SU552907A3 - The method of obtaining antibiotics - Google Patents

Description

(54) METHOD FOR OBTAINING ANTIBIOTICS

The microorganism used to produce the antibiotic of the present invention was isolated from a sample of the night in Eginta. Have you ever grown potato and agar agar and found that it belongs to the class of actinomycetes that produce sporangia, similar to the sporangia of the species Actinoplanes. Therefore, various media used for growing these species were used for its growth. Culture suspensions were prepared by crushing pieces of culture removed from agar-agar plates and grown in small tubes with the addition of sterile distilled water and bringing the culture to 5 ml.

Then these suspensions were grown in test tubes, on plates or in Petri slats on various media. The incubation temperature was 28 ° C. Results were evaluated in 14-22 days. This new culture (Pfizer F.D. 24090) was brought to the American Type Culture Collection in Rockville, Maryland, and March 1974 and was named Actinoplanes auranticolor ATCC 31011.

The following media were used to identify the culture:

1) 2% agar-agar in tap water;

2) "Artifact-carrot agar-agar for

M.P. Leschevalier (1968);

3) sucrose agar-agar of čapek according to Waxman (1961);

4) glucose-aspartic agar-agar according to Waxman (1961);

5) agar-agar with extract and malt;

6) Hickey and Tresner agar-agar;

7) potato glucose agar-agar;

8) starch agar-agar;

9) latina;

10) tyrosine agar-agar;

11) iron-peptone agar-agar Difco;

12) Difco milk removed;

13) dextrose nitrate broth;

14) organic compound and nitrate based broth;

15) ATCC environment 172, American Catalog of crop types;

16) using carbon.

Signs that characterize this culture.

The growth on the medium containing agar-agar in tap water is weak, the colonies are thin, flat, about 9D2 (very weak pink); air mycelium is absent, mycelium substrate is from colorless to 9D2, there is no soluble pigment.

Sucrose agar-agar of čapek. Growth is moderate to good; colonies are flat, about 9G6 (light orange); aerial mycelium is absent; substrate mycelium about 9G6; soluble pigment is missing.

Glucose-aspartic agar-agar. Growth is moderate, eminent, rough colonies, about 9G9 (light orange);

aerial mycelium is absent, soluble pigment is absent.

Agar-agar with extract and malt. No growth is observed.

Agar-agar Hickey and Tresner. Growth from moderate to good, colonies slightly elevated and rough, approximately 9F9 (dull orange), a weak whitish coating on the surface, the substrate is about 918 mycelium, pale brown soluble pigment.

Potato-glucose agar-agar. Growth is moderate, the colonies are elevated, blackish, about 9L9 (light orange), there is no aerial micelle, the substrate is about 9L9 mycelium, there is no soluble pigment.

Tyrosine agar-agar. Low to moderate growth, flat colonies, approximately 13A10 (dull reddish orange color) aerial mycelium is absent, mycelium substrate is approximately 10D11, brown soluble pigment.

Gelatin. Growth is moderate, colonies are flat, approximately 9K12 (reddish-orange); traces of whitish plaque, substrate mycelium approximately 9K12, there is no soluble pigment.

Starch agar agar. Growth is moderate to good, the colonies are elevated, approximately 9Q10 (orange), a weak whitish coating, the substrate of the mycelium is approximately 9Q10, light yellow soluble pigment.

Starch undergoes weak hydrolysis, the degree of liquefaction of gelatins is high, nitrates do not reduce to nitrites in any medium containing nitrates even after 22 days (growth is very weak in dextrose-nitrate broth, but good in broth containing organic substances and nitrate).

The formation of hydrogen sulfide is negligible. In iron-peptone agar-agar, the formation of a soluble pigment is not observed. There is no coagulation or hydrolysis in milk, even after 22 days. Tyrosine is not dehydrated.

Growth in the ATCC 172 medium is observed at a temperature of from 21 to 37 ° C, the best growth at a temperature of from 28 to 37 ° C, at a temperature of 45 ° C growth is not observed.

Arabinose, fructose, mannitol, raffinose, rhamnose, sucrose and xylose are consumed, inositol is not consumed. There is no smell on any of the media.

The sporangium is formed only on potato-carrot agar-agar. This forms a palisade layer, 5.5; -11X4.5-8 microns in width and 9-12 microns in height. The colonies are numerous, irregular in shape and emit spores with a gradual softening. Sporangia on potato-carrot agar-agar after three weeks of incubation releases spores for several hours at a temperature of about 2 ° C if pieces of colonies are immersed in a small amount of solution: 1 g of glucose and 1 ml of Tween 80 in 1 l of water. The spores form chains of irregular shape in snorangia, but, being freed from the sporangium, they become -subgloboznymi and have a width of 1.6-2.2 mm before the elliptical form 1.6-2.2X1.1 - 1.6 microns. Almost all of them are mobile. The cultivation of A. auranticolor ATCC 31011 preferably takes place in an aqueous nutrient medium at 26-36 ° C in a submerged aerobic state with mixing. Nutrient media suitable for its cultivation include those which include a source of assimilable carbon, such as sugars, starch and mela-cass; The sources of organically bound nitrogen, such as casein, the enzymatic product of the breakdown of casein, soybean flour, cottonseed meal, peanut flour and wheat gluten. Growth sources can also include waste products from distilleries, fish meal and yeast extract, as well as salts, such as sodium chloride and calcium carbonate, and trace elements, such as iron, magnesium, zinc, cobalt and manganese. If excessive foaming occurs during culture, a defoamer, such as vegetable oils or silicones, can be introduced into the nutrient medium. The aeration of the medium in the tanks when the culture is grown by the deep method is preferably carried out at a rate of about 1.2-2.0 volumes of free air per volume of broth per minute. The stirring speed is maintained with the help of billers usually used in the fermentation industry. An agent for the preparation of antibiotics can be obtained by using a culture from a plate on a medium, for example, ATCC 172, mentioned above. This culture can be used to inoculate either the contents of the flasks on the agitator, or the contents in tanks for inoculation, or embryos from flasks subjected to shaking can be introduced into the tanks. In flasks on a shaker, the growth of the culture usually reaches its maximum after about 4 days, while when inoculated in tanks, the period from 2 to 3 days is most beneficial. Significant antibiotic activity is achieved in the final fermentation stage in about 20-30 hours. The antibiotic production process during the fermentation process is usually controlled by biological testing of the broth using the sensitive strain Staphylococcus aureus. It uses a standard plate test method, in which the zone of inhibition surrounding a circle of filter paper saturated with broth is taken as a measure of antibiotic activity. After the antibiotic activity of the fermentation broth has reached the desired level, products are isolated either from the whole broth or from the filtered broth. In the latter case, the mycelium is removed by filtration or centrifugation. You can also use the method of thin-layer chromatography on silica gel. This method serves to analyze a mixture of antibiotics obtained in a fermentation medium, and makes it possible to determine the composition of the raw and purified materials extracted by extraction from the fermenting broths. The separation of the components of a mixture of antibiotics depends on the content of antibiotics in the system. Too small bactericidal activity makes it impossible to detect those components of the antibiotic that are present in small quantities; too high bactericidal activity leads to a resistance effect with the resultant unsatisfactory separation. The thin-layer chromatography system is a mixture of chloroform and ethanol (9: 1). After development, thin-layer chromatograms can be viewed in ultraviolet light at a wavelength of 254 mmk and 366 mmk. Bioavtographic detection of bactericidal components should be carried out by superimposing a thin-layer chromatogram on agargar with pharmaceutical substances into which the germs of the sensitive SZ strain are inserted. aureus or other sensitive organism. The main components of the antnbiotics mixture secreted by A. auranticolor ATCC 31011 are a number of macrocyclic lactones and depsipeptide antibiotic components. The appearance or absence or percentage composition of a mixture of these antibiotics varies from fermentation to fermentation and is a function of voemueniya, pH, medium composition, etc. When you observe the words given in the examples below, the main bactericidal components of the mixture of antibiotics are compounds No. 37,277 (depsipeptnd) and no. 39 926 (macrocyclic lactone), while antibiotic components present in small amounts include compounds No. 37 932 (depsipeptide) and no. 35 763 (macrocyclic lactone). The components of a mixture of antibiotics can be separated and separated from the fermentation broth using a variety of methods, including solvent extraction, countercurrent Craig distribution, column chromatography, or a combination of these methods. When extracting antibiotics from a broth, it is common to use various organic solvents, such as chloroform ethyl acetate and methyl isobutyl ketone. Solvent extraction is preferably carried out by extracting the broth twice with a pH of 7 with a solvent volume of 1 / 3-1 / 2 of the broth volume, from which it is desirable to isolate a mixture of antibiotics. The most recommended method for isolating and recovering components of a mixture of antibiotics is as follows. The unclarified or clarified broth is adjusted to a pH of about 7 and extracted with two portions of methyl isobutyl ketone, the volume of which is about 1/3 to ½ of the volume of extractable broth. The extract in the solvent is concentrated in vacuo, and the concentrate is degreased by extraction with heptane or petroleum ether. After that, the non-fat extract in a solvent is dried to dryness in a vacuum. The solids are subjected to a counter-current Craig distribution (6 taelines) using toluene (5 parts), ethanol (2 parts) aqueous (phosphate buffer solution with a pH of 4.5 (3 parts). The separated layers form the upper and lower phases of the co-current distribution system After distribution, the layers are ana lyzed by thin layer chromatography. The separated fractions are evaporated to dryness in a vacuum. Solids containing hepsipeptides are dissolved in chloroform, treated with activated wood. The coal is filtered, and evaporated in The residue obtained after evaporation of chloroform is dissolved in acetone. The solids precipitated by the addition of heptane are dissolved in a small amount of chloroform and added to the column by silica gel, buffered with pH, prepared in the presence of chloro Pol and n-propanol in the ratio 99: 1. Kolonkv exhibit the same system of solvents under pressure of 5600 N / m. Separate areas of the column is controlled by thin layer chromatography. The fractions containing the separated depsipeptides are combined, evaporated in vacuo, and their contents crystallized from apetone-heptane. Fractions obtained by a countercurrent method containing macrocyclic lactones, combined, evaporated in a vacuum, and the solid substances are dissolved in ethyl pane. The solution is stirred with silica gel. FILTER, and the solvent is removed in vacuo. The solution was dissolved in ethyl acetate and precipitated with hexane. Solid substances are dissolved in a small amount of chloroPoM and chromatographed under a pressure of 5600H / i on a column of silica gel, buffered to a pH of 6.0 and manufactured in the presence of ethylene ketone. For manifestation, a system of ethyl acetate, tetrahydrofuran, and hexane is used in a ratio of 80:20:20. Column portions are monitored by thin layer chromatography. The fractions containing separated macrocyclic lactones are combined and evaporated in vacuo. Separate fractions are treated with an additional countercurrent Craig distribution and / or column romatography using different systems. Careful monitoring of each purification stage makes it possible to isolate the individual macrocyclic lacgon in a sufficiently pure state, so that the fractions in the solvent can be evaporated to dryness to obtain pure components. A. auranticolor ATCC 31011 forms at least four depsipeptides (of which the main compound No. 37277 and the secondary compound are 37 932) and four macrocyclic lactones. (Compound No. 36 926 - main, No. 35763 - side). However, the primary components are depsipeptides. Raw mixtures of antibiotics, obtained directly from the broth, and purified individual components have a wide range of antibacterial properties. The organisms that are not capable of reproduction in the presence of antibiotics include: Salmonella typhasa, Shigella dysenteriae, Escherichia coli, Klebsiella bacterium, Staphylococcus ai.ireus. Streptococcus pyogenes. Streptococcus faecalis, Diplocoocus pneimoniae. Bacillus subtiiis, Corynebacterium diphtheriae, Clostridium septicum, Brucella abortus, Neisseria sioca, Lactobacillus acidophilus ,. Pasteurella multocida. Antibiotics encompassed by the present invention, both in the form of a crude mixture and in the form of purified individual components or their mixtures, can be used in the treatment of various infectious diseases in humans and in animals. As a rule, these antibiotics are administered orally in daily doses of 0.5-1.0 g or parterally in doses of 100-500 mg, depending on the type and severity of the infectious disease and the characteristics of the patient. They can be administered alone or in combination with a pharmaceutically acceptable carrier, and both single and multiple doses can be used for treatment. Tablets containing various additives (sodium citrate, calcium carbonate and dicali iyphosphate), as well as various disintegrants such as starch, alginic acid and some complex silicates, can be used for oral administration, with binders, such as polyvinylpyrrolidone, sucrose, gelatin and gum arabic. In addition, lubricants such as magnesium stearate, sodium laryl sulfate and talc can be added, which often provide advantages when pelletizing. Solids of a similar type can also be used as fillers in soft and hard gelatin capsules. Recommended materials include lactose and high molecular weight polyethylene glycols. When using suspensions and / or elixirs, an important ingredient may

sweetening agents or flavoring agents together with such diluents as water, ethanol, propylene glycol, glycerin, and various combinations of these substances.

Antibiotic solutions in sesame or peanut oil or in aqueous propylene glycol can be used with parenteral administration.

Individual antibiotics exhibit bactericidal activity, which is mainly bacteriostatic. However, crude mixtures of antibiotics or mixtures of purified depsipeptides and purified macrocyclic lactones show a synergistic activity that is essentially bactericidal.

In individual tests of the main macrocyclic lactone — Compound No. 36,926 (A) —and the main depsipeptide — Compound No. 37,277 (B) —or combinations thereof (by diluting in test tubes) against various organisms, minimal inhibitory concentrations were detected. The results are given in table. .

Table 1

Similar results were obtained by individual testing of a side macrocyclicolactone compound

No 35 763 (A) and side depsipeptide - jNb 37 932 compounds (B), as well as when testing various combinations: А-А, ВВ, (А + В), (А + В), (А + В), ( A + B).

The maximum synergistic activity in combinations of purified macrocyclic lactones and deppeptides was achieved at a ratio of about 1-2: 1. Approximately the same ratios were observed for fermentation broths A. auranticolor ATCC 31011 and for selected crude mixtures of antibiotics.

This phenomenon is the opposite of that observed for other synergistic mixtures of antibiotics, for which synergistic factors are observed in fermentation broths and raw mixtures at bead-optimum ratios.

The data obtained in vivo, when administered orally and subcutaneously, when conducting experiments on mice injected with the strain Staphylococcus aiireus 01A005, are presented in Table. 2

The antibiotics encompassed by the present invention can be used as agents to stimulate the growth of poultry and animals due to the high bactericidal spectrum and in the treatment of dysentery in sweat.

due to significant activity against the anaerobic spirochete causing this disease. Raw growth promoting activity

antibiotic mixtures are determined on young pigs for 40 days. The average weight gain per day, feed consumption and efficacy were significantly improved in animals treated with antibiotics compared with the control animals (, 01). The results are shown in Table. 3. Similar results were obtained for other mixtures of antibiotics used in an amount of 10-100 parts per 10 parts of feed.

Similar results were also obtained using pure individual compounds Nos. 36 926, 37 932 and 37 277 or mixtures of pure compounds. Table 3 The efficacy of antibiotic use to stimulate growth was demonstrated in experiments on chickens fed with antibiotics. They noted a significant improvement in weight gain compared with control chickens (, 01). The results are presented in table. 4. Table 4 The same results were obtained when using other crude mixtures of antibiotics, the compositions of which are given below, or using individual pure compounds Nos. 36 926, 37 932 and 37 277 or mixtures of pure compounds. The prophylactic efficacy of the antibiotics covered by the present invention is determined in pigs experimentally infected with dysentery. Feed containing antibiotics is administered within 28 days. The results are presented in Table. 5. Table 5 Similar results were obtained with the introduction (10-100 parts per 10 feed) of pure individual compounds No. 36 926, 5 10 15 20 25 30 35 40 45 50 55 60 65 37 932 AND 37 277 or mixtures of pure compounds Example I. Prepare a sterile aqueous medium composition (g / l): Glucose10 Soluble starch20 Yeast extract5 Enzymatic casein dehydrogenation product 5 Calcium carbonate 1 pH7 The cells from the plate with A. auranticolor ATCC 31011, grown on ATCC 172 medium, are transferred into several 300 m. lilithyr cone Erlenmeyer flasks. each of which contains 50 ml of this medium and which is shaken on a rotating separator for 3-4 days at 28-30 ° C. Aliquots of 5 ml of the grown inoculum are transferred to 300 ml Eolenmeyer conical flasks, each containing 100 ml of the above-described sterile medium. After shaking for 3–4 days at 28–30 ° C, 5–10% of the grown monk was transferred to a four-liter fermentation tank, salt-containing 2 liters of sterile medium of composition (g / l): Yeast extract 2.000 Glucose 10,000 Liquid after corn soaking 1 (ml) Casein enzymatic product dehydrogenation 5.000 Cobalt bivalent chloride 0.002 pH7.0 Fermentation is carried out for 20-30 hours at 28-30 ° C, moving at a speed of 1700 rpm and aeration - 1 volume of air per 1 volume of broth in 1 min. If necessary, the pH of the entire broth is set at 7 and extracted twice with methyl isobutyl ketone (1 / 3-1 / 2 of the volume of the broth). The extract in a solvent is concentrated in vacuo and degreased by extraction with petroleum ether. The broth activity, the solvent extract and the subsequent fractions are monitored by thin layer chromatography on silica gel using an evolving system consisting of chloroform-ethanol (9: 1), and observations are made in the ultraviolet region of the spectrum at a wavelength of 254 mmk and 336 mmk. The fat-free concentrate in a solvent is dried to dryness in vacuo. The solids are counter-current to Craig distribution on six plates using toluene (5 parts), ethanol (2 parts), aqueous phosphate buffer solution (pH 4.5; 3 parts). After distribution, the layers are monitored by thin layer chromography. Depsipeptide (compound No. 37 277) is concentrated in the upper layer, mainly on the zero plate. The second depsipeptide (compound No. 37 932) is isolated from the upper plates — the 1st, 2nd, and 3rd. Macrocyclic lactone (Compound No. 35,763) ishodigs mainly in the lower layers of the 0th and 1st plates. Connection No. 36 926 concentrate in the lower phases of the plates of the 2nd, 3rd, 4th and 5th. The upper phase of the zero plate containing compound No. 37,277 is dried to dryness in vacuo, dissolved in chloroform and stirred for a time with activated carbon. The solution is filtered and dried under vacuum. The residue is dissolved in acetone, solid substances are precipitated, and heptane is added. The precipitated solids are dissolved in a small amount of chloroform and chromatographed on a column of silica gel, buffered to a pH of 6.0, prepared in the presence of a mixture of chloroform with n-propanol (99: 1). The column is developed with the same system at a pressure of 5,600 N / m. Fractions from the column are monitored by thin layer chromatography. The fractions containing the isolated compound No. 37,277 were combined, evaporated in vacuo, and the compound crystallized from acetone-heptane. Compound No. 37,277 does not have a distinct melting point. Decomposition starts at 140-150 ° C. The compound is insoluble in diethyl ether, hexane, heptane and water. It is slightly soluble in acetone and benzene and readily soluble in methanol, ethanol, chloroform and methylene chloride. Analysis of compound No. 37,277 yields the following approximate results (%): C, 60.91; H 5.98; N 10.45; O (by difference) 22.66. Compound No. 37,277 is optically active and has a rotation angle of + 11 ° (, 0 ethanol). The absorption maxima in the ultraviolet region are at wavelengths (mmc): 225, 274, 282, 303 and 355; e}, 309.3; 36.67; 45.01 and 20. Chloroform solution shows a characteristic absorption in the infrared region at the following wavelengths (μm): 3.05; 3.40; 5.70; 5.77; 6.07; 6.62; 6.82 and 7.67. The fractions obtained in countercurrent distribution and containing compounds Nos. 35 763 and 36 926 are evaporated in -vacuum, the residue is dissolved in ethyl acetate, and the solution is stirred with silica gel. The filtered solution is evaporated in vacuo, the residue is dissolved in ethyl acetate, the solids are precipitated by adding hexane. The precipitated solids are dissolved in a small amount of chloroform and chromatographed on a silica gel column, buffered to pH 6.0, prepared in the presence of ethyl acetate. The column was developed with a mixture of ethyl acetate, tetrahydrofuran, and hexane (80:20:20), saturated with an aqueous phosphate buffer solution with a pH of 6.0, under a pressure of 5600 N / m2. The first 17 of the fractions obtained (each with a volume of 20 ml) contain a yellow oil, which is discarded. Fractions 26-40 have a high content of compound No. 36 926. Fractions 41-50 are mainly mixtures of compounds 36 36 and 35 763. Fractions 26-40 are combined, concentrated in vacuo and again chromatographed on silica gel with the selection of fractions according to 20 ml. The first 15 fractions contain yellow oil, which is discarded. Fractions 16-30 are mainly Compound No. 36,926. Fractions 31-40 contain a mixture of compounds No. 36 962 and No. 35 763. Fractions 16-30 are evaporated in vacuo, the residue is dissolved in a small amount of chloroform and introduced into a silica gel column buffered to pH 6.0, made in the presence of chloroform. The column is developed with a mixture of chloroform and ethanol (95.5-4.5% by volume) under a pressure of 9100 N / m and 160 fractions of 6 ml are collected. Fractions 60-90 contain only compound No. 36 926. These fractions are combined and evaporated in vacuo. The residue is dissolved in a minimum amount of ethanol and precipitated with diethyl ether to obtain pure amorphous compound No. 36,926. Compound No. 36,926 is soluble in methanol, ethanol, chloroform and methylene chloride, insoluble in diethyl ether, hexane and heptane. It does not have a distinct melting point, decomposition begins at a temperature of 100 ° C. The analysis established the following average ratios (%): C 57.89; H 6.78; N 8.04; O (by difference) 27.29. The molecular weight, as determined by the mass spectrum with high resolution, is 501, the molecular formula is C2bPz5YzO7. Compound No. 36 926 has optical activity with a rotation angle of about - 130 ° C (, 0, ethanol). The maximum absorption in the ultraviolet region of the spectrum in an ethanol solution is at a wavelength of 214 kM; 723.8. A potassium bromide tablet shows characteristic absorption maxima in the infrared region at the following wavelengths (µm): 2.95; 3.40; 5.75; 5.98; 6.23; 6.58; 6.87; 7.45; 8.25; 8.38; 8.80; 9.08; 10.15; 10.35; 11.10 and 13.30. Compound No. 35,763 is isolated and purified in the same manner as compound No. 36926. The pure compound is soluble in methanol, ethanol, chloroform and methylene chloride, insoluble in diethyl ether, hexane and heptane. It does not have a distinct melting point, decomposition begins at a temperature of 100 ° C. The analysis gives the following average ratios (%): C, 61.29; H 6.73; N 8.83; O (by difference) 23.15. Molecular weight, determined by high resolution mass spectrum, composition