DK143713B - PROCEDURE FOR THE PREPARATION OF ANTIBIOTIC SUBSTANCE N-ACETYL-THIENAMYCINE AND SALTS THEREOF - Google Patents

Description

(19) DENMARK

§J

| J | (12) PRESENTATION DD H3713B

PATENT AND TRADEMARKET DIRECTORATE

(21) Application No. 4972/76 (51) lnt.CI.3 C 12 P 17/18 (22) Filing date 3 Nov. 1976 C 07 D 487/04 (24) race day Nov 3 1976 (41) Aim. accessed May 22, 1977

(44) Presented 28. s ep. 198I

(86) International application # - (86) International filing day (85) Continuation day - (62) Master application no -

(30) Priority 21. November 1975; 6;? 4? 01, US

(71) Applicant MERCK & CO. INC., Rahway, US.

(72) Inventor jean Sawyer Kahan, US: Frederick Marvin Kahan, US:

Robert Thomas Goegelman, US: Edward Olley Stapley, US: m · fl · (74) Hofman-Bang & Boutard full-time engineering firm.

(54) Process for preparing an antibiotic substance N-acetyl-thl = enamycin and its salts.

The present invention relates to a process for the preparation of the novel antibiotic N-acetyl-thienamycin having the formula I as claimed in claim 1 and to non-toxic pharmaceutically acceptable salts thereof. These compounds are active against both Gram-positive and Gram-negative bacteria.

The discovery of the antibiotic properties of the penicillin has greatly QQ stimulated interest in this area, which has resulted in Ό the discovery of many other valuable antibiotics, such as: - other penicillins, cephalosporins, streptomycin, bacitracin, 2 tetracyclines, chloramphenicol and erythromycin . None of these - antibiotics are active against all clinically important pathogenic bacteria. For example, some are mostly only active towards

Gram-positive bacteria. Furthermore, acquired resistance due to extensive use of known antibiotics for the treatment of bacterial infections has given rise to serious resistance problems.

The deficiencies of known antibiotics have therefore stimulated further research to find other antibiotics that are active against a larger range of pathogens as well as against resistant strains of particular microorganisms.

The present invention aims to prepare a novel antibiotic, hereinafter referred to as N-acetylthienamycin.

This is achieved by the method according to the invention, which is characterized by the characterizing part of claim 1.

Thus, the subject antibiotic, N-acetyl-thienamycin, is produced by cultivation under controlled conditions by the microorganism Streptomyces cattleya. Furthermore, when growing Streptomyces cattleya in nutrient medium, thienamycin is produced. The preparation of thienamycin by fermentation of Streptomyces cattleya is described. knows in Danish publication no. 138 66j5.

Based on extensive taxonomic studies of Streptomyces cattleya isolated by a soil sample, this microorganism has been identified as an actinomycet. A culture of this is deposited in the permanent cultural collection at Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, 111., under the registration designation NRRL 8057.

The morphological and cultural characteristics of Streptomyces cattleya are shown in Table 1 below.

TABLE 1

Morphology - The sporophores are compact spirals that appear as lateral and terminal branches of the aerial mycelium. The spores are ellipsoidal to cylindrical in shape, 0.9, e x 1.2 / t in size, occur in chains of more than 10.

Growing Properties

Tomato paste-oatmeal agar

Vegetative growth - reverse tan, flat, widespread;

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

Soluble pigment - nothing.

Czapek Dox agar (sucrose nitrate agar)

Vegetative growth - colorless, flat, scattered;

Aerial mycelium - sparse, pinkish white;

Soluble pigment - nothing.

Egg albumin agar

Vegetative growth - tan with gray-orchid hue, flat, scattered; Aerial mycelium - orchid (10 gc) mixed with lighter shades of orchid and some white;

Soluble pigment - nothing.

Glycerol-Asparagine Agar

Vegetative growth - back-tan with gray-pink hue, flat, scattered;

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

Soluble pigment - nothing.

Yeast extract glucose + salt agar

Vegetative growth - tan with gray-pink hue;

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

Soluble pigment - nothing.

Yeast extract malt extract agar Vegetative growth - tan;

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

Soluble pigment - nothing.

4 \ 43713

Pepton-iron-yeast extract-agar Vegetative growth - tan;

Air mycelium - nothing;

Soluble pigment - slight brown tinting of medium;

Melanin - negative; E ^ S production - negative.

nutrient

Vegetative growth - light tan;

Air mycelium - nothing;

Soluble pigment - nothing.

Næringsstivelsesagar

Vegetative growth - cream for tan;

Air mycelium - nothing;

Soluble pigment - nothing;

Hydrolysis of starch - moderate.

Næringsgelatine agar

Vegetative growth - cream colored;

Air mycelium - nothing;

Soluble pigment - nothing;

Melting of gelatin - moderate.

Gelatin cuttings Vegetative growth - tan;

Air mycelium - nothing;

Soluble pigment - nothing;

Melting of gelatin - moderate.

potato Pieces

Vegetative growth - moderate, tan;

Aerial mycelium - sparse, greyish-pinkish-white;

Soluble pigment - nothing.

Loeffler's blood serum

Vegetative growth - cream colored;

Air mycelium - nothing;

Soluble pigment - nothing;

Melting - none.

Skim milk agar

Vegetative growth - tan;

Aerial mycelium - sparse, whitish;

Soluble pigment - slight brown tinting of medium;

Casein hydrolysis - positive.

5 143713

Litmus milk

Vegetative growth - tan to brown;

Air mycelium - nothing;

Color - no soluble pigment, litmus indicator turns bluish; Coagulation and / or peptonization - partial peptonization, becomes alkaline.

Skimmed milk

Vegetative growth - tan;

Air mycelium - nothing;

Soluble pigment - nothing;

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

Tyrosine agar

Vegetative growth - tan;

Aerial mycelium r mixture of orchid (10 gc) and white;

Soluble pigment - nothing;

Decomposition of tyrosine - positive.

All the above measurements and observations were made after three weeks of cultivation at 28 ° C, med. unless otherwise noted. The pH of the media used in these studies was approximately neutral, namely pH = 6.8-7.2. The color designations in the specification are in accordance with the definitions in the Color Harmony Manual, ed. (1958), Container Corporation of America, Chicago,

Illinois.

Streptomyces cattleya was also tested for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown on a synthetic base medium (Pridham and Gottlieb) containing 1% of the carbohydrate at 28 ° C for three weeks. The pH of the medium used was approximately neutral (6.8 - 7.2). Table 2 shows the utilization of these carbohydrate sources by Streptomyces cattleya: + indicates good growth, - poor growth and - no growth for the carbohydrate concerned.

6 TABLE 2

Glucose ............ +

Arabinose ............ -

Cellulose ............ -

Fructose ............. ί

Inositol ............. -

Lactose .............. -

Xylose ............... Ϊ

Maltose .............. -

Mannitol .......... +

Mannose .............. -

Raffinose ............ -

Rhamnose ............. -

Sucrose ............ The amount of growth at different temperatures, the oxygen demand and the effect of the microorganism on nitrate are as follows:

Temperature range (yeast extract-glucose + salt-agar) 28 ° C - Good 37 ° C - Moderate 50 ° C - No growth

Oxygen Requirements (Yeast Extract Glucose Glucose + Salt Agar)

aerobic

Nitrate reduction - positive.

7 1437 13

The antibiotic N-acetyl-thienamycin is formed by aerobic fermentation of a suitable nutrient medium under controlled conditions after inoculation with the microorganism Streptomyces cattleya. For this fermentation, the usual aqueous media is used, which is also used in the preparation of other antibiotics. Such media contain sources of carbon, nitrogen and inorganic salts which are assimilated by the microorganism.

In general, carbohydrates such as sugars, e.g. glucose, fructose, maltose, sucrose, xylose and mannitol, and starches such as grains, e.g. oats, rye, cornstarch and cornmeal, either alone or in combination as sources of assimilable carbon in the nutrient medium. The exact amount of carbohydrate in the medium depends in part on the other constituents of the medium, but usually carbohydrate is used in an amount between 1 and 6% by weight of the medium. Such carbohydrate sources may be used individually or more may be combined in the medium. In general, many different proteinaceous materials can be used as a source of nitrogen in the fermentation process. Suitable nitrogen sources are, for example, yeast hydrolysates, primary yeast, soybean meal, cotton seed oil, casein hydrolysates, corn molding liquid, distiller's solubles or tomato paste. The nitrogen sources are used either alone or in combination in amounts of 0.2 to 6% by weight of the nutrient medium.

The culture medium must also contain inorganic nutrient salts containing sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride and carbonate ions. Furthermore, the medium must contain trace metals such as cobalt, manganese, iron and magnesium.

The fermentation is carried out at temperatures between 20 and 37 ° C, but optimum results are obtained at temperatures between 22 and 30 ° C. The pH of the nutrient in the cultivation of Streptomyces cattleya in the preparation of N-acetyl-thienamycin can vary between 6.0 and 8.0.

Although N-acetyl-thienamycin can be formed both in surface and submerged cultures, it is preferred to carry out the fermentation in the submerged state.

8 143713

For small-scale fermentation, it is appropriate to seed a suitable nutrient medium with the antibiotic-producing culture and then transfer to a production medium so that fermentation can proceed at a constant temperature of 24 ° C with shaking for several days.

The fermentation is initiated in a sterile flask containing the medium after one or more steps to develop seed cultures. The nutrient medium for this is any suitable combination of carbon and nitrogen sources. Shake the flask at a constant temperature of approx. 28 ° C for one or two days, or until growth is satisfactory, and a portion of the growth formed is used for inoculation of either one more stage graft culture or the production medium. If intermediate graft flasks are used, they are developed in the same way; that is, a portion of the contents of the flask from the last inoculation stage is used for inoculation of the production medium. The inoculated flasks are shaken at constant temperature for several days and after the end of the incubation period the contents of the flasks are centrifuged or filtered.

For large-scale preparation, it is preferred to carry out the fermentation in suitable tanks equipped with agitators and means for aerating the fermentation medium. By this method, the nutrient medium is worked up in the tank, which is sterilized by heating to temperatures of about 120 ° C. After cooling, the sterilized medium is seeded with a precultured seed culture of the production medium and the fermentation is allowed to elapse for a period of time, e.g. 3-5 days while shaking and / or aerating the nutrient medium while maintaining the temperature at approx. 24 ° C. This method of preparing N-acetyl-thienamycin is particularly suitable for the preparation of large amounts of antibiotic.

ZZ5i® £ ®_2 £ _ ^ ®? In ^ ®_M®5§kaber_for_N-acetyl thienamycin

An NMR spectrum at 100 MHz for N-acetyl-thienamycin shows the following peaks: 1.27, d, 3H, J &6.5; in 1.98, s, 3H; <4. 2.94 m, 2H; 9 3.17, m, 2H; * 3.38, t, 2H, J = 6.5; in 3.38, m, 1H; 4.20 m, 2H.

The NMR spectrum of a combined solution of N-acetyl-thienamycin formed by fermentation and by acetylation of thienamycin is indistinguishable from the individual solutions. The AfA max dependence on the pH is definitely a pK. of 3.3 - 0.1 for the COOH group α pen in N-acetyl-thienamycin.

By paper electrophoresis in 0.1M potassium phosphate buffer, pH = 7, using Schleicher and Schuell No. 2043-B paper with a voltage gradient of 50 v / cm, both N-acetyl-thienamycin obtained by fermentation and obtained by acetylation of thienamycin, walk 2.7 cm to the anode over 20 minutes at 10 ° C. The antibiotic is located by bioautography on Vibrio percolans, ATCC 8461 (without intermediate drying of the paper) and migration is measured; from the point of application to the center of zone inhibition. :

Thin-layer chromatography using cellulose-coated sheets and a solvent of ethanol: water, 70:30 reveals an Rf of N-acetyl-thienamycin obtained by fermentation and obtained by acylation of thienamycin of 0.7, determined by bioautography on Vibrio percolans, ATCC 8461. The R 1 value refers to the distance from the starting point to the center of bioactivity divided by the distance from the starting point to the solvent front.

The IR spectrum of N-acetyl-thienamycin is shown in the drawing.

N-acetyl-thienamycin further exhibits the antibiotic spectrum profile described below. The sample uses the Bauer-Kirby disc diffusion method, which is modified only by using 2 mm depth of the agar plates. The results, expressed by the diameter in millimeters of zone inhibition, are shown in Table 3. This table contains the antibiotic spectrum profile for N-acetyl-thienamycin and for the material obtained by acetylation of thienamycin.

ια ί43713

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N-Acetyl-thienamycin exhibits activity in vivo against Gram-negative and Gram-positive organisms and is therefore well suited for controlling bacterial infections in humans and animals. In determining the activity in vivo, N-acetyl-thienamycin is dissolved in and diluted with 0.01M sodium phosphate, pH = 7.0, to obtain five quadruplicate concentrations of the active substance for testing. White Swiss female mice with an average weight of 21 g were infected intraperitoneally with the test organism suspended in the fluid. The number of organisms injected was determined by a standard plate counting technique. At the time of infection, at the point and six hours later, some of the mice were treated intraperitoneally with the antibiotic. Five mice were used at each concentration of the drug tested. Control samples of five mice for each of several dilutions of the infected culture were included in each sample to calculate the number of organisms that were lethal to 50 # of the infected untreated mice (LD ^ q). This calculation was made using survival data on the seventh day after infection, at which time the amount of drug intended to protect 50 # of infected mice (ED ^ 0) was also calculated.

All animals that received this effect and were not treated with the antibiotic died within 48 hours of infection. The efficacy of N-acetyl-thienamycin is shown in Table 4: TABLE 4

Efficacy studies (mice) a

Organism_LD, 0 νβ ^ ο ^ Γ m50 mg / li «ά ° 3ΐ5 °

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Staphylococcus aureus 2949 13 i.p. x 2 0.050 aCD-1, female mice, body weight 21 g ^ Indicates treatment at time of infection and again 6 hours later c Weight of N-acetyl-thienamycin based on rated E ^ cm

12 T43713 N-Acetyl-thienamycin is a valuable antibiotic that is active against various Gram-positive and Gram-negative bacteria and is therefore used in human and veterinary medicine. The compounds of the invention can be used as antibacterial drug to treat infections caused by Gram-positive or Gram-negative bacteria, e.g. against Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae.

The antibiotic compounds formed by the invention can further be used as feed additives for animals, for the preservation of feed and as disinfectants. For example, it can be used in aqueous compositions at concentrations of 0.1 to 100 parts or preferably at concentrations of 1-10 parts of the antibiotic per day. million parts of solution to prevent and inhibit the growth of harmful bacteria on medical and dental instruments and as a bactericide for industrial use, e.g. in aqueous paints and in paper mill water to inhibit the growth of harmful bacteria.

The antibiotic produced according to the invention can be used in the form of various pharmaceutical preparations as the sole active ingredient or in combination with one or more other antibiotics or with one or more pharmacologically active substances.

An example of the former is an aminocyclitol antibiotic such as gentamycin which can be added to expand the anti-microbial spectrum and reduce any chance of development of resistant organisms. Examples of the latter include diphenoxylate and atropine, which can be combined in dosage forms for the treatment of gastroenteritis. The antibiotic can be used in capsule form or as tablets, powders or liquid solutions or as suspensions or elixirs. It can be administered orally, topically, intravenously or intramuscularly.

In the treatment of bacterial infections, the subject compound can be administered orally or parenterally in a manner known per se in amounts of 2 to 600 mg / kg / day, preferably 5-100 mg / kg / day, conveniently distributed three or four times per day. day. Thus, unit doses may be used, e.g. containing 25, 250, 400, 13, or 1,000 mg of active ingredient with suitable physiologically acceptable carrier or diluent. The dosage units take the form of liquid preparations, such as solutions or suspensions, or solids in tablets or capsules. It will be appreciated that the optimal dose in any given case will depend on the nature and severity of the infection to be treated and that smaller doses are used for pediatric use as directed by the physician.

Non-toxic, pharmaceutically acceptable salts of N-acetylthienamycin, e.g. pharmacologically acceptable salts formed with inorganic and organic bases, including metal salts derived from alkali metal or alkaline earth metal hydroxides, carbonates or bicarbonates such as sodium, potassium, ammonium and calcium, and salts derived from primary, secondary or tertiary amines such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower-alkanolamines, lower alkylene diamines, N, N-diaralkyl-lower-alkylenediamines, aralkylamines, amino-substituted lower-alkanols, N, N-di-lower-alkylamino-substituted lower alkanols , amino-, polyamino- and guanidino-substituted lower alkanoic acids and nitrogen-containing heterocyclic amines. Representative examples include salts derived from sodium hydroxide, ammonium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine, lysine, protamine, phin, benzylamine, ethylenediamine, N, Ν'-dibenzylethylenediamine, diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol, theophylline and N-methylglucamine.

The salts in question can be prepared in a manner known per se.

For example, the monosalts, such as the monosodium salt, are obtained by treating an equivalent of sodium hydroxide with an equivalent of compound (I) in a suitable solvent. Also mixed salts with divalent cations can be prepared by combining one mole of a divalent base with one mole of the product (I) plus one equivalent of another acid. Instead, salts can be obtained by treating an equivalent of a base with a divalent cation, such as calcium hydroxide, with an equivalent of the product (I). The salts in question are pharmacologically acceptable non-toxic derivatives which can be used as active ingredient in suitable pharmaceutical form. Furthermore, they can be combined with other drugs to form compositions with a broad spectrum of activity.

The fermentation liquids prepared by the process of the invention contain antibiotic with activities between 0.1 and 4 µg per day. ml. Antibiotic preparations can be purified and the antibiotic can be extracted in various ways. One such method consists in passing the filtered liquid containing N-acetyl-thienamycin, preferably adjusted to a pH between 4 and 5, through a column of strong cation exchange resin. Examples of such resins are the sulfonate type with styrene-divinylbenzene matrix, e.g. polystyrene nuclear sulfonic acid resin Dowex 50 x 2 (manufactured by Dow Chemical Co., Midland, Michigan), on sodium cycle. Other representative members of this group of highly cation exchange resins are Dowex 50 x 4, Dowex 50 x 8 (manufactured by Dow Chemical Co., Midland, Michigan), Amberlite IR 120 (manufactured by Rohm & Haas Co., Philadelphia, Pennsylvania), Duolite C25D (manufactured by Chemical Process Co., Redwood City, California),

Permutit Q (manufactured by Permutit Co., Birmingham, New Jersey),

Ionac C-249 (manufactured by Ionac Chemical Co., Birmingham, New Jersey) and Amberlite 200.

The effluent from the cation exchange resin containing the antibiotic N-acetyl-thienamycin can be further purified, if desired, by other purification methods. It is noted that thienamycin remains adsorbed on the strong cation exchange resin. Accordingly, these two antibiotics can be clearly separated.

One such procedure consists of adsorbing N-acetyl-thienamycin on a strong basic anion exchange resin. Typical examples of such highly basic anion exchange resins are those having a styrene-divinylbenzene matrix, e.g. polystyrene nuclear quartz-near ammonium resin Dowex 1x2 (manufactured by Dow Chemical Co., Midland, Michigan), on the chloride cycle. Other representative highly basic ion exchange resins are the following: Duolite A-40, A-42, A-101, A-102 and A-114 (manufactured by Chemical Process Co., Redwood City, California); Amberlite IRA-400, IRA-401 and IRA-410 (manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania).

15 143713

The N-acetyl-thienamycin contained in the eluate can be further purified if the eluate is passed through a column packed with an intermediate polarity acrylic ester polymer such as XA.D-7 or 8, or through the polyester non-polar hydrophobic cross-linked divinylbenzene polymers such as XAD-1, 2 and 4, preferably XAD-2.

(XA.D-1, 2, 4 »7 and 8 are manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania).

One method of preparing further purified N-acetyl-thienamycin consists of conducting gel filtration through polyacrylamide gel having a pore size that excludes molecules with a molecular weight greater than 1800, such as Bio-Gel P-2 (manufactured by Bio Rad. Richmond, California).

The process of the invention is illustrated below by some examples. The following Table 5 thus illustrates a process scheme for the purification of N-acetyl-thienamycin.

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Test Procedure for Antibiotic N-Acetyl-Thlenamyein I. Bioassays

Samples of antibacterial activity are performed by the disk diffusion method described below using either Vibrio percolans ATCC 8461 or Staphylococcus aureus ATCC 6538P as the test organism.

Plates containing Vibrio percolans ATCC 8461 are prepared as follows:

A lyophilized culture of Vibrio percolans ATCC 8461 is suspended in 15 ml of a sterile medium containing 8 g / l Difco broth and 2 g / l yeast extract in distilled water (hereinafter referred to as NBYE). The culture is grown for 18 hours on a rotary shaker at 28 ° C. This culture is used for grafting the surface of slant cultures containing 1.5 $ agar in NBYE, and the grafted slant cultures are grown for 18 hours at 28 ° C and then stored in a refrigerator.

The chilled slant cultures from a single lyophilized culture are used for up to four weeks after preparation as follows:

With a graft needle, graft material is transferred from the slant culture and dispersed in 50 ml NBYE in a 250 ml conical flask. The culture is grown for 18 hours in a rotary shaker at 28 ° C and then diluted to a concentration which gives $ 50 transmission at 660 nm.

A 33.2 ml portion of this diluted culture is added to 1 liter of NBYE containing 15 g of agar and maintained at 46 ° C. The grafted agar-containing medium is poured into 100 x 15 mm plastic petri dishes, 5 ml per ml. bowl, cool and maintain at 2-4 ° C for up to 5 days prior to use.

Plates containing Staphylococcus aureus ATCC 6538P are prepared as follows: An 18 hour old growth of the test organism,

Staphylococcus aureus ATCC 6538P, in nutrient liquid plus $ 0.2 yeast extract, is diluted with nutrient liquid plus $ 0.2 yeast extract for a suspension with $ 55 transmission with a wavelength of 660 nm.

This suspension is added to Difco nutrient agar supplemented with 2.0 g / L Difco yeast extract at 47 ° C to 48 ° C to prepare a mixture containing 33 »2 ml of the suspension per liter of agar, 5 ml of this suspension is poured into Petri dishes with a diameter of 85 mm and the formed plates are cooled and maintained at 4 ° C until use (maximum 5 days).

Samples of the antibiotic are diluted to a suitable concentration in phosphate buffer at pH = 7. Slices of filter paper, 6.55 or 12.7 mm in diameter, are dipped in the sample solution and placed on the surface of the sample plates. The plates are incubated at 37 ° C for 18 hours and the zone of inhibition measured as mm diameter. The inhibition zone measured in mm determines the relative activities.

II. Hydroxylamine extinguishable absorption

The ratio of absorbance measured at 301 nm, which may be due to the content of antibiotic substance in the impure sample, is determined by selectively quenching this absorption (with associated inactivation of antibiotic activity) after reaction with dilute hydroxylamine.

Samples containing antibiotic are prepared in 0.01M potassium phosphate buffer at pH = 7 and with an initial between 0.1 and 1.0. Freshly prepared neutralized hydroxylamine (NHgOH'HCl. Plus NaOH to a final pH of 7) is added to a final concentration of 10mM and the reaction is allowed to proceed at room temperature for at least 30 minutes. The resulting A ^ q-j is subtracted from the initial reading (after correction for dilution with added reagent) and gives hydroxylamine quenchable absorption. Solutions of pure N-acetyl-thienamycin exhibit a hydroxylamine-quenchable absorption of 96.0 EXAMPLE · 1

A tube with lyophilized culture of Streptomyces cattleya NRRL · 8057 is opened aseptically and the contents are suspended in a tube containing 0.7 ml of sterile Havis salts of the following composition: 19 143713

Davis-aalte

Sodium citrate 0.5 g k2hpo4 7.0 g KH2PO4 5.0 g (nh4) 2 SO4 1.0 g

MgSO4-7H2O 0.1 g

Distilled H2 O 1000 ml

A 0.2 ml portion of this suspension is used to inoculate a slant culture of Medium A (plus agar) with the following composition:

Medium A

Yeast Autolysate (Ardaminx) 10.0 g

Glucose 10.0 g + Phosphate buffer 2.0 ml

MgSO4.7H2 O 0.05 g

Distilled H 2 O 1000 ml pH is adjusted to 6.5 with NaOH xArdamine: Yeast Products Corporation + Phosphate - buffer solution kh2po4 91.0 g

Na2HPO4 95.0 g

Distilled H2 O 1000 ml

For slanting cultures: add agar - 25.0 g / l.

The seeded slant culture is incubated for 28 days at 28 ° C and then left at 4 ° C.

A portion of the spores and aerial mycelia for this slanting culture is used to inoculate an aerated 250 ml conical flask containing 50 ml of Medium A (without agar). This seed flask is shaken at 28 ° C in a shaker at 220 rpm. per minute (50 mm stroke) for 2 days, after which time the growth is satisfactory.

Stir 250 ml conical flasks, each containing 40 ml Medium B, inoculated with 1 ml per ml. flask of the growth from the inoculum. Medium B

20 143713 has the following composition:

Medium B

Stomach flour 20.0 g

Distiller's Solubles 10.0 g

Sog bean flour 15.0 g

Sodium citrate 4.0 g

CaCl2-2H2O 0.5 g

MgSO4-7H2O 0.1 g

CoC12 * 6H20 0.01 g equal SO4 * 7H20 0.01 g ^ Polyglycol 2000 0.25 fo vol.

Distilled Η £ 0 1000 ml

pH: adjusted to 6.5 with NaOH

^ Polyglycol 2000: Dow Chemical Co.

These 15 production flasks are shaken at 28 ° C on a shaker at 220 rpm. per minute (50 mm stroke) for 55 hours.

A collected portion (after 50 hours) of the liquid from 15 flasks is mixed and a portion of it is centrifuged for sample.

Prior to the sample, the pH of the centrifuged liquid is adjusted to 6.5 from 5.9 with sodium hydroxide.

Tests were performed on Staphylococcus aureus A1CC 6538P and Vibrio percolans AlOC 8461 sample plates with 12.7 mm slices dipped in the centrifuged liquid.

The test results are as follows: AICC 6538P activity ATCC 8461 activity (mm zone) (mm zone)

39/44 SH 35/44 SH

SH = slightly cloudy 200 ml of the filtered liquid is adjusted to a pH of 8.0 and adsorbed on 10 ml Dowex 1x2 resin on chloride cycle at 2 ml / min, collecting the effluent in 10 x 20 ml fractions.

21 143713

The adsorbate is eluted with 90 # methanol; 10 # water; 3 # ammonium chloride v / v / w, collecting the eluate in 10 x 5 ml fractions. The eluate fractions 1-6 are combined and evaporated in vacuo to remove the methanol. The concentrate is tested by the slice diffusion procedure with a 12.7 mm slice containing 100 μΐ of antibiotic solution against Staphylococcus aureus MB-2985, obtaining a zone diameter of 28 mm.

Samples of MB-2985 are prepared as follows:

An 18-hour culture of MB-2985, cultured in cardiac-cardiac infusion medium at 37 ° C with shaking, is diluted 20,000 times and rinsed on the surface of 10 ml of cardiac-cardiac infusion agar in an 85 mm diameter Petri dish. Discs of 12.7 mm in diameter containing 100 µl of antibiotic solution are placed on the plates, which are then incubated for 18 hours at 37 ° C. The inhibition zones are read in mm.

EXAMPLE 2

A lyophilized culture tube of Streptomyces cattleya NRRL 8057 is opened aseptically and the contents are used to inoculate an aerated 250 ml conical flask containing 50 ml of Medium A having the following composition:

Medium A

Yeast Autolysate (ArdaminX) 10.0 g

Glucose 10.0 g + Phosphate buffer 2.0 ml

MgSO4-7H20 0.05 g

Distilled H2 O 1000 ml

pH: adjusted to 6.5 with NaOH

xArdamine: Yeast Products Corporation + Phosphate Buffer Solution kh2po4 91.0 g

Na2HPO4 95.0 g

Distilled HgO 1000 ml 22 143713

This seed flask is shaken at 28 ° C on a shaker at 220 rpm. per minute (50 mm stroke) for 2 days to achieve satisfactory growth.

15 250 ml conical flasks, each containing 40 ml Medium B, are seeded with 1 ml per flask of growth from the inoculum flask. Medium B has the following composition:

Medium B

Corn flour 20.0 g

Distiller's Solubles 10.0 g

Soybean flour 15.0 g

Sodium citrate 4.0 g

CaCl 2 * 2H 2 O 0.5 g

MgSO4-7H2O 0.1 g

CoCl2-6H20 0.01 g

DeSO4-7H20 0.01 g ^ Polyglycol 2000 0.25 $ per vol.

Distilled H2 O 1000 ml

pH: adjusted to 6.5 with NaOH

^ Polyglycol 2000: Dow Chemical Co. *

These flasks are shaken at 28 ° C on a shaker at 220 rpm. per minute (50 mm stroke) for 3 days, taking samples during the fermentation. The tests are performed on standard Staphylococcus aureus ATCC 6538P and Vibrio percolans ATCC 8461 test plates using 12.7 mm slices, dipped in the centrifuged liquid. The pH of this liquid is adjusted prior to the sample as given in the following table. The results are as follows:

Age (hours) 48 55 72 ATCC 6538P activity 33 / 39h 31 / 38h 21 / 27h ATCC 8461 activity 35sh / 46h 36sh / 44h 33sh / 38h pH, initial 5.2 5.1 4.8 pH, adjusted 6.1 6 , 2 6.9 sh = slightly cloudy h = cloudy 23 143713

After 53 hours, the liquids are collected and filtered to give 590 ml of filtrate at a pH of 5.9. The pH of the filtrate is adjusted to 7.0 and 5.9 mg of ethylenedinitrile-tetraacetic acid (EETA) is added.

580 ml of the above filtrate at a pH of 7.0 are adsorbed on 130 ml of Eowex 1 x 2 resin on the chloride cycle, whereby the effluent is collected in 2 x 290 ml fractions. The aqueous surfactate is then washed with 130 ml of deionized water. One washed adsorphate is stored in a cold room for 18 hours and then eluted with 5 $ sodium chloride solution, collecting 6 x 50 ml fractions.

The yield as a percentage of the original activity, as determined by the plate-plate method, is shown in the following table:

Vibrio percolans Staphylococcus aureus Fraction ATCO 8461 ATCC 6538P

Eluate Fractions 1-5 26 $ 1 $

One antibiotic substance N-acetyl-thienamycin is detected in fractions 1-5 of the NaCl eluate. Fraction 4 was tested by the 12.7 mm diameter disc duct fusion procedure containing 100 µl of antibiotic solution against Staphylococcus aureus MB 2985 to give a zone of 29 mm. Surfaces containing MB-2985 are prepared by the process of Example 1.

EXAMPLE 3

A lyophilized culture tube of Streptomyces cattleya NRRL 8057 opens aseptically and the contents are suspended in 0.8 ml of sterile Eavis salts of the following composition: 24 143713

JAVIS salts

Sodium citrate 0.5 g K2HPO4 7.0 g

^ 2 ^ 4 3.0 S

(nh4) 2so4 1.0 g

MgSO4.7H2O 0.1 g

Distilled H2 O 1000 ml

This suspension is used for grafting four slant cultures of Medium A (plus agar) with the following composition:

Medium A

Yeast Autolysate (Ardaminz) 10.0 g

Glucose 10.0 g + Phosphate buffer 2.0 ml

MgSO4-7H20 0.05 g

Distilled HgO 1000 ml

pH: adjusted to 6.5 with NaOH

xArdamine: Yeast Products Corporation + Phos ~ phat-puf fer solution kh2po4 91.0 g

Na2HPO4 95.0 g

Distilled HgO 1000 ml

For oblique cultures; add agar - 25.0 g / l

The grafted slanting cultures are grown for a week at 28 ° C and then stored at 4 ° C.

Ten ml of Medium A (without agar) is aseptically transferred to one of these slant cultures, the spores and air mycelia are scraped into the suspension and 1.2 ml of this suspension is used to inoculate three 2 liter aerated conical flasks containing 500 ml of Medium A (without agar ). These seed flasks are shaken at 28 0 on a shaker at 160 rpm. per minute for 24 hours, thereby satisfying growth.

25 1437 13 The growth of these seed flasks is collected and used for inoculation of a 756 liter stainless steel fermentation vessel containing 467 liters of Medium A (without agar). This tank is operated at 28 ° C at a stirring rate of 130 rpm. per minute and an air flow of 283 liters per minute. per minute for 24 hours. Anti-foaming agent, Polyglycol 2000 (Dow Chemical Corp.) is used if necessary, but not more than 0.15¾. PH determinations are carried out as follows:

Age, hours 0 24 pH 6.3 6.4454 liters of the fermentation material from this seed tank is used for inoculation of a 5,670 liter stainless fermentation container containing 4.082 liters of Medium E, with Medium E having the following composition:

Medium E

Cerelose 25.0 g

Corn molding liquid (wet basis) 15.0 g

Distiller's Solubles 10.0 g

Cotton seed medium (Pharmamedia) 5.0 g

CoClg'SH ^ O 0.01 g

CaCO 3 (after pH adjustment) 3.0 g

Polyglycol 2000 0.25 #

Waterworks water 1000 ml

pH: adjusted to 7.3 with NaOH

This tank is operated at 24 ° C at a speed of 70 rpm. per minute and an air flow of 1550 liters per minute. per minute for 138 hours. More anti-foaming agent, Polyglycol 2000, is added if necessary, but no more than 0.1 #. Antibacterial tests are performed with the following result: 26 143713

Age pH ATCO No. 6633 (5/8 "washers) (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 6.7 36 132 6.7 41 138 6.7 39 The fermentation liquid of 4,082 liters is filtered by means of a 75 cm filter press and added filtration aid in the amount of 4 $ w / v. 46 g of ethylenedinitrile-tetraacetic acid (EDIA), sodium salt, is added to the filtrate, the solution is cooled to 6 ° C, adjusted to a pH of 4.5 - 0.2 and maintained at 6 ° C.

The cold filtrate is introduced into a 480 liter column of Dowex 50 x 4 Na +, 20-50 mesh at a rate of 48 liters / minute. After a flow of 1400 liters has passed, 18.9 liters of drains are collected, whose pH is adjusted to 7.08 with sodium hydroxide and the product is stored at 5 ° C.

A 3.8 cm column packed with 300 ml Dowex 1x2, 50-100 mesh, chloride cycle resin is prepared and washed with 600 ml deionized water at 5 ° C. 4 liters of cold Dowex 50 x 4 drain are passed through the column at a rate of 30 ml / minute. Wash the column with 300 ml of 25 μΜ EDTA. The antibiotic N-acetyl-thienamycin is eluted at 5 ° C at a rate of 15 ml / min with 900 ml of 5 $ sodium chloride solution containing 0.01M potassium phosphate buffer, pH = 7.0, and 25 μ 25 EDTA. 14 fractions of 75 ml are collected and tested for biological activity by the slice diffusion procedure. Eluate fractions 3-9, amounting to 525 ml, are collected and concentrated under vacuum to 115 ml. The concentrate contains $ 65 of the total bioactive material applied to the column of Dowex 1 x 2 Cl ".

27 1437 13

The eluate concentrate from the column is applied at 5 ° C to a 3.8 cm diameter column packed with 450 ml of pre-washed XAE-2. The column is prewashed in order of 4 column volumes:

1) Q, 001M EEIA

2) IN NaOH

3) Deionized Η £ θ 4) IN KC1 5) Deionized H2 O 6) Methanol 7) Acetone 8) Deionized water and prior to use with 2250 ml of 5 $ NaCl solution containing 25 µM EETA.

After the sample is applied to the column, it is followed by two 25 ml portions of water. The column is developed at 5 ° C with deionized water at a flow rate of 10 ml / minute. A first fraction contains 400 ml and 11 additional 75 ml fractions are collected. The pH of each fraction is adjusted to between 6.9 and 7.13 with 1N sodium hydroxide or 1N HCl. Fractions 3-9 containing 461 of the total bioactive material applied to column XAE-2 are combined to a total volume of 490 ml. A 45 ml sample is taken for bioassays by the standard disk diffusion procedure against Staphylococcus aureuB ATCC 6538P. One remaining 445 ml is concentrated under a vacuum to 50 ml.

One 50 ml XAE-2 eluate concentrate is pumped at 5 ° C into a 1.5 cm column packed with 40 ml pre-washed Eowex 1 x 4 Cl ", minus 400 at 5 ° C and a rate of 1 xol / min. Eowex 1x4 Cl "minus 400 (defined by decantation from water) resin is washed column-wise prior to use with 240 ml of 0.2M sodium chloride solution containing 0.005 M ammonium chloride and 0.1 mM ammonia at a rate of 1 ml / minute and then with 120 ml of deionized water at the same speed.

After the sample is applied to the column, it is followed by two 5 ml portions of deionized water. The column is developed at 5 ° C at a rate of 0.92 ml / min with 0.07M sodium oxyuride containing 0.0Q5M ammonium chloride and 0.1 mM ammonia. Fractions of 8.6 28 to 3.3 ml are collected. The fractions obtained after collecting 60Q ml of eluate and even 710 ml are combined and this portion contains $ 98 of the total bioactive material which was "applied to the column of Dovrex 1x4. This portion is evaporated in vacuo to 2 ml.

The concentrate from Dowex 1x4 is applied to a 2.2 cm diameter column packed with 200 ml Bio-Gel P-2, 200-400 mesh, with an exclusion limit of 1800 Daltons (defined prior to use in decantation from distilled water).

The column of Bio-Gel P-2 is washed prior to use with 225 ml of 1M sodium chloride followed by 100 ml of deionized water. The column is developed at 5 ° C with deionized water and a flow rate of 1 ml / minute and 2 ml fractions are collected, Fractions from 104 ml to 128 ml eluate containing 83 $ of the hioactivity applied to the column of Bio-Gel P-2 , combine and evaporate to 1.58 ml.

A 50 ml column of XAD-2 (1.6 cm x 27 cm) is prepared and pre-washed column by 200 ml of 1 mM EDTA, 1N sodium hydroxide, deionized water, 1N hydrochloric acid, deionized water, methanol, acetone and deionized water. The Bio-Gel P-2 concentrate containing 44.4 hydroxylamine extinguishable optical density units is applied to the column with XaD-2 at 5 ° 0 followed by two 2 ml portions of deionized water. The column is washed with deionized water at a rate of 1 ml / minute until the UV absorption at 300 nm of the wash liquids is reduced to 0.060. The column is eluted with 50 $ methanol in deionized water at a rate of 1 ml / minute and 1 ml fractions are collected. Fractions with absorption at 300 nm above 0.1 are combined and evaporated in vacuo to give the product, N-acetyl-thienamycin containing 11.6 hydroxylamine extinguishable O.D. units.

EXAMPLE · 4

A tube of lyophilized culture of Streptomyces cattleay NRRL · 8057 opens aseptically and the contents are suspended in 0.8 ml of sterile Davis salts of the following composition; 29 1 A3 7 13

Davis-ealte

Sodium citrate 0.5 g K2HPO4 7.0 g kh2po4 5.0 g (nh4) 2 SO4 1.0 g

MgSO4.7H2O 0.1 g

Distilled H2 O 1000 ml

This suspension is used for grafting four slant cultures of Medium A (plus agar) with the following composition:

Medium A

Yeast Autolysate (Ardaminx) 10.0 g

Glucose 10.0 g + Phosphate buffer 2.0 ml

MgSO4-7H20 0.05 g

Distilled Ηγ, Ο 1000 ml

pH: adjusted to 6.5 with NaOH

xArdamine: Yeast Products Corporation + Pho Sphat Buffer Solution KH2PO4 91.0 g

Na2HPO4 95.0 g

Distilled H2 O 1000 ml

For oblique cultures: add agar - 25.0 g / l

The grafted slanting cultures are grown for a week at 28 ° C and then stored at 4 ° C.

Ten ml of Medium A is aseptically transferred to one of these slant cultures, the spores and air mycelia are scraped into suspension and 1.2 ml of this suspension is used to inoculate three 2 liter buffered conical flasks containing 500 ml of Medium A (without agar).

These seed flasks are shaken at 28 ° C in a shaker at 160 rpm. per minute for 24 hours, thus achieving satisfactory growth.

The growth from these seed flasks is collected and used to inoculate a 756 liter stainless steel fermentation container containing 467 liters of Medium A (without agar). This tank is operated at 28 ° C at a speed of .150 rpm. per minute and an air flow of 285 liters per minute. per minute for 24 hours. Anti-foaming agent, Polyglycol 2000 (Dow Chemical Corporation) is used if necessary, but in an amount not exceeding $ 0.1. PH determinations are performed with the following result:

Age, hours 0 24 pH 6.5 6.4 454 liters of the growth from this seed tank is used for inoculation of a 5,670 liter stainless steel fermentation vessel containing 4,082 liters Medium E of the following composition:

Medium E

Cerelose 25.0 g

Maize cast water (wet basis) 15.0 g

Distiller's Solubles 10.0 g

Cotton seed medium (Pharmamedia) 5.0 g ΟοΟΙ ^ βΗ ^ Ο 0.01 g

CaCO 3 (after pH adjustment) 5.0 g

Polyglycol 2000 $ 0.25

Waterworks water 1000 ml

pH: adjusted to 7.3 with NaOH

This tank is operated at 24 ° C with a stirring rate of 70 rpm. per minute and an air flow of 1550 liters per minute. per minute for 158 hours. Additional anti-foaming agent, Polyglycol 2000, is added, if necessary, but not more than $ 0.1. Antibacterial tests are performed with the following result: 31 143713

Age pH ATCC No. 6633 (3/8 ”Assives) (mm)

O, 6.9

24 6.3 O

36 6, '0 O.

48 5.9 °

60 6.0 23 72 5.9 84 6.0 21 96 6.2 108 6.5 35 120 6.6 36 152 6.7 41 158 6.7 39 The fermentation liquid of 4,082 liters is filtered using a 65 cm filter press while adding filtration aid in an amount of 4 # w / v. Here 46 g of ethylenedinitrile tetraacetic acid (EDTA), sodium salt are added to the filtrate. The hood is cooled to 6 ° C and the pH is adjusted to 4.5 - 0.2, then maintained at 6 ° G. The cold filtrate is applied to a column of -480 liters of Dowex 50 x 4 sodium +, 20-50 mesh at 48 liters / minute. After passing a flow of 1400 liters, 18.9 liters of drains are collected, the pH of which is adjusted to 7.08 with sodium hydroxide and then left at 5 ° C.

A 3.8 cm diameter column packed with 300 ml Dowex 1x2, 50-100 mesh, chloride cycle resin is prepared and washed with 300 ml deionized water at 5 ° C. Ere liters of cold drain from Dowex 50 x 4 are passed through the column at a rate of 30 ml / minute.

The column is washed with 350 ml of 25 µM EDTA and then eluted at 5 ° C with 900 ml of 5 # sodium chloride solution containing 0.1M Tris-HO1, pH = 7, and 25 µM EDTA at a rate of 15 ml / minute. 75 ml erections are collected and tested by the slice diffusion procedure against Staphylococcus aureus ATCC 6538P. Fractions 4-10 containing 47 # of the applied bioactivity are added to 42.5 ml of the sample taken from bio-samples from the first portion of XAD-2 described in Example 3. These combined fractions are evaporated in vacuo to 100 ml and the pH is adjusted to 6.32 with hydrochloric acid.

32 143713

A 5.8 cm diameter column packed with 450 ml of XAE-2 resin is pre-washed column by row with 4 column volumes: 1) 0.001M ΕΕΪΑ

2) 1N NaOH

3) Deionized E ^ O

4) 1N HCl

5) Deionized E ^ O

6) Methanol 7) Acetone 8) Deionized water

And washed prior to use with 2250 ml of 5 $ sodium chloride solution containing 25 M EDTA. The above concentrate is applied to the XAE-2 column and followed by two 5 ml portions of deionized water. The column is developed at 5 ° C at a rate of 10 ml / min with deionized water. A first fraction contains 40 ml and subsequent 75 ml fractions are collected and tested by the slice diffusion procedure. Fractions 9 to 15 containing 22 µl of the hioactivity applied to the XAE-2 column are collected and evaporated in vacuo to 56 ml.

A 21 cm x 1.7 cm column packed with 40 ml Eowex 1 x 4 01 ”, minus 400 mesh (defined by decantation from water) is washed column wise prior to use with 240 ml 0.2M NaCl containing 0.005M ammonium chloride and 0.1mM ammonia at a rate of 1 ml / minute and then with 120 ml of deionized water at the same rate.

The concentrate from XAE-2 is applied to the column followed by two 2 ml portions of deionized water and then two 2 ml portions of elution buffer. The column is eluted at 5 ° 0 with a solution of 0.07M sodium chloride containing 0.005M ammonium chloride and 0.1mM ammonia at a rate of 1 ml / minute. 10 ml fractions are collected and tested by the slice diffusion method. Eluate fractions from 524 ml to 647 ml containing practically 1005¾ of the applied hioactivity are combined and evaporated in vacuo to 2.3 ml. The concentrate contains 36.4 hydroxy lamin extinguishable optical density units.