DE2450813C2 - Antibiotic FR-02A - Google Patents

Description

; £! * = 320 ; £ Jl = 180;

an inert carrier in a growth promoting agent for ΉεΓε.

h) Rf = 0. M4 in thin layer chromatography on a silica gel plate while developing with a solvent system of 80% Chloroform, 20% methanol, and 1% concentrated ammonium dioxide;

i) Rf = 0.9 on paper chromatography below Development in the solvent system of 70% isopropanol and 30% phosphate buffer solution (0.01 molar), pH 6.0;

j) nuclear magnetic resonance spectrum with a doublet at 1.21, 131 and 4.63 τ and a singlet at 4.87 τ,

as well as the pharmaceutically acceptable, non-toxic salts thereof.

2. Process for the preparation of the antibiotic FR-02A according to claim 1, characterized in that Streptomyces lactamdurans NRRL 3802 is grown aerobically in an aqueous nutrient medium, whereupon the antibiotic formed FR-02A

a) from the entire fermentation liquid with a water-immiscible polar organic solvents or

b) from the fermentation liquid from which the solids have been separated with a water-immiscible polar organic solvents or

ς) from the solids separated from the fermentation liquid with a polar £> o organic solvents

extracted.

3. Use of the antibiotic FR-02A or a pharmaceutically safe, non-toxic f> 5 see salt thereof along with a non-toxic, pharmaceutically acceptable excipient in a therapeutic agent or together with The invention relates to the method defined in claim 1 antibiotic, said in claim '· £ process for producing this antibiotic and the use of this antibiotic, as she calls the patent claim 3

FR-02A is an antibiotic that is effective against both gram-positive and gram-negative bacteria acts and as a result are used to treat a wide variety of animal infections can, in particular, FR-02A is effective against PPLO in chickens, pigs and cattle. It is also effective against mouse coccidiosis and against the most prevalent types of chicken coccidiosis. It also works Antibiotic subcutaneously against air suculitis caused by M. gallisepticum in chickens and orally in Mice against systemic infections caused by Bordetella bronchiseptica. Furthermore, FR-02A can be used as growth-promoting agents can be used in animals such as chickens, pigs and cattle.

The antibiotic FR-02A is obtained by cultivating the already known microorganism Streptomyces lactamdurans NRRL 3802 under controlled conditions in a fermentation liquid and extracting the whole fermentation liquid obtained with a water-immiscible polar organic solvent. The fermentation can take place in media that contain suspended nutrients, or be carried out in predominantly clear media that are practically free of suspended nutrients.

If the fermentation is carried out in media containing suspended nutrients, this will be found Antibiotic both in the fesl; ! open of the mycelium and the suspended nutrients as well as in the fermentation liquid. The antibiotic is isolated from the solids by removing the solids from the The fermentation liquid is separated by filtration, centrifugation or in some other way and the solid cake extracted with an organic solvent, preferably a polar organic solvent. The antibiotic that was left in the liquid from which the solids were previously separated is isolated from the liquid by extraction with a water-immiscible polar organic solvent. It should be noted that the total amount of solids in the fermentation liquid is reduced by delayed harvesting, and a lesser amount of the antibiotic is found in the solids recovered from the liquid

Fermentation media that do not contain suspended nutrients contain most of the FR-02A in the predominantly clear fermentation liquid. In these cases, all of the liquid is used a water-immiscible polar organic solvent, such as chloroform, extracted to the Win antibiotic. But you can also remove any solids, such as mycelium, from the fermentation liquid separate before the latter with the water-immiscible polar organic solvent extracted. Further, in order to recover any remaining FR-02A, the mycelium separated from the fermentation liquor is extracted with a suitable polar organic solvent.

The preferred method for obtaining the antibiotic according to the invention is the cultivation of the known microorganism Streptömyces lactamdurans NRRL 3802 down controlled ^ Be ^^ a medium which contains suspended solids ^ or. in a clear medium that is free from; suspended solids is, and extracting the whole; Liquid with a water-immiscible * polar organic solvent. The extraction is carried out by adjusting the pH of the liquid to an acidic range and adding the solvent to the liquid. After mixing, the solids are separated from the liquid. The liquid is left to stand until the solvent layer has separated out The solvent layer is stripped off, washed with water, dried with a suitable drying agent and evaporated in vacuo. The residue is washed with a non-polar organic solvent, such as petroleum ether or hexane, and air-dried. This gives the antibiotic FR-02A. " ^R

A preferred method for obtaining the antibiotic FR-02A is to cultivate the known one Microorganism: Streptömyces lactamdurans NRRL 3802 under controlled conditions in one Medium Containing Suspended Nutrients The solids including the mycelium and suspended nutrients are filtered off from the fermentation liquor after the filter cake is so dry has been blown as possible, it will turn into a polar » Stir in organic solvent and filter off again. After washing the filter cake 3 times with The combined solvent extracts and washing liquids are further solvent in vacuo evaporated, leaving an aqueous slurry. The pH value becomes acidic Range adjusted The aqueous slurry is made with a non-polar organic solvent such as Petroleum ether, hexane and the like, washed until the washing liquid »!! are colorless. Then the aqueous Slurry extracted with a polar organic solvent. The solvent extract is extracted dried, filtered and evaporated in vacuo, the antibiotic FR-02A remaining as a residue According to the method according to the invention, for. B. the antibiotic FR-02A ίίι about 40 to Preserved 50 percent pure form.

The other method can be fermentation in a medium which is practically free of suspended nutrients. The mycelium will be carried out filtered off from the fermeni: ation liquid and with extracted with a polar organic solvent. The extract is dried and in vacuo to Evaporated to dryness. The residue is shaken with a non-polar organic solvent such as petroleum ether, hexane and the like. The organic solvent is decanted after the Residue brought to settling by 2Leritrifugation Has. The residue is air-dried and the antibiotic FR-02A is obtained. -

An even better method for obtaining the antibiotic according to the invention is to cultivate the Microorganism Streptömyces lactamdurans NRRL 3802 under controlled conditions in one Fermentation medium and extraction of the medium after separating the solids, namely the mycelium or the mycelium and suspended nutrients. The pH of the fermentation liquid is adjusted to the acidic area set and the solids, namely either just mycelium or mycelium and suspended Nutrients are separated The fermentation liquid, which is free of solids, is mixed with a Water immiscible polar organic solvent mixed. After mixing, the Layers separate from each other. The organic layer is peeled off, dried with a suitable desiccant and dried to dryness in vacuo evaporated The residue is with a non-polar organic solvent such as petroleum ether or Hexane, washed and air dried, being the antibiotic FR-02A remains

If the extractions in the procedures described above use a water immiscible polar organic solvents are carried out, one can use solvents such as alkyl esters of lower alkanecarboxylic acids, such as methyl formate, ethyl formate, methyl acetate, ethyl acetate, n-butyl acetate, isobutyl acetate, ethyl propionate, ketones, wh cyclohexanone, or lower halogenated hydrocarbons such as chloroform, methylene chloride, carbon tetrachloride, ethylene dichloride, l-chloro ^ -dimethylpropane, tetrachlorethylene or bromoform.

When the above-described extraction processes are carried out with a polar organic solvent, solvents such as lower Alkyl esters of lower alkanecarboxylic acids, such as methyl formate, ethyl formate, methylacetate, ethyl acetate n-butyl acetate, isobutyl acetate, ethyl propionate, ketones, such as acetone, methyl ethyl ketone or cyclohexanone, or lower halogenated hydrocarbons such as chloroform, Methylene chloride, carbon tetrachloride, ethylene dichloride, 1 -Chlor ^ -dimethylpropane, tetrachlorethylene or Bromoform.

The microorganism not only produces FR-02A, but, as is known from the literature, produces Streptömyces lactamdurans NRRL 3802 the antihioticum cephamycine (see »Antimicrobial Agents and Chemotherapy ", September 1972, pp. 122-131, Vol. 2 Ni 3," Cephamycins, a New Family of ^ -Lactam Antibiotics ", as well as BE-PS 7 64 160). Since FR-02A-, in with Water-immiscible organic solvents are highly soluble, while Cephamycin C is in with water Immiscible organic solvents are as good as insoluble, these two substances can easily be separated separate from each other, and obtained by extracting the fermentation liquid with one with water immiscible solvents of either antibiotic in one of the other antibiotic not contaminated form.

The antibiotic FR-02A isolated from the fermentation liquid is of course carried out further Chromatography on a molecular sieve and subsequent chromatography on a surface-active one Adsorbent cleaned. A suitable molecular sieve for this is a cross-linked dextran like that Molecular sieve »Sephadex LH-20«. FR-02A can be made from this molecular sieve with a lower alkanol. how Methanol. A suitable surface-active adsorbent is a hydrophobic, nonionic, macroporous, crosslinked with divinylbenzc-I Copolymer of styrene which is commercially available under the names "Amberlite XAD-I to XAD-12". A Preferred resin for purification of FR-02A is "XAD-2". To elute the adsorbed FR-02A suitable solvents are aqueous solutions of lower alkanols, e.g. B. aqueous solutions of

Methanol, ethanol, isopropanol, butanol and the like. The preferred solvent for eluting FR-02A made from "XAD-2" resin is a 50 percent solution of isopropanol in water.

The microorganism that produces FR-02A is the already known strain Streptomyces lactamdurans NRLL 3802. It was isolated from a soil sample and placed in the Northern Utilization Culture Collection Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture ίο (formerly Northern Regional Research Laboratories), Peoria, Illinois 61604, for the duration without restrictions on accessibility and is available to the Available to the public under culture number NRRL 3802. is

Full taxonomic and morphological studies of Streptomyces lactamdurans can be found in the BE-PS 7 64 ίδΟ. Aiii reason νυϊι cäxünüriiisciieri Research has identified Streptomyces lactamdurans as a new Actonomycete. It was found, that it belongs to the genus Streptomyces and has many characteristics of the well-known. Species Streptomyces fradiae exhibits biochemically the two almost completely agree; morphologically, however, are important Differences exist. So is z. B. the color of the aerial mycelium of S. fradiae sea mussel pink, whereas S. lactamdurans is creamy due to this Different and other characteristic features became the species name of the microorganism Streptomyces lactamdurans given.

FR-02A is used in the aerobic fermentation of a suitable aqueous nutrient medium. «; under controlled Conditions created by inoculation with the organism Streptomyces lactamdurans NRRL 3802. Aqueous media such as those used for the generation of others Antibiotics used * are also suitable for the manufacture of the antibiotic FR-02A. Such media contain sources of carbon, nitrogen and inorganic sources that can be assimilated by the microorganism Salts. The choice of medium is not critical and the fermentation can be carried out in media that contain suspended nutrients or those that are predominantly clear and practically free of suspended nutrients.

In general, carbohydrates such as sugars, e.g. B. dextrose, glucose, arabinose, maltose, raffinose, xylose, mannitol and the like, and starches such as grain, ζ. B. oats, rye, corn starch, corn flour and the like, in the nutrient medium either alone or in combination as sources for assimilable carbon so are used. The exact amount of the carbohydrate source or sources in the medium will depend in part on the other components of the medium; however, in general the amount of carbohydrate will vary between about 1 and 6 percent by weight of the medium. A single substance can be used as the C source, or several C sources can be combined with one another in the medium. Many protein substances can be used as N sources in the fermentation process. Suitable N sources are e.g. B. nutrient broth, yeast extract, yeast hydrolyzates, primary yeast, soybean meal, cottonseed meal, casein hydrolyzates, corn steep liquor, soluble pulp ingredients or tomato paste and the like. The nitrogen sources may be used either alone or m combination with one another the S ⁶ are applied in amounts of from about 0.2 to 6 weight percent of the aqueous medium.

The fermentation processes described in the examples

media are only used to explain the multitude of different possible media.

The fermentation takes place at temperatures of about 20 to 37 "C; for the best results however, the fermentation is preferably carried out at temperatures of 24 to 32 ° C. Of the pH of the nutrient medium for growing and producing the culture of Streptomyces lactamdurans of the antibiotic FR-02A is said to be in the range of about 6.0 to 8.0.

On a small scale, fermentation of the antibiotic is conveniently carried out by inoculate a suitable nutrient medium with the culture producing the antibiotic and after the transfer on the production medium the fermentation is allowed to proceed for several days in the shaking machine at a constant temperature of about 28 "C. At the end of the iiikuuaiiuiiszcii, the fviycei and the suspended nutrients can be centrifuged or filtered off and with a solvent can be extracted, or the entire liquid can be extracted with chloroform or with Extract water immiscible liquids. But you can also use the fermentation liquid extract after having the solids consisting of mycelium or of mycelium and suspended nutrients, has separated from the same.

Dk fermentation is carried out on a small scale in a sterilized flask by one-step, two-step, three-step or four-step inoculum development. Any suitable combination of C and N sources can be used as the nutrient medium for the vaccination stage. The inoculation flask is shaken in a constant temperature chamber at about 28 "C for 1 to 2 days, and some of the culture obtained is used to test either a second-stage inoculation medium or the product medium the inoculum developed in these essentially in the same way; that is, part of the contents of the flask is used for inoculating the production medium.The inoculated flasks are shaken for several days at constant temperature, and at the end of the incubation period the antibiotic FR-02A, as already described , isolated

On a large scale, the fermentation is preferably carried out in suitable fermenters with a stirrer and devices for aerating the fermentation medium are equipped. After this Method, the nutrient medium is prepared in the fermenter and heated to temperatures up to about 120 ° C sterilized After cooling, the sterilized medium is cultured with a previously Inoculate the culture culture inoculated and fermentation for several days, e.g. 2 to 4 days, with movement and / or aeration of the nutrient medium and pause carried out at a temperature of about 28 ° C. The yield of FR-02A is generally from 20 to 200 mg per liter of cultivation volume, determined by bioanalysis.

Analytical method

Analyzes are carried out by the test disc plate method using 9.5 mm filter paper discs. The analysis plates are carried out with Difco nutrient agar and 2.0 g Difco yeast extract each Liter made in an amount of 10 ml per plate Cultivation of the test organism Vibrio percolans (American Type Culture Collection

ATCC 8461) in nutrient broth with a content of 0.2% yeast extract becomes a Diluted suspension with a permeability of 40% at a wavelength of böO πιμ. Before watering this suspension is added to the medium in an amount of 20 ml per liter of the plates.

The assay plates are (maximum 5 "Oge) maintained at 4 ° C until use. After laying the saturated with the antibiotic test discs, the plates are incubated for 16 to 24 hours at 28 ⁰ C. The ι ο inhibition zones are read in mm diameter and to determine the relative strength or, if compared with a purified reference standard, the strength in y / ml. Analyzes of the FR-02A in fermentation fluids, mycelium and the suspended nutrients separated from the fermentation fluids as well as in the fermentation fluids free of nutrients are carried out according to After extracting the FR-02A with a suitable solvent, the analyzes of solutions containing FR-02A at a concentration of 200 μg / ml show zones of inhibition of 16 mm when using 9.5 mm discs Analysis is carried out quantitatively, 50 to 100 μg / ml antibiotic can be detected. '

FR-02A is active against gram-negative and gram-positive bacteria, coccidia and species of Mycoplasma. In vitro FR-02A is effective against E acervulina, Bordetella, Streptococcus faecalis. Streptococcus faecum, Streptococcus agalactiae, Streptococcus pyogenes, Proteiij vulgaris, M. hyorhinis, M. synoviae, M. arthritidis, M. gallisepticum and species of Pasteurella. There was also activity against Vibrio percolans (ATCC 8461), Salmonella gallinarum (MB 1287). Escherichia coli (MB 1418), Klebsieila pneumoniae (MB 1264), Pseudomonas stutzen (MB 1231) and (MB 2765), Bacillus subtilis (MB 964) and (MB 7971 Staphylococcus aureus (MB 108), (MB 210) and (MB 703) as well as against Pseudomonas aeruginosa (MB 3210)

FR-02A can be used both as an antibiotic and as a growth-promoting agent for animals.

When FR-02A is used as an antibiotic, the particular way of administration to animals is not particularly crucial; all currently used or used to treat infected or Methods available to animals at risk of infection are satisfactory.

FR-02A can be used as an antibiotic, e.g. in the form of pharmaceutical preparations which also contain an organic or inorganic, solid or liquid pharmaceutical diluent, which is suitable for enteral, parenteral or local administration Suitable extenders are substances who do not react with the antibiotic, e.g. B. water, Gelatin, lactose, starches, sfearyl alcohol, magnesium ss stearate, talc, vegetable oils, benzyl alcohol, resins, Propylene glycol, polyalkylene glycols, white petrolatum, Cholesterol or other known medicinal excipients. The pharmaceutical preparations can, for. B. as Tablets, coated tablets, ointments, creams or capsules or in liquid form as solutions, suspensions or emulsions. You can be sterilized and / or auxiliaries, such as preservatives, stabilizers, wetting or emulsifying agents, solubilizers, salts for regulating the osmotic pressure or Buffer, included. -

If the antibiotic is to be presented in dry, solid unit dose form, then one is used Capsules, pills or tablets that contain the desired amount of antibiotic. These dosage forms are prepared by intimately and uniformly the active ingredient with suitable finely divided diluents, fillers, disintegration-promoting agents and / or Binders such as starch, lactose, talc, magnesium stearate, vegetable resins and the like. Such Unit dose formulations may vary depending on factors such as the type of host animal being treated, the The severity and type of infection and the weight of the host animal, in terms of their total weight and theirs The content of FR-02A can vary within wide limits. The antibiotic can be used in amounts of about 5 to 100 mg per kg of body weight can be administered.

The invention also includes the non-toxic, pharmaceutically acceptable salts of FR-02A, e.g. B. the alkali and alkaline earth salts, such as the salts of sodium, potassium, ammonium and calcium, or salts with organic bases such as triethylamine, N-Äihyipiperidin and Dibenzyiäthylendiamin.

In addition to being an antibiotic, the FR-02A can also be used as a feed additive to prevent the growth of To speed animals such as chickens, sheep and cattle. By using FR-02A, reduces the time it takes for animals to reach the weight required for sale.

When FR-02A is used as a growth enhancer for animals, it can be used as part of feed or be given in solution or suspension in drinking water.

When FR-02A is used as an ingredient in feed, it is first formulated as a feed additive In such feed additives is FR-02A in relatively high concentrations in an intimate distribution in one contain inert carriers or diluents. The feed additive can be added directly to the feed or be processed into a premix in a dilution or mixing stage. Is an inert carrier one that does not react with the antibiotic and that can be safely administered to the animals. A carrier is preferably used which is or can be a component of the diet of the animals. Typical carriers or diluents suitable for such mixtures are e.g. B. dried distillery residues, corn flour, citrus flour, fermentation residues, ground oyster shells, wheat milling waste, soluble molasses, corn on the cob flour, edible bean mill charge, soy groats, crushed limestone, and the like. The antibiotic is after methods such as stirring, grinding or Agitation, intimately dispersed in the vehicle, mixtures containing the antibiotic in concentrations of about 5 to Contain 50 percent by weight, are particularly suitable as feed additives.

Examples of typical feed additives that FR-02A in Dispersion contained in a solid carrier are:

FR-02A kg (A) Wheat milling waste 5 FR-02A 95 (B) Corn distillery residues 50 50

These and similar feed additives are made by evenly mixing the antibiotic with the Carrier made

The feed additive can be added directly to the feed or by further diluting or mixing with one

orally administrable carrier can be processed into a premix. Mixtures containing the antibiotic in Containing concentrations of 0.03 to 5 percent by weight are particularly suitable as premixes. These premixes are made by mixing the antibiotic evenly with an orally administered Carrier mixes.

Such additives or premixes are added to the feed in such quantities that the finished Lining the FR-02A in for speeding up the ι ο Contains the desired concentration for growth. For chickens, FR-02A is used at a final concentration of 50 Up to 300 g per ton of feed is given in order to achieve the desired acceleration of growth. In the case of pigs, including pigs infected with M. hyorhinis, FR-02A can be used in the Food can be given in similar concentrations.

In the above description of the invention, reference is primarily made to solid mixtures where the FR-02A is in the feed additive, the so-called premix or in the finished feed is in admixture with an edible carrier. This is the preferred method of administering FR-02A. Another method is to dissolve or dissolve the FR-02A in the animals' drinking water suspend. The amount that will be suspended in the water without risk of excessive settling can is limited. You can therefore also add emulsifiers or surfactants.

The feed additives and finished animal feed mixes that contain FR-02A can also contain vitamins, Contain other antibiotics and growth-promoting agents or other nutrients.

FR-02A is against poultry PPLO in amounts in Effective range from 5 to 100 mg / kg. A preferred range for a single dose is 35 to 45 mg / kg. For the sake of simplicity, the preferred method of administering the antibiotic is for Treatment of PPLO in missing the FR-02A with the animal feed. For the fight against PPLO is a preferred range from 0.0055 to 0.02 Weight percent of the feed.

For the treatment of aerial sacculitis in chickens, the ED ₅₀ value is 40 to 100 mg / kg. Accordingly, the dose of FR-02A can vary in the range from 10 to 150 mg / kg.

A solution or suspension for subcutaneous injection used to treat air suction Chickens can be made in the following ways:

Ampoule with subcutaneous solution or suspension containing 20 mg FR-02A

FR-02A 20 mg _ ₅

Diluent:

sterile water for injection 2 cm ³

The preferred method for treating coccidiosis is to have the FR-02A in the feed in a concentration of about 0.05 to 2 percent by weight of the feed

The correct dose depends largely on the condition and weight of the Host animal, and for the treatment of PPLO and coccidiosis, oral administration is preferred The FR-02A is preferably used as a growth-promoting agent in admixture with the feed presented

example 1

Manufacture of the antibiotic FR-02A

by shaking bowl fermentation of Streptomyces lactamdurans N RRL 3802

A freeze-dried culture is used as the inoculation material.

The freeze-dried culture is placed in a 250 ml Erlenmeyer flask fitted with three deflectors which contains 40 ml of first-stage inoculation medium:

First stage inoculation medium

Primary yeast in distilled water to adjust the pH
with NaOH to 7.0

1%

The first stage seed flask and the subsequent second stage piston and the production flasks are incubated in a 220 U / min working shaker at 28 ⁰ C.

After 2 days in the first-stage medium, 1 ml of the first-stage inoculum is used to inoculate 40 ml of the two-stage inoculation medium in a 250 ml Erlenmeyer flask equipped with three deflectors used.

Second stage inoculation medium

»Ardamine YEP« (99F) 1%

in distilled water;
pH to 7.0 with NaOH
set.

The second-stage inoculation flask is incubated for one day, after which ten, not with baffles equipped 250 ml Edenmeyer flasks, each 40 ml Production medium contained, inoculated with 1 ml of this culture each.

Production medium Primary yeast 1% Soluble slurry ingredients 3 / o Glycine 0.05% L-phenylalanine 03% Cornstarch 2.0% Dimethylformamide l.OVoL-% "Mobil par-S" 0.25% by volume as an anti-foaming agent in distilled water; pH to 7.0 with NaOH set

A sodium thiosulfate solution is prepared by dissolving 12 g of Na ₂ S ₂ O ₃ · 5 H ₂ O in 100 ml of distilled water. This solution is filter-sterilized, after which 1 ml of the solution is added to each of the 10 production flasks

The 10 flasks are incubated for 4 days at 28 ° C. and then harvested

Isolation of FR-02A

The pH of 400 ml of the thus obtained Fermentation liquid is adjusted to 5 with hydrochloric acid, whereupon 2 parts by volume of chloroform are added Add liquid After thorough mixing, the liquid is filtered through a filter plug To accelerate the filtration, water is added. The filtrate is allowed to stand until the chloroform layer appears The chloroform layer has separated

stripped off, washed twice with 500 ml of water each time, dried over anhydrous magnesium sulfate and im Evaporated to dryness in vacuo. The solid residue is mixed with 500 ml of petroleum ether, filtered off, vnit Washed 50 ml of petroleum ether and air-dried. 144 mg of FR-02A are obtained. The FR-02A is built into the The ammonium salt is converted by a solution of the FR-02A in 50 ml of water, which is mixed with ammonium hydroxide has been adjusted to a pH of 10, is freeze-dried. After freeze drying the product weighs 140 mg.

Since FR-02A accumulates together with the mycelium and the suspended nutrients, the mycelium and first filter off the suspended nutrients from the fermentation liquid and remove the FR-02A from the filter cake with a polar, water-miscible organic solvent such as acetone, extract. This method of isolating FR-02A is carried out as follows:

The mycelium and the suspended nutrients are filtered off from 400 ml of the total fermentation liquid obtained in the fermentation described above. The filter cake is stirred with 40 ml of acetone for 30 minutes and filtered again. The cake is then washed with 15 ml of acetone. The combined acetone extracts and washings are evaporated in vacuo at 30 ⁰ C to drive off the acetone. The residue is taken up in 15 ml of water. The pH is adjusted to 4.0 with hydrochloric acid

An equal volume of hexane is added and the mixture is stirred for 10 minutes. After settling, the hexane becomes decanted and thrown. Extract twice more with equal volumes of hexane, the last extract being colorless

The aqueous slurry is then extracted with an equal volume of chloroform. Then extracted one more time with an equal volume and combines the second extract with the first. the aqueous phase is discarded.

The chloroform extract is dried over anhydrous sodium sulfate, filtered and added in vacuo 106 mg FR-02A evaporated

Chromatography of the fermentation process described above Material on "Sephadex LH-20" is determined that the molecular weight of the FR-02A is about 1000 Das material obtained from this treatment is again, this time on »Amberlite XAD-2«, chromatographies, to get an analytically pure sample.

Chromatography on "Sephadex LH-20" gel

0.5 ml of a methanol solution containing 106 mg of the FR-02A obtained according to Example 1 are applied to a 75 ml layer (1.25 cm in diameter and 56 cm in height) of “Sephadex-LH-20” gel in methanol . The column is developed with methanol. The eluate flow is monitored with the aid of a differential refractometer and the diagram thus obtained shows 3 maxima. Fractions are analyzed in a 1 to 10-fold dilution with 12.7 mm paper flakes on agar diffusion test plates inoculated with Vibrio percolans. Zones of inhibition are observed which correspond to the established third mass maximum (Kd = 0.3). The fractions corresponding to the third mass maximum are combined and evaporated to give 43 mg of FR-02A-If the product is analyzed with Vibrio percolans according to the biological agar diffusion method described above, it gives a zone of inhibition of 16 mm at a concentration of 200 μg / ml. The ultraviolet absorption in a phosphate buffer solution with a pH of 7.0 gives the following values;

λ ™ ,. 327 nm; Egg * = 216
A _mat . 232 nm; E \ * _m = 464

If the product obtained according to Example 1 is in ίο water at a pH value of 9 (which is obtained by adding dilute sodium hydroxide solution is set), the insoluble material is filtered off and the filtrate is freeze-dried cleaning with "Sephadex LH-20" gel can be omitted.

Chromatography on Amberlite XAD-2 resin

The 43 mg of product obtained in the purification with "LH-20" are further processed by chromatography

112 cm high) "Amberlite XAD-2" resin in 50 percent Isopropanol purified in water. The starting material is acidified to a pH value of 2 in order to convert it into the to convert free acid before it is introduced into the column. The column is driven with the help of a differential

monitored by a refractometer, and the diagram obtained shows 2 mass maxima. Biological analyzes performed by agar diffusion with 6.4 mm slides using Vibrio percolans show that those centered at Kd 3.5-4.0

Mass maxima contain the antibiotic. The fractions that correspond to these Ko-values will verienigt together and evaporated to dryness in vacuo. This gives 16.4 mg of the antibiotic FR-02A. FR-02A is obtained as a pale yellow solid material that is resistant to normal handling and is purely analytical.

Example 2

Manufacture of the anti-diotic FR-02A

by shake flask fermentation of Streptomyces lactamdurans NRRL 3802

A freeze-dried cult is used as inoculation. The freeze-dried culture is put into one with three Deflection organs provided 250 ml Erl8nmeyerkoiben entered the 40 ml first-stage inoculation medium of the contains the following composition:

First stage inoculation medium

Primary yeast in distilled water, 1% with NaOH to pH set from 7.0

The first-stage inoculation flask as well as the subsequent second-stage flask and the production flask become incubated in a shaker operating at 220 rpm at 28 ° C

After 2 days of incubation in the first stage medium

1 ml of the first-stage inoculum is used for inoculating 40 ml of the second stage vaccine medication in one with three Deflection organs provided, 250 ml Erlenmeyer flasks used

Second stage inoculation medium

»ArdamineYEP« (99F) 1%

in distilled water;
adjusted to pH 7.0 with NaOH

The second-stage inoculation flask is incubated for a day, after which ten, not with deflecting organs provided 250 ml Erlenmeyer flasks, each 40 ml Production medium included, mh per 1 ml of each Culture inoculates the production medium has the following composition:

Production medium Corn steep water 2 pounds wt% Cerelose 5.6% by weight

»Proflo« 2.8% by weight

Glycerine 1.4% by volume

Dimethylformamide 1.4% by volume in tap water, with NaOH adjusted to a pH of 73

0.2% 1.0%

A drop of anti-foaming agent (»P-2000«) is added to each flask before being autoclaved

A sodium thiosulfate solution is prepared by dissolving 6.25 g of Na ₂ S ₂ Os-5 H ₂ O in 100 ml of distilled water. This solution is filter-sterilized, after which 0.5 ml of this sodium thiosulfate solution is added to each of the production flasks after the autoclave treatment

The ten production flasks (400 ml in total) are incubated for 5 days at 28 ° C. and then harvested

The contents of the ten flasks are combined, the pH is adjusted to 5.5 with hydrochloric acid, whereupon 500 ml of chloroform are added and the mixture is stirred for </ ₂ hours at room temperature. The mixture is then centrifuged to separate the phases and the aqueous phase is discarded. The chloroform layer is dried over anhydrous magnesium sulfate and the solvent is evaporated off in vacuo. The residue is shaken with 30 ml of hexane.After the residue is centrifuged off, the hexane is decanted for the biological analysis, the pH value is then reduced to 83 with hydrochloric acid and the solution is diluted with water at a pH value of 83 for the analysis after the disc test. that the yield of the fermentation flask 327 γ FR-02A per ml of fermentation liquid or a total of 130 mg

Example 3 Manufacture of the antibiotic FR-02A

by shake flask fermentation from Streptomyces lactamdurans NRRL 3802

A freeze-dried culture is used as the inoculation material.

The freeze-dried culture is placed in a 250 ml Erlenmeyer flask equipped with three deflectors entered, which contains 40 ml of first-stage inoculation medium of the following composition:

First stage inoculation medium

Primary yeast in distilled water, 1% with NaOH to pH set from 7.0

The first-stage inoculation flask and the subsequent production flask are in one at 220 rpm

working shaker at 28 ° C

After two days of incubation in the first-stage inoculation medium, ten do not have diverting organs equipped 250 ml Erlenmeyer flasks, each 40 ml Contain production medium, which is practically free of suspended nutrients, each with ImI des first-stage inoculation medium The production medium has the following composition:

Production medium Nutrient broth (Difco) Yeast extract (Difco)

Dextrose

in distilled water.

A drop of tree preventive agent (»P-2000«) is added to each flask before being autoclaved

The ten production flasks are then incubated for 5 days at 28 ° C. and harvested

Isolation of FR-02A

The contents of the ten production flasks (a total of 400 ml) are combined and the pH value is increased with hydrochloric acid 5.5 adjusted and the fermentation liquid filtered, to remove the mycelia a The clear filtrate is with 500 ml of chloroform mixed and stirred for 1/2 hour. Then the mixture is centrifuged to lock the phases separate and the aqueous phase is discarded. The chloroform layer is dried over anhydrous magnesium sulfate and in vacuo to dryness evaporated. The residue is washed with 30 ml of hexane, whereupon the Receives antibiotic FR-02A

Example 4 Manufacture of the antibiotic FR-02A

by shake flask fermentation from Streptomyces lactamdurans NRRL 3802

A freeze-dried culture is used as the inoculation material.

The freeze-dried culture is placed in a 250 ml Erlenmeyer flask fitted with three deflectors which contains 40 ml of first-stage inoculation medium of the following composition:

First stage inoculation medium Primary yeast in distilled water, 1%

with NaOH to pH set from 7.0

The first-stage inoculation flask and the subsequent production flask are incubated ^{at 28 s C in a shaking machine operating at 220 rpm}

After two days of incubation in the first-stage inoculation medium, ten do not have diverting organs equipped 250 ml Erlenmeyer flasks, each containing 40 ml production medium, which is practically free of suspended nutrients is inoculated with 1 ml each of the first-stage inoculation medium.The production medium has the following composition:

Production medium Nutrient broth (Difco) Yeast extract (Difco)

Dextrose

in distilled water.

0.8% 0.2% 1.0%

A drop of anti-foaming agent (»P-2000«) is added to each flask before being autoclaved

Then the ten production flasks are harvested 5 days at 28 ^o Cmkubiert and

Isolation of FR-02A

The contents of the ten production flasks (a total of 400 ml) are combined and the pH value is increased with hydrochloric acid 5.5 adjusted and the mixture filtered to win the mycelium. The mycelium is mixed with 500 ml of chloroform extracted The chloroform extract is dried over anhydrous magnesium sulfate and in vacuo to Evaporated to dryness. The residue is washed with 30 ml of hexane, and dried in air the antibiotic FR-02A is obtained.

Example 5 Manufacture of the antibiotic FR-02A

by shake flask fermentation by Streptonsyces! actsmdurans NRRL3802

A freeze-dried culture serves as inoculation well.

The freeze-dried culture is put into one with three 250 ml Erlenmeyer flasks fitted with deflection organs entered, the 4OmI first-stage inoculation medium of the contains the following composition:

First stage inoculation medium

Primary yeast in distilled water, with NaOH to pH set from 7.0

1%

Production medium Nutrient broth (Difco) Yeast extract (Difco)

Dextrose

in distilled water.

03% 0.2% 1.0%

Example 6 Production of the antibiotic FR-Ö2A

by shake flask fermentation . by Streptomyces lactam durans NRRL3802

A freeze-dried culture is used as the inoculation material. The freeze-dried culture is placed in a 250 ml ErIenmeyer flask equipped with three deflectors entered, which contains 40 ml first-stage inoculation medium of the following composition:

15th

First stage inoculation medium

Primary yeast in distilled water, with NaOH to pH set from 7.0

1%

The first-stage inoculation flask and the following one second stage piston and the production piston v ^ xlen incubated in a shaker operating at 220 rpm at 28 ° C

After 2 days of incubation in the first stage Medium is I mi of the first-stage inoculum for Inoculating 40 ml of the second stage inoculation medium in

one with three deflectors, 250 ml Erlenmeyer flask used

30th

The .ratstufige seed flask and the subsequent production flasks are incubated in a 220 U / min working shaker at 28 ⁰ C

After 2 days of incubation in the first stage Impfmediuin be ten not provided with deflection members 250 mI-Erlenmeyer flask containing 40 ml of production medium which is substantially free <o suspended nutrients ml with 1 of the first stage seed medium inoculated The production medium has the following composition :

Each flask is used before autoclaving a drop of anti-foaming agent (»P-2000«) added

Then the ten production flasks are at for 5 days Incubated at 28 ° C and harvested.

Isolation of FR-02A

The contents of the ten production flasks (in total 400 ml) is combined and the pH value is adjusted to 54 with acidic acid. After the addition of 500 ml of chloroform the mixture is 1/2 hour at room temperature then the mixture is centrifuged to separate the phases and the aqueous phase is discarded. The chloroform layer is dried over anhydrous magnesium sulfate and im Evaporated to dryness in vacuo. The residue is washed with 30 ml of hexane and air-dried. This is how you get the antibiotic FR-02A.

Second stage inoculation medium

»Ardamine YEP« (99F) in distilled water, with NaOH to pH set from 7.0

1%

45

50

The second-stage inoculation flask is incubated for one day, after which you do not have to do ten with deflectors equipped 250 ml Erlenmeyer flasks, each containing 40 ml of production medium, with 1 ml of each Culture inoculated The production medium has the following composition:

Production medium Corn steep water 2.8% by weight Cerelose 5.6% by weight

"Proflo" 2fi % by weight

Glycerine 1.4% by volume

Dimethylformamide 1.4% by volume in tap water, with NaOH adjusted to a pH of 73

A drop of anti-foaming agent (»Ρ> ~ · 800«) is added to each flask before being autoclaved

By dissolving 6.25 g NaiS ^ · 5 H ₂ O in 100 ml distilled water, a sodium thiosulfate solution is prepared This solution is filter sterilized, followed by adding to each of the production flask by autoclaving 04 ml of sodium thiosulfate solution

The ten production flasks (a total of 400 ml) are incubated for 5 days at 28 ° C. and then harvested

Isolation of FR-02A

The contents of the ten flasks are combined, the pH adjusted to 54 with hydrochloric acid, and the mycelium and the suspended nutrients are filtered off from the fermentation liquid. The filtrate is with 500 ml of chloroform mixed and stirred for an hour. The mixture is centrifuged to lock the phases separate, whereupon the aqueous phase is discarded. the

The chloroform layer is dried over anhydrous magnesium sulfate and the solvent is dried in vacuo aborted. The residue is only 30 ml of hexane washed. The antibiotic FR-02A is obtained by air drying.

Physical Properties

The elemental analysis of the FR-02A provides the following result:

C = 60.98%, H = 7.60%, N = 2.60%.

15th

The empirical formula is C59H90-95N2O21. This stands in agreement with the molecular weight of 1168 as determined by mass spectrometry.

In the form of its ammonium salt, the FR-02A is shown in Table 20 I AjIr ₀ I ₂₀ ! and iB chloroform soluble. At pH 7.0 or more, it is sparingly soluble in water. The ultraviolet spectrum of the ammonium salt in water shows the following characteristics:

X _n ^ 233 nm; £} L = 320 = 180

The FR-02A is a practically pure material because it is at thin layer chromatography only provides a single spot and when monitoring the analysis in the "LH-20" column is in the "XAD-2" column with the refractometer a single profile of Gaussian shape gives ■■

. To further characterize the FR-02A in vitro, ssine activity data are collected using a Profile of the antibiotic spectrum bestüvmt Zur To carry out this test, a drop of approximately 0.015 ml of the antibiotic is placed on the Surface of inoculated complex agar plates applied. The FR-02A is in solution in 10 percent methanol, which does not create any inhibition zone for anything. The results can be found in Table I as inhibition zones in millimeters.

Profiles of the antibiotic spectrum of FR-02 A at Agar diffusion test

Microorganism, MB For FR-O2A, s = 19xlO ³ .

The nuclear magnetic resonance spectrum of the antibiotic FR-C2A is recorded at 100 MHz in CDCI ₃ as the solvent with tetramethylsilane (TMS) as the internal standard. Representative features of the spectrum are doublets bti J, 21.1.31 and 4.63 τ and a singlet at 4.87T.

The infrared absorption spectrum of the antibiotic FR-02A in Nujol is shown in Fig. 1 FR-02A shows a characteristic absorption in the infrared of the spectrum at the following wavelengths, expressed in cm- ':

Broad band at 3400,

strong bands at 1640, 1460, 1380, 1080 and 1020, protruding bands at 1550, 1505, 1240, 1195, 940,860,720,620.

FR-02A was examined by various analytical methods with the following results:

1. "LH-20" column in
Methyl alcohol

(abandoned as NH " ⁺ salts) / C _D = 0302

2. "XAD-2"column;
50 percent isopropanol
in water

(abandoned as a free acid);

Exit at DV. = 3.5 to 4.0

3. Thin layer chromatography on silica gel, eluted with
a mixture of

80 parts of chloroform,

20 parts of methyl alcohol and

1 part concentrated NH ₄ OH R _f = 0.344

4. Paper chromatography
with a mixture of
70 parts isopropanol and
30 parts of phosphate buffer

(pH = 6.0; 0.01 molar) Rr = 0.9

Diameter of the zones of inhibition, mm

FR-02A 1 rag / ml

30 Bacillus sp. 633
Proteus vulgarus 1012
Pseudomonas aeruginosa 979
Serratia marcescens 252

Staphylococcus aureus 108
Bacillus substilis 964
Sarcina lutea 1101
Staphylococcus aureus 698 *)
Streptococcus faecalis 753

Alcaligenes faecalis 10
Brucella bronchiseptica 965
Salmonella gallinarum 1287
Vibrio percolans 1272
Xanthomonas vesicatoria 815
Escherichia coli 1418
Ps. Stutzen 1231
Klebsieila pneumoniae 1264
Aerobacter aerogenes 835
Erwinia atroseptica 1159
Corynebact. pseudodipht. 261
S. aureus 3032
S. aureus 2756
Strep, faecium 2820
Proteus vulgaris 838
E. coli 60

45

50

55

60

S. aureus (res. Erythromycin) 1909

18th

10

Ϊ0Η

1OH

11th

19th

28

12H

11th

19th

18th

13th

21

13th

15th

10

16

13th

12th

27

12th

23

20th

18th

10

16

H - cloudy zone.

*) Resistant to streptomycin, streptothricin, neomycin and viomycin.

When examined on mice, the antibiotic FR-02A shows a lower toxicity and in vivo a wide range of antibacterial activity. An example of the results for a gram-negative and a a gram-positive bacterium are summarized in Table II.

j; j ,. 19th 24 50 813 therapy ED ₅₀ 20th toxic Administration 1.73 > 5.0 § Tables IP 0.247 5.0 m in vivo test results with FR-02A?) on mice IP ToxMtät **) H infection - Tolerate S microorganism Administration 5.0 H Proteus vulgaris' IP 1.25 §§ Strep, pyogenes J
I- ■ ■ ^; ! IP

*) The. Values in the current table are expressed in mg per test animal. Both the infection and the administration of the therapeutic Do"; j; c of FR-02 A occurs introperitoneaL FR-02 A occurs once at the time of infection and then again after 6 hours.

**) The toxicity tests with FR-02 A on uninfected mice show that two doses of 2.5 mg per test animal, which are administered every 6 hours, are tolerated by the animals, higher doses, namely 5 to 10 mg per test animal, but are toxic.

1 sheet of drawings

Claims (1)

Patent claims: 1. Antibiotic FR-02A, characterized in that it is a pale yellow solid with following properties is: a) soluble in lower alkyl esters of lower alkanecarboxylic acids, ketones and chloroform, slightly soluble in water and insoluble in hexane; ^J b) ammonium salt soluble in alcohol, chloroform and moderately soluble in water with pH values of 7.0 or more; c) Composition: 6038% carbon, 7.60% hydrogen, 2.60% nitrogen and 28.82% Oxygen; d) molecular weight about 1'ό8; e) Equivalent formula CSgH ₉ O-SeN ₂ O ₂₁ ; f) "Traviolet absorption in phosphate buffer solution of pH 7.0 A _(rat .327nm; £ l ^ = 216 A _mÄ 232nm; £ "i ^ = 464; g) Ultraviolet absorption of the ammonium salt in water: