ -
3 mice. As an example the results for a gram-negative
4 bacterium and a gram positive bacterium are presented in
5 Table 2.
~ '
:
6 TABLE 2
7In Vivo Test Results With FR-02A in Mice
.
8 Infection Therapy ' Toxicity
.~ ._ ._ _ I ___
ED
9 Organism Route Route 50 Tolerated Toxic
.. ..... -- ~,
10 Proteus vulgaris IP IP 1.73 5.0 ~5.0
11 Strep. pyogenes IP IP 0.247 1.25 5.0
12 ~ Values in the table are expressed in mg./mouse.
13 Both infections and therapeutic doses of FR-02A were ad-
14 ministered intraperitoneally. FR-02A was administered at
time of infection and again 6 hours later.
16 ~ Toxicity studies of FR-02A in noninfected miGe
17 indicate that two doses of 2.5 mg./mouse, 6 hours apart are
18 tolerated; but higher doses i.e. 5 to 10 mg./mouse is toxic.
:
,
~ .
:
` . ' '
' ' ''.
-33-
. , .
.. . .. . . . . . ..
15523Y '.
~039~S
The antibiotic FR-02A is known under the generic term
"efrotomycinlland corresponds to the following structure:
UH O ~ ~ OH
7~ o 3 0~ OH H5C2 ~ CH3
CH3 0
,~0~
CH3 0 ~ OH
~ ~ OCH3
CH30~oCH3
OH
:'; '
' ' " '
- 33A - -
, .~.
t~,-~.
-
