CH637960A5 - The antibiotic deacetyl 890A10 - Google Patents

Description

The invention relates to the new antibiotic Desacetyl 890Aio, which is active against both gram-positive and gram-negative bacteria and which is formed by treating 890Aio with an N-acetyl-890Aio-amidohydrolase, which is produced by soil microorganisms, the soil microorganisms by enrichment processes be isolated. The invention further relates to a method according to which 890Aio is deacetylated enzymatically.

The discovery of penicillin's remarkable antibiotic properties has stimulated a great deal of interest in the field, through which many other valuable antibiotic compounds have been found, such as other 40 penicillins, streptomycin, bacitracin, tetracyclines, chloro-amphenicol, erythromycins and similar compounds. In general, the antibacterial activity of each of these antibiotics does not include certain clinically important pathogenic bacteria. For example, some are only mainly active against gram-positive types of bacteria. The resistance acquired through the widespread use of the existing antibiotics in the treatment of bacterial infections has caused a serious resistance problem to arise.

The disadvantages of the known antibiotics have therefore stimulated further research with the aim of finding other antibiotic substances which are active against a broader range of pathogens and also against resistant strains of particular microorganisms.

The invention relates to a new antibiotic agent. The invention relates in particular to a new antibiotic compound which is referred to in the present application as "desacetyl 890Aio". The invention encompasses the antibiotic in dilute forms, as raw concentrates and in 60 pure forms.

The object of the present invention is to provide a new and useful antibiotic which is highly effective in inhibiting the growth of various gram-negative and gram-positive microorganisms. According to the invention, a method for producing this new antibiotic substance by enzymatic. table deacetylation of compound 890Aio be provided.

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55

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The novel antibiotic compound of the present invention is produced by hydrolysis of the N-acetyl group of 890Aio using an amidohydrolase that can hydrolyze the N-acetyl group. A suitable source of an amidohydrolase with this ability are strains of the microorganism Protaminobacter ruber which supply amidohydrolase. The special enzyme produced by Protaminobacter ruber is N-acetyl-890Aio-amidohydrolase, a member of the subgroup of enzymes called EC3.5.1 according to the recommended enzyme nomenclature of the International Union of Pure and Applied Chemistry and International Union of Biochemistry will.

The microorganism with which the deacetylation process can be performed has been isolated from a soil sample and, based on taxonomic studies, has been identified as belonging to the Protaminobacter ruber species and has been assigned MB-3528 to the depository of Merck & Co., Inc., Rahway, New Jersey. One of his cultures was indefinitely in the Culture Collection of the Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agri-cultural Research Service, U.S. Department of Agriculture, Peoria, Illinois. It was assigned the deposit number NRRL B-8143.

The morphological and cultural properties of Protaminobacter ruber NRRL B-8143 as well as its carbon and nitrogen utilization and use and biochemical reactions are as follows:

The nutrient broth cultures are uniformly cloudy without cuticles.

The pigment production does not depend on the light or the tested temperatures (28 and 37 ° C). The pigment is soluble in 5 acetone, but insoluble in water or chloroform.

Growth on nutrient agar and brain-heart infusion agar in aerobic conditions is somewhat slow, but good at 28 ° C. The growth is moderate to good, but slower at 37 ° C. No growth takes place at 50 ° C.

10th

Use of carbon and nitrogen sources

Using basic salt media with ammonium sulfate as the nitrogen source, growth is good with Arabiosis; moderate with xylose and poorly with dextrose, fructose, is mannose, rhamnose, lactose, maltose, sucrose, raffiosis, cellulose, inositol and mannitol.

N-acetylethanolamine can be used as the only source of carbon and nitrogen.

No acid or gas is formed from dextrose or lactose in OF Basal Medium 20 (Difco Laboratories, Detroit, Michigan) under aerobic or anaerobic conditions.

Biochemical reactions

The biochemical reactions are based on standard procedures as described in Manual of Microbiological Methods, published by the Society of American Bacteriologists, McGraw Hill Book Co., New York, 1957.

Morphology 30

The cells are rod-shaped with rounded ends, 0.9 to 1.2 x 2.3 to 4.6 microns, and occur individually or in pairs. 24- and 48-hour cells stain gram-negative with a granular appearance. The granules, especially the polar granules, turn black with Sudan Black B 35. The cells are mobile at 28 ° C, but motility is questionable at 37 ° C.

Cultural characteristics 40

Nutrient agar colonies are initially thin, punctiform, semi-transparent and colorless. They then become somewhat convex, opaque, smooth, undamaged at the edge, somewhat dry in consistency and are pink to pink-red pigmented.

Catalase - positive oxidase - negative starch is not hydrolyzed casein is not hydrolyzed gelatin is not liquefied

Litmus milk remains unchanged in its consistency, but becomes slightly alkaline indole - negative H2S - negative after 7 days

Nitrates are not reduced urease-positive

Lysine and ornithine decarboxylase - negative.

890Aio is the name given to the antibiotic of the structure

) so, h s-ch2-ch2nh-c-ch3

cooh is assigned.

890Aio, its description and method for its manufacture- November 17, 1976).

Position in DT-OS 27 51 303.7 from November 16 The novel antibiotic desacetyl

1977 (corresponding to U.S. Serial No. 742,958, filed on 890Aio has the structural formula

637960

and is produced by enzymatic hydrolysis of 890Aio using amidohydrolase, which is present in species of the Protaminobacter genus.

ch3-: gh-

<j> s03h f

The method according to the invention comprises the cleavage of the N-acetyl group of the compound of the structure

V,

fi cooh

-ch2-ch2-nh-c-ch3

and is carried out by intimately contacting or treating the compound with an amidohydrolase capable of hydrolyzing the N-acetyl group. In particular, the process according to the invention for the N-deacetylation of 890Aio consists in the intimate treatment of this compound with the amidohydrolase, N-acetyl-890Aio-amidohydrolase.

An unexpected homology between N-acetylethanolamine and 890Aio shows up, whereby extracts of microorganisms with the enzyme, N-acetylethanolamine amidohydrolase, are in many cases able to hydrolyze 890Aio.

The compound 890Aio is produced by fermentation of broth with the microorganism Streptomyces flavogriseus.

Due to extensive taxonomic research, the strain of microorganisms used in the present invention has been identified as belonging to the species Streptomyces flavogriseus and has been designated MA-4638 by the depository of Merck & Co., Inc., Rahway, N.J. One of his crops has been permanently listed with no restrictions on availability in the Culture Collection of the Northern Regional Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, III., And is publicly accessible under the deposit number NRRL 11 020.

Streptomyces flavogriseus MA-4638 produces 890Aio antibiotic, which is isolated from the fermentation broth in essentially pure form.

The morphological and cultural properties of Streptomyces flavogriseus MA-4638 are listed in the following table.

morphology

Spore carriers branch out, straight-chain to tortuous chains of spores that form tufts. The chains are longer than 10 spores. The spores are spherical to oval -0.9jiXl, 2p. (970x).

Cultural characteristics

Oatmeal Agar (ISP Medium 3)

vegetative growth - reversed-yellow-dark yellow lined with brown, curled

Air mycelium - light gray lined with medium gray soluble pigment - none

Czapek dox agar (sucrose nitrate agar)

vegetative growth - reverse-brown lined with dark brown

Air mycelium - medium gray, velvety soluble pigment - slight browning of the medium

Egg albumin agar vegetative growth - reversed-yellow-dark yellow lined with brown

Luftmycelium - medium gray, mixed with yellowish gray (2dc) and greyish money (2db)

soluble pigment - light yellowish-dark yellow is glycerin-asparagine agar vegetative growth - reversed brown aerial mycelium - velvety, light gray with yellowish tone (2dc) soluble pigment - light dark yellow inorganic salt starch agar (ISP Medium 4) 20 vegetative growth - reversed -greenish-yellowish-dark-yellow

Luftmycelium - velvety, medium gray with yellow tone (3fe) soluble pigment - very light dark yellow yeast extract-dextrose + salt agar 25 vegetative growth - reverse-dark brown

Air mycelium - dark gray mixed with a lighter gray soluble pigment - none

Yeast extract-malt extract agar (ISP Medium 2) 30 vegetative growth - reverse brown air mycelium - velvety, dark gray lined with a lighter gray soluble pigment - no skim milk agar 35 vegetative growth - dark yellow air mycelium - sparse, whitish soluble pigment - slightly browning Medium hydrolysis of casein - good litmus milk

40 vegetative growth - moderate growth ring, dark yellow

Air mycelium - no color - purple

Coagulation and / or peptonization - complete pepto-45 nization, becoming alkaline skimmed milk vegetative growth - moderate growth ring, dark yellow

Luftmycelium - not so soluble pigment - light brown

Coagulation and / or peptonization - complete peptonization, becoming alkaline

Nutrient agar vegetative growth - reverse dark brown 55 aerial mycelium - dark gray lined with grayish white soluble pigment - slight browning of the medium decomposition of tyrosine - positive

Peptone-iron-yeast extract agar 60 vegetative growth - dark yellow aerial mycelium - whitish, moderately soluble pigment - no melanin - no HsS formation - negative

65 Nutrient agar vegetative growth - reverse-light grayish brown aerial mycelium - light gray lined with dark gray.

soluble pigment - none

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Nutrient starch agar vegetative growth - dark yellow lined with gray aerial mycelium - medium gray soluble pigment - no starch hydrolysis - good nutritional gelatin agar vegetative growth - dark yellow lined with gray aerial mycelium - grayish-white soluble pigment - no liquefaction of gelatin - good

Potato cones vegetative growth - dark yellow aerial mycelium - medium to dark gray soluble pigment - none

Loeffler's blood serum vegetative growth - cream-colored aerial mycelium - no soluble pigment - no liquefaction - none

Vegetable growth - dark yellow aerial mycelium - no soluble pigment - no liquefaction of gelatin - complete.

All of the above readings were made after three weeks of incubation at 28 ° C unless otherwise stated. The pH of the media used in these tests was approximately neutral, namely pH 6.8 to 7.2. The color names used in the description are based on the definitions of Color Harmony Manual, 4th Edition (1958), Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus MA-4638 is also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism is grown on a synthetic base medium (Pridham and Gottlieb) containing 1% carbohydrate at 28 ° C for 3 weeks. The pH of the media used in these tests is approximately neutral (6.8 to 7.2). Table I lists the use of these sources of carbohydrates by Streptomyces flavogriseus MA-4638, where + growth, ± indicates poor or questionable growth and - no growth compared to the negative control (no carbon source).

Table I

glucose

+

Maltose

+

Arabinose

+

Mannitol

+

Cellulose

-

Mannose

+

Fructose

+

Raffinose

-

Inositol

-

Rhamnose

+

Lactose

+

Sucrose

±

Xylose

+

The amount of growth with changes in the temperature and oxygen demand of the microorganism are as follows:

Temperature range (yeast extract dextrose + salt agar) 28 ° C - good vegetative and air growth 37 ° C - good vegetative growth, no air hyphae 50 ° C - no growth.

Oxygen requirement (sting culture in yeast extract dextrose + salt agar)

aerobic.

The present invention is not limited to the organism Streptomyces flavogriseus or to organisms which completely meet the above growth and microscopic properties which have been given for illustration purposes Measures such as X-ray radiation, ultraviolet radiation, nitrogen mustard, exposure to bacteriophages and the like are formed.

io 890Aio is formed during aerobic fermentation under controlled conditions of suitable aqueous nutrient media that are inoculated with a strain of the organism Streptomyces flavogriseus. Aqueous media, such as those used to form other antibiotics, are suitable for the production of 890Aio. Such media contain sources of carbon, nitrogen and inorganic salts which are assimilable by the microorganism.

In general, carbohydrates such as sugar, e.g. 20 dextrose, glucose, fructose, maltose, sucrose, xylose, mannitol and the like, and starches such as dextrin or such as cereals or grains such as e.g. Wheat, rye, corn starch, corn flour and the like, either alone or together, can be used as sources of assimilable carbon in the nutrient medium. The amount of carbohydrate normally varies between about 1 and 6% by weight of the medium. These carbon sources can be used individually or several such carbon sources can be mixed in the medium. In general, many proteinaceous materials can be used as nitrogen sources in the fermentation process. Suitable nitrogen sources include e.g. Yeast hydrolysates, primary yeast, soybean meal, cottonseed meal, hydrolysates of casein, corn steep liquor, stillage or soluble substances from the manufacture of brandy or similar. The preferred source is stillage or soluble substances from the production of spirits. The sources of nitrogen, either alone or together, are used in amounts ranging from about 0.2 to 6% by weight of the aqueous medium.

40 Among the inorganic nutrient salts that can be incorporated into the culture media are the usual salts that give sodium, potassium, ammonium, calcium, magnesium, phosphate, sulfate, chloride, carbonate and similar ions . Also included are trace metals such as 45 cobalt, manganese and iron.

The media described in the examples are only examples from a variety of media that can be used, and this is not intended to be a limitation.

The fermentation takes place at temperatures in the range of about 20 to 37 ° C, but for optimal results it is preferred to carry out the fermentation at temperatures of about 23 to 28 ° C. The initial pH of the nutrient media suitable for growing the strains of Streptomyces flavogriseus culture and producing the antibiotic 890Aio can vary from about 6.0 to 8.0.

Although the 890Aio antibiotic is produced by both surface and submerged cultures, it is preferred to carry out the fermentation in the submerged state.

60 Small-scale fermentation of the antibiotic is conveniently carried out by inoculating a suitable nutrient medium with the culture producing the antibiotic and, after being transferred to a production medium, fermenting at a constant temperature of 65 to 24 ° C on a shaker for several days.

The fermentation is carried out in a sterilized flask of the nutrient medium through one or more stages of the germ

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development initiated. The nutrient medium for the seed stage can be any suitable combination of carbon and nitrogen sources. The inoculation flask is shaken in a constant temperature chamber at about 28 ° C for a day or until growth is satisfactory, and part of the resulting growth is used to inoculate second stage inoculum or the production medium. Inoculation flasks of the intermediate stages, when used, are developed in essentially the same way, i.e. some of the flask contents from the last inoculation stage are used to inoculate the production medium. The inoculated flasks are shaken at constant temperature for several days and the contents of the flask are centrifuged or filtered towards the end of the incubation period.

For working on a large scale, it is preferred to carry out the fermentation in suitable tanks which are equipped with a stirring or agitation device and a device for aeration of the fermentation medium. According to this method, the nutrient medium is prepared in the tank and sterilized by heating at temperatures up to about 120 ° C. After cooling, the sterilized medium is inoculated with a previously grown inoculum of the producing culture and the fermentation can be carried out for a period of time, e.g. 1 to 6 days, with stirring and / or aerating the nutrient medium and maintaining the temperature at about 22 to 26 ° C. This method of producing the 890Aio antibiotic is particularly suitable for the production of large quantities of the antibiotic.

Physical and chemical properties of the antibiotic 890Aio

The 890Aio antibiotic is an acidic compound that migrates towards the positive pole during electrophoresis at neutral pH. With a gradient of 50 V / cm in 0.03 M potassium phosphate buffer, pH 7.1, the antibiotic migrates 8.0 cm in 30 minutes compared to a 4.0 cm hike for 890Ai. The disodium salt is a white or light yellow powder after lyophilization from an aqueous solution. Under acidic conditions in aqueous solution, the antibiotic is unstable and the free acid form has not yet been isolated.

The disodium salt of the antibiotic 890Aio has an absorption maximum at 299 nm and a minimum at 243 nm at neutral pH in water. The E% at 300 nm of the most purified preparation of the disodium salt is 214. The ratio A300 / A250 is 3.33 for the purest sample and the ratio A300 / A220 is 2.05. The presence of minor contaminants in this sample suggests that the corresponding ratios are slightly higher for a sample of the highest purity. More than 94% of the absorption at 300 nm can be extinguished by reaction with hydroxylamine at neutral pH. The absorption at 250 nm also decreases in the reaction with hydroxylamine, and the ratio of the absorption decrease at 250 nm to the decrease at 300 nm is. about 0.16. The reaction with hydroxylamine, followed by the A3oo decrease under the conditions described in the part “hydroxylamine reaction”, is obviously first order with a half-life at room temperature of 23 to 60 seconds.

Measured against a standard of the antibiotic 890Ai, the antibiotic 890Àio has 174 bioassay units (bioanalysis units) / HAEA300 unit. HAEA300 is described in the section “Hydroxylamine Reaction”.

Table II lists the 100 MHz nuclear magnetic resonance spectral signals from 890Aio in D2O at 32 ° C. The chemical shifts are listed in ppm, based on HOD, at 4.70 8 and 32 ° C and the coupling constants in Hertz.

Table II

CHsCH

1.55 (3H, D, 6.5 Hz)

CH3CO

2.02 (3H, S)

10 C (6) -H

3.89 (IH, D, D, Ì6-5 = 5.4 Hz, Jmi = 9.2 Hz)

C (S) -H

-4.34 (1H, D, T, Js-6 = 5.5 Hz, Jj-i = 9.5 Hz)

C (8) -H

—4.8 (partially covered by the HOD line)

C (l) -H2

~ 3.14 (lH, D, D, ~ 9.2 + ~ 18Hz)

-3.33 (1H, D, D, ~ 10 + 18 Hz)

15 -CH2NH

3.43 (2H, T, 7 Hz)

-CH2-S-

3.03 (2H, M)

The mass spectrum of TMSi-890Aio is characterized by the. Characterized 20 fragments listed in Table III.

Table III

25th

30th

m / e 86

227.0224 C5H15SO4SÌ2, calculated 227.0230

241

300/1

339.1325 C15H25NO4SÌ2, calculated 339.1322 342.1444 C15H26N2O3S Si, calculated 342.1433 368 440 458

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40

Physical and chemical properties of Desacetyl 890Aio

Table IV shows the antibacterial spectrum profile of Desacetyl 890Aio.

Table IV

ASP No. organism, MB ■ / = / (ATCC ^)

Inhibition zone diameter, mm

45

50

1

Bacillus sp. 633

28

2nd

Proteus vulgaris 1012

5

3rd

Pseudomonas aeruginosa 979

0

4th

Serratia marcescens 252 (990)

9

5

Staphylococcus aureus 108 (6538 P)

23

6

Bacillus subtilis 964 (6633)

31

7

Sarcina lutea 1101

21st

8th

Staphylococcus aureus 698

20th

9

Streptococcus faecalis 753

0

10th

Brucella bronchiseptica 965 (4617)

0

11

Vibrio percolans 1272 (8461)

27th

12

Proteus vulgaris 838 (21100)

14

13

Escherichia coli1418

12

14

Pseudomonas stutzeri 1231 (11607)

16

15

Klebsiella pneumoniae 1264

8th

16

Aerobacter aerogenes 835

9

17th

Erwinia atroseptica 1159 (4446)

11

18th

Pseudomonas aeruginosa 2824

14H

19th

Corynebacterium pseudodiph261 (9742)

14

20th

Escherichia coli 60 (9637)

10th

21st

Streptococcus faecium 2820

0

22

Streptococcus agalactiae 2875

23

23

Proteus vulgaris 2112 (Episom)

15

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Table IV continued

ASP No.

Organism, MB -j ^ (ATCC j = j)

diameter

the inhibition zone, mm

24th

Proteus mirabilis 3126

0

25th

Vibrio percolans 1272 + 2 x 105 ji / ml

Penicillinase

10 '

26

V.percolans 1272 + lactamase from

Enterobacter MB2646

28

27th

Micrococcus flavus 369 (10240)

17th

In Table V, the 300 MHz nuclear magnetic resonance (NMR) spectrum signals of deacetylated 890Aio are determined relative to the internal standard DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfonate) in D2O (24 ° C ) listed.

Table V

H

8a ch3

1.51 (6.2) b cms

3.14

CH2N

3.43

--H

c, <^

3.07 (18.0, 8.5)

~ --H 3.43 (18.0.9.0)

Ha

3.88 (5,6,8,9)

H5

4.36

Ms

4.7-4.9 (estimated)

• 'Shifts, relative to the calculated position of the inner DSS b the values in brackets are the coupling constants. 35

The mobility of the deacetylated 890Aio is determined on Dowex-1. About 100 (ig desacetyl 890Aio are applied to a column (0.7 x 15 cm) made of Dowex-1 x2 (Cl_), 0.074 to 0.037 mm (200 to 400 mesh), with an ionic strength of less than 0.01 M) and eluted with a solution containing 0.10 M NaCl + 0.011 M NH4C1 + 0.0001 M NHa at pH 7.2 in deionized water The main peak of Desacetyl 890Aio occurs between the eluted volumes of 65 ml and 124 ml with a maximum at an eluted volume of 109 ml 4s.

Desacetyl 890Aio has a maximum absorption in the ultraviolet spectrum at 296 nm.

The mobility of Desacetyl 890Aio is determined by thin layer chromatography and electrophoresis. so

In the thin layer chromatography of Desacetyl 890Aio on Eastman Chromagram 6064 cellulose sheets at 23 ° C in 66% (vol / vol) ethanol, the antibiotic shows an Rr value of 0.68, determined by bioautography.

When Desacetyl 890Aio is electrophoresed on Whatman ss 3MM chromatography paper using a buffer containing 35.2 g KH2P04 + 57.2 g NaaHPO ^ l at 47 V / cm for 50 min, the antibiotic Desacetyl 890Aio moves 65 mm in the direction on the positive electrode. Under the same conditions, the antibiotic 60 890Aio moves 118 mm in the direction of the positive electrode. The mobilities are measured by bioautography using chloramphenicol as a reference marker, the mobility of which is assumed to be zero.

Desacetyl 890Ato, the compound according to the invention, 65 is a valuable antibiotic which is active against various gram-positive and gram-negative bacteria. Accordingly, it is used in human and veterinary medicine. The compound according to the invention can be used as an antibacterial drug for the treatment of infections caused by gram-positive or gram-negative bacteria. e.g. to sensitive strains of Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The antibacterial material according to the invention can furthermore be used as an additive for animal feed or animal feed additives, for the preservation of food or feed and as a disinfectant. For example, it can be used in aqueous preparation in concentrations in the range from 0.1 to 100 parts of antibiotic / million parts of solution or preferably in concentrations in the range from about 1 to 10 parts of antibiotic / million parts of solution to destroy and inhibit the growth of harmful bacteria on medical and dental devices or devices and as a bactericide in industrial applications, for example in water-based paints and in white water from paper mills to inhibit the growth of harmful bacteria.

The antibiotic of the invention can be used in any of a variety of pharmaceutical preparations as the only active ingredient or together with either one or more other antibiotics or with one or more pharmacologically active compounds. As an example of the former, an aminocyclite antibiotic such as gentamicin can be co-administered at the same time so that any chance that resistant organisms will be preserved is minimized. As an example of the latter, diphenoxylate and atropine can be combined in dosage forms intended for the therapy of gastroenteritis. The antibiotic can be used in capsule form or as tablets, powder or in the form of liquid solutions or as suspensions or elixirs. It can be administered orally, topically, intravenously or intramuscularly.

Tablets and capsules for oral administration can be in unit dosage forms and they can be conventional excipients or diluents such as binders, e.g. Syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; Fillers, e.g. B. lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; Lubricants, e.g. Magnesium stearate, talc, polyethylene glycol, silicon dioxide; Disintegrant e.g. Potato starch, or acceptable wetting agents such as sodium umlauryl sulfate. The tablets can be coated or coated using methods which are well known per se. Oral, liquid preparations can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, etc., or they can be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can contain additives known per se, such as suspending agents, e.g. Sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated, edible fats; Emulsifiers, e.g. Lecithin, sorbitan monooleate or acacia; non-aqueous vehicles which may include edible oils, e.g. Almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; Preservatives, e.g. Methyl or propyl p-hydroxybenzoates or sorbic acid. The suppositories become common suppository bases, e.g. Cocoa butter or other glycerides.

Compositions for injection may be in unit dose form in ampoules or in multiple dose containers with added preservative. The

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Compositions can be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles and can contain formulation auxiliaries such as suspensions *, stabilizers and / or dispersants. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable carrier, e.g. sterile, pyrogen-free water, before use.

The compositions may also be in suitable forms for absorption through the mucous membranes of the nose and throat or bronchial tissues and may conveniently be prepared in the form of powders or liquid sprays or inhalants, lozenges, paints for the throat, etc. For the medication of the eyes or ears, the preparations can be in the form of individual capsules, in liquid or semi-solid form, or they can be used as drops, etc. Topical applications can be prepared in hydrophobic or hydrophilic base materials as ointments, creams, lotions, paints, powders, etc.

In addition to a carrier, the preparations or compositions according to the invention can contain other constituents, such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity regulators or flavoring agents or flavorings and the like.

In veterinary medicine, such as in the treatment of poultry or chicks, cows, sheep, pigs and the like, the preparations can e.g. be prepared as intramammary preparations in either long-acting or quick-release base materials.

The dose to be administered depends to a large extent on the condition of the subject to be treated, the weight of the host and the type of infection, the route and the frequency of administration. The parenteral route is preferred for generalized infections and the oral route for intestinal infections.

In the treatment of bacterial infections in humans, the compounds according to the invention are preferably orally or parenterally divided according to methods known per se for antibiotic administration in an amount of about 2 to 600 mg / kg / day and preferably about 5 to 100 mg / kg / day Cans, e.g. three to four times a day. The compound of the invention can be administered in unit doses, e.g. 25,250,330,400 or 1000 mg of active ingredient with suitable physiologically acceptable carriers or diluents. The dose units are in the form of liquid preparations, such as solutions or suspensions, or as solids in tablets or capsules. The optimal dose in a given case will depend on the type and severity of the infection to be treated, and smaller doses will be used for infant purposes. All of these settings are familiar to the person skilled in the art.

Analysis and assay procedures for antibiotics 890Aio

I. Bioassay (bioanalysis)

An agar plate disk diffusion method is used using Vibrio percolans ATCC 8461 as the test organism. A purified sample of the 890Ai antibiotic is used as a standard. Antibiotic 890Ai is made according to the procedure described in Example 4.

Plates containing Vibrio percolans ATCC 8461 are made as follows.

A lyophilized culture of Vibrio percolans'ATCC 8461 is suspended in 15 ml of a sterilized medium containing 8 g / 1 Difco broth and 2 g / 1 yeast extract in distilled water, "broth-yeast extract" (hereinafter referred to as NBYE). The culture is incubated overnight on a rotary shaker at 28 ° C. This culture is used to inoculate the surface of slant cultures containing 1.5% agar in NBYE, and the inoculated slant cultures are incubated overnight at 28 ° C and then stored in a refrigerator.

The slanted cultures stored in the refrigerator, which are produced from a single, lyophilized culture, are used for up to 4 weeks from their preparation as follows: an eyelet of inoculum from the slanted culture is in 50 ml NBYE, which is contained in a 250 ml Erlenmeyer flask, dispersed. The culture is incubated overnight on a rotary shaker at 28 ° C and then diluted to a density which gives 50% transmission at 660 nm. A 33.2 ml portion of this diluted culture is added to 11 NBYE containing 15 g agar and kept at 46 ° C. The inoculated medium containing agar is poured into 100 x 15 mm plastic petri dishes, 5 ml / dish, cooled, and stored at 2 to 4 ° C. for up to 5 days before use.

Filter paper disks with a diameter of 1.27 cm are immersed in the solution to be analyzed and placed on the agar. Alternatively, the slices can be loaded by pipetting 1/10 ml solution onto a dry slice and then the slice can be placed on the agar. The diameter of the zone of inhibition is determined after the appropriate incubation (12 to 24 h at 25 ° C.). If necessary, the solutions to be analyzed are diluted in 0.05 M potassium phosphate buffer, pH 7.4 “potassium phosphate buffer” (hereinafter referred to as KPB), or in deionized water.

The effectiveness or potencies are calculated as follows: An inclination is determined by determining the zone diameter of a solution of the antibiotic 890Aio and a four-fold dilution (in KPB) of this solution. Two slices of each concentration are analyzed on a single plate and the average zone size is determined at each concentration. The slope is equal to half the difference in the average zone sizes. The potencies or efficacies are then calculated according to the following formula:

Potency (units / ml) =

(Potency of the standard) x dilution x 10 ([D-Dj ^ \

\ Tilt /

Therein mean: D the average diameter of the zones formed by the unknown substance, Ds the average diameter of the standard zones and "dilution" the degree to which the unknown sample was diluted before analysis. If no standard is used, Ds is assumed to be 25 mm and (potency of the standard) is assumed to be 1 unit / ml when measuring against Vibrio percolans ATCC 8461. Pure 890Ai is defined to have a potency of 250 units / hydroxylamine-quenchable absorption unit at 300 nm when used as a standard.

II. Analysis methods for determining the «890 assay units»

A common agar plate diffusion method is used using Vibrio percolans ATCC 8461 as a test organism. Cephaloridine is used as the standard. The plates containing Vibrio percolans ATCC 8461 are made as follows. A culture by Vibrio

5

10th

IS

20th

25th

30th

35

40

45

50

55

60

65

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percolans ATCC 8461 is incubated in a nutrient broth yeast extract overnight on a rotary shaker at 28 ° C and then diluted to a density of 60% permeability at 660 nm. A 33.2 ml portion of this diluted culture is added to 11 medium containing a nutrient agar plus 0.2% yeast extract and kept at 46 ° C. The inoculated medium containing agar is poured into 100 x 15 mm plastic petri dishes, 10 ml / dish, cooled, and kept at 2 to 4 ° C. for up to 5 days before use.

The concentration of cephaloridine, which is equivalent to 1 unit / ml 890Ai, is determined by analysis on plates made as described above but containing 5 ml of inoculated medium / plate as follows. Four concentrations of cephaloridine give the standards -3.12.6, 25, 12.5 and 25 mcg / ml, with 12.5 mcg / ml serving as the reference solution. The zone diameters on a 5 ml plate for the standard are as follows.

Concentration (mcg / ml) Zone diameter (mm)

3.12 16.8

6.25 22.3

12.5 25.0

25 29.6

One unit is defined as the amount of antibiotic per ml that gives a 25 mm zone of inhibition on a 5 ml plate as described in Part I, above. In this assay, therefore, a concentration of 12.5 mcg / ml cephaloridine is considered equivalent to 1 unit 890Ai / ml. Since the slope for the line for cephaloridine is 4.0, the potency calculations of the sample are made using a slope of 4.0.

III. Hydroxylamine reaction

Antibiotic 890Aio reacts with hydroxylamine to give a substance in which the absorption at 300 nm is markedly reduced. This provides a basis for the quantitative analysis of the 890Aio antibiotic.

The solution to be analyzed is added to 0.05 M potassium phosphate, pH 7.4, by adding 1/20 volume of a solution containing 0.8 M foHPC and 0.2 M KH2PO4. Then 1/100 volume of 1 M hydroxylamine hydrochloride is added and the absorption at 300 nm is measured at intervals from Vi to 2 min. The reaction is carried out at room temperature. First order kinetic values are assumed and the half-life is determined from the decrease in absorption during the first 10 minutes. The time after which no further absorption decrease should be observed is calculated from this half-life and the observations are continued beyond this time. If no further decrease is observed after this time, the total absorption decrease (correcting for the dilution effect and the absorption of hydroxylamine) is taken as the “hydroxylamine-quenchable absorption at 300 nm (HAEA300)”. If an absorption decrease is observed after this time, the rate of the background absorption decrease is calculated, and the decrease observed at this time is corrected for the background decrease, assuming that the background decrease is linear with time. The corrected value is then recorded as HAEA300.

The number of HAEAaoo units is equal to the HAEA300, multiplied by the volume in ml.

The following examples illustrate the invention and in particular methods by which the products according to the invention are produced.

example 1

Process for the isolation of N-acetyl-890Aio-amidohydrolase-forming organisms

A 1% (w / v) suspension of fertile turf is prepared by suspending 1 g of turf in 100 ml of sterile phosphate buffer saline, the phosphate buffer saline having the following composition:

Phosphate buffer saline

NaCl 8.8 g

1 M phosphate buffer, pH 7.5+ 10 ml distilled water 1000 ml

+ 1 M phosphate buffer, pH 7.5: 16 ml 1 M KH2PO4 are mixed with 84 ml 1 M K2HPO4. The pH of the phosphate buffer is adjusted to 7.5 by adding small amounts of either 1 M KH2PO4 or 1 M K2HPO4.

Aliquots of this 1% stock or stock suspension are used to make 10, 100 and 100 fold dilutions.

1 ml parts of the stock suspension or 1 ml parts of the 10, 100 and 100-fold dilutions are added to 2 ml parts of sterile 1.0% agar solutions at 48 ° C. The mixtures are quickly poured over the surface of sterile 85 mm diameter petri dishes containing 20 ml of Medium A. Medium A has the following composition.

Medium A

KH2PO4 3.0 g

K2HPO4 7.0 g

MgS04 0.1 g distilled water 1000 ml

N-acetylethanolamine solution + 8.5 ml

+ N-acetylethanolamine solution: N-acetylethanolamine is diluted tenfold in water and sterilized with a membrane. This solution is added after treatment in an autoclave.

For solid media: 20 g agar are added.

The petri dishes are incubated at 28 ° C for 18 days. Colonies that are well isolated are removed and streaked onto medium B. Medium B has the following composition.

Medium B

Tomato paste or pulp 40 g ground wheat flour 15 g distilled water 1000 ml the pH is adjusted to 6 using NaOH.

For solid media: 20 g agar are added.

Individual clones are selected and grown on slant cultures of medium B at 28 ° C. for 2 days.

Part of the growth of the slant cultures is used to inoculate a 250 ml Erlenmeyer flask containing 50 ml Medium A, a 250 ml Erlenmeyer flask containing 50 ml supplemented Medium B (supplemented after treatment in an autoclave with 0.4 ml of a membrane-sterilized solution from N-acetylethanolamine, diluted tenfold s

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with water) and a 250 ml Erlenmeyer flask containing 50 ml Medium C. Medium C has the following composition: '

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn steep liquor (on a wet basis) 5 g distilled water 1000 ml pH adjusted to 7 with NaOH or HCl

N-acetylethanolamine solution + 8.5 ml

+ N-acetylethanolamine solution: N-acetylethanolamine is diluted tenfold with water and sterilized against a membrane. This solution is added after the treatment in an autoclave.

The flasks are shaken for 4 days at 28 ° C on a 220 rpm shaker (5.08 cm = 2 "vibration amplitude). A 30 ml portion from each flask is centrifuged at 8000 rpm for 15 minutes. The excess portion is removed leaving only enough to give a thick suspension of cells and media solids. Half of the suspension is subjected to ultrasound disruption using a Branson Instrument Model LS-75 Sonifier with a 1.27 cm (Vi inch) probe Input power is set to position # 4 and four consecutive 15 sec cycles of radiation are used while the suspension is cooled in ice water during and between breakdowns to test for the presence of N-acetyl-890Aio-amidohydrolase activity a 10 ml portion of the sonicate is mixed with 25 ml of an 890 Aio solution containing 500 jig / ml. Control samples containing the antibiotic and buffer alone and sonicated Traded cells and buffers without antibiotics are also checked. After overnight incubation at 28 ° C, 10 ul amounts are applied to Schleicher and Schuell # 2043-B paper. A voltage gradient of 50 V / cm is applied over 30 min using 0.03 M potassium phosphate buffer, pH 7.1. The paper is placed on a Staphylococcus aureus ATCC 6538P plate and incubated at 37 ° C for 18 h.

The assay plates are prepared as follows: An overnight growth of the assay organism Staphylococcus aureus ATCC 6538P in nutrient broth plus 0.2% yeast extract is diluted with nutrient broth plus 0.2% yeast extract to a suspension with a permeability of 60% at a wavelength of 660 nm . This suspension is added to Difco nutrient agar, which was supplemented with 2.0 g / 1 Difco yeast extract, at 47 to 48 ° C., so that a composition is obtained which contains 33.2 ml of the suspension / 1 agar. 40 ml of this suspension are poured into 22.5 cm x 22.5 cm petri dishes, and these plates are cooled and kept at 4 ° C until used (maximum 5 days).

The unchanged bioactive 890Aio stain migrates 8 cm towards the positive pole. A new bioactive stain due to Desacetyl 890Aio is found and it migrates 4 cm towards the positive pole. Control incubation mixtures of antibiotic plus buffer and cell sonicate plus buffer do not result in a bioactive material that migrates 4 cm in the direction of the positive pole.

Example 2 Deacetylation of 890Aio

Part of the growth of a slant culture of Protaminobacter ruber MB-3528 is used to inoculate a 250 ml

Erlenmeyer flask containing 50 ml Medium C used. Medium C has the following composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn spring water or soaking

water (on a wet basis) 5 g distilled water 1000 ml pH with NaOH or HCl to 7

set

N-acetylethanolamine solution + 8.5 ml

+ N-acetylethanolamine solution: N-acetylethanolamine is diluted tenfold in water and sterilized against a membrane. This solution is added after the treatment in an autoclave.

The flask is shaken for 4 days at 28 ° C on a 220 rpm shaker (5.08 cm vibration amplitude). A 25 ml portion from the flask is centrifuged at 8000 rpm for 15 min. The supernatant is removed and the cells on the surface of the media solids are scraped off in 0.5 ml of 0.05 M potassium phosphate buffer, pH 7.4. The resulting suspension is subjected to ultrasonic disruption using a Branson Instrument Model LS-75 Sonifier with a 1.27 cm probe at setting 4 during four 15 sec intervals while cooling the suspension in ice water during and between the disruptions. A 10 µl portion of the sonicate is mixed with 25 ml of an 890 Aio solution containing 500 µg / ml. Control samples containing antibiotic and buffer alone and sonicated cells and buffer without antibiotic are also tested After overnight incubation at 28 ° C., 10 μl amounts are applied to Schleicher & Schuell No. 2043-B paper, using a 0.03 M potassium phosphate buffer, pH 7.1, a voltage gradient of 50 V / cm during 30 min The paper is placed on a Staphylococcus aureus ATCC 6538P assay plate and incubated at 37 ° C. for 18 hours.

• The assay plates are produced as follows: An overnight growth of the assay organism, Staphylococcus aureus ATCC 6538P, in nutrient broth plus 0.2% yeast extract is combined with nutrient broth plus 0.2% yeast extract to a suspension with a permeability of 60% at a wavelength of 660 nm diluted. This suspension is added to Difco nutrient agar, supplemented with 2.0 g / 1 Difco yeast extract, at 47 to 48 ° C. to produce a composition which contains 33.2 ml suspension / 1 agar. 40 ml of this suspension are poured into 22.5 cm x 22.5 cm petri dishes, and these plates are cooled and kept at 4 ° C until used (maximum 5 days).

In addition to the unchanged bioactive 890Aio spot that moves 8 cm in the direction of the positive pole, a new bioactive spot due to desacetyl 890Aio is found that moves 4 cm in the direction of the positive pole. Control incubation mixtures of antibiotic plus buffer and cell sonate plus buffer do not result in a bioactive material that moves 4 cm in the direction of the positive pole.

Example 3

Manufacture of the antibiotic 890Aio

Two frozen ampoules, each containing 2 ml of MA-4638 inoculum, are slowly thawed and the contents are transferred aseptically into two inoculation flasks, each containing 500 ml of medium C.

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The inoculation flasks, 21 shake flasks with triple deflection elements, are equipped with a side arm, which is closed with cotton or cotton wool.

Medium C

autolyzed yeast (Ardamine +) 10.0 g

Glucose 10.0g

MgSCU • 7H2O 0.05 g

Phosphate buffer ++ 2.0 ml distilled water 1000 ml the pH is adjusted to 6.5 with NaOH before sterilization

+ Ardamine: Yeast Products, Inc.

++ phosphate buffer solution:

KH2PO4 91.0 g

Na2HP04 95.0 g distilled. Water 1000 ml

The inoculated inoculation flasks are shaken for 24 to 30 h at 28 + 1 ° C on a 210 rpm gyroscope (5.08 cm vibration amplitude).

The growth from the inoculation flasks is used to inoculate two 141 glass fermentation devices containing 101 production medium (Medium D plus 0.15% soybean oil, vol / vol).

Medium D

Dextrin (CPC modified starch) 40.0 g of soluble substances from the

Brandy production 7.0 g

Yeast extract 5.0 g

C0CI2 • 6H2O 50.0 mg distilled water 1000 ml the pH is adjusted to 7.3 with NaOH before sterilization

The fermentation devices are operated at 24 ° C using a stirring rate of 640 rpm (approximately Kd 5.5) and an air flow of 0.5 WM for 72 hours. Defoamer, Hodag-MF (Hodag Chemical Corp.), is used as needed, but not more than 0.1%.

The contents of the two fermentation devices are combined after the fermentation attempt; you get about 201.

200 ml parts of the batch are centrifuged in a Servali RC-2B centrifuge at 9000 rpm for 20 min. The supernatant liquid is filtered through a 1 cm layer of Hyflo Super-Cel in a 33.02 cm (13 inch) Lapp funnel with a cotton or cotton filter. The filtrate has a bioactivity of 20 units / ml.

The filtered broth (181) is applied to a column (8.2x29 cm) of Dowex-1 x2 (Cl ") 0.297 to 0.149 mm (50-100 mesh) at 150 ml / min. The column is then deionized with 11 Water and then with 1510.15 M

NaCl + 0.01 M tris-HCl buffer, pH 7.0, +25 µM neutral EDTA in 50% MeOH at 150 ml / min.

The antibiotic 890Aio is then eluted with 3.2% NaCl + 0.02 M tris-HCl buffer, pH 7.0, +25 ml neutral EDTA in 80% MeOH at 100 ml / min. Fractions are collected as follows: one fraction of 11, four fractions of 500 ml each and 12 fractions of 11 each. The bioactivity and the absorption at 220 nm are determined by each fraction, and the fractions, the bioactivity / A22o ratios above 0 .6 units / A22o unit and which also contain more than 10% of the total activity obtained per fraction are combined for further purification. Fractions 4 to 8 are thus combined, which contain a total of 11% of the bioactivity used.

The combined fractions are concentrated to 190 ml under reduced pressure, the precipitated salt is washed with deionized water, and the wash water is added to the supernatant liquid, bringing the final volume to 220 ml. The pH of the concentrate is adjusted to 6.5 with HCl, and the concentrate is placed on a column (6.0 x 59 cm) made of Amberlite XAD-2, which has been washed beforehand with 8160% aqueous acetone and then 161 deionized water was applied. After the application is complete, the column is rinsed with 5 x 10 ml portions of deionized water and the antibiotics are eluted with deionized water at 35 ml / min. The fractions are collected as follows: 1 fraction of 500 ml, followed by 7 fractions of 250 ml each, followed by 4 fractions of 500 ml.

The bioactivity and the HAEA304 values are determined on fractions 3 to 12. Fractions 4 to 9 with a HAEA304 / A220 ratio above 0.01 are pooled for further purification. Fraction 3, which contains about 'A of the salt used, is also added to the combined material. The combined material contains 469 HAEA304 units.

The combined fractions are diluted to 4.11 and applied to a column (2.15 x 42 cm) of Dowex-1x4 (Cl "), minus 400 mesh or -0.037 mm, at 2 ml / min. The column is used with Wash 100 ml of 50% methanol and the antibiotic is eluted with 0.25 M NaCl + 0.01 M NH4C1 + 0.0001 M NH3 in 80% MeOH at about 2 ml / min. 8 to 12 ml fractions collected.

Bioactivity and absorption at 220 nm, 260 nm and 300 nm are determined for every fifth fraction and HAEA300 is determined for the bioactive peak fractions. The 890Aio bioactivity occurs in fractions 85 to 150 with a maximum in fraction 120. Fractions with H AEA300 / A300 values above 0.05 are combined for further purification. Fractions 105 to 133 are combined in this way, they contain a total of 130 HAEA300 units. The combined fractions 105 to 133 are diluted to 1900 ml with deionized water and the pH is adjusted to 7.6. The sample is applied to a column (2.15 x 42 cm) made of Dowex-1 x 2 (C1_) minus 400 mesh = -0.037 mm, previously with 3 13% NaCl in 50% methanol and then with 50 ml of 50% methanol was washed. After the entire sample was applied, the column was washed with 100 ml of 30% methanol and eluted at 2 ml / min with 0.26 M NaCl + 0.005 M NH4CI + 0.0002 M NH3 in 30% methanol. Fractions of 10 to 12 ml are collected.

The main peak of the antibiotic 890Aio occurs in fractions 230 to 290 with a maximum in fraction 260. The fractions with an A300 / A2so ratio above 1.10 and an A300 / A22o ratio above 0.83 are used for further purification united. Fractions 240 to 275 are pooled in this way, they contain 91.6 HAEAsoo units.

The combined fractions 240 to 275 are concentrated to 10 ml under reduced pressure. The concentrate is separated from the precipitated salt by pipetting off. The salt is washed with 2 ml of deionized water and the wash water is added to the concentrate. The combined 12 ml concentrate and wash water are placed on a column (2.15 x 76 cm) made of Bio-Gel P-2 (0.074 to 0.037 mm = 200-400 mesh), which was previously washed with 30 ml saturated NAC1 in deionized water, followed by 1500 ml of deionized water and 50 ml of 0.05 mM NH3 in deionized water. After the application is fully5

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the column is rinsed with 3 x 1 ml amounts of 0.05 mM NHs in deionized water for rinsing and the antibiotic is eluted with 0.05 mM NH3 in deionized water at 0.73 ml / min. Fractions of 3.65 ml each are collected.

The main peak of the antibiotic 890Aio occurs in the fractions 29 to 50 with a maximum in the fraction 36. The fractions with an A3oo / A25o ratio above 1.9 and also with an A300 / A220 ratio above 1.45 are for the Lyophilization and NMR analysis combined. Fractions 36 to 45 are combined in this way, they contain 50.8 A3oo units, 45.1 of which can be extinguished by hydroxylamine.

The combined fractions 36 to 45 are concentrated to 1 ml under reduced pressure and 5 ml of D2O are added. The sample is again concentrated to 0.835 ml and 0.825 ml thereof are transferred to a glass ampoule and freeze-dried and lyophilized. The lyophilized sample contains 48.2 A3oo units and 4.6 mg solids.

Example 4 Preparation of Antibiotic 890Ai A tube of lyophilized culture from Streptomyces flavogriseus MA-4600a is opened aseptically and the contents are suspended in a tube containing 1.5 ml of sterile medium A of the following composition:

Medium A

Yeast extract 10.0 g

Glucose 10.0 g

MgSÛ4-7H20 0.05 g

Phosphate buffer + 2 ml distilled water 1000 ml

+ Phosphate buffer solution:

KH2PO4 91.0g

Na2HP04 95.0 g distilled. Water 1000 ml

This suspension is used to inoculate a 250 ml Erlenmeyer inoculation flask with triple deflectors, which contains 54 ml of inoculation medium B of the following composition.

Medium B

autolyzed yeast (Ardamine +) 10.0 g

Glucose 10.0g

MgSCU • 7H2O 0.05 g

Phosphate buffer ++ 2.0 ml distilled water 1000 ml the pH is adjusted to 6.5 with NaOH

+ Ardamine: Yeast Products Corporation ++ phosphate buffer solution:

KH2PO4 91.0g

Na2HP04 95.0 g distilled water 1000 ml

The inoculation flask is closed with cotton or cotton wool and shaken for 30 hours at 28 ± 1 ° C on a 220 rpm gyro shaker (5.08 cm vibration amplitude).

Fifty 250 ml Erlenmeyer production flasks without baffles, each containing 40 ml production medium C, are inoculated with 1 ml per flask of the broth from the inoculation flask. The production flasks are closed with cotton or cotton wool.

Medium C

Tomato paste or pulp 20.0 g primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

5 C0CI2 • 6H2O 5.0 mg distilled water 1000 ml

The pH is adjusted to 7.2 to 7.4 with NaOH.

10 After the inoculation, the production flasks are incubated for 3 days at 28 ± 1 ° C while shaking on a 220 rpm gyroscope (5.08 cm vibration amplitude). The flasks are analyzed for activity against standard Vibrio percolan's ATCC 8461 assay plates using 1.27 cm assay discs that are immersed in the centrifuged fermentation broth samples. The samples are diluted with 0.05 M phosphate buffer, pH 7.4. The results are summarized in the following table.

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pH 6.4

Vibrio percolans

(1/100 dilution) Assay 23 mm

25 890 assay, units / ml 103

The entire broth is centrifuged in 200 ml parts in polycarbonate bottles at 9000 rpm for 15 min; 1600 ml of combined supernatant solution with a potency of 104 units / ml are obtained. Add 0.5 ml of neutral EDTA.

The centrifuged broth is on a Dowex-1 x2 (Cl "), 0.297 to 0.149 mm (50-100 mesh) column,

Layer dimensions 3.8x22 cm, adsorbed with a flow rate of 35 6 to 20 ml / min. The column is rinsed with 100 ml deionized water and with 11 deionized water containing 50 g sodium chloride, 0.02 M tris-HCl buffer, pH 7.0 and 25 (in neutral EDTA) at a flow rate of 6 ml / min elutes Fractions of 10 ml 40 are collected.

Antibiotic 890Ai occurs in fractions 13 to 81 with a maximum in fractions 25 to 33,

counted from the first application of the salt eluate. Fractions 24 to 41 with the highest 45 biopotency / A2oo ratios are pooled for further treatment. The combined fractions have a total of 29,000 units or 17% of the bioactivity applied.

The Dowex eluate is concentrated to 10 ml. The 50 pH is adjusted to 6.5 with dilute hydrochloric acid, the concentrate is applied to a column made of XAD-2, layer dimensions 3.3 x 36 cm, which previously each with 2160% aqueous acetone, deionized water and 5 % (w / v) sodium chloride was washed in deionized water. The sample is eluted with deionized water at a flow rate of 6 ml / min. The 40 to 260 ml fractions are collected.

The activity of the antibiotic occurs in fractions 6 to 14, which thus ranges from 220 to 2560 ml of eluted volume. The peak occurs at fractions 9 and 10, which range from 370 to 590 ml of eluted volume. Fractions 9 to 12, which make up 370 to 1060 ml of the eluted volume, have the highest ratios of HAEA300 / A220 and are combined for further treatment 65. These fractions contain 36,600 units; this corresponds to 126% of the apparent activity applied.

The combined fractions 9 to 12 are made up to 100 ml

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concentrated. The concentrate is applied to a column of Dowex-1 x4 (Cl_), -0.037 mm (minus 400 mesh), layer dimensions 2.2x41 cm, at a flow rate of 2 ml / min. The column is rinsed with 50 ml deionized water and eluted with 310.07 M NaCl + 0.005 M NH4C1 + 0.0001 M NH3 in deionized water at a flow rate of 2 ml / min. 10.8 ml fractions are collected starting with the first application of the eluent.

The main peak of antibiotic 890Ai occurs in fractions 181 to 217 with a maximum in fraction 198. Fractions 186 to 210, which contain a total of 114 absorption units at 300 nm, are combined.

The combined fractions are concentrated to 4.0 ml and the pH is adjusted to 7.3 by the addition of 16 (11M NaOH). The concentrate is placed on a column of Bio-Gel P-2.0.074 to 0.037 mm (200-400 mesh), layer dimension 2.15 x 70 cm, applied and washed with 3 x 1 ml washing solutions from deionized water and eluted with deionized water at 0.96 ml / min. The fractions of 3.85 ml are collected .

The main peak of the 890Ai antibiotic occurs in fractions 24 to 44 with a maximum in fractions 33 and 34. Fractions 27 to 38 with the highest A3oo / A245 ratios are pooled for lyophilization 5. These combined fractions have a total of 72 A3oo units.

To carry out the lyophilization, the combined fractions are concentrated to 3.0 ml, and the pH of the concentrate is adjusted to 7.5 by adding 10 ml of 0.1 M 10 NaOH. The sample is divided into two parts of 1.50 ml each, and the parts are quickly frozen separately and lyophilized from 14 ml glass ampoules with a screw cap. Each sample contains 1.73 mg 890Ai; this corresponds to 35.8 A3oo units.

is a culture of Streptomyces flavogriseus called MA-4600a at de Merck & Co., Inc. The depository has been permanently and without limitation as to availability in the Culture Collection of Northern Regional Laboratories, Northern 20 Utilization Research and Development Division,

Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, and is open to the public under number NRRL 8140.

B

Claims (8)

637960 2. Process for the preparation of the deacetyl 890Aio of the structure s-ch2-ch2-nh2 cooh characterized in that the compound 890Aio of the structure -ch2- ^ ch2-nh- I, treated with an enzyme that can hydrolyze the N-acetyl group. 2nd PATENT CLAIMS 1. The antibiotic Desacetyl 890Ato of the structure 0S03H ch3 ~ Ch- / _N • S-CH2-CH2-NH2 cooh and pharmaceutically acceptable salts thereof. 3. The method according to claim 2, characterized in that the enzyme is an amidohydrolase. 4. The method according to claim 3, characterized in that the amidohydrolase is an N-acetylethanolamine amido hydrolase. 5. The method according to claim 3, characterized in that the amidohydrolase is an N-acetyl-890Aio-amidohydrolase. 6. The method according to claim 2, characterized in that the method is carried out with an amidohydrolase, which is produced by an amidohydrolase-producing strain of the microorganism Protaminobacter ruber. 7. The method according to claim 2, characterized in that 890Aio is treated intimately with the enzyme N-acetyl-890Aio-amidohydrolase, which is formed by an amidohydrolase-forming strain of the microorganism Protaminobacter ruber. 8. Antibacterial preparation, characterized by a content of an antibacterial, effective amount of the compound desacetyl 890Aio according to claim 1.