DE2652678A1 - ANTIBIOTICA AND METHOD OF MANUFACTURING THE SAME - Google Patents

Description

Dr. Karus-A. Ci-auns Ü

UVi ^ UMir.a \ ^ g ₁ HOVEMBER 1976

15 837

MSRCK & CO., INC. Rahway, New Jersey 07065, V.St.A.

Antibiotics and methods of making the same

The discovery of the remarkable antibiotic properties of. Penicillin has aroused great interest in this area, which has led to the discovery of many other valuable anti-blotch substances; such antibiotics are, for example, other penicillins, streptomycin, bacitracin, tetracyclines, chloramphenicol, erythromycins and the like. In the aligeaieinar) the antibacterial activity of any of these antibiotics does not extend to certain clinically important pathogenic bacteria. For example, some are mainly only effective against gram-positive types of bacteria. In the course of the widespread use of previously known antibiotics to treat bacterial infections, diabetic resistance has led to the emergence of difficult resistance problems.

Hence the deficiencies of the known antibiotics have one more Research has encouraged finding other antibiotics that are effective against a wider range of pathogens as well active against resistant strains of special microorganisms

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The invention relates to two new antibiotics. In particular, relates they focus on the new antibiotic substances referred to here as Desacetyl-890A-j and Desacetyl-890A. the Invention encompasses the antibiotics in diluted forms, as raw concentrates and in pure forms.

The invention is based on the object of providing new and valuable antibiotics which are highly effective Way to inhibit the growth of various gram-negative and gram-positive microorganisms. Another job The invention is to provide a process for the preparation of these new antibiotics by enzymatic deacetylation of the compounds 890A ^ or 890A ^ available. In the end the invention is based on the object of providing a process for deacetylating N-acetyl thienamycin place.

The new antibiotic substances according to the invention will be by hydrolysis of the N-acetyl group of 890A * and 890A ^ produced with the help of an amidohydrolase, which is able to hydrolyze the N-acetyl group. Suitable sources for an amidohydrolase exhibiting this ability are amidohydrolase-producing strains of the microorganism Protaminobacter ruber. The enzyme produced by Protaminobacter ruber is N-acetylthienamycin amidohydrolase, a representative the subgroup of enzymes that are classified according to the recommended enzyme nomenclature of the "International Union of Pure and Applied Chemistry "and the" International Union of Biochemistry "as E.C. 3.5.1.

The microorganism capable of carrying out the deacetylation process was isolated from a soil sample and classified as to that based on studies

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Species identified as belonging to Protaminobacter ruber. In the Cultural Collection from Merck & Co., Inc., Rahway, N.J., he is with MB-3528 has been designated. A culture of the microorganism is permanent in the Northern culture collection Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois and assigned number NRRL B-8143.

The morphological and cultural characteristics of Protaminobacter ruber NRRL B-8143, as well as the details on the utilization of carbon and nitrogen and the biochemical reactions of the microorganism are below specified:

Morphology - The cells are rod-shaped with rounded ends, 0.9-1.2 χ 2.3-4.6 μ in size and occur individually or in pairs. 24 and 48 hour old cells are stained gram negative with a granular appearance. The grains, especially the polar grains, are colored black by Sudan Black B. The cells are motile at 28 ° C, but at 37 ° C motility is questionable.

Culture characteristics - Colonies on nutrient agar are initially thin, punctiform, semi-transparent and colorless; then they become low-convex, opaque, smooth, edge entire, somewhat dry in consistency and pigmented pink to pink-red.

Cultures in nutrient broth are uniformly cloudy without membranes.

The pigment production is from light or the temperatures studied (28 C and 37 C) independently. The pigment is in

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15837> 2S52678

Acetone soluble, but insoluble in water or chloroform.

At 28 C, growth takes place on nutrient agar and brain-heart-infusion agar a little slow under aerobic conditions, but good; at 37 C growth is moderate to good, but slower; no growth takes place at 50.degree.

Utilization of C and N sources

When using a basal salt medium with ammonium sulfate as the N source, growth is good with arabinose, with xylose moderate and with dextrose, fructose, mannose, rhamnose, lactose, maltose, sucrose, raffinose, cellulose, inositol and mannitol bad.

N-acetylethanolamine can be used as the only C and N source will.

In OF-Basal Medium (Difco Laboratories, Detroit, Michigan) no acid or gas is formed from dextrose or lactose under aerobic or anaerobic conditions.

Biochemical reactions

The biochemical reactions are based on standard methods, those in "Manual of Microbiological Methods" edited by the Society of American Bacteriologists, McGraw-Hill publisher Book Co., New York, 1957.

Catalase - positive

Oxidase - negative

Starch is not hydrolyzed. Casein is not hydrolyzed. Gelatin is not liquefied

Litmus milk remains unchanged in consistency, but becomes somewhat alkaline after 7 days

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Indole - negative

HpS - negative

Nitrates are not reduced. Urease - positive

Lysine and ornithine decarboxylase - negative.

N-acetylthienamycin, 890A ₁ and 890A, are the names for isomers of the antibiotic of the structural formula

CH ₁ -CH (OH). . . "

³ 1 Λ S-CH ₀ -CH ₀ -NH-C-CH ₃

OOH

N-acetylthienamycin, its description, and a method for making the same are disclosed in U.S. Patent Application Serial No. 634,301 filed Nov. 21, 1975. 890A ₁ and 890A ^, their description and method of making the same are disclosed in U.S. Patent Application Serial No. 634,300 dated Nov. 21, 1975.

The new antibiotics according to the invention, namely Desacetyl 890A ₁ and Desacetyl 890A ,, are isomers of the structural formula

CH, -CH (OH)

³ I ^x S-CH ₂ -CH ₂ -NH ₂

COOH

and are produced by enzymatic hydrolysis of 890A ₁ or with the aid of an amidohydrolase, which occurs in species of the genus Protaminobacter.

The new method according to the invention is based on the Abspal-

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tion of the N-acetyl group of the compounds of the structural formula

CH, -CH (OH), S \?

³ I \ S-CH ₂ -CH ₂ -NH-C-CH ₃

COOH

and consists in bringing the compounds into intimate contact with an amidohydrolase which is capable of Hydrolyze N-acetyl group. In particular, the process according to the invention enables the N-deacetylation of N-Acety! Thienamycin, 890A-] and 890A * through close contact of these Compounds with the amidohydrolase N-acetylthienamycin amidohydrolase.

There has been a surprising homology between N-acetylethanolamine and N-acetylthienamycin found by making extracts of microorganisms with the hitherto not yet described enzyme N-acetylethanolamine amidohydrolase capable in many cases are to hydrolyze N-acetylthienamycin. It was also found that amidohydrolases, which are able to hydrolyze N-acetylthienamycin, also have the ability to Antibiotics 890A ^ and 890A ^ in their new deacetyl forms convict. Another novel feature of the invention relates to the method for obtaining such microorganisms which consists in choosing those strains that are able to use N-acetylethanolamine for their growth, and these microorganisms then examined for the presence of N-acetylthienamycin amidohydrolase.

The antibiotic thienamycin, which is produced according to the new N-deacetylation process obtained from N-acetylthienamycin is a valuable antibiotic. Its description, manufacture

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Development and commercial exploitation are in the USA patent application Serial No. 526,992 dated November 25, 1974 disclosed. The antibiotics Desacetyl - 890A- | and Desacetyl-890A ^ are new and valuable antibiotics.

A feature of the invention is the novel deacetylation of N-acetylthienamycin. There are two sources of N-acetyl thienamycin. N-acetylthienamycin is produced by fermentation of a liquid with the microorganism Streptomyces cattleya NRRL-8057 manufactured. This microorganism also produces thienamycin, which can then be chemically N-acetylated.

On the basis of extensive classifying investigations, the microorganism isolated from a soil sample became Streptomyces cattleya identified as Actinomycete and in the culture collection by Merck & Co., Inc., Rahway, N.J., designated as MA-4297. A culture of this microorganism is permanent in the culture collection of Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, and has the Identification number NRRL 8057 obtained.

The classification keys for the genus Streptomyces and the culture descriptions of the species Streptomyces in "Bergey's Manual of Determinative Bacteriology" (7th edition, 1957) and in "The Actinomycetes", Volume II (1961) by S.A. Waksman and in "Cooperative Descriptions of Type Cultures of Streptomyces "by E.B. Shirling and D. Gottlieb," International Journal of Systematic Bacteriology ", Vol. 18, pp. 69-189 (1968), Vol. 18, pp. 279-392 (1968), Vol. 19, p 391-512 (1969) and Volume 22, pages 265-394 (1972), were after

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of a species of Streptomyces searched the similar
Has morphological and cultural characteristics as MÄ-4297. However, there is none in these classic manuals
Species of Streptomyces described with the orchid-colored pigmentation of the aerial mycelium, the morphological features and the absence of diffusible pigment, which together represent the distinguishing features of MA-4297.
For this reason the identification of a new species of Streptomyces was justified and necessary.

The characteristic morphological properties and cultural properties from Streptomyces cattleya are summarized in the following table.

Morphology r- The sporophores are compact spirals that
appear as side and end branches on the aerial mycelium. The spores are ellipsoidal to cylindrical in shape,
have a size of 0.9μ x 1.2 u and occur in chains of more than 10.

Cultural features
Tomato porridge oatmeal agar

Vegetative growth - back - brownish, flat, self

spreading;

Aerial mycelium - orchid colors (10 gc) mixed with white;

Soluble pigment - none.

Czapek Dox agar (sucrose nitrate agar)

Vegetative growth - colorless, flat, spreading;
Aerial mycelium - sparse, white with a pink tinge;
Soluble pigment - none.

Egg albumin agar

Vegetative growth - brownish with a gray-orchid tinge, flat, spreading;

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Aerial mycelium - orchid-colored (10 ge), mixed with lighter ones Tones of orchid colors and some white; Soluble pigment - none.

Glycerine asparagine agar Vegetative growth - reverse side - brownish with gray-pink Approach, flat, spreading; Aerial mycelium - orchid-colored (10 gc) mixed with a little white; Soluble pigment - none.

Yeast extract-glucose + salt-agar Vegetative growth - brownish with gray-pink tinge; Aerial mycelium - orchid-colored (10 gc), mixed with pink-white; Soluble pigment - none.

Yeast Extract-Malt Extract Agar Vegetative growth - brownish; Aerial mycelium - orchid-colored (10 gc), mixed with pink-white; Soluble pigment - none.

Peptone Iron Yeast Extract Agar Vegetative growth - brownish; Aerial nyc - none; Soluble pigment - weak browning of the medium; Melanin - negative; HpS development - negative.

Nutrient agar

Vegetative growth - light brownish; Aerial mycelium - none; Soluble pigment - none.

Nutrient starch agar

Vegetative growth - cream-colored to brownish; Aerial mycelium - none; Soluble pigment - none; Hydrolysis of starch - moderate.

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Nutrient gelatin agar

Vegetative growth - cream colored; Aerial mycelium - none; Soluble pigment - none; Liquefaction of gelatin - moderate.

Vertical gelatin tube Vegetative growth - brownish; Aerial mycelium - none; Soluble pigment - none; Liquefaction of gelatin - moderate.

Potato wad

Vegetative growth - moderate, brownish; Aerial mycelium - sparse, grayish-pink-white; Soluble pigment - none.

Loeffler's blood serum Vegetative growth - cream-colored; Aerial mycelium - none; Soluble pigment - none; Liquefaction - none.

Skimmed milk agar

Vegetative growth - brownish; Aerial mycelium - scanty, whitish; Soluble pigment - weak browning of the medium; Casein hydrolysis - positive.

Litmus milk

Vegetative growth - brownish to brown; Aerial mycelium - none; Color - no soluble pigment, litmus becomes bluish;

Coagulation and / or peptonization - partial peptonization, becomes alkaline.

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Skimmed milk

Vegetative growth - brownish;

Aerial mycelium - none;

Soluble pigment - none;

Coagulation and / or peptonization - partial peptonization, becomes alkaline.

Tyrosine agar

Vegetative growth - brownish;

Aerial mycelium - mixture of orchid colors (10 gc) and white; Soluble pigment - none;

Decomposition of tyrosine - positive.

Unless otherwise stated, all of the above characteristics were observed after three weeks of incubation at 28 ° C. The media used in these studies were approximately neutral (pH 6.8 to 7.2). The ones in the description The color names used correspond to the definitions in "Color Harmony Manual", 4th edition (1958), published from Container Corporation of America, Chicago, Illinois.

Streptomyces cattleya was also examined for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown for three weeks at 28 ° C. on a basal synthetic medium (Pridham and Gottlieb) which contained the carbohydrate in question in a concentration of 1 % . The media used in these studies were approximately neutral (pH 6.8 to 7.2). Table I shows the utilization of these carbohydrates by Streptomyces cattmeya, where + means good growth, - bad growth and - no growth on the carbohydrate in question.

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Table I.

Glucose + maltose -

Arabinose - mannitol +

Cellulose - mannose

Fructose - raffinose

Inositol - rhamnose

Lactose - sucrose xylose

For the extent of growth with temperature change, den The following applies to oxygen demand and the effect of the microorganism on nitrate:

Temperature range (yeast extract-glucose + salt agar); 28 ° C - good
37 ° C - moderate
50 ° C - no growth

Oxygen demand (needle inoculum in yeast extract-glucose-

Salt agar);
aerobic.
Nitrate reduction - positive.

With regard to the production of thienamycin, the invention does not apply to the microorganism Streptomyces cattleya Microorganisms that fully meet the above growth characteristics and microscopic features are limited. The invention Rather, it also includes the use of mutants obtained from the organisms described above by various means, such as X-rays, ultraviolet rays, N-mustard gas, exposure to phage and the like.

Thienamycin becomes more suitably aqueous during aerobic fermentation Culture media under controlled conditions by inoculation produced with the organism Streptomyces cattleya. For the her-

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Thienamycin can be prepared using the same aqueous media that are suitable for the production of other antibiotics are. Such media contain C and M sources that can be assimilated by the microorganism, as well as inorganic salts.

Another feature of the invention is the novel deacetylation of 890A ₁ and 89OA ₅ . 890A ₁ and 89OA ₅ are produced by fermentation of a liquid with the microorganism Streptomyces flavogriseus NRRL 8139. The antibiotic 890A ₁ can also be produced by fermentation of Streptomyces flavogriseus NRRL 8140.

On the basis of extensive classifying studies, it was found that the strains of microorganisms of the species Streptomyces flavogriseus and these are in the Culture Collection of Merck & Co., Inc., Rahway, N.J., as MA-4434a and MA-4600a. A culture from each of these tribes is irrevocable and permanent in the culture collection from Northern Regional Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, and has the identification number NRRL 8139 and 8140, respectively.

Streptomyces flavogrisrus NRRL 8139 produces both antibiotics 890A ₁ and 890A ^, which are isolated in practically pure form from the fermentation fluid. Streptomyces flavogriseus NRRL 8140 produces the antibiotic 890A ₁ with no detectable amounts of 890A-

3 '

The characteristic morphological features and cultural features of Streptomyces flavogriseus NRRL 8139 are compiled in the overview below.

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Morphology - The sporophores are branching, straight to winding spore chains that form tufts. The chains have a length of more than 10 spurs. The spores are spherical to oval - 0.9w χ 1.2μ (at 970x magnification).

Cultural features

Oatmeal agar

Vegetative growth - back side - yellowish-brownish, parchment-like growth;

Aerial mycelium - light gray with a medium gray border; Soluble pigment - none.

Czapek Dox agar (sucrose nitrate agar)

Vegetative growth - back side - brown with a dark brown margin;

Aerial mycelium - medium gray, velvety;
Soluble pigment - light browning of the medium.

Egg albumin agar

Vegetative growth - back - yellowish-brownish

with brown border;
Aerial mycelium - medium gray, mixed with yellowish gray (2dc)

and grayish yellow (2db);
Soluble pigment - light yellowish-brownish.

Glycerine asparagine agar

Vegetative growth - back - yellowish-brownish,

flat, spreading;
Aerial mycelium - velvety, light gray with a strong yellowish tone

to gray (2dc);
Soluble pigment - none.

Inorganic Salts Starch Agar

Vegetative growth - back side - brown; Aerial mycelium - medium gray, velvety;
Soluble pigment - light yellowish-brownish.

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Yeast extract dextrose + salt agar

Vegetative growth - back side - brown with very dark brown Edge;

Aerial mycelium - dark gray, mixed with light gray, velvety; Soluble pigment - none.

Yeast Extract-Malt Extract Agar Vegetative growth - back side - dark brown; Aerial mycelium - dark gray, velvety; Soluble pigment - none.

Skimmed milk agar

Vegetative growth - brownish; Aerial mycelium - scanty, greyish; Soluble pigment - slight browning of the medium; Casein hydrolysis - good.

Litmus milk

Vegetative growth - moderate growth ring, dark brownish; Aerial mycelium - none; Color - purple;

Coagulation and / or peptonization - complete peptonization; becomes alkaline, pH 8.2.

Skimmed milk

Vegetative growth - moderate growth ring, brownish; Aerial mycelium - none; Soluble pigment - brownish;

Coagulation and / or peptonization - complete peptonization; becomes alkaline, pH 8.0.

Tyrosine agar

Vegetative growth - back side - dark brown; Aerial mycelium - dark gray; Soluble pigment - weak browning of the medium; Decomposition of tyrosine - none.

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Peptone Iron Yeast Extract Agar Vegetative growth - brownish; Aerial mycelium - scanty, greyish; Soluble pigment - none; Melanin - none;
HpS development - none.

Nutrient agar

Vegetative growth - back - light grayish brown with

darker gray-brown border; Aerial mycelium - light gray with a dark gray border; Soluble pigment - none "

Nutrient starch agar

Vegetative growth - brownish with a gray border; Aerial mycelium - medium gray with a dark gray border; Soluble pigment - none; Hydrolysis of starch - good.

Nutrient gelatin agar

Vegetative growth - colorless with a dark gray border; Aerial mycelium - grayish-white; Soluble pigment - none; Liquefaction of gelatine - good »

Potato wad

. Vegetative growth - good growth, severely wrinkled; Aerial mycelium - gray to greenish gray; Soluble pigment - slight browning of the medium.

Loeffler's blood serum

Vegetative growth - cream colored; Aerial mycelium - none;
Soluble pigment - none; Liquefaction - none.

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Vertical gelatin tube

Vegetative growth - cream colored;
Aerial mycelium - none;
Soluble pigment - none;
Liquefaction of gelatin - good.

Unless otherwise stated, all of the above characteristics were observed after three weeks of incubation at 28 ° C. The media used in these studies were approximately neutral (pH 6.8 to 7.2). The ones in the description The color names used correspond to the definitions in "Color Harmony Manual", 4th edition (1958), published from Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus NRRL 8139 was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown for three weeks at 28 ° C. on a basic synthetic medium (Pridham and Gottlieb) which contained the carbohydrate in question in a concentration of 1 % . The media used in these studies were approximately neutral (pH 6.8 to 7.2). Table II shows the utilization of these carbohydrates by Streptomyces flavogriseus NRRL 8139, where + means good growth, - poor growth and - no growth on the carbohydrate in question.

Table II

Glucose + maltose +

Arabinose + mannitol +

Cellulose - mannose +

Fructose + raffinose -

Inositol - rhamnose +

Lactose + sucrose -

Xylose +

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The following applies to the extent of growth with temperature change and the oxygen demand of the microorganism:

Temperature range (yeast extract-dextrose + salt agar); 28 ° C - good

37 ° C - good vegetative growth; no aerial hyphae 50 ° C - no growth

Oxygen demand (needle inoculum in yeast extract-dextrose-salt agar);
aerobic.

The characteristic morphological features and cultural features of Streptomyces flavogriseus NRRL 8140 are compiled in the overview below.

Morphology - The sporophores are branching, straight to tortuous chains of spores that form tufts. Chains are more than 10 spurs in length. The spores are round to oval - 0.9p x 1.2u (at 970x magnification).

Cultural features

Oatmeal agar

Vegetative growth - back side - yellowish-brownish with a dark brown border;

Aerial mycelium - light gray with a medium gray border; Soluble pigment - none.

Czapek Dox Agar (Sucrose Nitrate Agar) Vegetative growth - back side - brown with dark brown Edge;

Aerial mycelium - medium gray, velvety; Soluble pigment - none.

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Egg albumin agar

Vegetative growth - back side - greyish-brownish with strongly yellow-brownish sections;

Aerial mycelium - medium gray, grayish-white and yellowish-gray (2dc) sections;

Soluble pigment - very light brownish.

Glycerine asparagine agar Vegetative growth - yellowish-brownish; Aerial mycelium - scanty, greyish; Soluble pigment - none.

Inorganic Salts-Starch-Agar Vegetative growth - back side - greyish cream-colored; Aerial mycelium - medium gray, velvety; Soluble pigment - none.

Yeast extract dextrose + salt agar Vegetative growth - back side - dark brown; Aerial mycelium - dark gray, mixed with light gray, velvety; Soluble pigment - none «

Yeast Extract-Malt Extract Agar Vegetative growth - back side - dark brown; Aerial mycelium - dark gray, velvety; Soluble pigment - none.

Peptone Iron Yeast Extract Agar Vegetative growth, brownish; Aerial mycelium - none; Soluble pigment - none; Melanin - none; HpS development - none.

Nutrient agar

Vegetative growth - light brownish; Aerial mycelium - none; Soluble pigment - none.

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Nutrient starch agar

Vegetative growth - cream colored; Aerial mycelium - none; Soluble pigment - none; Hydrolysis of starch - good »

Nutrient gelatin agar

Vegetative growth - cream colored; Aerial mycelium - none; Soluble pigment - none; Liquefaction of gelatin - good.

Vertical gelatin tube Vegetative growth - brownish; Aerial mycelium - none; Soluble pigment - none; Liquefaction of gelatin - completely.

Skimmed milk agar

Vegetative growth - brownish; Aerial mycelium - none; Soluble pigment - none; Casein hydrolysis - good.

Litmus milk

Vegetative growth - brownish growth ring; Aerial mycelium - none; Color - brownish-purple;

Coagulation and / or Peptonization - Complete Peptoni " ization, becomes alkaline, pH 8.0.

Skimmed milk

Vegetative growth - brownish, moderate growth ring; Aerial mycelium - none; Soluble pigment - light brown;

Coagulation and / or peptonization - complete peptonization, becomes alkaline, pH 8.5.

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is 837 IH 2852678

Potato wad

Vegetative growth - good, brownish; Aerial mycelium - very sparse, whitish; Soluble pigment - none »

Loeffler's blood serum

Vegetative growth - cream colored;
Aerial mycelium - none;
Soluble pigment - none;
Liquefaction - none «

Tyrosine agar

Vegetative growth - brownish;

Aerial mycelium - none;

Soluble pigment - slight browning of the medium; Decomposition of tyrosine - very weak.

Unless otherwise noted, all of the above observations have been made carried out after three weeks of incubation at 28 ° C. The media used in these studies were approximate neutral (pH 6.8-7.2). The color names used in the description correspond to the definitions in "Color Harmonv Manual, "4th Edition (1958), published by Container Corporation of America, Chicago, Illinois.

Streptomyces flavogriseus NRRL 8140 was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown for three weeks at 28 ° C. on a basal synthetic medium (Pridham and Gottlieb) which contained the carbohydrate in question in a concentration of 1 % . The media used in these studies were approximately neutral (pH 6.8-7.2). Table III shows the utilization of these carbohydrates by Streptomyces flavogriseus NRRL 8140, where + means good growth, - bad growth and - no growth on the carbohydrate in question.

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Table III

Glucose + maltose +

Arabinose + mannitol +

Cellulose - mannose +

Fructose + raffinose -

Inositol - rhamnose +

Lactose + sucrose

Xylose +

The following applies to the extent of growth when the temperature changes and the oxygen demand of the microorganism:

Temperature range (yeast extract dextrose + salt agar); 28 ° C - good

37 ° C - moderate vegetative growth; no aerial hyphae 50 ° C - no growth

Oxygen demand (needle inoculum in yeast extract-dextrose-salt agar);

aerobic.

The generation of 890A ,. and 890A ^ is nicVit on the microorganism Streptomyces flavogriseus or restricted to microorganisms that have the above growth characteristics and microscopic Fully match characteristics. Rather, the invention also encompasses the use of mutants derived from the above-described organisms by various means, such as X-rays, ultraviolet rays, N-mustard gas, exposure phage and the like.

^ and 890A ^ are used when under controlled conditions carried out aerobic fermentation generated suitable aqueous nutrient media with strains of the organism Streptomyces flavogriseus have been inoculated. For the production of

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890A ₁ and 890A ^ are aqueous media, such as those used to produce other antibiotics. Such media contain C, N sources and inorganic salts which are assimilated by the microorganism.

The compounds according to the invention Desacetyl-890A ₁ and Desacetyl-890A ^ are valuable antibiotics which are active against a wide variety of gram-positive and gram-negative bacteria and are therefore used both in human medicine and in veterinary medicine. The compounds according to the invention can be used as antibacterial medicaments for the treatment of infections caused by gram-positive or gram-negative bacteria, for example against Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The antibacterial agents according to the invention can also be used as additives to animal feed, for preserving food and as disinfectants. For example, they can be used in aqueous compositions at concentrations of about 0.1 to 100 parts antibiotic per million parts solution, or preferably at concentrations of about 1 to 10 parts antibiotic per million parts solution, to prevent the growth of harmful bacteria on medical and dental care Destroying and inhibiting equipment and as bactericides for technical purposes, e.g. in water-based paints, in the white water of paper mills and to inhibit the growth of harmful bacteria.

The antibiotics according to the invention can be in the most varied pharmaceutical preparations as the sole active ingredient or in combination with one or more other antibiotics or used with one or more pharmacologically active substances. As an example of the former

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15 837

Case can use an aminocyclitol antibiotic, such as gentamicin administered together with the antibiotics according to the invention be to reduce the possibility of the appearance of resistant organisms to a minimum. As an example of the latter case, one can use diphenoxylate and atropine in dosage forms combine for the treatment of gastrointestinal diseases. The antibiotics can be in the form of capsules, tablets, powders, liquid solutions or as suspensions or elixirs. You can take it orally, topically, or intravenously administered intramuscularly.

Tablets and capsules for oral administration can be in unit dosage form and conventional excipients such as binders, Z ₀ B ₀ syrup, acacia resin, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone, fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine, lubricants , for example “magnesium stearate, talc, polyethylene glycol, silicon dioxide, agents that promote disintegration, such as potato starch or harmless wetting agents such as sodium lauryl sulfate. The tablets can be by conventional methodical '■, to be coated. Orally administered liquid preparations ^ - can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs etc. or as ^% ; dry product to be mixed with water or other suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, for example sorbitan syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate or acacia oil, such as non-aqueous edible oils Almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol, preservatives, e.g. p-hydroxybenzoe-

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acid methyl or propyl ester or sorbic acid. Suppositories contain conventional suppository bases, such as knuckle-nut or other glycerides.

Means for injection may be available in unit dose form in pull-on form or in multiple dose form in r.hüber with the addition of preservatives ft;: .- te7.lt -.ve rc en. Dia I'ittel may have in oily or aqueous vehicles, and ^v orniuli? Rj-.Tsnittel the form of Suspension'η, solutions, emulsions, such as suspending, stabilizing and / or dispersing agents. The 'active ingredient can also be in powder form and prior to use with a. suitable carrier, e.g. 3. sterile, pyrogen-free Water, to be turned on.

The agents may also be used in for absorption through the mucous membranes of the nose and throat or the erchial tissue in a suitable form, e.g. as a powder or liquid spray or inhalant, lozenge, rickets, medication etc. For the treatment of the eyes or ears kc ·.: ~ n the preparations as individual capsules, in liquid or semi-liquid fermentation Form provided or ver ". ·" -> rivet as drops etc. will. For local application, they can be used in hydrophobic or hydrophilic bases in the form of ointments, creams, lotions, paints, powders, etc.

In addition to a carrier, the means according to the invention can be other Components such as stabilizers, binders, oxidation retardants, Preservatives, lubricants, suspending agents, agents affecting viscosity or flavorings and the like.

In veterinary medicine, as in the treatment of chickens, cows, sheep, pigs and the like, the agents

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709822/1031 _{BAD grandmaW} AL

e.g. formulated as preparations for injection into the Erust that are canceled for long-term effects or the V. 'active ingredient set free quickly.

The adequate dose depends largely on the condition of the tiare to be treated.?., its weight and the type of infection, the type and frequency of administration, with parenteral administration in general ··; Infections and oral administration for intestinal infections preferably virex.

For the treatment of bacterial infections in patients, the compounds according to the invention are administered orally or by conventional methods for borimide treatment with antibiotics in amounts of about 2 to 600, preferably about 1 ^liter ~> administered up to ICO mg / kg / day and preferably divided into single doses, which are administered three to four times per day. Unit dose formats can be used, e.g. 2.5, 250, 330, 400 or 1000 mg V / The dose units are in the form of liquid preparations, such as solutions or suspensions, or as solid substances in tablets or capsules. The optimal dose depends on the individual case according to the type and severity of the injection, whereby smaller doses are used for the treatment of children; all such rules of measurement are known to a person skilled in the art.

The antibiotic thienamycin can be presented in the same way like the antibiotics Desacetyl- ~ 89ÖA- and Desacetyl-890A ,,.

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BATH QÜQiiMAL

709 822/1031

Analytical method for antibiotic N-acetylthienamycin I. Bioanalysis

Analyzes for antibacterial activity are carried out using the following disc diffusion method Vibrio percolans ATCC 8461 or Staphylococcus aureus ATCC 6538P carried out as a test organism.

Plates with Vibrio percolans ATCC 8461 are made as follows manufactured:

A freeze-dried culture of Vibrio percolans ATCC 8-61 is suspended in 15 ml of a sterile medium which contains 8 g / l Difco nutrient broth and 2 g / l yeast extract in distilled water (hereinafter abbreviated from KnYE). The culture is incubated overnight in a rotating shaker at 28 ° C. This culture is used to inoculate the surface of veη slant tubes that contain 1.5% agar in Ν3ΥΞ. and the inoculated slant tubes are ^{incubated overnight at 23 "1} C and then stored in the refrigerator.

The from a single freeze-dried culture he r? .O. The cooled oblique tubes provided are used for a period of up to 4 weeks after their production as follows: A loop with inoculum from the oblique tube is dispersed in 50 ml NBYE in a 250 ml Erlenmeyer flask. The culture is incubated overnight in a rotating shaker at 28 ° C. and then diluted to a density that results in 50 percent light transmission at 66Ο nm. 33.2 ml of this diluted culture are added to a volume of 1 1 NBYS which contains 15 g of agar and is kept at 46 ° C. The inoculated, agar-containing medium is placed in plastic Petri dishes of 100 χ 15 mm in quantities of 5 ml per dish.

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poured, chilled and up to a point of 5 days before
kept at 2 to 4 C during use.

Plates with Staphylococcus aureus ATCC 6538P are made as follows manufactured:

An overnight culture of the analytical organizer. 5
Staphylococcus aureus ATCC 6538P in NährbrUhe containing 0.2% Hofe- extract is diluted with 0.2% nm Kefeextrakt containing Nöhrbrühe to a suspension having an optical transmittance of 55% at a wavelength of 660th These
Suspension is added to Difco nutrient agar, which has been supplemented with 2.0 g / 1 Difco yeast extract and is at a temperature of 47 to UQ C, a mixture being obtained which contains 33.2 ml of the Sue-pension. per liter of agar contains .. 5 parts
this suspension is poured into Petri dishes of 85 mm diameter and these plates are cooled and kept at 4 C until use (maximum 5 days).

Samples of the antibiotic to be analyzed are diluted to a suitable concentration with phosphate buffer solution of pH 7. Filter paper disks have a diameter of 6.55
or 12.7 mm are dipped into the test solution and applied to the
Surface of the analysis plates placed. The panels are
incubated overnight at 37 C, whereupon the diameter of the inhibition zone is determined in millimeters. The diameter of the Kemir.-.one in millimeters determines the relative strength.

II. Extinction that can be erased by hydroxylamine (absorbance)

The proportion of the absorbance measured at 301 nm which is attributed to the content of the antibiotic in impure samples can be, is dis-

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ORIGINAL INSPECTED
709822/1031

water absorbance (with simultaneous inactivation the antibiotic activity / activity) by reaction with dilute Determined by hydroxylamine.

Samples that contain the antibiotic to be examined are prepared in 0.01 molar Kiliumphocphatbuffer solution (pH 7) so that they have an initial A ₇ ^ ₁ between 0.1. and 1.0. Freshly prepared, neutralized hydroxylamine (NH ₂ OH-HCl + NaOH up to a final pH value of 1) is added up to a final concentration of 10 mmolar, whereupon the reaction takes place at room temperature for at least 30 minutes ": can, ^ f you _n a ^ thus obtained. value from the initial reading (after correction for dilution by the added reagent) is subtracted, we obtain the ausiöschbare by hydroxylamine Lxtinktion (absorbance). solutions of pure N-Acetylthienamycin show a by hydroxylamine extinction (absorbance) of 86.0 %.

Analytical method for thienamycin
I. Bioanalysis

Antibacterial activity analyzes are performed using the following disk diffusion method unless otherwise specified. The analysis plates are produced as follows: A culture of the analysis organism Staphylococcus aureus ATCC 6535? In Klihrbrühe containing 0.2 % yeast extract is diluted with 0 _F 2 % yeast extract containing nutrient broth to a suspension with an optical transmittance of 55 % at a wavelength of 660 nm. This suspension is added to Difco Xähr agar, which has been supplemented with 2.0 g / l Difco yeast extract and is at a temperature of 47 to 48 ° C., a mixture containing 33.2 ml of the suspension being obtained contains per liter of agar. 5 ml of this suspension are in petroleum

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shells of 85 mm in diameter are poured, and these plates are made refrigerated and kept at 4 C until use (5 days maximum).

Samples of the antibiotic to be analyzed are mixed with phosphate buffer solution diluted from pH 7 to a suitable concentration. Filter paper disks with a diameter of 12.7 πιτ. are immersed in the test solution and placed on the surface of the Analysis plates laid; two slices of each prob? are usually placed opposite each other on a plate. The plates are incubated overnight at 37 C, and the zone of inhibition is measured in millimeters in diameter Hernrr.zcri-2 measured in millimeters determines the relative strengths or, if it gets with a purified reference standard, like Cephalothin, is compared to the strength of the antibiotic in Units / ml. The activity unit has Cephalot'nin standard solutions of 8, 4, 2 and 1 y / ml as the basis. Sine unity is defined as the amount that, according to the calculation, produces the same inhibition as 1 γ cephalothin yes milliliter, this zone of inhibition having a diameter between 16 and 21 mn.

II. Extinction that can be erased by hydroxylamine (absorbance)

The fraction of the absorbance measured at 297 nm (absorbancc), which is attributed to the content of impure samples in the antibiotic by the selective extinction of this absorbance (with simultaneous inactivation the antibiotic activity) determined by reaction with dilute hydroxylamine.

Samples are prepared in 0.01 molar potassium phosphate buffer solution (pH 7.0) so that they have an initial A ~ _QV between 0.05 and 2.0. Freshly made, neutral! -

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ORIGINAL INSPECTED

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Sated hydroxylamine (NHpOH.HCl + NaOH up to one: "Enc-pH-value of 7) is used to a final concentration of 10 mmolar added, whereupon the reaction can be carried out at room temperature at least Allow 30 minutes to drain. Taking the Apqy value thus obtained from the original reading (after correction for If the dilution caused by the added reagent is subtracted, the extinction which can be canceled by hydroxylamine is obtained (absorbance). Solutions of pure thienamycin show one extinction (absorbance) of 94.5 56.

Analysis methods for 890A ^ and 890A ^

I. Microbiological analysis

An agar plate disc diffusion method is used using either Vibrio percolans ATCC 8461 or Salmonella gallinarum MB-1287 as the test organism. A purified sample of the antibiotic ^ 89CA ₁ is used as the standard.

Plates containing Vibrio percolans ATCC S461 are made as follows manufactured:

A freeze-dried culture of Vibrio percolans ATCC 8451 is suspended in 15 ml of a sterile medium, which 8 g / l Difco nutrient broth and 2 g / l yeast extract in distilled Contains water and is called "nutrient broth yeast extract" (hereinafter abbreviated as NBYE). The culture is overnight incubated in a rotating shaker at 28 C. This culture is used to cover the surface of Inoculate slants containing 1.5% agar in NBYE, and the inoculated slants are kept overnight at 28 ° C incubated and then stored in the refrigerator.

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The chilled slant tubes made from a single freeze-dried culture will be used for periods of up to Used 4 weeks after their production as follows: An eyelet with inoculum from the slanted tube is in one 250 ml Erlenmeyer flask dispersed in 50 ml Γ »Ί; ϊΞ. A culture is overnight in a rotating shaker at 28 " incubated and then diluted to a density that gives 50 percent light transmission at 660 nm. 33.2 ml of this diluted culture are added to a volume of 1 1 Ν3ΥΞ, which contains 15g agar and is kept at A6C. The inoculated agar-containing medium is poured into plastic Petri dishes of 100 χ 15 mm in sizes of 5 ml per dish, refrigerated and held at 2 to 4 ° C for up to 5 days prior to use.

The plates containing Salmonella gallinarum MB-1287 are made manufactured as follows:

A sealed tube containing frozen and freeze-dried cells of Salmonella gallinarum MB-1287 in skimmed milk, which has been sealed under vacuum, is opened and inoculated into 15 ml of brain-heart broth. The cells are allowed to grow overnight at 37 ° C. without shaking, and the culture is inoculated into inclined tubes containing 0.8 % BBL nutrient broth + 0.2% Difco yeast extract + 1.5 % agar. After overnight growth at 37 ° C., a loop of the culture is transferred from the inclined tube into a flask containing 50 ml of 0.8 percent nutrient broth + 0.2 percent yeast extract. The flask is shaken overnight, and 1 liter of 0.8 % nutrient

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Broth + 0.2 % yeast extract + 1.5 % agar, which has previously been sterilized and cooled to 48 ° C., is inoculated with 20 ml of this polish. The inoculated KBYE agar is immediately placed in a petri dish :! Poured from plastic of 100 χ 15 mm (5 ml per dish), and the plates are kept at 0 to 5 C until use.

Filter paper discs with a diameter of 12.7 cm are dipped in the solution to be analyzed and placed on the agar. Another method is to load the discs by pipetting 0.1 ml of the solution onto a dry disc and then placing the disc on the agar. After a suitable incubation (for Salmonella gallinarun ΓΟ-1257 9 to 18 hours at 37 ° C, for Vibrio percolans ATCC 8461 12 to 24 hours at 25 C), the diameter of the Hsmmzone is measured. If necessary, dilutions of the solutions to be analyzed must be prepared in 0.05 molar potassium phosphate buffer (pH 7, ^; hereinafter abbreviated as KPB) or in demineralized water.

The strength is calculated as follows: A slope is determined by measuring the zone diameters of a solution of the antibiotic 890A ₁ or 890A ^ and a four-fold dilution of this solution (in KPB). Zv / egg slices of each concentration are analyzed on a single plate and the mean zone size is determined at each concentration. The gradient is equal to half the difference in the mean values of the zone sizes. The strength is then calculated according to the following equation:

Strength (units / ml) =

-D ₃ ] log 2

Slope (strength of the standard) χ Dilution χ 10

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15 837

In the above equation, D means the mean diameter of the zones formed by the unknown sample, D _{- means} the mean diameter of the standard ^{zones, and "Dilution 11" means} the degree to which the unknown sample has been diluted prior to analysis. If the test organism Vibrio porcolans ATCC 8 · 'ν6ΐ is used as the test organism, it is determined that pure 890Λ -, one Sv -. '. rke of 250 units per unit of the extinction (absorbance) which can be quenched by hydroxylamine at 300 nz>. , wc-rir. it is used as a standard. Accordingly, the strength of the aritibiotic 890A is ₇ , 31 Units per unit of the hydroxylamine-erasable absorbance at 300 nT ;.

For analyzes on plates with Salmonella gallinarun MB-I287, the slope is determined in the same orphan as with Vibrio porcolans ATCC 8461. If no standard is used, only relative strengths are calculated, '' because no slope is measured, a value of 2.3 ram is assumed. The strength calculations are carried out as in the analysis with Vibrio percolsns ATCC 8461. If no 890A ₁ standard is used, one can work with a control of penicillin G at 250 γ / ml in order to prove that the sensitivity of the organisms is normal Area lies.

II. Analytical method for the determination of "890 angular units"

The conventional disk flattening method is used with disks with a diameter of 12.7 mm. You work with routinely poured 10 ml per plate, inoculate with Vibrio percolans (ATCC 8461) and use cephaloridin as the standard. Four concentrations of cephaloridin, namely 3.12, 6.25, 12.5 and 25 r / ml, make up the standard, the concentration of 12.5 7 / ml being used as the reference solution. The zone through

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MMftlNAL WSPECTEO 709822/1031

15 837

knife on a 5 ml plate for the standard are the following

the:

Concentration, , 12 γ / ml 3 , 25 6th • - 12th

Zone durationar, ism

16.δ 22.3 25.0 29.6

A unit is defined as the amount of the antibiotic that which creates a pinch zone of 25 mm on a 5 ml slat. Therefore, in this analysis, a concentration of cephaloridin of 12.5 µg / ml becomes equivalent to one unit of 890A viewed. Since the slope of the line for cephaloridin is 4.0, the strength calculations for a sample are below Made use of a slope of 4.0.

III. Hydroxylamine reaction

The two antibiotics 890A ₁ and 890A ^ react with hydroxylamine to form a substance which greatly reduces the absorbance at 300 nm. This provides a basis for quantitative analysis on the antibiotics and

The solution to be analyzed is adjusted to 0.05 molar in a potassium phosphate solution (pH 7.4) by adding 1/20 volume part of a solution of 0.8 molar K ^ ₁ HPO <and 0.2 molar KHpPO ^ . I / 100 parts by volume of 1 molar hydroxylamine hydrochloride are then added and the absorbance at 300 nm is determined at intervals of 1/2 to 2 minutes. The reaction is carried out at 22-28.degree. A kinetic reaction of the first order is assumed and the half-life is determined from the decrease in absorbance during the first 10 minutes. From this half-life, the point in time is estimated beyond which there will be no further decrease in the absorbance

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709822/1031

(absorbance) should be observed, and the observations are continued beyond this point in time, if no further decrease is observed beyond this point in time, the total decrease in absorbance (corrected for the dilution effect and the absorbance (absorbar.ce) of hydroxylamine) is set as "absorbance at 300 nm which can be eliminated by hydroxylamine" (hereinafter abbreviated as HAEA ^ ₀₀ ) observed decrease at this point corrected for the decrease in background absorbance, assuming that the decrease in background absorbance. runs linearly over time. The corrected value is then recorded as ΗΛΕΑ.

The number of HAEA, ₀₀ units is equal to the value of multiplied by the volume in ml.

example 1

Method for isolating N-acetylthienamycin amidohydrolaso producing organisms

Make a suspension of 1 g of fertile turf soil in 100 ml of sterile phosphate buffer saline solution of the following Composition ago:

Phosphat buffer saline solution

NaCl 8.8 g

1 molar phosphate buffer, pH 7.5 * 10 ml distilled water 1000 ml

* 1 molar phosphate buffer, pH 7.5 16 ml 1 molar KH ₂ PO ^ solution are mixed with 84 ml 1 molar KpHPO / solution. The pH of the phosphate buffer is _{adjusted to 7.5 by adding a little 1 molar KH 2} PO ^ solution or 1 molar K ₂ HPO ^ solution.

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7 0 9 8 2 2/1031

Portions of this 1% stock soil suspension are used to make 10-fold, 100-fold and 1000-fold dilutions.

Portions of the stock suspension of 1 ml each or portions of the 10-fold, 100-fold and 1000-fold dilutions of 1 ml each are added to 2 ml of sterile, 1.0% agar solution at 15 ° C. The mixtures are quickly poured over the surface of sterile Petri dishes of 85 mm diameter, each 20 ml medium A contain the following composition:

Medium A

KH ₂ PO ₄ 3.0 g

K ₂ HPO ₄ 7.0 g

MgSO ₄ 0.1 g

distilled water 1000 ml N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution

N-acetylethanolamine is 10 times with water diluted and membrane sterilized. This solution is added after the autoclave treatment.

For solid media , add 20 g of agar.

The Petri dishes are incubated at 28 ° C. for 18 days. Well insulated Colonies are selected and streaked onto medium B. Medium B has the following composition:

Medium B

Tomato puree 40 g

Ground oatmeal 15 g

distilled water 1000 ml

pH adjusted to 6 with NaOH. For solid media , 20 g of agar are added.

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15 837

Individual clones are selected and grown on medium 3 slant tubes at 28 ° C for 2 days.

A portion of the slant culture is used to create a 250 ml Erlenmeyer flask containing 50 ml of medium A, one 250 ml Erlenmeyer flask containing 50 ml of supplemented medium B. (supplemented after the autoclave treatment with 0.4 ml of a membrane-sterilized Solution of N-acetylethanolamine diluted 1C times with water), and a 250 ml Srlenmeyer flask, containing 50 ml of medium C to inoculate. Medium C has the the following composition:

Medium C

Dextrose 20 g Pharmamedia S g Corn steep water (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 10-fold with water and membrane-sterilized. This solution will added after autoclaving.

The flasks are shaken for 4 days at 28 ° C. at 220 rpm (stroke 5.1 cm). 30 ml of each flask contents are centrifuged for 15 minutes at 8000 rpm. The excess is removed leaving only enough to form a thick suspension of cells and medium solids. Half of the suspension is sonicated in a Branson sonicator, model LS-75, with a 12.7 mm probe to disrupt the cells. The output power is set to position No. ₀ 4, and you work with four successive sound reinforcement cycles each

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15 seconds, the suspension being cooled in ice water during and between the individual sonication processes. In order to examine the whole-cell preparation or the sonication product for N-acetylthienamycin amidohydrolase activity, proportions of both suspensions are analyzed by incubating 5 μl of these suspensions with solutions containing 20 μl of a solution of 1.586 mg N-acetylthienamycin per ml of 10 mm potassium phosphate buffer solution ( pH 7) and 10 μl of 0.2 molar potassium phosphate buffer solution "(pH 7.4). Control experiments are carried out on the one hand with antibiotic and buffer alone and on the other hand with cell suspensions which do not contain antibiotic. After these mixtures have been incubated at 28 ° C. overnight - 2 iil portions are removed and applied to cellulose-coated thin-layer chromatography plates (TLC plates), which are then developed with a mixture of 70 % ethyl alcohol and 30 % water.After drying in air, the TLC plates are opened for 5 minutes Analysis plates from Staphylococcus aureus ATCC 6538P applied,

The TLC plates are removed and the analysis plates over-, o

J l

The analysis plates are produced as follows: A culture of the analysis organism Staphylococcus aureus ATCC 6538P, developed overnight in nutrient broth with a yeast extract content of 0.2 % , is diluted with broth containing 0.2% yeast extract to form a suspension which, at a wavelength of 660 nm has an optical transmission of 60 % . This suspension is added to Difco nutrient agar, which is supplemented by 2 g of Difco yeast extract per liter and is at a temperature of 47 to 48 C; the mixture obtained in this way contains 33.2 ml of the suspension per liter of agar. 40 ml of this suspension are poured into Petri dishes of 22.5 × 22.5 cm, and these plates are cooled and until

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709822/1031

Use (maximum 5 days) kept at 4 ° C.

The activity of N-acetylthienamycin amidohydrolase in the incubated Mixtures result from a bioactive range at R-. 0.44-0.47 due to thienamycin. That unreacted, bioactive N-acetylthiena-eycin appears at R-P 0.7-0.89. The procedure of this example enables the Isolation of N-acetylethanolamine amidohydrolase producing Microorganisms with N-acetylthienamycin amidohydrolase activity.

Example 2 Deacetylation of N-acetylthienamycin

Make a suspension of 1 g of fertile turf soil in 100 ml of sterile phosphate buffer saline solution of the following Composition ago:

Phosphate buffer saline solution

NaCl 8.8 g

1 molar phosphate buffer, pH 7.5 * 10 ml distilled water 1000 ml

* 1-molar phosphate buffer _y pH 7.5 16 ml of 1-molar KH ^ PO; -solution are mixed with 84 ml of 1-molar KpHPO / -solution. The pH of the phosphate buffer is increased by adding about 1-molar KH ₂ P0 ^ Solution or 1 molar K ^ HPO ^ solution set to 7.5.

Portions of this 1% stock soil suspension are used to make 10-fold, 100-fold and 1000-fold dilutions.

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709822/1031

Portions of the stock suspension of 1 ml each or portions of the 10-fold, 100-fold and 1000-fold dilutions of 1 ml each are added to each 2 ml of sterile, 1.0% agar solution at 43 ° C. The mixtures are quickly poured over the surface of sterile Petri dishes of 85 mm diameter, each Contain 20 ml of medium A of the following composition.

Medium A

KH ₂ PO ₄ 3.0 g

K ₂ HPO ₄ 7.0 g

MgSO ₄ 0.1 g

distilled water 1000 ml

N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution

N-acetylethanolamine is 10 times with water diluted and membrane sterilized. This solution is added after the autoclave treatment.

For solid media , add 20 g of agar.

The Petri dishes are incubated at 28 ° C. for 18 days. A well isolated colony is selected and streaked onto a Petri dish with medium B. Kedium B has the following composition:

Medium B

Tomato puree 40 g

ground oat flour 15 g

distilled water 1000 ml

pH adjusted to 6 with NaOH. For solid media , add 20 g of agar.

A single clone is selected and kept at 28 C for 2 days cultured in an oblique tube with medium B. Part of the culture this oblique tube is on the surface of six

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Slanted tubes made with medium B are spread on. These slanted tubes are incubated at 28 ° C. for 2 days. This culture is identified as Protaminobacter ruber and in the culture collection of Merck & Co _t., Was Ine, ranway, New Jersey, resigned under the name MB 3528.

A portion of the slant culture of Protaminobacter ruber MB-3528 is used to inoculate a 250 ml Erlenmeyer flask containing 50 ml of Medium C of the following composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g corn steep water

(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 10 times with water diluted and membrane sterilized. This solution is added after the autoclave treatment.

The flask is operated for 4 days at 28 ° C at 220 rpm (stroke 5.1 cm) shaken. 25 ml of the contents of the flask are centrifuged at 8000 rpm for 15 minutes. The supernatant liquid will removed, and the cells on the surface of the medium solids are dissolved in 0.5 ml of 0.05 molar potassium phosphate buffer (pH 7.4) scraped into it. The suspension thus obtained is in a Branson sonicator, model LS-75, with a 12.7 mm probe at the # 4 setting of the ultrasound treatment subjected to rupture the cells. You work with four sound reinforcement cycles of 15 seconds each

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to 2652ÖV8

and cools the suspension in ice water during and between the individual treatment periods. 10 μl of the sonication product are mixed with 25 μl of a solution containing 840 μl /: nl N-acetylthienamycin in 10 mmolar potassium phosphate buffer solution (pH 7) and incubated at 28 ° C. overnight. Control experiments are carried out on the one hand with antibiotic and buffer alone and on the other hand with sonicated cells and buffer without antibiotic. After overnight incubation at 28 ° C., 2 μl each are applied to TLC plates coated with cellulose, which are developed with a mixture of 70 % ethyl alcohol and 30 % water. After drying in air, the TLC plates are placed on analysis plates from Staphylococcus aureus ATCC 6538P for 5 minutes.

The analysis plates are prepared as follows: A culture of the analysis organism Staphylococcus aureus ATCC 6538P, developed overnight in nutrient broth with a yeast extract content of 0.2 % , is diluted with nutrient broth containing 0.2% yeast extract to form a suspension which, at a wavelength of 660 nm has an optical transmission of 60 % . This suspension is added to Difco nutrient agar, which is supplemented by 2 g of Difco yeast extract per liter and is at a temperature of 47 to 48 C; the mixture obtained in this way contains 33 »2 ml of the suspension per liter of agar. 40 ml of this suspension is poured into Petri dishes measuring 22.5 x 22.5 cm, and these plates are cooled and kept at 4 C until use (5 days at most).

The TLC plates are removed and the analysis plates are incubated at 37 ° C. overnight. Except for the bioactive stain for unreacted N-acetylthienamycin observed at R-0.7-0.89 one has a bioactive spot at R ~ 0.44-0.47 which is on

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709822/1031

Thienamycin is due - The control incubation mixtures of antibiotic + buffer, sonicated cell suspension + buffer and a sample of antibiotic + buffer to which the sonicated cell suspension was added immediately before the TLC analysis show no bioactive material at R _f 0, 44-0.47c

Example 3

Deacetylation of 890A ₁

Part of the slant culture of Protaminobacter ruber MB-3528 is used to add a 250 ml Erlenmeyer flask inoculate the 50 ml of medium C of the following composition contains:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn steeple (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 10 times with water diluted and membrane sterilized. This solution is added after the autoclave treatment.

The flask is operated for 4 days at 28 ° C at 220 rpm (stroke 5.1 cm) shaken. 25 ml of the contents of the flask are centrifuged at 8000 rpm for 15 minutes. The supernatant liquid will removed, and the cells on the surface of the medium solids are dissolved in 0.5 ml of 0.05 molar potassium phosphate buffer (pH 7.4) scraped into it. The suspension thus obtained is in a Branson sonicator, model LS-75, with a 12.7 mm probe with setting No. 4 of the ultra

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709822/1031

sonicated to disrupt the cells. You work with four sonication cycles of 15 seconds each and cool the suspension in ice water during and between the individual treatment periods. 10 μΐ of the sonication product are _{mixed with 25 μΐ of an 890A 1} solution that contains 4.85 optical density units at 300 nm per ml that can be erased with hydroxylamine Antibiotic. After overnight incubation at 28 ° C. , portions of 5 μl each are applied to a cellulose-coated TLC plate which is developed with a mixture of 70 % ethanol and 30 % water. After air drying, the TLC plate is placed on a Staphylococcus aureus ATCC 6538P analysis plate for 5 minutes.

The analysis plates are prepared as follows: An overnight culture of the analysis organism Staphylococcus aureus ATCC 6538P, developed in nutrient broth with a yeast extract content of 0.2%, is diluted with nutrient broth containing 0.2 % yeast extract to form a suspension which, at a wavelength of 660 nm has an optical transmission of 60 % . This suspension is added to Difco nutrient agar, which is supplemented by 2 g of Difco yeast extract per liter and is at a temperature of 47 to 48 ° C; the mixture obtained in this way contains 33.2 ml of the suspension per liter of agar. 40 ml of this suspension is poured into Petri dishes measuring 22.5 x 22.5 cm, and these plates are cooled and kept at 4 C until use (5 days at most).

The TLC plate is removed and the analysis plate is incubated at 37 ° C. overnight. In addition to the spot at R _f 0.7-0.89 for the unchanged bioactive 890A ₁ , there is a new bioactive spot

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observed at R _f 0.44-0.47, which is due to deacetyl-890A ^. Control incubation mixtures of antibiotic + buffer as well as sonicated cells + buffer do not produce any bioactive material at R _{£ 0.44-0.47.}

Example 4

Deacetylation of

The antibiotic 890A ^ is according to that in Example 3 for the deacetylation by the 890A * described method deacetylated.

B, e.! Game 5

Production of

Six 250 ml Erlenmeyer inoculation flasks, each containing 50 ml medium C, are inoculated with a portion of a slant culture of Protaminobacter ruber MB-3528. Medium C has the following Composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn steeple (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 10 times with water diluted and membrane sterilized. This solution is added after the autoclave treatment.

The flasks are operated for one day at 28 ° C at 220 rpm (stroke 5.1 cm) shaken.

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42 2-liter production flasks, each containing 400 ml of medium C, are inoculated with 7 ml of the culture from these inoculation flasks. These production flasks are shaken for 6 days at 28 ° C. at 220 rpm (stroke 5.1 cm). The contents of the flask are combined and centrifuged at 8000 rpm for 15 minutes. The cells are scraped from the clump of medium solids and made up to a final volume of 1600 ml with 0.05 molar potassium phosphate buffer solution (pH 7.4). This suspension is centrifuged again at 8000 rpm for 15 minutes. The cells are again scraped from the clump of medium solids and made up to a final volume of 160 ml with 0.05 molar potassium phosphate buffer solution (pH 7.4). This suspension is cooled to 5 ° C. and portions of 15 ml each are successively subjected to the ultrasound treatment described in Example 1 in 15 second intervals until no further decrease in turbidity is observed when the suspension is diluted 500-fold in phosphate buffer saline solution the following composition is made:

Phosphate buffer saline solution

NaCl 8.8 g

1 molar phosphate buffer, pH 7.5 * 10 ml distilled water 1000 ml

* 1 molar phosphate buffer, pH 7.5 16 ml 1 molar KH ₂ PO, solution are mixed with 84 ml 1 molar KpHPO, solution. The pH of the phosphate buffer is adjusted to 7.5 by adding a little 1 molar KHpPO solution or 1 molar KpHPO, solution.

_{250 mg of antibiotic 890A 1 are} added to 1 liter of 0.005 molar potassium phosphate buffer solution (pH 7.4). This mixture is mixed with 160 ml of the sonication extract of Protaminobacter ruber, which contains N-acetylthienamycin amidohydrolase. That

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Mixture is slowly added with a magnetic stirrer for 20 hours Stirred at 28 ° C. Then the mixture is at 10,000 g for 15 minutes centrifuged and the supernatant liquid removed, cooled to 5 C and with acetic acid to a pH of 4.5 - 0.2 set. The separation of the Desacetyl-890A-, the non-hydrolyzed antibiotic and other ingredients of the reaction mixture is carried out as follows, using a Schel ^ end diffusion bioanalysis against Staphylococcus aureus ATCC 6538P and measurements of the with Hydroxylamine erasable absorbance at 297 nm (as under the heading "Analytical Methods for Thienamycin" is used to monitor the performance of cleaning procedures.

The acidified, centrifuged reaction mixture is at a rate of 12 ml / min on 120 ml Dowex-50 χ 4 in the Sodium form (300-840 u) adsorbed. The adsorbate is washed with 120 ml of demineralized water and then with a Eluted at a rate of 6 ml / min with 2% aqueous pyridine solution. As soon as 75 ml of eluate has run through the column are, the following 240 ml are combined, concentrated to 25 ml and the concentrate is adjusted to a pH of 7.

The 25 ml concentrate are dispensed at a rate of 1 ml / min on 25 ml of Dowex-1 χ 2 resin in the chloride form (150-300 u) adsorbed. The resin comes out at the same speed eluted with demineralized water. After 25 ml has passed through the column, the following 50 ml combined, neutralized to pH 7 and concentrated to 10 ml. This concentrate is made up with acetic acid adjusted to pH 6.3 and placed in a bed of Dowex 50 χ 8 resin (37-74 u) in the 2,6-lutidinium form, which has a diameter of 1 cm and a height of 50 cm.

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and previously with 0.1 molar 2,6-lutidine acetate buffer (pH 6.3) has been brought into equilibrium. Plan elutes with the buffer at a rate of 1 ml / min. As soon 25 ml of eluate have leaked from the column are as follows 35 ml combined and freeze-dried.

The freeze-dried solids are dissolved in 0.5 ml of 0.1 molar 2,6-lutidine acetate buffer (pH 7.0). The solution is placed in a column of Bio-Gel P-2 (37-74 _; u) which has a diameter of 1 cm and a height of 50 cm and which has previously been equilibrated with this buffer. Then the gel is developed with the same buffer at a rate of 0.5 ml / min. After 25 ml of eluate has passed through the column, the following 10 ml are combined and freeze-dried.

The freeze-dried solids are dissolved in 4 ml of distilled water and placed in a diameter column of 1.7 cm entered, loaded with 90 ml of prewashed XAD-2 and equilibrated at 5 C with distilled water has been brought. Before use, the XAD-2 is successively with 1) 5 parts by volume of 1N NaOH and then with demineralized water until the drain reacts neutrally, 2) 5 parts by volume of 1N HCl and then with demineralized Water until the effluent reacts neutrally, 3) 5 parts by volume of methanol, acetone, 0.001 molar tetrasodium EDTA solution and finally washed with distilled water. After the sample has been applied, two portions are poured for every 2 ml of distilled water. The column is filled with distilled water at a rate of 2 ml / min washed. The eluate is collected in fractions of 4 ml each. Fractions 25 to 58 are combined and freeze-dried; one obtains deacetyl-890A, ..

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example Production of N-acetylthienamycin by fermentation

A tube with a freeze-dried culture of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents in 0.8 ml of sterile Davis salts of the following composition suspended:

Davis salts 0.5 G Sodium citrate 7.0 G K ₂ HPO ₄ 3.0 G 1.0 G ( TVTT T ι G! C \
V V \ Π / J nüU / 0.1 G MgSO ₄ .7H ₂ O 1000 ml distilled water

This suspension is used to inoculate four slant tubes with medium A (with agar) of the following composition:

Medium A

Yeast autolysate (Ardamine *) 10.0 G glucose 10.0 G Phosphate buffer ** 2.0 ml MgSO ₄ -7H ₂ O 0.05 G distilled water 1000 ml

pH adjusted to 6.5 with NaOH. * Ardamine: Yeast Products Corporation

** Phosphate buffer solution

KH ₂ PO ₄ 91.0 g

Na ₂ HPO ₄ 95.0 g

distilled water 1000 ml
For slanted tubes, add 25 »0 g agar per liter,

The inoculated slant tubes are incubated for one week at 28 ° C and then stored at 4 C.

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10 ml of medium A (without agar) are aseptically applied to one of these Transfer oblique tubes, the spores and aerial mycelium are scraped into suspension, and 1.2 ml of this suspension are added used to turn three 2 1 Erlenmeyer flasks provided with deflectors to inoculate, the 500 ml medium A (without agar) contained. These inoculation flasks are 24 hours at 28 C with 160 rpm shaking, whereupon sicti a satisfactory Culture has developed «

The cultures from these inoculation flasks are pooled and used to inoculate 467 liters of Medium A (without agar) in a 756 liter stainless steel fermenter. This fermenter operates at 28 ° C. for 24 hours, stirring at 130 rpm and passing 283 liters of air per minute through it. Antifoam agent (Polyglycol 2000 from Dow Chemical Corp.) is added as needed, but not in amounts greater than 0.1%. The pH determinations have the following results:

Age, h 0 24

pH 6.3 6.4

454 1 of the culture in this seed fermenter are used to 4082 1 Medium E in a 5670 1 stainless steel fermenter. Medium E has the following composition:

Medium E

Cerelose

Maize steep water (based on Hass) soluble sludge components Cottonseed medium (Pharmamedia) CoCl ₂ * 6H ₂ O

CaCO ^ (after pH adjustment) Polyglycol 2000
tap water

pH adjusted to 7.3 with NaOH.

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25.0 G 15.0 G 10.0 ε 5.0 G 0.01 G 3.0 G 0.25 % 1000 ml

15 837 S ^

This fermenter is operated for 138 hours at 24 C with stirring at 70 rpm and air bubbling through at one speed operated at 1.54 m / min. Additional anti-foaming agent is used as required (Polyglycol 2000), but not in amounts of more than 0.1%. There will be antibacterial analyzes performed with the following results:

ATCC No. 6633 (9.5 mm discs), Age, h pH hemin zone, mm

0 6.9 0 24 6.3 0 36 6.0 0 48 5.9 0 60 6.0 23 72 5.9 - 84 6.0 21 96 6.2 - 108 6.5 35 120 6.6 36 132 6.7 41 138 6.7 39

The 4082 l fermentation liquid are filtered through a 76 cm filter press with the addition of a filter aid in an amount of 4 parts by weight per 100 parts by volume. 46 g of the sodium salt of ethylenedinitrile tetraacetic acid (EDTA) are added to the filtrate. The filtrate is cooled to 6 ° C., adjusted to a pH of 4.5-0.2 and kept at 6 ° C. The cold filtrate is introduced at a rate of 48 l / min into a 480 l column with Dowex-50 χ 4 Na (300-840 μ) . After an initial run of 1400 1, an outflow of 18.9 1 is collected, the pH of which is adjusted to 7.08 with sodium hydroxide solution and which is stored at 5 ° C »

A 3.8 cm diameter column filled with 300 ml of Dowex-1x2 (150-300 u) is filled in the chloride form, is washed with 600 ml of demineralized water at 5 ° C. 4 1 des

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cold effluent from the Dowex-50 χ 4 column are connected to a Speed of 30 ml / min passed through this column. Then the column is freed with 300 ml of 25 µmolar EDTA solution

to wash. The antibiotic N-acetylthienamycin is eluted at a rate of 15 ml / min at 5 ° C. with 900 ml of 5% saline solution which is 0.01 molar of potassium phosphate buffer (pH 7.0) and 25 µmolar of EDTA. 14 fractions of 75 ml each are collected and analyzed for their biological activity using the disk diffusion method. The eluate fractions No. 3 to 9, the amount of which is 525 ml., Are combined and concentrated to 115 ml in vacuo. The concentrate contains 65 % of the total bioactive material introduced into the Dowex 1 χ 2 Cl column.

The concentrate of the Dowex-1 χ 2 Cl ~ -Eluats is entered at 5 ° C in a column with a diameter of 3.8 cm, the is loaded with 450 ml of pre-washed XAD-2. The XAD-2 is added in columns one after the other with 4 column volumes each 1) 0.001 molar EDTA solution, 2) 1N NaOH, 3) demineralized Water, 4) 1N HCl, 5) demineralized water, 6) methanol, 7) acetone, 8) demineralized water and pre-washed with 2250 ml of 5% NaCl solution before use, which is 25 µmolar in EDTA.

After the sample has been introduced into the column, two portions are added, each with 25 ml of water. The column is developed with demineralized water at a flow rate of 10 ml / min at 5 ° C. A first fraction of 400 ml and then 11 further fractions of 75 ml each are collected. The pH of each fraction is adjusted to between 6.9 and 7.13 with 1N sodium hydroxide solution or 1N hydrochloric acid. Fractions No. 3 through 9, which contain 46% of the total bioactive material added to the XAD-2 column, are combined to a total volume of 490 ml. A sample of 45 ml is made

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taken for bioanalysis according to the standard disk diffusion method against Staphylococcus aureus ATCC 6538P. The remaining 445 ml are concentrated to 50 ml in vacuo.

The 50 ml concentrate of the XAD-2 eluate are at 5 C with at a rate of 1 ml / min into a column 1.5 cm in diameter filled with 40 ml of pre-washed Dowex-1 χ 4 Cl (particle size <37 u) is charged. The Dowex-1x4 Cl ~ resin (<37 u, determined by decanting water) is added at a rate of 1 ml / min before use in columns with 240 ml of 0.2 molar saline solution, which is 0.005 molar in NH-Cl and 0.1 mmolar in NH ^ OH, and then with washed at the same speed with 120 ml of demineralized water.

Following this sample, two portions of 5 ml each of demineralized water are added to the column. The column is developed at 5 C at a rate of 0.92 ml / min with 0.07 molar saline solution, which is 0.005 molar in NH ^ Cl and 0.1 mmolar in NH-OH. Fractions of 8.6 to 9.3 ml are collected. The fractions that begin with a drain volume of 600 ml and end with a volume of 710 ml are combined; they contain 98 % of the total bioactive material introduced into the Dowex-1 χ 4 column. This liquid is concentrated to 2 ml in vacuo.

The Dowex-1 χ 4 concentrate is placed in a column with a diameter of 2.2 cm, which is filled with 225 ml of Bio-Gel P-2 (37-74 u) loaded with an exclusion limit of 1800 Daltons (previously determined by decanting distilled water) is.

Before use, the Bio-Gel P-2 column is filled with 225 ml 1 molar saline solution and then with 100 ml of demineralizing

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washed in water. The column is developed with demineralized water at a rate of 1 ml / min at 5 ° C., with fractions of 2 ml each being collected. The eluate fractions collected from 104 to 128 ml contain 83 % of the bioactivity introduced into the Bio-Gel P-2 column and are combined and concentrated to 1.58 ml.

A column with 50 ml of XAD-2 (1.6 cm 27 cm) is made and in columns with 200 ml of 1 mmolar EDTA solution each, 1n sodium hydroxide solution, demineralized water, 1n HCl, demineralized Pre-washed water, methanol, acetone and demineralized water. The concentrate of the Bio-Gel P-2 eluate, which contains 44.4 optical density units that can be erased by hydroxylamine, is added to the XAD-2 column at 5 C, whereupon two portions of 2 ml demineralized water each are poured in. The column is at a speed of Washed 1 ml / min with demineralized water until the UV absorbance of the washing liquids is at 300 nm has decreased to 0.060 «The column is moving at a speed washed at 1 ml / min with 50% strength methanol in demineralized water, with fractions of 1 ml each being collected will. The fractions with an absorbance at 300 nm of more than 0.1 are combined and concentrated in vacuo. The product obtained is N-acetylthienamycin with 11.6 optical density units which can be erased by hydroxylamine.

Example 7 Production of N-acetylthienamycin by fermentation

A tube with a freeze-dried culture of Streptomyces cattleya MA-4297 is opened aseptically and the contents in 0.8 ml of sterile Davis salts of the following composition suspended:

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Davis salts 0.5 G Sodium citrate 7.0 G K ₂ HPO ₄ 3.0 G KH ₂ PO ₄ 1.0 G (NH ₄ ) ₂ S0 ₄ 0.1 G MgSO ₄ .7H ₂ O 1000 ml. distilled water

This suspension is used to contain four slant tubes Inoculate medium A (with agar) of the following composition:

Medium A

Yeast autolysate (Ardamine *) 10.0 g

Glucose 10.0 g

Phosphate buffer * 2.0 ml

MgSO ₄ .7H ₂ O 0.05 g

distilled water 1000 ml

pH adjusted to 6.5 with NaOH. * Ardamine: Yeast Products Corporation ** Phosphate buffer solution

KH ₂ PO ₄ water 1 91 0 G Na ₂ HPO ₄ 95 0 G distilled 000 ml

For slanted tubes, add 25.0 g of agar per liter.

The inoculated slant tubes are incubated at 28 ° C. for one week and then stored at 4 C.

10 ml of medium A are aseptically transferred to one of these oblique tubes, the spores and aerial mycelium become in suspension scraped, and 1.2 ml of this suspension are used to make three 2-liter Erlenmeyer flasks provided with deflectors inoculate, each containing 500 ml medium A (without agar). These Inoculation flasks are shaken at 160 rpm at 28 ° C for 24 hours, whereupon a satisfactory culture has developed.

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The cultures from these inoculation flasks are pooled and used to inoculate 467 liters of Medium A (without agar) in a 756 liter stainless steel fermenter. This fermenter operates at 28 ° C. for 24 hours, stirring at 130 rpm and passing 283 liters of air per minute through it. Antifoam agent (Polyglycol 2000 from Dow Chemical Corp.) is added as needed, but not in amounts greater than 0.1%. The pH determinations have the following results:

Age 0 24

pH 6.3 6.4

454 1 of the culture in this seed fermenter are used to 4082 1 Medium E in a 5670 1 stainless steel fermenter. Medium E has the following composition:

Medium E

Cerelose 25.0 g

Corn steep liquor (on a wet basis) 15.0 g

soluble pulp ingredients 10.0 g

Cottonseed Medium (Pharmamedia) 5.0 g

CoCl ₂ «6H ₂ O 0.01 g

CaCO ^ (after pH adjustment) 3.0 g

Polyglycol 2000 '0.25 %

Tap water 1000 ml

pH adjusted to 7.3 with NaHO.

This fermenter is operated for 138 hours at 24 ° C. with stirring at 70 rpm and air passing through at a speed of 1.54 m / min. If required, additional antifoaming agent (Polyglycol 2000) is added, but not in amounts of more than 0.1 % . Antibacterial analyzes are carried out with the following results:

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Age, h

pH

ATCC No. 6633 (9.5 inm discs), inhibition zone, mm

0 6.9 0 24 6.3 0 36 6.0 0 48 5.9 0 60 6.0 23 72 5.9 - 84 6.0 21 96 6.2 - 108 6.5 35 120 6.6 36 132 6.7 41 138 6.7 39

The 4082 l fermentation liquid are filtered through a 76 cm filter press with the addition of a filter aid in an amount of 4 parts by weight per 100 parts by volume. 46 g of the sodium salt of ethylenedinitrile tetraacetic acid (EDTA) are added to the filtrate. The filtrate is cooled to 6 ° C., adjusted to a pH of 4.5-0.2 and kept at 6 ° C. The cold filtrate is introduced at a rate of 48 l / min into a 480 l column with Dowex-50 χ 4 Na (300-840 μ) . After a forerun of 1400 l, an outflow of 18.9 l is collected, the pH of which is adjusted to 7.08 with sodium hydroxide solution and which is stored at 5 ° C.

A column of 3.8 cm in diameter filled with 300 ml of Dowex 1x2 (150-300 u) in the chloride form the is.t ₉ is washed with 300 ml of demineralized water at 5 C. 4 liters of the cold discharge from the Dowex 50 χ 4 column are passed through this column at a rate of 30 ml / min. The column is then washed with 350 ml of 25 µmolar EDTA solution and at 5 ° C. at a rate of 15 ml / min eluted with 900 ml of 5% NaCl solution, which is 0.01 molar

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TrisoHCl (pH 7) and 25 µmolar in EDTA. Fractions of 75 ml each are collected and analyzed against Staphylococcus aureus ATCC 653SP using the disk diffusion method. Fractions No. 4 to 10, which contain 47% of the introduced bioactivity, are added to 42.5 ml of the sample which had been taken for analysis in Example 6 from the pooled fractions from the first XAD-2 adsorption. These combined fractions are concentrated to 100 ml in vacuo and the pH is adjusted to 6.32 with hydrochloric acid.

A column with a diameter of 3.8 cm, which is charged with 450 ml of XAD-2 resin, is successively filled in columns with 4 column volumes of 1) 0.001 molar EDTA solution, 2) 1N NaOH, 3) demineralized water, 4 ) 1N HCl, 5) demineralized water, 6) methanol, 7) acetone, 8) demineralized water prewashed and washed with 2250 ml of 5% NaCl solution, which is 25 µmolar in EDTA, before use. The above concentrate is added to the XAD-2 column, whereupon two portions of 5 ml of demineralized water each are poured in. The column is developed with demineralized water at 5 ° C. at a rate of 10 ml / min. A first fraction of 40 ml and further fractions of 75 ml each are collected and analyzed using the disk diffusion method. Fractions Nos. 9 to 15, which contain 22% of the bioactivity added to the XAD-2 column, are combined and concentrated in vacuo to 56 ml.

A 21 cm χ 1.7 cm column filled with 40 ml of Dowex-1x4 Cl ~ (<37 u, determined by decanting water) charged is, before use at a rate of 1 ml / min with 240 ml of 0.2 molar NaCl solution, the 0.005 molar in NH ^ Cl and 0.1 mmolar in NH / OH, and then

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washed at the same speed with 120 ml of demineralized water.

The XAD-2 concentrate is introduced into the column, whereupon two portions of 2 ml each of demineralized water and then Pour two portions of 2 ml elution buffer each. The column is at a rate of 1 ml / min at 5 C with a 0.07 molar NaCl solution, the 0.005 molar of NH ^ Cl and 0.1 mmolar in NH ^ OH, eluted. There are factions too 10 ml each are collected and analyzed using the disk diffusion method. The eluate fractions collected from 544 to 647 ml apparently contain 100% of the entered bioactivity and are combined with each other and in vacuo to 2.3 ml constricted. The concentrate contains 36.4 hydroxylamine-erasable optical density units.

A 2.2 cm χ 62 cm column filled with 225 ml Bio-Gel P-2 resin (37-74 u) with an exclusion limit of 1800 Daltons is charged, is charged with 225 ml of 1 molar saline solution and then with 100 ml of demineralized water before use washed. The Dowex-1 χ 4 concentrate is added to the column, whereupon two portions of 2 ml demineralized water each are poured in. The column is at 5 C with one rate of 1 ml / min with demineralized water, with fractions of 2 ml each being collected, which then analyzed by the disk diffusion method. The fractions from 124 to 129 ml, the 7.04 by hydroxylamine Erasable optical density units contain, are combined and in vacuo to 2 ml of an aqueous solution of the Concentrated product N-acetylthienamycin. Those from 117 to 123 ml and fractions collected from 130 to 139 ml of eluate are combined to form a solution of the product N-acetylthienamycin, the 13.7 optical density that can be erased by hydroxylamine

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contains units. By deacetylating the thus obtained According to the method of Example 2, the antibiotic thienamycin is obtained.

Example 8 Manufacture of thienamycin

A tube with a freeze-dried culture of Streptomyces cattleya MA-4297 is opened aseptically and the contents in 50 ml of sterile medium A suspended in a 250 ml Erlenmeyer flask equipped with a deflector. Medium A has the following Composition:

Medium A

Yeast autolysate (Ardamine *)
glucose
Phosphate buffer **
MgSO ₄ = 7H ₂ O 10,
10,
2,
0, 0
0
0
05 G G
G
ml
G distilled water 1000 G ml ml Corporation 91.0 pH adjusted to 6.5 with NaOH. 95.0 * Ardamine: Yeast Products 1000 ** Phosphate buffer solution KH ₂ PO ₄ Na ₂ HPO ₄ distilled water

The inoculated flask is at 28 ° C for 48 hours with 220 rpm (Stroke 5.1 cm) shaken. From the 48 hour old fermentation liquid 40 ml are removed aseptically and mixed with 40 ml sterile, 20% by volume aqueous glycerine. Quantities of 2 ml of this mixture are pipetted into sterile, 3.53 ml ampoules, then frozen out and in the Stored vapor phase of a freezer operated with liquid nitrogen.

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> 15 837 β 3

The frozen contents of the ampoule are used to make a diverter equipped 250 ml Erlenmeyer flask to inoculate the Contains 50 ml of medium A. This inoculation flask will last for 24 hours 28 ° C shaken at 160 rpm «

10 ml portions taken from this inoculation flask are used to convert 2-liter Erlenmeyer flasks provided with a deflector to inoculate, each containing 500 ml of medium A. These inoculation flasks are shaken at 160 rpm at 28 ° C. for 24 hours.

1000 ml of the combined contents of this seed flask are used to 467 1 of medium A in a 756 1 fermentor to inoculate stainless steel "In this fermenter, the fermentation is 24 hours at 28 C, a stirring speed of 130 rev / min and an air flow rate of 283 l / min. If necessary, Polyglycol 2000 (Dow Chemical Corp ”) is added as an anti-foaming agent , but not in an amount greater than 0.1%. pH and dextrose content measurements have the following results

Age, h mg / ml 6th 0 12th 4th 24 pH 8th A. 6> 1 6.6 Dextrose, ,1 8th, 8.1

453 l of this culture are used to make 4082 l of medium E in to inoculate a 5670 1 fermenter made of stainless steel «, Medium E has the following composition?

Medium E

Cerelose 25 _sec 0 g

Corn steep water (on a mass basis) 15s> 0 g

soluble pulp ingredients 10.0 g

Cottonseed Medium (Pharmamedia) 5.0 g

CoCl ₂ .6H ₂ O 0.01 g

CaCO, (after pH adjustment) 3.0 g

Polyglycol 2000 0.25 %

Tap water 1000 ml

pH adjusted to 7.3 with NaOH «

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In this fermenter, the fermentation is carried out for 144 hours at 24 ° C. with a stirring speed of 70 rpm and an air flow rate of 1.54 m / min. If required, Polyglycol 2000 is added as an anti-foaming agent , but not in quantities greater than 0.1%. The centrifuged fermentation liquid is analyzed against Staphylococcus aureus ATCC-6538P by the standard disk diffusion method. The results can be found in the following table under the heading "Antibiotic activity against ATCC 6538P". In addition, analyzes are carried out by the disk diffusion method using 9.5 mm filter paper disks and 10 ml analysis plates; the results of these analyzes are given in the table below under the heading "Antibiotic activity (10 ml plates)".

The 10 ml analysis plates are prepared as follows: A culture of the analysis organism Staphylococcus aureus ATCC 6538P, developed overnight in nutrient broth with a yeast extract content of 0.2%, is diluted with nutrient broth containing 0.2% beefy extract to form a suspension which at one wavelength of 660 nm has an optical transmission of 40 % . This suspension is added to Difco nutrient agar, which is supplemented by 2 g of Difco yeast extract per liter and is at a temperature of 47 to 48 C; the mixture obtained in this way contains 33.2 ml of the suspension per liter of agar. 10 ml each of this suspension are poured into Petri dishes with a diameter of 85 mm, and these plates are cooled and kept at 4 ° C. until use (maximum 5 days).

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age pH Dextrose, mg / ml Antibiotic
activity
against
ATCC 6538P,
mm inhibition zone Antibiotic
activity
(10 mm
Plates),
min inhibition zone O 6.6 22.2 12th 6.3 20.2 24 5.8 18.0 0 36 6.0 13.2 21.5 48 6.0 8.6 21.5 60 5.7 6.4 26.5 72 5.8 2.7 25.5 84 6.2 0.3 27.5 96 6.4 0.2 36.0 108 6.4 0 35.0 120 6.3 41.5 37.0 132 5.8 37.5 144 5.9 43.0 37.5

The 4082 l fermentation liquid are filtered through a 76 cm filter press after adding 5 parts by weight of filter aid per 100 parts by volume. The filtrate is mixed with 12 g of the sodium salt of ethylenedinitrilotetraacetic acid. The filtrate is cooled to 6 ° C., adjusted to a pH of 4.5-0.2 and kept at 6 ° C. The cold filtrate is adsorbed on 480 1 Dowex-50 χ 4 Na (300-840 u) at a rate of 48 l / min. The adsorbate is washed with 480 l of demineralized water and then eluted with 2% aqueous pyridine at a rate of 24 l / min. Three fractions of 300 liters, 520 liters and 240 liters, respectively, are collected and analyzed at a pH of 7.0. The analyzes show that the eluate fractions contain 4 %, 16 % and 6 % of the bioactivity entered into the Dowex 50 χ 4 Na column. The eluate fraction no. 2 is concentrated to 48 l and adjusted to a pH of 7.

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The 48 l of concentrate are adjusted to a pH of 7.3 and adsorbed on 76 l of Dowex-1 χ 2 in the chloride form (150-300 u) at a rate of 7.6 l / min. The resin is eluted with demineralized water at the same rate. Four fractions, two at 48 l each, one at 70 l and one at 48 l, are collected. The fractions are adjusted to a pH of 7. Analyzes show that 68 % of the initial bioactivity is contained in the 70 l fraction. This fraction is concentrated to 18 liters at a pH of 7.0 and filtered through a "Millipore" filter with pores of 0.45 ρ. The filtrate is freeze-dried in dishes; 99 g of product with a strength of 310 units / mgo are obtained

10 g of the freeze-dried solids are taken up in 0.1 molar 2,6-lutidine acetate buffer (pH 6.3). 125 ml of this solution are adjusted to a pH value of 6.3 with acetic acid and transferred to a column of Dowex-50 χ 8 in the 2,6-lutidine form (particle size 37-74 n, dimensions 7.6 χ 142 cm) entered that has been previously equilibrated with the buffer. The column is developed at a speed of 25 ml / min with 0.1 molar buffer solution. After a forerun of 3 liters, 200 fractions of 20 ml each are collected. Every fourth of fractions Nos. 36 to 192 is analyzed at a dilution of 1: 200. The bioactivity is found in fractions Nos. 56 to 192, reaching a maximum in fractions Nos. 92 to 96. The Fraction Nos. 80 to 136 are combined and brought ml by the addition of 590 ml of demineralized water to a volume of 1760 » The combined, dilute solution, which contains 62 % of the initial bioactivity introduced into the Dowex 50 χ 8 column, is freeze-dried.

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The freeze-dried solids are in 0.1 molar 2,6-lutidine acetate solution (pH 7.0) dissolved. The solution (27 ml) is placed in a 5 x 112 cm column with Bio-Gel? -2 (37-7 ^ u), which was previously filled with 0.1 molar buffer solution has been brought into balance. Then the gel is applied at a rate of 10 ml / min with the same buffer solution developed.

The process is carried out with a recording differential refractometer (Meccomatic) monitored. The development continues until 105 fractions of 20 ml each have been collected are. Each of Fractions Nos. 70 to 93 is analyzed at a dilution of 1: 300. The bioactivity is found in fractions No. 73 to 82 and reaches a maximum in fractions No. 77 and 78. Fractions No. 75 to 80 are freeze-dried to 90 mg antibiotic with an average strength of 10,000 units / mg.

The 90 mg of freeze-dried solids are taken up in 4 ml of 0.01 molar potassium phosphate buffer solution (pH 7). This solution, which contains 596 hydroxylamine-erasable optical density units (this measure of the thienamycin content is described in Section II on "Analytical Methods for Thienamycin"), is placed in a column with a 1.7 cm in diameter loaded with 90 ml of pre-washed XAD-2 and stored at 5 ° C before use 180 ml of 0.01 molar potassium phosphate buffer solution (pH 7) has been equilibrated one after the other with 1) 5 parts by volume of 1N NaOH and then demineralized water until the drain reacts neutrally, 2) 5 parts by volume of 1N HCl and then demineralized Water until the drain reacts neutrally, 3) 5 parts by volume of methanol, acetone, 0.001 molar tetrasodium EDTA

- 66 709822/1031

15 837 JO

Solution and finally washed with distilled water. A vacuum is applied to all of these solvents prior to use «

After the sample has been introduced into the column, two portions of 2 ml each of phosphate buffer solution are poured in. The column is developed at 5 ° C. with the buffer solution at a flow rate of 2 ml / min. Eluate fractions of 4 ml each are collected. The fractions collected after 100 ml of eluate have passed through 253 ml are combined with one another and in a rotating evaporator under vacuum
concentrated to 6 ml.

steamer under vacuum at a temperature below 10 C

This solution, the 436 with hydroxylamine erasable optical Contains density units, is placed in a column with a 1.7 cm diameter, which is filled with 90 ml, as described above, prewashed XAD-2 and equilibrated at 5C with distilled water. According to the task Two portions of 2 ml of distilled water each are poured into the sample. The column is at a speed of Developed 2 ml / min with distilled water. Eluate fractions of 4 ml each are collected. The factions that after passage of 100 ml of eluate to passage of 151 ml are collected, are combined and concentrated in a rotary evaporator to a volume of 2.73 ml, and this solution is freeze-dried. 6.49 mg of thienamycin are obtained. The eluate fractions obtained between 152 ml and 345 ml are combined and concentrated in a rotary evaporator to a volume of 3.34 ml and freeze-dried. They give 11.53 mg of thienamycin. These fractions contain a total of 369 hydroxylamine-erasable optical Density units. This corresponds to a 3.1-fold cleaning

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of the material fed into the first XAD-2 column and provides a calculated strength of 31,000 units / mg. The spectrophotometric analysis of a sample of this product

shows eJ = 253, measured in phosphate buffer solution (pH 7) ι cm

at 297 nm.

A sample of 10 g of the 99 g of freeze-dried solids obtained in the above-described cleaning with Dowex-1 χ 2 is taken up in 0.1 molar 2,6-lutidine acetate buffer (pH 6.3). The solution (125 ml) is adjusted to a pH value of 6.3 with acetic acid and entered into a 7.6 χ 142 cm column with Dowex-50 χ 8 in the 2,6-lutidine form, which was previously ins Balance has been brought about. The column is developed at a rate of 35 ml / min with 0.1 molar buffer solution. After a forerun of 3.6 l, 200 fractions of 20 ml each are collected. Every fourth of fractions No. 6 to 194 is analyzed at a dilution of 1: 200. The bioactivity is found in fractions No. 18 to 178 and reaches a maximum in fractions No. 62 to 82. Fractions No. 42 to 102 are combined and brought to a volume of 1920 ml by adding 640 ml of demineralized water. The combined, dilute solution contains 63 % of the bioactivity introduced into the Dowex 50 χ 8 column and is freeze-dried.

The freeze-dried solids are in 0.1 molar 2,6-lutidine acetate buffer solution (pH 7.0) dissolved. This solution (25 ml) is poured into a 5 x 112 cm column with bio-gel P-2 (37-74 ji) entered previously with 0.1 molar buffer solution has been brought into balance. Then the gel is applied at a rate of 10 ml / min with the same buffer solution developed.

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The process is carried out with a recording differential refractometer (Meccomatic) monitored. The development continues, up to 125 fractions of 20 ml each have been collected. Each of fractions Nos. 70 to 89 is at one dilution analyzed from 1: 300 »The bioactivity is found in fractions no. 72 to 81 and reaches a maximum in the Fraction No. 77. Fractions No. 75 to 79 become 100.5 mg of antibiotic at a strength of 8320 units / mg freeze dried.

The 100.5 mg of freeze-dried solids are taken up in 4 ml of 0.01 molar potassium phosphate buffer solution (pH 7). This solution, which contains 692 hydroxylamine-erasable optical units, is placed in a column 1.7 cm in diameter loaded with 90 ml of pre-washed XAD-2 and before use with 180 ml of 0.01 molar potassium phosphate buffer solution (pH 7) is brought to equilibrium at 5 C. Before use, the XAD-2 will be sequentially with 1) 5 parts by volume of 1N sodium hydroxide solution and then with demineralized Water until the drain reacts neutrally, 2) 5 parts by volume of 1N HCl and then with demineralized Water until the drain reacts neutrally, 3) 5 parts of the room Methanol, acetone, 0.001 molar tetrasodium EDTA solution and finally washed with distilled water. Vacuum is applied to all of these solvents prior to use.

After the sample has been added to the column, two portions of 2 ml each of phosphate buffer solution are poured in. The column is developed with the buffer solution at a flow rate of 2 ml / min at 5 ° C. Eluate fractions collected at 4 ml each. The fractions beginning after 109 ml of eluate and ending with the 309th milliliter are collected and united with one another. To this

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In a certain eluate, the 11.53 mg of the above-described, antibiotic purified with XAD-2 added to the 186 with Hydroxylamine contain erasable optical density units. The combined eluate is used together with the added antibiotic concentrated to 7 ml in a rotary evaporator under vacuum at a temperature below 10 C.

This solution, which contains 720 optical density units which can be erased by hydroxylamine, is placed in a column measuring 1.7 cm Diameter entered, which is loaded with 90 ml, as described above, pre-washed XAD-2 and before use at 5 C has been equilibrated with distilled water. After the sample, two portions of 2 ml each are distilled Water poured into the column. The column is distilled at a rate of 2 ml / min Water developed, with eluate fractions of 4 ml each being collected will. The fractions collected from the passage of 109 ml of eluate to the passage of 301 ml of eluate are combined combined and concentrated to 10.3 ml in a rotary evaporator. This solution, the 589 by hydroxylamine Containing erasable optical density units becomes 23.6 mg of antibiotic with a calculated strength of 30 140 units / mg freeze dried.

The antibiotic thienamycin produced in this way is a white amorphous solid substance with a fibrous consistency. A sample if heated in a glass capillary at a rate of 3 C / min, it undergoes decomposition without a liquid phase occurs. The following stages are observed: Softening takes place at 130 to 140 ° C with volume contraction of the solid substance and continues to 170 to 174 C; in the latter area the M is colored material yellow; Sintering and progressive color deepening up

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too reddish brown is observed in the range from 180 to 200 C, and finally charring occurs at 205 C, whereby remaining traces of solids remain.

Another sample of this material shows when spectrophotometric Analysis of an absorbance maximum (absorbance

peak) at 296.5 nm with an E ¹ ^ _ = 268.2. The elementary

ι cm

analysis gives the following results: 1) weight loss of 5.67% on drying for 4 hours at room temperature under vacuum and 2) composition: 47.68 % carbon, 6.22 % hydrogen, 11.48 % nitrogen. These results agree with the empirical formula C ^ H ^ gNpO ^ S. (NH,) _Q ^ q ; the calculated composition corresponding to this empirical formula is C = 47.68 %, H = 6.13 %, _t N = 11.52 %, S = 11.57 % and 0 = 23.1 %. The polarimetric analysis of a sample of 1 mg / ml in 10 mmolar potassium phosphate buffer solution shows a specific optical rotation [α] _Q +80. The infrared spectrum of a suspension of this sample in Nujol shows characteristic absorption maxima at 1765 cm, 1650-1550 cm " ¹ , 2800-2500 cm" ¹ and 3500-3100 cm " ^1. An NMR spectrum at 100 MHz of a sample of this product dissolved in D ₉ O, shows a doublet at ο 1.275, a doublet pair at

r Γ r ~

ο 3.39 and multiples at ό 3.15 and ö 4.20; these maxima

are characteristic of thienamycin.

The thienamycin thus obtained can according to the method of the example 8 can be acetylated to N-acetylthienamycin.

Example 9 Acetylation of thienamycin

10.9 mg of thienamycin are freshly prepared for 10 minutes at 0 ° C in a mixture of 1 ml of dry dimethylformamide and 2 ml.

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set acetic anhydride stirred. The dimethylformamide and acetic anhydride are repeatedly (5- to 6 times) washing with 25 to 40 ml of hexane each and last time Washing with hexane removed after adding 1 ml of dry ethyl ether. The crude sample of N-acetylthienamycin is in 20 ml demineralized water, which is 100 µmolar in Tris base [Tris (hydroxymethyl) aminomethane] and 35- ^ molar of HCl, solved. After dissolving the sample, the pH is 7.9. the Solution contains 244 absorbance units at 298 nm, and a 1.27 cm analytical disk containing 0.1 ml of a Contains a 1000-fold dilution of the sample, creates a zone of inhibition when incubated on plates with ATCC 8461 at 25 ° C a diameter of 23 mm.

This sample is placed in a column (bed dimensions 1.3 cm 14 cm) of Dowex-1 χ 4 (Cl ") (particle sizes <37 u) entered. The column is washed with 10 ml of demineralized water and the antibiotic N-acetylthienamycin with an NaCl 0.07 molar, NH ^ Cl 0.005 molar and NH ^ 0.0001 molar Solution eluted in demineralized water. At a flow rate of 0.7 ml / min, fractions become too 6.1 ml each collected »The main peak of the UV absorbance at 298 nm appears in fractions no. 36 to 50 with a maximum in fraction No. 40. The fractions Nos. 38 to 46 are combined and contain a total of 107 absorbance units at 298 nm combined fractions are concentrated in a rotating evaporator under reduced pressure to 2 ml and with 5 μl 1 molar Sodium hydroxide solution added.

This concentrate is in a column (bed dimensions 2.2 χ 80 cm) entered with Bio-Gel P-2 (37-74 u). The sample is made with two portions of 1 ml demineralized water each

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washed in and with a flow velocity of 0.6 ml / min eluted with demineralized water. Be there Fractions of 3.04 ml each collected.

The main peak of the UV absorbance (UV absorbance) at 300 nm appears in fractions No. 58 to 64 with a maximum in fraction No. 60. Fractions No. 59 to 62 contain 83.7 A ^ QQ units and are combined with one another. A 2.2 A ^ QQ units equivalent part is taken as a reference sample and the remainder was concentrated to 1.5 ml and freeze-dried in a 14 ml glass ampoule to give 3.9 mg of N-acetylthienamycin.

^nm ' ^E max / ^E min. = ⁴ ' ⁴⁵ ' ^E% 301 ⁱⁿ demineralized

tem water = 208.

By deacetylating this starting material by following the procedure of Example 2, the antibiotic thienamycin is obtained.

Example 10

Manufacture of the antibiotic 890A,

A tube with a freeze-dried culture of Streptomyces flavogriseus MA-4434a is aseptically opened and the contents suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts

Sodium citrate 0.5 g

K ₂ HPO ₄ 7.0 g

KH ₂ PO ₄ 3.0 g

(NH ₄ ) ₂ S0 ₄ 1.0 g

MgSO ₄ .7H ₂ O 0.1 g

distilled water 1000 ml.

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This suspension is used to contain four slant tubes Inoculate medium A, which has the following composition:

Medium A 20.0 g Glycerin 5.0 g Primary yeast 15.0 g Fish meal 1000 ml distilled water 20.0 g Agar

pH adjusted to 7.2 with NaOH.

The inoculated slants are incubated for one week at 27 to 28 ° C and then at 4 to 6 ° C until used canceled (not longer than 21 days).

10 ml of medium B of the following composition: medium B

Yeast autolysate (Ardamine *) 10.0 g

Glucose 10.0 g

Phosphate buffer ** 2.0 ml

MgSO ₄ "7H ₂ O 50 mg

distilled water 1000 ml

pH adjusted to 6.0 with HCl or NaOH. * Ardamine: Yeast Products Corporation

** Phosphate buffer solution

KH ₂ PO ₄ 91.0 g

Na ₂ HPO ₄ 95.0 g

distilled water 1000 ml

are aseptically transferred to one of these oblique tubes, the spores and aerial mycelium are scraped into suspension, and 3.3 ml of this suspension are used to inoculate a 2-capacity Erlenmeyer flask provided with a deflector, which contains 500 ml medium B. This inoculation flask is operated at 160 rpm for 36 hours at 28 ° C

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Shaking machine (stroke 5.1 cm) shaken, after which the culture has developed satisfactorily «

The culture from this inoculation flask is used to make a 189 l stainless steel fermenter containing 160 l medium B. This fermenter will run 24 hours operated at 28 ° C with stirring at a speed of 150 rpm with an air flow of 85 l / min. A Antifoam agent (Polyglycol 2000 from Dow Chemical Corp.) is used according to Redarf, but not in larger quantities Concentration than 0.1% "The pH values are the following:

Age, h 0 12 pH 6.3 6.35

43 1 of the culture of this seed fermenter are used to inoculating a 756 liter stainless steel fermenter containing 467 liter medium C of the following composition:

Medium C

Tomato puree 20.0 g

Primary yeast 10.0 g

Dextrin (Amidex) 20.0 g

CoCl ₂ * 6H ₂ O 5.0 mg

distilled water 1000 ml

pH adjusted to 7.2-7.4 with NaOH.

This fermenter is operated for 92 hours at 25 ° C. with stirring at a speed of 146 rpm with an air flow of 255 l / min. If necessary, additional antifoaming agent (Polyglycol 2000), but not more than 0.1 %, is added. Antibacterial analyzes against Salmonella gallinarum MB 1287 and Vibrio percolans ATCC 8461 are carried out with the following results:

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Age, h O 12 24 36 48 60 72 84 92

pH 6.6 6.7 6.65 6.3 6.0 6.4 6.15 6.5 6.5

MB-1287, mm S 19 26 28 32

ATCC 8461, mm _{Q ΟΛ o} , _o r ^ _n

(1-10) - - - ^s """ ^{21 24 2b 30}

890A unit

th / ml NA NA 6.8 13.5 24.9 24.3

The 890A units / ml are determined as described in Section II on "Analytical Methods for the Antibiotics 890A ₁ and 890Ay".

473 1 fermentation liquid are cooled to 5 C and centrifuged in a centrifuge (Titan P-9). 22.7 kg of Celite are added to 378.5 liters of the supernatant liquid and the suspension is filtered through a 45.7 cm filter press (Shriver). The 344.4 l of filtrate are adsorbed on a column which contains 26.5 l of Dowex-1 χ 2 (Cl ") with particle sizes of 150 to 300 μ , and the column is washed with 37.85 l of demineralized water. The mixture the antibiotics 890A ₁ and 89OA ₃ are mixed with 113.5 liters of 5% sodium chloride solution, which is 0.01 molar in Tris-HCl buffer (pH 7.0) and 25 nmolar in neutral EDTA, in demineralized water Fractions of 18.9 l each are collected The mixture of antibiotics 890A ₁ and 890A ^ appears in fractions 3 to 6 with a maximum strength in fractions 4 and 5. Fractions 4 , 5 and 6, which contain 8% of the activity of the filtered fermentation liquor, are combined and concentrated to 7.6 liters under reduced pressure.

The 7.6 l concentrate are placed in a column that 37.85 1 XAD-2 and previously with 189 1 60% aqueous acetone, then with 189 1 demineralized water and finally

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borrowed has been washed with 189 l of 5% saline solution. The adsorbate of this concentrate is eluted with 142 l of demineralized water. Three fractions of 9.5 liters and six fractions of 18.9 liters are collected. The activity appears in fractions # 1 to 6 with a maximum strength in fraction # 3. Fractions # 4 and 5, which contain 64% of the activity loaded into the XAD column, or 370,000 units, are pooled.

Fractions Nos. 4 and 5 are concentrated to 120 ml by evaporation under reduced pressure. The pH is adjusted to 6.5 and the concentrate is placed in a column (7 x 50 cm) of XAD-2, which was previously filled with 8 liters of 60% aqueous acetone solution, then with 4 liters of demineralized water and finally with 8 1 5% saline solution has been washed in demineralized water. The sample is allowed to drain up to the level of the adsorbent bed and the column is washed three times with 20 ml of demineralized water each time and each time allowed to drain to the level of the bed. The antibiotic is then eluted with 7 liters of demineralized water at a flow rate of 40 l / min. Eight fractions of 200 ml each and then 14 fractions of 400 ml each are collected. The antibiotic activity appears in fractions No. 4 to 19, which contain 77% of the bioactivity entered into the column (determined against Salmonella gallinarum MB1287); a maximum of the activity is found in fractions no. 6 to 8.

The ratio of the bioactivity of the fractions against Vibrio percolans ATCC 8461 to the HAEA, _QQ value of these fractions is determined. Those fractions in which the ratio is about 250 contain the antibiotic 890A ₁ , and the-

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those fractions in which the ratio is about 31 contain the antibiotic 89OA ^. Accordingly, the Fractions No. 10, 11, 12 and 13, which contain most of the antibiotic 890A ^, for further processing together united.

The combined fractions Nos. 10 to 13 obtained from the second XAD-2 column are concentrated to 40 ml and transferred to a column (bed dimensions 2.15 cm 40 cm) of Dowex-1 χ 4 (Cl ") with particle sizes below 37 The antibiotic is eluted with 3 liters of a 0.075 molar NH / Cl solution and a 0.001 molar NH solution in demineralized water at a flow rate of 3 ml / min. Fractions of 9 ml each are collected percolans ATCC 8461 determined antibiotic activity appears in fractions No. ₀ 165 to 234 with a maximum activity in fractions No. 195 to 201.

Fractions No. 186 to 213 are combined; they contain a total of 472 A ^ _0Q units, of which 312 can be erased by reaction with hydroxylamine.

The combined fractions are concentrated to 4.2 ml in a rotary evaporator under reduced pressure. The concentrate is placed in a column with Bio-Gel P-2 (37-74 u; bed dimensions 2.15 cm × 60 cm). The sample is up to the level of the Let the bed run off, washed twice with 1 ml of demineralized water each time and each time run off to bed level calmly. The column is then eluted with demineralized water at a rate of 1 ml / min, whereby one collects fractions of 2.6 ml each.

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The antibiotic 89OA ₇₅ is eluted in fractions Nos. 38 to 48; the maximum activity, determined by the absorbance at 300 nm which can be extinguished by hydroxylamine, occurs in fraction no. Fractions 40 to 43 are combined; they contain 260 A ^ _0Q units, 173 of which can be erased with hydroxylamine.

In order to remove the remaining contamination of the antibiotic 890A ^ by the antibiotic 890A ₁ , the combined desalted fractions No. 40 to 43 are treated with 50 μl of penicillinase [Difco "Bacto-Penase", containing 500,000 units per ml (1000 LU / ml, where the term LU refers to Levy units; 1000 LU inactivate 500,000 units of penicillin G)] and 0.1 ml of 1 molar Tris-HCT buffer solution (pH 7.5) are added. The reaction mixture is left to stand for 6 hours at 23 ° C. and 3 ml of demineralized water and 15 ml of methanol are then added. This mixture is placed in a column (2.15 x 44 cm bed dimensions) of Dowex-1 χ 2 (Cl ") (particle size below 37 μ) , which is filled in 50% methanol and filled with 2 1 50 vol"% The antibiotic 890A ^ is eluted with 2 liters of 50% methanol which is 0.05 molar in sodium chloride, 0.005 molar in NH ^ Cl and 0.0001 molar in NH ^ Fractions of 9.2 ml each were collected. The main maximum of the UV absorbance, measured at 300 nm, occurs in fractions No. 74 to 88. Fractions No. 78 to 85 are combined Of methanol under reduced pressure, the total value of absorbance units of these fractions at 300 nm is 97.6, of which 87.5 can be extinguished by reaction with hydroxylamine.

The combined fractions No. 70 to 85 are in a rotating Evaporator reduced to 3.92 ml under reduced pressure.

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concentrated, and the concentrate is placed in a column of Sephadex-G-10 (bed dimensions 2.15 x 70 cm), which is washed with 4 ml of 4 molar ammonia solution and then equilibrated with 0.15 mmolar ammonia solution in demineralized water has been brought. After washing twice with 1 ml of 0.02 mmolar NH, solution, the antibiotic 890A is eluted with 0.02 mmolar NH, solution at a flow rate of 0.8 ml / min. First 49 fractions of 2.45 ml each and then 20 fractions of 3.33 ml each are collected. The main peak of absorbance (absorbance) at 300 nm appear in Fraction Nos fractions No. = 35 to 53.. 38 to 46 having the highest A, ₀₀ / A2 ^ ₅ values, are combined for further processing. The combined fractions have a total of 65 absorbance units at 300 nm. The combined fractions are concentrated to 2.84 ml in a rotary evaporator under reduced pressure, divided into two equal parts and separately quick-frozen and freeze-dried in 14 ml glass ampoules. This is how you get the antibiotic 890A ,. One sample contains 32.0 «

in 0.88 mg, the other sample contains 31.9 in 0.82 mg ο The first sample, which contains 32.0 A, Q ₀ units, shows the following peaks in the NMR analysis:

1.29 (d, J = 6.5); 1.98 (s); 3.42 (d of d, J = 5 and J = 2.4); ^ 4.01-4.28 (m); 3.14 (d of d, J = 5 and J -9); 3.39 (t, J = 6.5); 3.92 (d of t, J = ^ 4 and J = 6 ").

The table above shows the 100 MHz NMR signals for the sodium salt of 890A, in D ₂ O in relation to sodium 2,2-dimethyl-2-silapentane-5-sulfonate as the internal standard; the chemical shifts are in ppm and the coupling

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lation constants are given in Hz, and multiple values are also included specified.

By deacetylating the material obtained in this way by the method of Example 4, the antibiotic deacetyl-890A ^ is obtained.

Example 11

Manufacture of the antibiotic 890A ₁

A tube with a freeze-dried culture of Streptomyces flavogriseus MA-4600a is aseptically opened and the contents 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

MgSO ₄ .7H ₂ O 0.05 g

Phosphate buffer * 2 ml

distilled water 1000 ml

* Phosphate buffer solution

KH ₂ PO ₄ 91.0 g

Na ₂ HPO ₄ 95, above

distilled water 1000 ml.

This suspension is used to add a 250 ml Erlenmeyer inoculation flask fitted with three baffles inoculate the 54 ml inoculation medium B of the following composition contains:

Medium B

Autolyzed yeast (.Ardamine *) 10.0 g glucose 10.0 g

MgSO ₄ .7H ₂ O 0.05 g

Phosphate buffer ** 2 ml

distilled water 1000 ml

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pH adjusted to 6.5 with NaOH

* Ardamine: Yeast Products Corporation

** Phosphate buffer solution

KH ₂ PO ₄ 91.0 g

Na ₂ HPO ₄ 95.0 g

distilled water 1000 ml.

The inoculation flask is closed with a cotton plug and 30 hours at 28 ί 1 ° C in a
5.1 cm) shaken at 220 rpm.

30 hours at 28-1 C in a rotary pouring machine (stroke

20.0 G 10.0 G 20.0 S. 5.0 mg 1000 ml

50 Erlenmeyer production flasks without deflectors, each 250 ml and each contain 40 ml of production medium C, are mixed with 1 ml of the fermentation liquid obtained from the inoculation flask inoculates. The production flasks are closed with cotton plugs »

Medium C

Tomato puree primary yeast dextrin (Amidex) CoCl ₂ · 6Η ₂ 0 distilled water

pH adjusted to 7.2-7.4 with NaOH.

After inoculation, the production piston 3 are days at 28 - 1 ⁰ C with shaking in a 220 U / min working Drehschüttelmaschine (stroke cm 5.1) were incubated. The flasks are analyzed for activity against standard analysis plates with Vibrio percolans ATCC 8461 using 1.27 cm disks which are immersed in centrifuged fermentation fluid samples. The samples are diluted with 0.05 molar phosphate buffer solution (pH 7.4). The results are as follows:

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Cultural age, h 72

pH 6.4

Vibrio percolans ,

Dilution 1/100,

Inhibition zone, mm 23

890 analysis units / ml 103

The 890A units / ml are determined as described in Section ^W II. Analytical Methods for 890A ^ and 890Αχ ".

The entire fermentation liquid is in portions of 200 ml in polycarbonate bottles for 15 minutes at 9000 rpm centrifuged to give 1600 ml of pooled supernatants having a strength of 104 units / ml. For this 0.5 ml of neutral EDTA solution are added.

The centrifuged fermentation liquid is passed through a column at a flow rate of 6 to 20 ml / min with Dowex-1 χ 2 (Cl ") (150-300 u, bed dimensions 3.8 χ 22 cm) adsorbed. The column is washed with 100 ml of demineralized water and with a flow rate of 6 ml / min eluted with 1 l of demineralized water which contains 50 g of sodium chloride and 0.02 molar of Tris-HCl buffer (pH 7.0) and 25 µmolar of neutral EDTA. It will Fractions of 10 ml each collected.

The antibiotic 890A ₁ appears in fractions No. 13 to 81 with a maximum in fractions No. 25 to 33, calculated from the first infusion of the salt eluate. Fractions Nos. 24 to 41, which have the highest ratios of bioactivity to Ap2o, are pooled for further processing. The pooled fractions contain a total of 29,000 units or 17% of the abandoned bioactivity.

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The Dowex eluate is concentrated to 10 ml, the pH value with dilute Hydrochloric acid adjusted to 6.5 and the concentrate placed in a column with XAD-2 (bed dimensions 3.3 x 36 cm), the previously demineralized with 2 liters of 60% aqueous acetone Washed water and a solution of 5 parts by weight of sodium chloride in 100 parts by volume of demineralized water has been. The sample is eluted with demineralized water at a flow rate of 6 ml / min, collecting fractions from 40 to 260 ml.

The antibiotic activity appears in fraction Nos. To 14 220 to 2560 ml of the eluted volume range, the maximum is contained in fractions No. ₀ 9 and 10, which extend from 370 to 590 ml of the eluted volume. Fractions No. 9 to 12, which comprise 370 to 1060 ml of the eluted volume, have the highest HAEA ^ _OO / Ap2o ratios and are pooled for further processing. These fractions have 36,600 units, which is 126 % of the apparent abandoned activity.

The combined fractions No. 9 to 12 are concentrated to 100 ml, and the concentrate is at a flow rate of 2 ml / min in a column with Dowex-1 χ 4 (Cl ") entered, in which the adsorbent has particle sizes below 37 ρ and bed dimensions of 2.2 χ 41 cm. the Column is washed with 50 ml of demineralized water and at a rate of 2 ml / min with 3 l Table salt 0.07 molar, 0.005 molar NH ^ Cl and NH ^ 0.0001 molar solution eluted in demineralized water. Starting with the first task of the eluent, be 10.8 ml fractions collected.

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The main peak of antibiotic activity 890A-. appears in fractions No. 181 to 217 with a maximum in fraction No. ₀ 198. Fractions No. 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 adding 16 μl of 1 molar sodium hydroxide solution set. The concentrate is placed in a column with Bio-Gel-P-2 (37-74 u; bed dimensions 2.15 x 70 cm) and Washed in three times with 1 ml of demineralized water each time. The column is at a speed of 0.96 ml / min eluted with demineralized water. Fractions of 3.85 ml each are collected.

The main peak of antibiotic 890A ₁ activity appears in fractions # 24 to 44 with a maximum in fractions # 33 and 34. Fractions # 27 to 38, which have the highest A ^^ / ApAc ratios, become united for freeze drying. These combined fractions contain a total of 72 A ^ _0Q units.

To carry out the freeze-drying, 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 molar sodium hydroxide solution. The sample is divided into two parts of 1.50 ml each, which are then quickly frozen and freeze-dried separately from 14 ml glass ampoules with screw caps. Each sample contains 1.73 mg 890A ^, corresponding to 35.8 A ^ ₀₀ units.

By deacetylating the material obtained in this way by the method of Example 3, the antibiotic deacetyl-890A _{1 is obtained} .

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Agents which contain the AntiMotica according to the invention, namely Desacetyl-890A-] and Desacetyl-890A ,, as well as agents which contain thienamycin, can be administered in various dosage forms, for example in solid or liquid oral dosage form. The agents, regardless of whether they are liquid or solid, contain 0.1 to 99% active ingredient per unit dose; the preferred range is about 10 to 60 %. The agent generally contains about 25 to 1000 mg of active ingredient, based on the total weight of the agent; however, dose levels in the range from about 250 to 1000 mg are generally used. For parenteral administration, the unit dose is usually in the form of a pure compound in a somewhat acidified sterile aqueous solution or in the form of a powder intended to be dissolved. Typical blends can be made using the following procedures:

Capsules per capsule

400 mg

Lactose, U.S.P., for filling capsules No. 0 sufficient amount, approximately 475 mg each.

In the example above, the active ingredient and diluent are mixed to form a uniform mixture, which is then filled into hard gelatine capsules no. 0 by hand or by machine. Mixing and filling is preferably done in a room with a relative humidity of less than 40 %.

Tablets . per tablet

Desacetyl-egOA ^! 330 mg

Calcium phosphate 192 mg

Lactose, U.S.P. 190 mg

Corn starch 80 mg

Magnesium stearate 8 mg

800 mg.

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In the example above, the active ingredient is calcium phosphate, the lactose and about half of the corn starch mixed together. The mixture is made with a 15% corn starch slurry granulated, roughly sieved and then sieved again through a sieve with a mesh size of 1.19 mm. After adding the rest of the Corn starch and the magnesium stearate, the mixture is compressed into tablets which have a diameter of about 1.27 cm and weigh 800 mg each.

Another method is to mix the active ingredient with the calcium phosphate, the lactose and half of the corn starch. The mixture is processed into compacted, tablet-like masses in a high-performance press. These become grains crushed from a diameter of about 1.2 mm. Then you add the rest of the corn starch and the magnesium stearate and compresses the mixture into tablets about 1.27 cm in diameter and 800 mg in weight.

Lyo form (for injection) per ampoule

Desacetyl-egOA ^ 25 mg

With water for injections, U.S.P.,

made up to 5 ml.

In the above example, the active ingredient is used in the specified ratio in a sufficient amount of water for injections dissolved. The solution is passed through Selas candles or "Millipore" membrane filters filtered to sterilize them. The solution is distributed into sterile ampoules. The Amuppen are with their contents are frozen and the water is aseptically removed by freeze-drying. The the sterile dry solid containing ampoules are sealed aseptically.

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709822/1031

15 837 ^ A

To regenerate a solution for parenteral administration, add 5 ml of sterile water for injections to the contents to an ampoule. '

Oral liquid forms

Desacetyi-890A.j

Sucrose

glucose

Sodium benzoate

concentrated orange oil

made up with purified water, U.S.Po.

The sucrose and glucose are dissolved in about 400 ml of water with heating. The solution is cooled and initially with sodium benzoate and then with concentrated orange oil. The solution is made up to about 900 ml with water, whereupon the antibiotic is added. The solution is clarified by filtering through a coarse filter.

each 1000 ml 1.0 G 600.0 G 250.0 G 1.0 G 0.2 ml 1000.0 ml

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70 9822/103 1

Claims (7)

Merck & Co., Inc. ^ c- - ^ y Claims 2. Desacetyl-890A ₃ 3. Process for making the compound of structural formula CH, -CH (OH) COOH characterized in that one has a compound of the structural formula CH -.- CH (OH) ³ S-CH ₀ -CH ₉ -NH-C-CH- COOH brings into intimate contact with an amidohydrolase capable of hydrolyzing the N-acetyl group. 4. The method according to claim 3, characterized in that an N-acetylthienairiycin amidohydrolase is used as the amidohydrolase used. - 89 - 7 0 3 8 2 2/1031 15 837 5. The method according to claim 3, characterized in that an N-acetylethanolamine amidohydrolase is used as the amidohydrolase used. 6. Process for obtaining microorganisms with N-acetylthienamycin amidohydrolase, characterized in that those microorganisms are selected for breeding, capable of utilizing N-acetylethanolamine, and these microorganisms for the presence of Iv-acetylthienamycin amidohydrolase examined. 7. The method according to claim 3, characterized in that it is carried out with an amidohydrolase, which by a Amidohydrolase producing strain of the microorganism Protaminobacter ruber has been produced. 8. A process for the preparation of thienamycin, characterized in that that N-acetylthienamycin is in intimate contact with the amidohydrolase-producing strain of The microorganism Protaminobacter ruber produces the enzyme N-acetylthienamycin amidohydrolase. 9. A process for the preparation of deacetyl-890A> i, characterized in that that one 890A-, in intimate contact with the strain of the microorganism that produces an amidohydrolase Protaminobacter ruber produced the enzyme N-acetylthienamycin amidohydrolase brings. 10. A process for the production of deacetyl-890A ^, characterized in that 89Ok ^ is brought into intimate contact with the enzyme N-acetylthienamycin amidohydrolase produced by an amidohydrolase-producing strain of the microorganism Protaminobacter ruber. - 90 703822/1031 837 ^ 2 B B? G 7 11. Agent, characterized in that there is an antibacterially effective amount of the compound Desacetyl-890A ^ and one Contains non-toxic, pharmaceutically acceptable carrier. 12. Agent, characterized in that there is an antibacterially effective amount of the compound Desacetyl-SSOA ^ and one Contains non-toxic, pharmaceutically acceptable carrier. - 91 - 7 0 9 n.7? I 1 0 3 1