CS199264B2 - Process for preparing streptomycin antibiotic mm 13.902 - Google Patents

Description

The present invention relates to a method of making a streptomycin antibiotic, designated MM 13.902, of the structural formula

HO _s SO

4-C-NH-CO-Cfy COOf-f fr by the chemical designation 3 - [(E) -2-acetarnidoethenylthio] -6- (1-sulfatoethyl) -7-oxo-1-azabicyclo [3.2.0] hept-2- en-2-carboxylic acid, optionally in the form of a salt isolated from some strains of Streptomyces, in particular Streptomyces olivaceus after cultivation. This potent antibiotic, designated MM 13.902, has been found to act synergistically with penicillins and cephalosporins.

British Patent 1,363,075 discloses that a useful β-lactamase inhibitor can be isolated by fermentation of some strains of Streptomyces olivaceus. Until the disclosure of the present invention, it was considered that the material disclosed in British Patent Specification 1,363,075 was substantially pure. However, we have found that this is not the case and that it is possible to isolate from this material a component which is contained in it in a smaller amount and is characterized by a powerful antimicrobial activity. This new fraction is designated MM 13.902 and is now considered to be responsible for the antibacterial efficacy disclosed in British Patent Specification 1,363,075, although on the other hand it is associated with only a very small fraction (3-lactam the inhibitory activity of this material is clear that this application does not speak of anything contrary to the subject-matter of British Patent 1,363,075. Other β-lactamase inhibitors are known to arise from the action of Streptomyces strains, for example, as in the case of strains listed in German patent application 2,340,005, but these materials, otherwise known, have not been found to exhibit potent antibacterial activity, such as the antibiotic MM 13.902.

Antibiotic MM 13.902 is a carboxylic acid of the solid character, characterized in the form of substantially pure sodium salt by the following properties:

very soluble in water, soluble in methanol, substantially insoluble in chloroform, diethyl ether and hydrocarbons, characterized by an ultraviolet absorption spectrum at an absorption maximum over a length of about 305 nm (Figure 1) at a concentration of 0,4% by weight in a freshly prepared potassium bromide disk, it is characterized by a characteristic infrared spectrum with absorption maxima in the wavelength range - about 3450, 2950, 1750, 1620, 1510, 1400 and 1260 cm ¹ (Figure 2), characterized by a characteristic NMR— - the spectrum in D2O, which spectrum is characterized, inter alia, in Figure 3, by a pair of low-pole doublets centered at about 2.85 S and 4.00 µ and coupling constants of approximately 14 Hz;

a doublet centered at approximately 8.55 S and a sharp singlet at about 8.00 δ, further characterized by antibacterial activity against various species, including, but not limited to, strains of Staphylococcuc aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis Salmonella typhi and Pseudomonas aeruginosa, when mixed with ampicillin, show synergistic activity against certain organisms, including Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Proteus morganii and Staphylococcus aureus Russell.

Pure disodium salt MM 13 902 is an inhibitor of δ lactamase, and is characterized by an I 50 (as previously described) of between 0.001 (µg / ml and 0.0001 µg / ml against Escherichia coli B-11 δ-lactamase.

When tested on cellulose using thin layer chromatography, using disodium salt of MM 13902, gave the following additional approximate _Rf values:

n-butanol / isopropanol / water 7: 7: 6 (by volume) R _f = 0.72, isopropyl alcohol / water 7: 3 (by volume), R _£ = 0.85, n-butanol / ethanol / water 7: 7 :

: 6, _Rf = 0.81 n-propanol / water 4: 1, R _f = 0.75.

In another aspect, MM 13.902 can be characterized as a sulfur-containing antibacterial active ingredient, which salts are formed during cultivation of Streptomyces olivaceus ATTC 31126, and solutions in the form of aqueous disodium salt solutions have a maximum absorption in the ultraviolet region of about 305 nm and at 225 nm. 1 as shown in FIG. 1 below.

Otherwise, MM 13.902 can be characterized as an antibacterially active substance which, in the form of substantially pure disodium salt, has an infrared spectrum as depicted in Figure 2 using a freshly prepared pressed potassium bromide disk containing 0.4% by weight of the active ingredient, with NMR The spectrum of this disodium salt is shown in Figure 3 using a freshly prepared solution in heavy water (D2O).

The MM 13.902 material is not very stable in acid form and therefore the preferred embodiment of the process of the present invention is the preparation of MM 13.902 salts. Suitable salts of the antibiotic MM 13.902 include those with 2 equivalents of base, and the most preferred salts are those formed with pharmacologically acceptable ions such as sodium, potassium, calcium, magnesium, aluminum, commonly substituted ammonium ions, and the like. Particularly suitable salts are alkali metal salts such as sodium and potassium salts, for example the disodium or dipotassium salts.

According to the process of the present invention, the antibiotic MM 13.902 and its salts are prepared having a purity of at least 50%, more preferably a purity of at least 70% and preferably a purity of above 80%, for example 90 to 100%.

Most preferably, the active ingredient-containing compositions of the present invention comprise the sodium or potassium salt of the antibiotic MM

13.902, for example, the disodium or dipotassium salt of the antibiotic MM 13.902.

Such formulations may be prepared in a form suitable for oral, topical or parenteral use, for example, tablets, capsules, creams, syrups, powders which can be used for further processing, as well as sterile forms suitable for injection or infusion. These preparations may contain conventional pharmaceutically acceptable excipients such as diluents, binders, colorants, flavors, preservatives, tablet disintegrating agents and the like, always in accordance with conventional pharmaceutical practice and using procedures well known to those skilled in the art of preparation. with antibiotics well known, such as in the case of penicillins or cephalosporins.

The MM 13.902 antibiotic may be the only therapeutically active ingredient in the composition of the present invention, or may be there together with another? -Lactamas antibiotic. Suitable β-lactamase antibiotics include those which are susceptible to β-lactamases and also have some intrinsic resistance to β-lactamases, such as ampicillin, amoxycillin, benzylpenicillin, phenoxymethylpenicillin, propicillin; cephaloridine, cefoxidine, cefalo> , cephalexin, carbenicillin, ticarcillin and esters, hydrolyzable in vivo of the compounds such as phenyl-y, tolyl · or indanyl carbenicillin or tikars cillin, or acetoxymethyl, pivaloyioxymethylestery or phthalidyl ^esters; ampicillin benzylpenioilinu, amoxyeilinu, cephaloridine, cephaloglycin, and the like.

The ratio of the antibiotic MM 13,902 or its salt to the (3-lactam antibiotic) is 0b · ¹ between 10: 1 and 1: 10, for example between 3: 1 and 1: 3.

Total amount of antibacterial-effect? The amount of active agent in any dosage form is usually between 50 and 1500 ^; mg, and is usually 100 to 1000 mg.

Preferred unit dosage formulations of the invention may be administered one or more times per day, for example two to four times per day, for the treatment of diseases of the urinary tract, respiratory tract, soft tissues and the like. Thus, these compositions can be used in the treatment of diseases such as bronchitis, gonorrhea, otitua media, mastitis and the like.

The method for producing the antibiotic MM 13.902 or salts thereof is characterized in that the solution of the MM 4550 complex as further described herein is chromataphically separated into fractions containing essentially the solution of the antibiotic MM 13.902 or the corresponding salt, and further fractions, from which of the corresponding solution isolates the antibiotic MM 13.902 or its corresponding salt.

By designating a fraction containing essentially a solution of the antibiotic MM 13.902 or the corresponding salt, it is meant that either the only antibiotic active material in said solution is the antibiotic MM 13.902 or the corresponding salt, or that other antibiotic active material is present in the solution, but lower amounts compared to MM 13.902 or a salt thereof. Suitably, the antibiotic MM 13.902 or salt thereof represents 70% more preferably 80% and most preferably 90 to 100% of the antibiotic active material contained in said fraction. It has been found that the most suitable method for determining the fractions in which the antibiotic MM 13.902 or its salt is substantially contained is by monitoring the ultraviolet spectrum of each sample. Those fractions that exhibit ultraviolet spectrum, similar to those in Figure 1, generally contain the antibiotic MM 13.902 or a salt thereof, and are substantially free of other antibiotic active agents, such as the antibiotic MM 4550, as further described herein. . Generally speaking, fractions with appreciable ultraviolet absorption at about 305 nm exhibit desirable purity.

Typically, the procedure is used to isolate the antibiotic MM 13.902 in the form of its disodium salt. If a salt with one equivalent base of the antibiotic MM 13.902 or the free acid form of the antibiotic MM 13.902 is required, then it can be prepared by acidifying the biphasic salt of the antibiotic MM 13.902, since generally the monobasic salts of the antibiotic MM 13.902

13.902 as well as the free acid form of the same antibiotic are not stable enough to isolate such compounds from the reaction mixture, as is the case with the MM 4550 complex described below. Thus, the monobasic salts of the antibiotic or the free acid form cannot be readily obtained. poor stability of these substances.

As stated herein, we believe that chromatographic isolation of the antibiotic MM 13.902 is best accomplished using salts of the antibiotic MM 13.902 such as the disodium salt. The salts of the antibiotic MM 13.902 are more soluble in aqueous solvents than in highly lipophilic solvents, and as a result, the use of aqueous solvent systems in the purification chromatography apparatus used in the present invention is preferred.

Electrolyte solutions, buffered to approximately neutrality, have been shown to be suitable, together with polar backing materials such as basic ion exchange resins. Thus, an aqueous sodium chloride solution, buffered to about pH 7 with a conventional buffer, such as a phosphate buffer, may be used together with a carrier material which may contain tertiary amino groups or quaternary ammonium groups. Suitable carrier materials have been found to be hazy ion exchange cellulose derivatives, as well as basic ion exchange crosslinked dextrans, and a particularly preferred carrier material is QAE Sefadex A25, especially when using a solvent system which is a 0.7 M sodium chloride phosphate solution. pH 7 buffer.

Further suitable for use with an inert carrier material such as silica gel or cellulose, a system comprising a mixture of water and an organic solvent / water miscible such as a lower alkanol, and a mixture of water and isopropyl alcohol, especially in the ratio of 3: 7 (isopropyl alcohol: water).

However, the product obtained using the ion exchange resins described above often contains a very high proportion of sodium chloride, so it is advisable to desalinate the collected solutions by passing through a column containing a lipophilic material on which MM 13.902 is adsorbed, not however sodium chloride. Suitable materials for such columns include polystyrenes such as Amber lit XAD 4, otherwise gel filtration using filtering agents such as cross-linked sefadex G 25 dextrans and polyacrylamide gels such as Biogel P2 may be used. The antibiotic can then be eluted from the column using water, aqueous methanol and the like.

After isolation of the expected solutions using the above procedure, the bibasic salt of the antibiotic MM 13.902 can be obtained in solid form by removing the solvent under mild conditions. It has been found that a suitable way to obtain such solids is to distil off the solvent under reduced pressure, followed by lyophilizing the pool of fractions containing the antibiotic MM.

13.902.

If desired, the above process may be carried out in two or more stages. For example, a solution of the MM 4550 complex may be separated into fractions containing the antibiotic MM 13.902 or its salts contaminated with other antibacterial agents up to a content corresponding to MM 13.902, whereupon a material containing, for example, about 50 to 60% from the antibiotic MM 13.902 or a salt thereof, and further chromatographing this material to obtain a fraction containing, for example, 90 to 100 why. antibiotics MM 13.902 or the corresponding salts.

For the purposes of the present invention, the term "kom199264 plex antibiotic MM 4550" is used to describe the material originally designated MM 4550 in British Patent 1,363,075. Repeating the examples in British Patent 1,363,075, it has been found that the MM 4550 complex is a an impure product containing, in varying proportions, the salts of antibiotic MM 4550, as described hereinafter, and salts of antibiotic MM 13.902, as well as substantial amounts of other substances. The MM 4550 complex is not characterized by the characteristic ultraviolet spectrum of the antibiotic MM 13.902. The Iso value, as further described herein, of the MM 4550 antibiotic complex when repeating the procedure from British Patent Specification 1,363,075 is typically less than 0.0004 µg / ml. The ratio of the salts of the antibiotic MM 4550 to the salts of the antibiotic MM 13.902 present in the complex of MM 4550 is, as we believe, very variable, and is believed to depend on factors such as the strain of the organism used and / or the isolation procedure used, but it has usually been found that said complex contains more MM 4550 compared to MM 13.902. The preparation of the MM 4550 complex is further described herein.

In this application, the term "MM 4550" is used to denote a substantially pure substance which is a potent inhibitor of β-lactamase and also has some antibacterial activity. The properties of the antibiotic MM 4550 are further described herein.

In another aspect, the present invention provides a process for the preparation of the antibiotic MM 13.902 and salts thereof, wherein the method is characterized by culturing a strain of Streptomyces producing the antibiotic MM 13.902, then isolating the antibiotic MM 13.902 or a salt thereof from the culture.

It has been found that the strain producing the antibiotic MM 13.902 is Streptomyces olivaceus, and is related to the organism, as will be described hereinafter.

The most suitable organism to be used is Streptomyces olivaceus, for example the ATCC strain 21 379, 21 380, 21 381, 21 382, 31 126 or the corresponding mutants.

A particularly preferred strain for use in the present invention is Streptomyces olivaceus ATCC 31126.

As used herein, the term "cultivation" means performing an aerobic growth of an organism in the presence of an assimilable. sources of carbon, nitrogen, sulfur and inorganic salts. Such aerobic growth may occur on a solid or semi-solid nutrient medium, or on a liquid medium in which the nutrient components are dissolved or suspended. The cultivation may be performed on an aerobic surface or in a submersible culture; the nutrient medium may be composed of a nutrient complex or may be chemically defined · It has been found in our view that nutrient media containing a nutrient complex such as yeast extract, soybean meal and the like are particularly suitable and as we are have found it very advantageous to add cobalt ions, sulfate and calcium carbonate.

It has been found that cultivation at 28 + 2 ° C achieves acceptable antibiotic yields, and can be described as a preferred mash cultivation time 2-3 days after. the start of fermentation.

The term "mutant", as used herein, includes any mutant strain, whether spontaneously generated or due to the action of an external agent, whether or not the agent is used intentionally. Suitable methods for preparing the mutating strains are described Η. I. Adler in "Radiation and Radioisotopes for Industrial Micro-organisms", Proceedings of a Symposium, Vienna 1973, p. 241, International Atomic Energy Authority, article "Techniques for the Development of Micro-organisms". Includes:

1. Ionizing radiation, such as X-rays and gamma rays, ultraviolet light, ultraviolet light together with a photosensitizing agent such as 8-methoxypsoralene, nitrous acid, hydroxylamine, pyrimidine and an analogous base such as 5-bromuracil, acridine compounds, alkylating agents such as mustard, ethyl methanesulfonic acid ester, hydrogen peroxide, phenols, formaldehyde, heating and so on.

2. Genetic procedures such as recombination, transformation, transduction, lysbgenization, lysogenic conversion, and selective procedures for spontaneous mutants.

It has been found that the use of a mutation promoting agent may result in an organism characterized by the ability to produce increased amounts of the desired antibiotics. For example, irradiation of cultures of Streptomyces olivaceus ATCC 21 379 followed by isolation of the resulting strain resulted in the highest bandwidth in the KAGtest, resulting in isolation of the Streptomyces olivaceus ATCC 31126 strain, as is. further described herein, which is the preferred strain for use in the process of the present invention. Strain 31126 is deposited in the Netherlands as CRS 155.75.

Generally speaking, all isolation and purification procedures are performed to produce the desired antibiotic at a non-elevated temperature, for example below 20 ° C and even more preferably below 12 ° C,

The expected product is usually obtained predominantly from the culture filtrate so that the preferred initial step in the isolation process is the removal of solids from the fermentation, for example by filtration.

The impure MM 13.902 antibiotic or salt thereof can be obtained from the clarified culture filtrate by adsorption of the MM 13.902 antibiotic or corresponding salt to an active adsorption material such as activated carbon or the like, followed by eluting the expected substance from the active material using a solvent such as aqueous. acetone. Typically, this procedure is performed using the MM 13.902 antibiotic salt with two equivalents of base.

Alternatively, the impure antibiotic MM 13.902 or salt thereof can be obtained from the culture filtrate by extraction using a lipophilic ammonium salt and a water-immiscible solvent. This process is often more efficient than the one described above. The antibiotic MM 13.902 can be obtained as a substituted ammonium salt by evaporation of the organic solvent under reduced pressure. Preferably, the initial solution of the substituted ammonium salt of the antibiotic MM 13.902 is then extracted back into the aqueous phase using an alkali metal iodide solution such as sodium iodide. The latter process variant usually results in the preparation of an aqueous solution of the impure salt of antibiotic MM 13.902 with two equivalents of base.

Streptromyces olivaceus and related organisms are characterized by the following primary properties:

They have sporulated aerial mycelium in green or yellow series, have sporophone morphology of either textiflexible or spiral type, have spores with a smooth surface and do not produce melanin.

Species with these properties are listed in Tables 5 to 5 below

9.

The impure forms of the antibiotic MM 13.902 or salts thereof, as described above, are usually purified by chromatographic procedures, hereinafter described with a view to preparing materials of satisfactory purity.

In the following description there is general information that can be used to isolate antibacterially active substances. The process of the invention is described in more detail in the examples.

Example 1

Determination of I50

I50 means the amount of material required to inhibit 50% of the hydrolysis of ampicillin by the enzyme (3-lactamase from Escherichia coli Bil, an organism containing the R-factor-controlled iodo-starch reagent E-lactamase from Escherichia coli buffer buffer inhibitor substrate) after incubation of the above mixtures for 15 minutes at 37 ° C

The reactions are monitored by recording the change in optical density at 590 nm and measuring the rate of reaction from the change in optical density after

100 seconds over time intervals of 3 to e (S-lactamase. This β-lactamase is classified as a type IIIa enzyme according to the classification of Rchmond and Sykes, Adv. In Microbiol. Physiol. 9, 31 (1973).

The rate of β-lactamase hydrolysis of ampicillin to penicilic acid can be monitored by an iodometric starch assay, measuring the rate of penicilic acid formation by subsequent decolourisation of the iodine-starch complex.

This procedure as well as the preparation of [beta] -lactamase are described by M. Cole, S. Elson and PD Fullbrook, Biochem. J. 127, 295 (1972). preincubate with enzyme for 15 minutes at 37 ° C before being added to ampicillin as substrate.

Here's how:

Reagents:

Buffer: 0.05 M potassium phosphate buffer solution, iodine starch solution: preparation see Novick,

Biochem J. 83 236 (1962) substrate: ampicillin 40 µg / ml in enzyme buffer (3-lactamase: preparation from Escherichia coli B 11, as described by Cole et al., J. J. 127, 295 (1972). of the enzyme preparation in the buffer was such that a decrease of about 0.3 units of optical density was obtained in an uninhibited reaction per 100 seconds. Other β-lactamase producing strains of Escherichia coli, particularly those characterized by the presence of R- a factor such as R. TEM.

Reaction conditions

Reactions were performed in 1 cm cuvettes at 37 ° C using an SP 800 Pye Unicam spectrophotometer. Four samples with corresponding blank experiments can be used in the instrument. The solid cuvette is used to control the reaction and does not contain an inhibitor, the second, third and fourth cuvettes are used for various dilutions of the inhibitor.

So

Blank sample cuvette

1.0 ml 1.0 ml 0.1 ml - 0.3 ml 0.4 ml 0.1 ml 0.1 ml 1.0 ml 1.0 ml

minutes. The inhibitor sample is diluted until a dilution is achieved that achieves a 50% reaction rate as compared to the control without inhibitor.

i 99 2 64

Example 2

KAG - test

This designation refers to the procedure for determining the presence of the β-lactamase inhibitor in the fermentation broth or during the isolation steps. The molten nutrient agar is inoculated with a suitable [beta] -lactamase producing strain of Klebsiella aerogenes at 45 [deg.] C. and then mixed with a sufficient quantity of sterile penicillin G solution to obtain a concentration of Θ4 g / ml penicillin G in the agar. Pour onto Petri dishes, and after solidification, the cylindrical metal knife is uniformly separated by sterile metal cuts in the agar layer, and the test solutions are cut into sections, and the plate is incubated at a constant temperature between 27 ° C and 37 ° C. the inhibitor in the test solution from the indentation to the agar and there inhibits the action of β-lactamase produced in Klebsellla cells, thus protecting penicillin G from β-lactamase action and thus from decomposition, and is present in concentrations sufficient to prevent growth In those parts of the agar to which the inhibitor did not diffuse at a sufficient concentration, penicillin G upset by the action of β-lactamase, and this allows the development of a dense growth of Klebsiella. Thus, clear circular zones are formed by inhibiting the growth of Klebsiella around the slots containing the Inhibitor, the size of each zone being dependent on the concentration of inhibitor in the test solution. The potency (expressed in units / ml) of the test solution is obtained by reference to the standard fit of the log concentration of the inhibitor concentration over the band average. This test can also be used in bioautography.

Example 3

Preparation of the MM 4550 complex, which can be compared to the product of British Patent Specification 1,363,075.

The Streptomyces olivaceus ATCC 21 370 strain was grown for 7 days at 28 ° C on a solid agar slice in a Roux flask. The agar medium has the following composition:

Quantity component in g / 1 '

Yeast extract 10.0 Glucose monohydrate 10.0 Agar 15.0 Tap water to liter

Yeast extract is identical to 'Yeatéx' and 'Agar', in both cases British products.

The pH of the medium is adjusted to 6.8 prior to sterilization. 50 ml of sterile, deionized water containing 0.02% Tween 80 '(i.e. polyoxyethylene sorbitan monooleyl ester) was added to the culture in a Roux flask, and the spores were suspended by shaking. The resulting spore suspension is added as a loop to a 75 liter sterile inoculation medium in a stainless steel fermenter 100. The composition of the sterile vaccine environment was:

Ingredient Quantity (g / l) soybean meal 10.0 glucose monohydrate 20.0 tap water to liter ¹ (Soybean meal is product At> kasoy 50).

To handle foaming, it is added to the fermentation medium prior to sterilization with 50 ml by volume of a 10% by volume solution of Pluronic L 81 (trademark), a block polymer of ethylene oxide and propylene oxide in soybean oil.

The medium is steam sterilized in a fermenter for 20 minutes at 120 degrees Celsius. The culture from the inoculation stage was stirred at 340 rpm with a 25 cm diameter disc mixer, introducing sterile air at 150 liters per minute. The culture apparatus is equipped with baffles, the temperature is controlled at 28 ° C, and after incubation under these conditions for 45 hours, 7.5 liters of this culture are added as a loop to the 150 L sterile fermentation broth contained in a 300 L stainless steel fermentation apparatus. liters. The fermentation medium has the following composition:

Component Quantity (g / 1) '

Soya bean meal (Arkasoy 50, see above) 10.0 glucose monohydrate 20.0 chalk (precipitated carbonate calcium) 0.2 cobalt chloride hexahydrate th 0.001 tap water make up to liter

To prevent foaming, 300 ml of a 10% solution of Pluronic L 81 in soybean oil is added. The fermentation liquid is clarified by centrifugation after 1 hour and then in the clarified mash. shows 50% inhibition in enzyme assay at a dilution of 1 per 100,000. 12 kg (wet weight) of ion-exchange cellulose in the Whatman DE 32 acetate cycle is a microcrystalline cellulose substituted with diethylaminoethyl groups.

The suspension is filtered and the MM 4550 complex is eluted from the modified cellulose using 12 liters of 0.5 M potassium sulfate solution. The extract is concentrated to a volume of 6 liters using an inclined evaporator from the film layer, using a vacuum and a temperature of not more than 30 ° C. A considerable amount of potassium sulfate precipitated by the addition of 12 liters of acetone, the solution was filtered and then concentrated to a volume of 200 ml under vacuum at a temperature below 30 ° C. The concentrate was applied to a 76 mm column on a 2 m Amberlit XAD-4 cartridge (a non-ionic polystyrene resin) eluting with water, collecting 140 ml fractions. 2.2 liters and concentrated to 275 ml by ultrafiltration using a 150 mm diameter Amicon UM-05 membrane under a nitrogen pressure of 4.2 atom.

2.2 g of brown powder (I50 0.004 .mu.g / ml).

From this powder, 1 g is dissolved in a liter of 0.2 M sodium sulfate solution, and the solution is mixed with a liter of 2% (w / v) acid tetra-n-butylammonium sulfate in dichloromethane. The solution in the solvent was spontaneously separated, cooled to -70 ° C, filtered from ice and concentrated to 20 ml. 400 ml of petroleum ether, b.p. 40-60 ° C, are added to the concentrate and the precipitate is separated by centrifugation. The precipitate is redissolved in 10 ml of dichloromethane, extracted with 10 ml of water containing 80 mg of barium iodide and 70 mg of barium carbonate, the phases are separated, the aqueous phase is filtered and the pH is adjusted to 6.5. Lyophilization of the solution yielded a yellow powder which was washed with acetone to dissolve excess barium iodide, then the pale yellow solid was centrifuged and dried in vacuo. Yield 13 mg. (Ϊ50 = 0.0004 jMg / ml).

Example 4

Evidence of adsorption of the culture filtrate on charcoal, which is useful for obtaining the materials described in British Patent 1 361 075.

The culture filtrate, as obtained from the procedure of Example 3, is characterized by band diameter; 17 mm by agar injection using a diffuse antibacterial test against Klebsiella aerogenes; with a diameter of 38 millimeters in the KAG test and an I50 value of 1 per 100 000. Under cooling to 5 ° C, 170 liters of clarified culture filtrate are percolated through a column of diameter

22.5 cm in which the activated carbon is up to 40 cm (Farnell BO, UK, particle size 60-80 mesh; the carbon is washed using a N hydrochloric acid solution, and buffered to 6 with phosphate buffer). The flow rate is 800 to 1000 milliliters per minute.

The charcoal is then eluted with 10 liters of deionized water, then the column is eluted at 20 ° C with 20% acetone. The active fractions, totaling 10 liters, are concentrated under a vacuum of temperature below 30 ° C to a volume of 6 liters, and lyophilized to give 282 g of crude MM 4550 complex; this preparation is characterized by an IC 50 value of 0.05 µg / ml. Regeneration of inhibitory enzyme activity corresponds to 22%.

Like other activated carbon with similar results, Darcogranular carbon (UK) is used.

Example 5

Demonstration of the usefulness of ion exchange chromatography of the culture filtrate in obtaining the material described in British Patent Specification 1,363,075.

DOWEX 21 K ion exchange resin (particle size 20 to 50 mesh, in chloride cycle) is used as a packer up to 6 cm (bed volume 4.7 ml) in a column with an internal diameter of 1 cm and is a polystyrene-divinylbenzene resin with basic groups ). The resin bed is washed using about 4 aliquots of 4.7 ml of 5% aqueous methanol, once again with 4.7 ml of distilled water, and using 2 liters of culture filtrate essentially as described in description 3; This was applied to the column and the impurities were washed with 50% aqueous methanol.

The MM 4550 complex was eluted from the resin with 5% sodium chloride in 50% aqueous methanol. In Table 1, below, the results are expressed in units of efficiency. The content of the MM 4550 complex in the culture filtrate is 8 units per ml. Fractions eluted from the column are tested using an antibacterial diffusion procedure against Klebsiella aerogenes, and units of activity are calculated using a standardized graph, which is evaluated by plotting the mean band versus units per ml for various dilutions of the culture filtrate (i.e. standard).

It can be seen that the use of the column is an effective way of concentrating the MM 4550 complex. Fractions 2 to 5 contain 60% efficiency in a total volume of only 37 ml. The total dry weight of these fractions is only 210 mg when 35 g of solids are introduced into the column.

Table 1

1B

Results of Dowex 21K chromatography

Sample PH Volume ml units / ml Total units culture filtrate 6.5 1970 8 15 760 percolate 1 6.9 550 under 1 under 100 2 7.0 525 under 1 under 100 3 7.0 595 under 1 under 100 4 7.0 300 under 1 under 100 eluate 1 7.8 7.0 84 588 2 7.8 7.0 328 2296 3 7.7 8.5 440 3740 4 7.7 10.0 320 2200 5 7.6 11.5 160 1840 6 7.5 11.0 80 880 7 7.6 14.2 66 937 8 7.6 20.0 33 660 - total 13 141 Example 6 Example 7 Proof of applicability extraction filtrate kul- Preparation of partially purified complex tury by ion pair at sourcing of materials an antibiotic comprising MM 4550 and MM according to British Patent Specification 1,363,075. 13.902 using acidic tetra-n-butylamo-

niumsulfate.

To 10 liters of clarified culture filtrate prepared as described in description 3, 240 g of sodium sulfate are added, and the solution is extracted for 30 minutes by stirring with 10 liters of a 2% solution of acidic tetra-n-butylammonium sulfate in dichloromethane. After phase separation, the solution in dichloromethane was cooled to 2 ° C, a small amount of suspended water was removed by filtration using Whatman 1 PS filter paper, then the dichloromethane solution was concentrated to 20 ml by evaporation in vacuo at below 30 ° C. Addition of 400 ml of petroleum ether, b.p. 40-60 ° C, precipitates a gum which contains the M 4550 complex.

The gum was separated by centrifugation, redissolved in 50 ml of dichloromethane, and the solution was extracted into 50 ml of water containing 1 g of barium iodide and 1 g of barium carbonate with shaking for 2 minutes. The solids present are filtered off, the two phases are separated, the aqueous portion containing the barium salt MM 4550 complex is acidified to pH 6.5, and then 317 mg of the MM 4550 crude preparation having an I50 = 0.001 [mu] g are lyophilized. ml.

The crude preparation of MM 4550 as obtained in Example 4 is redissolved in water, i.e. 12.5 g in 125 ml of water, and the solution is extracted using 125 ml of a 10% (by weight) solution of acidic tetra-n- butlyammonium sulfate in dichloromethane. The two phases were separated, and the dichloromethane solution was extracted while cooling to 2 ° C using 100 ml of water containing 190 mg of sodium iodide while slowly stirring and adjusting the pH of the aqueous layer to 6.4 by adding 2% sodium bicarbonate solution. The solution was further lyophilized and the solid was washed with acetone to give 88 mg of a mixture of sodium salts MM 4550 and MM 13.902 having a value of 150 = 0.0002 µg against Escherichia coli β-lactamase. Antibiotic regeneration = 70%.

The antibacterial activity of the mixture of amplcillin and the material prepared according to Example 7 is determined by serial dilution on nutrient agar. The minimum inhibitory concentrations as obtained are shown in Table 2.

Table 2

Synergism of ampicillin and the preparation of Example 7

Amplicillin Ampicillin + MM 4550 complex per se 0.1 µg / ml 1 µg / ml 10 µg / ml

Escherichia coli B 11 over 500 over 500 500 50 JT417 250 250 250 50 JT39 over 500 over 500 500 25 + Shigella Sonnei S239 125 125 125 25+ Klebsiella aerogenes 125 25 5+ below 0.1 l ⁺ IP282 50 50 12.5 0,5+ Proteus mirabilis 889 over 500 over 500 50 0.5 247 over 500 125 5.0 0.5 Proteus morganii F 50 50 25 0.5 Proteus rettgeri R110 250 125 25 + 0.25+ Staphylococcus aureus (Russell] 250 125 below 0.1 0.25 Staphylococcus aureus Russell H over 500 below 0.1 below 0.1 below 0.1 ⁺ · Under these concentrations causes himself vany pro mixtures of amoxycillin and complex complex MM 4550 partial inhibition MM 4550.

Similar synergistic effects were observed in Example 8

Preparation of a partially purified antibiotic complex containing MM 4550 and MM 13.902 using cetyldimethylbenzylammonium chloride.

The lyophilized product with an I50 = 0.02 µg / ml, eluted with acetone from a Farnell charcoal column as described in description 4, is dissolved in distilled water to a concentration of 13 mg / ml. The pH of the solution was adjusted to 6.5.

100 ml of this solution is extracted with an equal volume of a 0.1% solution of cetyl dimethyl benzyl ammonium chloride in dichloromethane, the phases are separated by centrifugation, and the organic phase is extracted back into 100 ml of a 0.05% sodium iodide solution. The phases are again separated, and any portion of dichloromethane in the separated aqueous phase is removed by keeping the aqueous solution under reduced pressure for 10 minutes.

The aqueous solution was lyophilized, the lyophilized product was washed three times with 50 ml of acetone each, after which the product was washed with acetone and dried in a vacuum dessicator to give a light brown powder, which was obtained in a yield of 4.3 mg; I50 = 0.02 .mu.g / ml.

Example 9

Preparation of partially purified antibiotic complex containing MM 4550 and MM 13.902 using gel filtration.

g of the crude MM 4550 complex of 150 = 0.2, prepared according to the procedure of Example 4, is chromatographed on Sepefex G25 (fine-grained) using a mixture of acetone and water (4: 6 by volume) to elute. The dimensions of the column are 2.5 cm by 32 cm, and elution is carried out with flow

1.5 ml per minute. The active fractions were combined, concentrated in vacuo at a temperature below 30 ° C, and lyophilized to give 22 mg of an amorphous, slightly brown solid with an I50 = 0.005 µg / ml. Regeneration corresponds to 88%, cleaning is 40 times. Example 10

Preparation of purified antibiotic complex containing MM 4550 and MM 13.902 using cellulose chromatography.

The MM 4550 antibiotic complex, weighing 610 mg, is prepared as described in Preparation 7 and chromatographed on a 2.5 cm by 32 cm cellulose column (Whatman CC 31) followed by elution with a 7: 3 mixture of isopropyl alcohol and water. at a flow rate of 1.5 ml per minute. Fractions with antibacterial activity were combined, concentrated in vacuo at below 30 ° C, and lyophilized to give a brown amorphous solid of antibiotic complex of 40 mg, 0507 µg / ml, in a yield of 40 mg. A very low I50 value indicates that an active material with improved purity was obtained.

On various occasions, a material with an IC 50 of 0.001 µg / ml was obtained using the above procedure. This material is characterized by the antibacterial activity shown in Table 3 below, as determined by a standard microtitration method with a sensitive mash using light stitches (1% of mash, overnight).

T a b u 1 k and 3

Antibacterial activity of a mixture of antibiotics MM 4550 and MM 13.902 with an Iso value of 0.001 µg / ml

Organism Minimum inhibitory concentration in µg / ml

Staphylococcus aureus (Oxford) 7.5 (Russell) 7.5

Streptococcus faeoalis 125

Bacillus subtilis 0.9

Escherichia coli (10418) 3.7 (bil) 15

Klebsiella aerogenes 1.8

Enterobacter cloacae 62

Proteus mirabilis 7.5

Providentia stuartii 7.5

Acinetobacter anitratus 0.9

Pseudotaonas aeruginosa 250

Serratia marcescens 7.5

Salmonella typhimurium 15

Shigella sonnei 7.5

Example 11

MM 4550

Antibiotic MM 4550 has the following characteristics: it is an acid-like solid and has the following properties in the form of the sodium salt:

1. The salt is very soluble in water, soluble in methanol, substantially insoluble in chloroform, diethyl ether and hydrocarbons.

2. In aqueous solution it is characterized by characteristic ultraviolet light with absorption maxima at about 238 nm and 287 nm.

3. The concentration by weight of 0.4% freshly prepared pressed disk of potassium bromide having characteristic infrared spectrum with absorption maxima inter alia at 3450, 2950, 1765, 1695, 1510, 1390 and. 1260 cm ^1st If another spectrum is measured about one week after the preparation of a compressed potassium bromide tablet, substantial changes in the spectrum appear, for example, to the extent that the broad and massive peak at 1765 cm < ^{1 &gt};

4. The salt has a characteristic NMR spectrum in heavy water; this spectrum is characterized, inter alia, by a pair of low-field doublets centered at approximately 2.45 τ and 3.65 τ with coupling constants of approximately 15 Hz; with a doublet centered at approximately 8.55 τ and a sharp singlet at approximately 7.95 τ.

5. When thin layer chromatography on cellulose were found these _Rf values in the following systems:

butanol / isopropanol / water eluant 7: 7: 6 R - 0.6 isopropyl alcohol / water 7: 3 v, R _f = 0.7, n-butanol / ethanol / water as the eluant 7: 7: 6; R _f = 0.7 and n-propanol / water as the eluant 4: 1, R _f = 0.6.

6. It has antibacterial activity against various species of microorganisms, inter alia against the strains Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Acinetobacter anitratus, Serratia marcescens and Shigella sonnei.

7. It is characterized by enzymatic inhibitory activity against [beta] -lactamasams originating in various species of microorganisms, inter alia in Escherichia coli, Klebsiella aerogenes and Staphylococcus aureus, with an Iso value (as defined below) of less than 0.0001 (tg / ml against β-lactamase from Escherichia coli Bil.

8. When mixed with ampicillin, synergism of action against certain microorganisms, including strains of Escherichia coli, Klebsiella aerogenes, Proteus mirabllis, Proteus morganii and Staphylococcus aureus Russell.

9. Amino acid analysis shows that said material is not a polypeptide or protein; N-aminoadipic acid was not detected after hydrolysis under acidic conditions;

10. React with Ehrlich's reagent (300 mg of p-dimethylaminobenzaldehyde is dissolved in a mixture of 54 ml of n-butyl alcohol, 9 ml of ethyl alcohol and 9 ml of concentrated hydrochloric acid) to give a blue color on paper after chromatography or on a chromatography plate thin layer.

11. The substance is generally not an enzyme poison and does not inhibit the following enzymes at concentrations higher than those required to inhibit (Escherichia coli 3-lactamase: monoamine oxidase, carbon dioxide anhydrase, dopa-decarboxylase, trypsin, chymotrypsin or urease.

Example 12

Preparation of complex MM 4550, and division of this complex into antibiotics MM 4550 and MM 13.902.

The isolation procedures, which have been described in detail in the description section, can also be used here with numerous variations in the sequence of sub-procedures. Particularly suitable sequencing of the processes is charcoal chromatography, paired ion extraction, and cellulose chromatography as further described herein:

The 340 L culture filtrate as obtained in Example 3 was percolated upward at a flow rate of 800 to 1200 ml per minute through a 22.5 cm diameter and 53 cm high column packed with Farnell BO activated carbon. This coal was previously used to adsorb the MM 4550 complex, and was regenerated by washing using the following components:

0.5 N sodium hydroxide solution, mixture of 0.2 N sodium hydroxide and acetone solution (3: 2), water, N hydrochloric acid solution, water, pH 6 phosphate buffer, and water.

Washing the column with water (20 L) washes the culture filtrate, then elutes with a flow of acetone / water (2: 8) downwards at a flow rate of 200 to 250 ml per minute. Fractions of 1 liter are collected. The fractions containing the MM 4550 complex were detected by an antibacterial diffusion test using Klebsiella aerogenes, pooled (11 L total), then concentrated under vacuum at a temperature below 30 ° C to a volume of 5.5 L. The recovery of the MM 4550 complex at this stage corresponds to 20%.

The concentrate was cooled to 2 ° C and added

5.5 L of a 2% (w / v) solution of acidic tetra-n-butylammonium sulfate in dichloromethane sub-cooled to -5 ° C, and the two phases are stirred for 2 minutes. Thereafter, the dichloromethane solution is separated and cooled to 0 ^C C is added to a liter of 0.6% sodium iodide solution. The two phases are mixed gently, the pH of the aqueous phase being gradually adjusted to 6.5-7.0 by the addition of a 2% aqueous solution of sodium bicarbonate. The aqueous phase is lyophilized after separation, the dried solid is extracted with dry acetone, which dissolves excess sodium iodide, the acetone is removed from the insoluble matter in vacuo, and 1.1 g of a brownish-colored powder are obtained as a residue. The overall recovery yield of MM 4550 at this stage is 10%. The purification, calculated on the total amount of dissolved solids in the culture filtrate, is 100-fold.

A 2.5 cm diameter column is packed with powdered cellulose (Whatman CC 31} in isopropanol / water (7: 3, v / v) to a height of 32 cm. Separately dissolve 500 mg of the brownish powder in 2 ml of isopropanol / water (7). Chromatography, using the same solvent and a flow rate of 1.5 ml per minute, fractions of 3 ml are collected from the column and combined with those characterized by an absorption maximum at 285 nm in the ultraviolet spectrum. Concentration and lyophilization yielded the MM 4550 complex. It is apparent from previous experiments that fractions characterized by absorption at 285 nm contain antibacterially active enzyme inhibiting material.

The yield of the lyophilized MM 4550 complex was 35 mg. The substance is amorphous, brown in color, and is characterized by an IC 50 value of 0.0001 µg / ml. The regeneration of the active material in this stage corresponds to a total of 3 why, the degree of purification is six times.

The antibacterial efficacy of this preparation is determined by a standard microtiter procedure, namely a muzzle sensitivity test and the use of a light eye. The minimum inhibitory concentration values are similar to those previously reported in Table 3.

By analysis of the preparation by thin-layer chromatography ("Chromatogram"), developing n-butyl alcohol, isopropyl alcohol, water (7: 7: 6), two active substances were found, one of R _f = 0.58, which was labeled MM 4550, and the second with a value _of R = 0.72 which was designated MM 13902. Both can be detected bioautographically using a variety of microorganisms including Bacillus subtilis, Staphylococcus aureus (Oxford), Staphylococcus aureus (penicillin resistant), Escherichia coli (penicillin-sensitive and resistant to Penicillin J, Salmonella typhimusus moristeus Pris.

In another experiment, the two substances were separated on a thin cellulose layer to produce isopropanol and water (8: 2), followed by extraction from the cellulose with phosphate buffer. Each extract was tested for inhibition, δ-lactamase, and both were found to inhibit Escherichia coli β-lactamase. Both substances also exhibit synergism with benzylpenicillin against Klebsiella aerogenes.

Table 4

Antibacterial efficacy of MM 4550 and MM winding with butyl alcohol / isopropyl alcohol system 13.902 as determined by bioautography (water / chlorine, 7: 7: 6 by volume) cellulose thin-layer mathography for organo-biography

Band in mm MM 4550, Band in mm MM 13.902

_Rf = 0.58 Rf = 0.70

Klebsiella aerogenes 20.0 20.0 Prouteus mirabilis 17.5 13.5 Proteus morganii 16.0 9.5 Salmonella typhi 19.5 21.0 Escherichia coli 10418 20.0 13.5 Bil 17.5 10.5 Enterobacter aerogenes 6.5 without band Staphylococcus aureus 13.0 4.0 (Russell) 12.0 4.0 Bacillus subtilis 24.0 15.0

Example 13

Preparation of complex MM 4550 and division of this complex into MM 4550 and MM 13.902.

1100 liters of culture filtrate from Streptocuccus olivaceus ATCC 31126 fermentation was percolated at a flow rate of 3.2 liters per minute at a flow rate of 3.2 liters per minute using a Darco granular activated carbon bed at pH 6.5 and 5 ° C. This coal has previously been used to adsorb the MM 4550 complex and has been regenerated for further use by washing with the following reagents:

0.5 sodium hydroxide solution, mixture of 0.2 N sodium hydroxide and acetone solution (3: 2), water, N hydrochloric acid solution, water, phosphate buffer pH 6, water.

Washing the column with water (60 liters) was washed out with the culture filtrate, and the column is eluted at 30 ⁰¹ C a mixture of acetone and water (8: 2 by volume) at a flow of 1.1 liters per minute. Collect 60 liters and collect 5 fractions of 10 liters each. Those fractions containing the antibiotic MM 4550 complex as assessed by the Klebsiella aerogenes antibacterial diffusion test are pooled, and the total volume of 40 liters is concentrated under vacuum at a temperature below 30 ° C to a volume of 32 liters. The recovery of MM 4550 (as complex) at this stage corresponds to 15%.

The concentrate is cooled to 5 DEG C., 16 liters of a 0.2% solution of cetyl dimethyl benzylammonium chloride in dichloromethane, also subcooled to 5 DEG C., are added, and the two phases are stirred for 5 minutes. The dichloromethane solution is separated, added at 5 ° C

3.75 liters of 0.4% sodium iodide solution, the two phases are again stirred for 5 minutes, after which the aqueous phase is separated and lyophilized. The solid was freed of excess sodium iodide by extraction with acetone and then drying of the solid residue insoluble in acetone gave 2.87 g of a brownish-colored powder. The overall yield of MM 4550 at this stage was 6%. The purification, calculated on all solid solutes in the culture filtrate, is sixty-six times.

The column with a diameter of 63 mm is packed with powdered cellulose (Whatman CC 31) in a mixture of isopropyl alcohol and water (7: 3, by volume) to a height of 300 mm. Dissolve 2.0 g of the brownish product in 5 ml of a 7: 3 mixture of isopropanol and water and chromatograph at a flow rate of 3 ml per minute. The fractions containing the MM 4550 complex as determined by the Klebsiella aerogenes assay were combined, concentrated by evaporation in vacuo at a temperature below 30 ° C, and then lyophilized to give 207 mg of a brown solid. The recovery of the MM 4550 complex at this stage corresponds to 51%, the degree of purification being five times.

The 16 mm diameter column was packed with pulverized cellulose (Whatman CC 31) in n-propyl alcohol / water (4: 1, v / v) to a height of 300 mm and 198 mg of the brown product was dissolved in water / n-propyl alcohol (1). 1), and chromatographed using n-propyl alcohol-water (4: 1, v / v) at a flow rate of 0.5 mL per minute, collecting 6 mL fractions, which are bioassayed on Klebsiella aerogenes, and for each fraction. Fractions 23 to 28, characterized by a maximum at 305 nm, contain the disodium salt MM 13.902, the fractions are combined, the solution is concentrated in vacuo, and the residue is freeze dried to give 30 mg of a solid. It has an R _{f of} 0.77 when thin-layer chromatography on cellulose in n-propyl alcohol-water (4: 1, v / v) .The zone can be detected by bioautography using Bacillus subtilis Fractions 29 to 40 with maxi UV absorption at 285 nm, containing MM 4550. Combined, concentrated in vacuo, and lyophilized to give 53 mg of a solid containing MM 4550 salt contaminated with a small amount of MM 13.902 salt.

Example 14

Organisms

The properties to produce MM 4550 were first detected in Streptomyces olivaceus cultures ATCC 21 379, ATCC 21 380, ATCC 21 381, and ATCC 21 382, which were isolated from soil samples from Spain, New Zealand, South Africa and Israel in each case. In a laboratory investigation, these cultures were found to be identical, and all were identified as Streptomyces olivaceus in the National Colleetion of Industrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, using the commonly used classification Hutter (1967, Systematic der Streptomyceten, S. Karger, Basel, 382 pages). Since then, the production of MM 4550 in other streptomyces has been observed, as shown in Table 4.

S. olivaceus was first described by Waksman (1919, Cultural Studies of Species of Actinomyces, Soil Sci., 871-215). Later in 1960, a group of Soviet workers described species with very similar characteristics, but under the name Actinomyces (Streptomyces) fulvoviridis, see Kuchayeva et al., Trud. Inst. Microbiol., Akad. Nauk USSR 8,226 (1960).

Microorganisms of the genus Streptomyces are extremely variable in terms of morphology and physiology, depending on the cultivation conditions. Numerous species have been described before this extraordinary variability has been identified, resulting in duplications and synonymous designations. In 1967, Hutter lists 25 species as synonyms of S. olivaceus, including S. fulvoviridis. To address this confusion in nomenclature and classification, the International Streptomyces Project began in 1962, see Shirling et al., Int. J. Syst. Bacteriol. 18, 69 (1968). The collaborators of this task conducted a series of studies and contributed to an accurate description of Streptomyces species under standardized conditions. Thus, descriptions of strains of 400 named species have been made, including Streptomyces olivaceus and Streptomyces fulvoviridis.

I. S. P. description Streptomyces olivaceus and S. fulvoviridis differ only in two characteristics:

(i) the form of sporophores; and (ii) the ability to use inositol as the sole carbon source.

Streptomyces olivaceus is described as follows:

Morphology of spore chains: spiral section with open spirals, permeating flexible spore chains, ie rectiflexible section. Chains of mature spores are usually long, often with more than 50 spores per string. Utilization of carbon sources: D-glucose, L-arabinose, D-xylose, i-inositol, D-mannitol, D-frucose and rhamnose are useful; no growth on sucrose and raffinose was observed.

Streptomyces fulvoviridis is described as follows:

Morphology of spore chains: rectiflexible section, mature spores in medium-short chains, 10 to 50 spores per chain.

Utilization of carbon sources: D-glucose, L-arabinose, D-xylose, D-mannitol, D-fructose and rhamnose are useful. No growth was observed, or only traces on i-inositol and raffinose.

The characterization of numerous cultures, named S. olivaceus or S. fulvoviridis, or synonymous as well as other species, was performed using standardized procedures and media (Shirling et al., Int. J. Syst. Bacteriol 16, 313 (1966)). as recommended by the International Streptomyces Project (ISP).

Cultures were from different sources. ATCC 21 379, 21 380, 21 381, 21 382 were isolated from the soil by production of the MM 4550 complex. Other strains were obtained from different culture collections for comparison purposes.

The results of the MM 4550 complex production, air mycelium color, sporophor shape, mycelium substrate color, soluble pigment production, melanin production and carbon utilization are shown in Tables 4 to 8. For all strains, the electron microscopic surface spores are smooth.

It is clear from the description of the ISP that the spiral growth of sporophores in Streptomyces olivaceus is variable in nature. Right spirals were observed with variable frequency in S. olivaceus species, such as ATCC 3335. Longer forms of sporophores. In ATCC cultures 21,379, 21,380, 21,381 or 21,382, no true spirals were observed, although sporophores were long, and tended to form spirals between the rectiflexible-type backgrounds. However, in two cases

S. olivaceus, NCIB 8238, and NCIB 8509 were the major part of the rectiflexible-type sporophores. In the case of NCIB 8238, they were of medium length while in the case of NCIB 8509 they were long.

The sporophores observed in ATCC 15,863, a S. fulvoviridis species, were shorter than those of isolated portions of ATCC 21,379, 21,380, 21,381 and 21,382 and 31126, and were of the exclusively rectiflexible type. In view of this, the isolated ATCC shares 21 379, 21 380, 21 381, 21 382 and 31126 correspond quite well, but not exactly, to the description of S. olivaceus.

The isolated proportions of ATCC 21 379, 21 380, 21 381 and 21 382 and 31126 both exhibit some variability with respect to the usefulness of inositol as a carbon source, another differentiating character according to the ISP species type description between S. olivaceus and S. fulvoviridis (Table 8 ). ATCC 31126 and ATCC 21 380 may utilize inositol, which is characteristic of S. olivaceus. However, this is generally not considered to be satisfactory because of the ability to utilize only one of the sugars. Such a difference can occur by mutating a single gene, and is often believed to be characteristic only of that or one strain. The difference in the ability to utilize sugars from Streptomyces olivaceus strains NCIB 8138, NCIB 8509, ATCC 21 549, ATCC 12 019 and ATCC 3335 gives reason to consider why many do not consider this to be a significant character of Species. Thus, ATCC 21 379, 21 380, 21 381 and 21382 and 31 126 are correctly referred to as Streptomyces olivaceus.

If further studies fail to arrive at: substantial and important differences between the cultures, which are simultaneously named S. olivaceus and S. fulvoviridis, they may be identified as a single species internationally. In such a case, the correct designation in terms of the priority rule would be Streptomyces olivaceus, including the cultures cited by Hutter as synonymous with S. olivaceus.

Examination of the filtrate of cultures of Streptomyccs' olivaceus strains and related species in the production of MM 4550 (complex) can be carried out as follows:

^with filtrates from cultures of said strains were applied as track on chromatography paper Whatman no. 1, at a dose of 20 μΐ with respect to the source and the cold overnight, followed by chromatography eluting with n-butanol-isopropanol-water (ratio 7 · 7: 6). The next series of paper chromatography is run in the cold overnight in n-butyl alcohol / glacial acetic acid / water (12: 3: 5), and a partially purified sample of the antibiotic MM 4550 complex is also run on both systems at the same time as the test. substance.

Alternatively, 25 ml of clarified mash can be extracted using 12.5 ml of a 0.2% solution of cetylbenzylammonium chloride in dichloromethane, the phases are separated, the organic phase is delayed, mixed with 2.5 ml of a 0.5% sodium iodide solution, and after shaking, the phases are separated, the aqueous phase is delayed, and used chromatographically, for example by applying 5µ to thin-layer chromatography bands using n-butyl alcohol / isopropyl alcohol / water 7: 7: 6. Table 5

The ability of cultures of Streptomyces olivaceus, Streptomyces fulvoviridis and similar species to produce MM 4550 (as complex) Culture Complex production

MM 4550

Streptomyces olivaceus

ATCC 21,379 +

ATCC 31126 +

ATCC 21380 +

ATCC 21381 +

ATCC 21,382 +

NCIB 8238 +

NCIB 8509 +

Streptomyces flavovirens

ATCC 3320 +

Streptomyces flavus

ATCC 3320 +

Streptomyces fulvoviridis

ATCC 15,863 +

Streptomyces argenteolus

ATCC 11009 +

Streptomyces sioyaensis

ATCC 13,989 +

Streptomyces lipmanii

NRLL 3584 + (Streptomyces olivaceus ATCC 21 549, ATCC 12 019 and ATCC 3335 do not produce MM 4550 complex; ATCC 15 863 is also in the depository as RIA 660).

1R92B4

Table 6

Streptomyces olivaceus, Streptomyces fulvoviridis and similar species, color and morphology of mature sporulating aerial mycelils on ISP after 14 days growth

Culture A B C D Morphology of sporophores and size

ATCC 21 379 grey grey 31126 gray / brown gray / brown 21 380 grey grey 21 381 grey grey 21 382 gray / white grey NCIB 8 238 gray / white grey 8 509 grey grey ATCC 21 549 grey grey 12 019 grey grey 3 335 grey grey 15 863 gray / white grey 3 320 gray / blue grey 3 369 grey grey 11 009 pale gray gray / brown 13 898 white / gray white

Explanatory notes:

A agar with starch and inorganic salts (ISP environment 4)

B malt and yeast extract agar (ISP environment 2)

C agar with glycerin and asparagine (ISP environment 5)

gray / white grey long, rf grey grey long, rf grey grey long, rf pale gray gray / white long, rf grey grey long, rf grey grey Medium, RF grey grey long, rf grey grey long, p grey grey long, rf / sec grey grey long, rf / sec gray / white grey medium, rf / s gray / green grey long, rf / sec grey pale gray / brown medium, rf / ra gray / white gray / green medium, rf / ra white / yellow gray / white short, p D agar with oatmeal ISP environment

3) rf rectiflexible ra retinaculiaperti with spirals

Table 7

Streptomyces olivaceus, Streptomyces fulvoviridis and similar species, the color of the mycelium in the reverse side substrate.

Culture . AND (B) C D ATCC 21,379 olive olive brown gray / brown olive green 31 126 olive brown olive brown Brown olive brown 21 380 olive olive green olive brown olive yellow 21 381 dark brown dark brown ' olive green olive green 21 382 olive olive Brown olive green NCIB 8,238 Brown olive / brown Brown olive yellow 8 509 Brown olive olive brown olive yellow ATCC 21549 brown / black brown / black brown / black brown / gray 12 019 Brown Brown Brown Brown 3 335 Brown Brown Brown grey 16 863 brown / gray dark brown olive brown olive yellow 3 320 yellow / brown olive / brown olive / brown orange / brown 3 369 brown / gray yellow-gray brown / green yellow 11009 black black / yellow black / yellow gray / green ATCC 13,989 Orange yellow yellow without color

For explanations, see Table 6

Table 8

Streptomyces olivaceus, Streptomyces ulvoviridis and related species: pigment production in culture.

Culture soluble AND nemelanoidní pigments D Formation of melanin ⁺⁾ (B) C ATCC 21379 - + (slightly yellow) - 31 126 - - - .—. __ 21 380 - - - __ _ 21 381 - - - -  _ 21 382 - - - - _ NCIB 8,238 + (slightly brown) - + (slightly brown) + (slightly brown) - 8 509 - - - + (light yellow) - ATCC 21,549 + (light brown) + (light red / brown) + (light brown) 12 019 -. - - - _ 3 335 - - - .— _ 15 863 - - - - -, 3 320 + (faint pink) - - - ATCC 3,369 - - - - - 11 009 - + (slightly brown) - - 13 989 + (slightly orange) - Explanatory Notes A, I 3, C and D: see Table 6 ⁺⁾ tested by nothing and on iron agar (: peptone, kvas1SP 6), agar s + = pigment is formed tyrosine (ISP 17), and on the muzzle with tryp- - = pigment not produced and yeast (ISP 1).

Table 9

Streptomyces olivaceus, Streptomyces fulvoviridis and related species, carbon utilization tests.

Culture rh raf chess

ATCC 21379 + - - 31 126 + - - 21 380 + - - 21 381 + -. - 21 382 + 4- - NCIB 8,238 + - —- 8 509 + ± - ATCC 21549 - -. - 12 019 + and + ATCC 3,335 4- - ί 15 863 + - -. 3 320 4- ' - + 3 369 + - ± 11 009 4 ~ - - 13 989 + +

1 + 1 + source is utilized source not utilized utilization is doubtful or very poor

ino glu xy fru Man ara 4- 4- 4- 4- + + 4- + 4 ~ + 4- + 4- 4- + 4- 4- 4- - 4- + 4- + - + 4- 4- 4- 4- - 4- + 4- 4- 4- - 4- + 4- + 4- 4- + + 4- + + 4- + + + + + + + 4 ~ 4_ + 4- - 4- 4- + 4- 4- - + + + 4- + + 4- + 4 ~ + + + + 4- + 4- + + • 4- 4 ~ 4- 4- - Explanatory notes: rh = rhamnosa, raf = raffinosa, chess - = sucrose, nec - inositol, glu = = glucó- for, xy = xylose, fru - fructose, man -

= mannitol, ara = arabinose.

Example 15

Preferred isolation procedure for MM 13.902 preparation.

The stock of Streptomyces olivaceus ATCC 31126 spores is maintained by storing in test tubes in dry soil in a sealed container with desiccant at 20 ° C. A small amount of storage medium (about 20 mg) is transferred aseptically to a 500 ml Erlenmeyer flask containing 100 ml of this medium component amount (g per liter) glucose monohydrate 20.0 soybean meal 10.0 ions dehydrated make up to liter

The pH is adjusted to 6.5 before sterilization. As soya bean meal, 'Arkasoy 50' is used.

The flask was incubated for 30 hours at 28 ° C on a rotary shaker (240 rpm). They are then used in the resulting vegetative growth of 2 ml as an eyelet on a solid agar slice in a Boux flask. Agar has the following composition:

ingredient quantity vegetable juice (V — 8) 20,0 ml agar 20,8 g ions dehydrated make up to liter

The pH is adjusted to 6.0 before sterilization. - Λ

The loop is spread over the agar surface by tilting the flask, which is then incubated at 30 ° C in a vertical position. After a two-day incubation, the excess liquid is withdrawn from the flask by pipette, and incubation is continued for an additional 4 days.

It has previously been found that the development of actinophage in sectioned culture is suppressed when used in a Roux culture flask.

50 ml of sterile, deionized water is added to the culture in a Roux flask, and the spores are suspended by shaking. And this spore suspension is then added as a loop to a 75 liter sterile inoculation medium in a 100 liter stainless steel fermenter. The composition of the medium is as follows: quantity component (g per liter) soya bean meal (Arkasoy 50) 10,0 glucose monohydrate 20,0 tap water make up to liter

To control foaming, 50 ml of a 10% solution of "Pluronic L 81" in soybean oil is added to the fermentation medium prior to sterilization.

The medium is steam sterilized in a fermenter at 120 ° C for 20 minutes. The culture is agitated at 140 rpm with a 18 cm disc mixer, and 75 liters of sterile air are allowed per minute through the feed tube.

The temperature is maintained at 28 ° C, and after incubation for 48 hours under these conditions, the contents of the vessel are added as a loop to 1500 liters of sterile fermentation broth in a 2000 liters stainless steel fermenter. The fermentation medium has the following composition:

component of quantity (g per liter) soya bean meal (Arkasoy 50) 10,0 glucose monohydrate 20,0 precipitated calcium carbonate 0,2 cobalt chloride hexahydrate 0,001 anhydrous sulphate, sodium 1,0 tap water make up to 1500 liters

Before sterilization, the pH is adjusted to 6.0 by addition of sodium hydroxide, and 3 liters of a 10% solution of "Pluronic L 81" in soybean oil are also added to prevent foaming. After sterilization, the pH is readjusted to a value of 7.0 with sterile sodium hydroxide solution, and the fermentation broth is stirred at 106 rpm, with two mixing discs of 48 cm diameter placed on the agitator shaft. The temperature is maintained at 30 ° C and the air intake is 1200 liters per minute. The fermentation is stopped after 60 hours, and the mash is clarified by centrifugation.

The resulting material has an activity of 340 units per ml. The assays are performed as described in description 2.

1050 liters of culture filtrate (340 units per ml) are extracted at 10 ° C and pH 8 using 310 liters of dichloromethane also cooled to 10 ° C containing 1200 g of cetyldimethylbenzylammonium chloride by pumping both liquids at a temperature of 10 ° C. the prescribed flow rates to the mixer. The phases are separated in a continuous centrifuge after mixing for about 2 minutes. 300 L of dichloromethane solution was extracted back with aqueous sodium iodide solution. This extraction was carried out in 4 batches using a total of 7 liters of water containing 210 g of sodium iodide. The phases are separated spontaneously, the pH of the aqueous phase is adjusted from 7.7 to 7.0 by the addition of hydrochloric acid, and after filtration, 7 liters of extract containing 21 900 units / ml in sodium iodide solution are obtained.

An ion exchange column was prepared containing Sephadex A 25 in 0.05 phosphate buffer (pH 7) containing 0.3 M sodium chloride in a 10 cm diameter glass column to a height of 40 cm. 7 liters of sodium iodide extract is percolated at 5 degrees Celsius with said sephade193216 4 times at a flow rate of 50 ml per minute. The column is eluted with a 0.7 M sodium chloride solution in 0.05 M phosphate buffer at pH 7 also at 5 ° C and at a flow rate of 25 ml per minute. From the eluate, set aside 2 liters (not required) and then collect 90 fractions of 100 ml each. The fractions are monitored by ultraviolet spectra, and those fractions which have an absorption maximum at 305 nm are pooled (fractions 50 to 62, total volume 1440 ml, 71,000 units per ml) and their pH is adjusted to 7. It was already here previously shown that the fractions with maximum absorption in this region contain MM 13.902.

To the total fraction of the above fractions, sodium chloride was added at a rate of 5 g per 100 ml, after which the combined fractions were percolated at 5 ° C with a diameter column

6.3 cm with Amberlit XAD 4 resin (Rohm & Haas Ltd). The packing in the column is up to 30 cm, flow rate 20 ml per minute. MM 13.902 adsorbs on resin under these conditions, inorganic impurities do not. MM 13.902 was eluted at room temperature with 200 ml of distilled water and then with 50% aqueous methanol. The eluent was concentrated to below 70 ° C under reduced pressure at 70 ° C, the pH of the residue was adjusted to 7, and lyophilized to give 1.62 g of a brown residue, a partially purified, MM 13.902 salt of 37,000 activity. units per mg.

1.0 g of partially purified antibiotic MM 13.902 disodium salt (37,000 units per mgj is chromatographed on a cellulose column (Whatman CG 31), 3.8 cm by 30 cm, eluting with n-propyl alcohol / water (4: 1, v / v) 170 ml of eluate is not needed and then 100 fractions of 15 ml each are collected The fractions containing the disodium salt MM 13.902 (No. 37 to -43) as assessed by ultraviolet absorption are The mixture is combined to a total volume of 89 ml, n-propyl alcohol is distilled off by evaporating to a temperature below 30 ° C under reduced pressure, and lyophilizing to give 219 mg of a yellowish powder having an activity of 73,000 units per mg.

This material is characterized by a absorption maximum at 305 nm with an Ε ϊ ™ of about 343. The infrared and NMR spectra of this material are shown in Figures 2 and 3 in each case. The antibacterial activity of the material is shown in Table 10. From elemental analysis of the material it is clear that the substance contains nitrogen, sulfur and sodium (among others), approximately in the ratio N: 2: Na = 2: 2: 2. The dichroism of disodium seli antibiotic MM 13.902 was determined on a Cary-61 spectropolarimeter at a concentration of 0.37 mg / ml at a length of 1 cm.

The results are as follows:

λ (nm j Έ / m 186 + 67,24.IO ' ⁴ 221 —67,24.10 ⁴ 286 - 3.3 .TO ⁴ 323 - 8.2. 10 ⁴ Table 10

Antibacterial activity of disodium salt MM 13.902 (microtiter procedure, heavy inoculum, 1/100 mash, overnight)

Organism Minimum inhibitory concentration of µg / ml

Bacilus subtillis A 0.15 Staphylococcus aureus 0.3 (Russell J 0.6 Streptococcus faecalis 3.1 Entenobaceter cloacae NI 25 Escherichia coli 10 419 0.15 JT 410 5 Klebsiella aerogenes 13 0.6 Proteus vulgaris WO 90 0.6 Prov. etuartii 0.07 Pseudomonas aeruginosa A 50 Saliffionella typhimurium CT 10 0.3 Serratia marcescens 2,5 Shigella sonnei 0.6 to 0.3 Haemophiius influenzae P 6 0.08 Neisseria gonorrhoeae 0.08

Claims (5)

A process for the production of a streptomycin antibiotic, designated MM 13.902 having the structural formula ho ₃ so c-nu-co-cm, Názvem named 3 - [(E) -2-acetamidoethenylthio] -6- (1-sulfatoethyl) -7-oxo-1-azabicyclo [3.2. O] hept-2-ene-2-carboxylic acid, optionally in salt form, consisting in aerobic cultivation of Streptomyces strains, in particular Streptomyces olivaceus, in a culture medium containing assimilable sources of carbon, nitrogen, sulfur and mineral salts under conventional conditions for cultivation of Streptomyces characterized in that the resulting mixture of antibiotics designated MM 4550 is chromatographically separated into fractions containing essentially the salt of antibiotic MM 13.902 with a maximum in the ultraviolet spectrum at 305 nm and another fraction, and the salt of antibiotic MM 13.902 is isolated from the solution by distilling off the solvent under reduced pressure lyophilizing. 2. The process of claim 1 wherein the chromatographic separation is carried out using an aqueous solution of an electrolyte buffered to approximately a neutral point together with a polar carrier material. 3. The process according to claim 1, wherein the separation is carried out using an aqueous solution of sodium chloride buffered to pH 7 together with a carrier material containing tertiary amino groups or quaternary ammonium groups. 4. A process according to claim 1 wherein the chromatographic separation is carried out using a solvent system which is a mixture of water and a C1-C4 alkanol together with an inert support material. 5. Process according to claim 1, characterized in that a mixture of water and isopropyl alcohol is used as the solvent system and sillkagel or cellulose is the inert carrier material.