DEP0012040MA - - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

Registration date: May 28, 1954. Advertised on February 2, 1956

GERMAN PATENT OFFICE

The present invention relates to a new valuable antibiotic called P.A. 105 contributes, and in particular its production by fermentation, processes for its extraction and concentration from raw solutions, e.g. B. from fermentation broth, on processes for its purification and production its salts. The invention includes on the one hand the diluted forms such as raw concentrates, and on the other hand, the pure crystalline forms of the antibiotic. These novel products are special advantageous for combating pathogenic, particularly gram-positive microorganisms.

The new antibiotic is formed when a new strain is grown under certain conditions a species of microorganism known as Streptomyces antibioticus. This strain was grown in a culture for the identification of such microorganisms commonly used nutrient solution. A culture of this microorganism was made in the "American Type Culture Collection" in Washington D. C. (America) under the name ATCC 11891 deposited. Identifying this new strain, identified in the applicant's collection as "Isolate No. 15784-1", was transferred to Hand of the "Manual of Determinative Bacteriology" by Bergey, 6th edition (1948).

The characteristics of the cultures of the new strain of Streptomyces antibioticus are * in the following table

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belle compiled. Unless otherwise noted, the experimental results are based on six Repeated attempts' incubated for 2 weeks

had been. The color designations marked with R correspond to the color specifications given by Ridgway in "Color Standards and Nomenclature ".

Table I.

Streptomyces antibioticus ATCC 11 891

10 Nutrient solution Growth
strength Color ' Soluble
pigment Remarks ¹⁵ glucose-asparagine agar moderate Mycelium formation in the air
and sporulation medium brown to
dark brown Back brown,
Conidial threads in
Tufts; none
Spirals; Spurs
0.65 Xi µ 20th
gelatin moderate white air mycelium,
low spores
education dark brown good liquefaction moderate Gray, waxy
Mycelium; no spurs
education dark brown no coagulation;
Hydrolysis, none
Change of the
. p _H value Skimmed milk (28 ⁰ ) moderate creamy white
ring light brown Back light brown Glucose Agar -. low to
moderate light brown, waxy
transparent bak
series growth,
no spore formation none Creamy reverse side
white, digested
Calcium malate Calcium malate
35 small amount white aerial mycelium;
low spore bil
dung, greyish-white none Back white up
light gray Synthetic agar .... small amount mouse gray (R)
Spore formation medium brown Back light brown Nutrient agar
40 low to
moderate light brown, waxy
transparent bacterium
line growth dark brown Back light brown. Emerson's Agar
45 small amount light gray to yellow, wax
kind wrinkled
Mycelium; no spurs
education light brown Cellulose 00
small amount white, air mycelium none Nitrates were not
reduced Glucose nitrate
C ₀ 'broth moderate dark brown
to black small amount Almost twilight gray (R),
Spore formation none Back dark yellow
to gray; no hy
drolysis Potato slices ..... yellow to brown, waxy
• like bacteria
growth; Spurs
education, almost
Hathi gray (R) Strength tokens
55

, Some of the distinctive differences between the new strain # 15 784-1 and the description

of S. antibioticus in Bergeys
Table II compiled.

»Manual« are in 125

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Table II

Nutrient solution

Strain ATCC 11 891 Description according to Bergey

gelatin

Litmus milk

All nutrient solutions that

organic substances

keep

gray, waxy mycelium; no spurs

(in skimmed milk, no litmus); creamy to dark brown Ring; Coagulation and hydrolysis

soluble light brown to dark brown pigment

The strain of S. antibioticus described in Bergey's "Manual" supplies the antibiotic "actinomycin", which, as can be seen from the following information, from the antibiotic »P. A. 105 «clearly is different.

The one relating to the manufacture of P.A. 105 The present invention is not limited to the use of this particular microorganism, nor to those organisms that correspond to the description above, as these are here only for explanation should serve. In fact, the invention also includes the use of biological variants, which differ from the organism described in various ways, e.g. B. by action from X-rays, ultraviolet rays, nitrogen mustard and the like.

The antibiotic P.A. 105 is highly effective against a large number of microorganisms. As already mentioned, its effectiveness against gram-positive organisms is particularly noteworthy. Against It is also effective in gram-negative organisms, but to a somewhat lesser extent. The following Table shows the antibiotic spectrum of P.A. 105 against various gram-negative and gram-positive microorganisms. The attempts at this were carried out by adding nutrient solutions containing various concentrations of the pure antibiotic with the respective bacterial species vaccinated.

The lowest concentration indicated in Table III, which just had an inhibitory effect, is the lowest concentration of the antibiotic (in y / cm ³ ) at which the growth of the microorganisms was barely prevented. Since the highest concentration used for this experiment was 100 γ ^{/ cm 2} , where the concentration apparently exceeded 100 γ / ecm, no precise value is given for the "lowest concentration just barely exerting an inhibitory effect". The tests were carried out under normal conditions.
Table IV gives the results of similar tests carried out in another laboratory with various other organisms or strains which differ from those mentioned in Table III, and also shows the spectrum of PA 105.

dark brown bacterial growth on the surface, with patches of gray aerial mycelium

thick, brownish ring on the milk surface; mouse-gray aerial mycelium with greenish Coloring, the growing cultures turn brown; no coagulation of the Milk; no clarification

soluble deep brown pigment

Table III Spectrum of the P.A.

Organism gram negative organisms

Bruceila bronchiseptica ...

A. aerogenes 2

E. coli 21

Proteus Sp. 1

Ps. Aeruginosa 173

Salmonella Typhosa 344 ..

K. pneumoniae 132

S. paratyphia A 134

B 139 .....

Listeria monocytogenes .. Neisseria gonorrheae

catarrhalis

meningitidis ....

gram-positive organisms Strep, faecalis 121 .......

Micrococcus pyogenes

var. aureus 5

209

M. pyogenes var. Albus 3.

B. subtilis 219

Diplococcus pneumoniae,

Type I

Type I, ATCC.

Type III

Type V

Erysioelothrix rhusio-

pathiae, 15 A

Egg

G-2

Cornybacterium xerose ... Clostridium perfringens ..

sporogenic ...

tetani,

Bacillus anthracis '......'.

Fungi Candida albicans 8,: .-.>.: .. _; ,

Barely inhibiting effect

exercising lowest

Concentration of P.A.

y / ccm

25th

> IOO

> IOO

> IOO

> IOO

> IOO

> IOO

> IOO

> IOO

6.25

3> I2

. 6.25

1.56

0.19 <o, i9

0.78

0.39

6.25

<o, i9 3.12 3.12

0.3

o, 7 o, 7 o, 5 6.25

o, 7

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In these tests the maximum concentration of the antibiotic was 900 μg / ccm.

Table IV Spectrum of P.A. 105, other microorganisms

organism
gram negative organisms

E. coli

E. coli

E. typhosa

Sahnonella sp.

Aerobacter aerogenes ..

S. paratyphosa

Klebsiella pneumoniae ..
Bruceila bronchisepticus

B. pyocyaneus

NA II SM resist

NA IV S. typhosa

E. typhi-murium

S. typhi-murium

S. newport

S. enteritidis

S. cholera suis

Proteus vulgaris

Barely inhibiting effect

exercising lowest

Concentration of P.A.

y / ccm

225 450 225 450 225> 0.00 225

7> o, oo

112 112 225 225 450 225 225 9OO organism
gram positive organisms

Barely inhibiting effect

exercising lowest concentration of P.A. 105 y / ccm

Strep, hemo

D. pneumoniae ..

Staph. H

B. subtilis

Enterococcus ...

Cl. welchii

Staph. aureus 235

mushrooms
Monilia albicans.

,> 90o

The new antibiotic also proved to be highly effective against such organisms as opposed to the common commercially available antibiotics were resistant. For example, were used to determine the just barely exerting an inhibitory effect lowest concentration certain resistant strains of Micrococcus isolated from clinical cases pyogenes, variant aureus, exposed to the action of the antibiotic P.A. 105 in the manner indicated. The lowest concentration that just had an inhibitory effect was used for comparison several other known antibiotics have also been identified. The test results are in Table V. specified.

Table V

Antibiotic used. Lowest concentration in y / ccm with an inhibitory effect

M. Pyogenes
var. aureus
tribe P.A. 105 Oxytetra
cyclin * Chlorotetra
cyclin chlorine
amphenicol Baci
tracin Neomycin penicillin Polymycin I. 0.78 > 100 100 . 6.25 56 3.12 6θ, Ο > 25Ο 2 3.12 > 100 56 . 6.25 28 3.12 15.0 > 250 3 1.56 > 100 50 6.25 28 1.56 7.5 > 25O 4th 1.56 > 100 100 12.5 14th 25.00 60.0 > 25O Ot 1.56 > ^I0 ° 50 6.25 14th 1.56 15.0 > 25O 6th 3.12 > 100 50 12.5 28 50.00 7.5o > 25O 7th 0.78 > 100 100TH 6.25 28 0.78 60.0 > 25O 8th 1.56 > 100 100 6.25 14th 1.56 3.75 > 250 9 0.78 > 100 50 TH 3.12 56 1.56 > 60.0 > 250 IO 0.78 > 100 > 100 6.25 14th 3.12 60.0 > 250 II 0.78 0.78 0.39 3.12 TH 28 6.0 3.75 . > 25C 12th 0.78 > 100 > 100 6.25 14th <o, 78 > 60.0 > 25O 13th 0.78 > 100 50 6.25 28 3.12 0.47 > 250 14th 0.78 > 100 > 100 6.25 14th <o, 78 60.0 > 25O 1.56 > 100 100 TH 3.12 7th <0.78 o, 93 > 25O 16 1.56 > 100 5 ° 6.25 56 <0.78 15.0 > 25O • ^τ 7 0.78 > 100 100 3.12 28 3.12 60.0 > 25O i8 0.78 > 100 100 3.I2TH 14th 3.12 60.0 > 25O

TH = partial inhibition. *) Is sold under the name »Terramycin«

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Apart from the fact that these attempts show the good effectiveness of the new antibiotic against this show resistant strains, they also show that the antibiotic P.A. 105 actually of oxytetracycline, chlortetracycline, chloramphenicol, bacitracin, neomycin, penicillin and Polymycin is demonstrably different.

As can also be seen from Table VI, which shows the results of mycobacteria Trials reproduces the antibiotic P.A. 105 also proved to be effective against a number of these Bacteria. In this case, test tubes with liquid Dubos nutrient solution were used as the experimental culture medium used. The antibiotic was added to the nutrient solution in the specified in the first column of the table Concentration levels added. The glasses containing such concentrations of the antibiotic with the nutrient solution were then inoculated with cultures of the various mycobacteria mentioned, whose designation is given at the top of each column in the table. The attempt then went on that the glasses were incubated under sterile conditions for 36 hours and then carried out to demonstrate the or no development of mycobacteria was observed. The lack of bacterial growth in the jars at the end of the test period, the sign (-) indicates that the development has taken place indicated by the sign (+).

The antibiotic proved itself in animal experiments

PA 105 as relatively less toxic. Was the antibiotic e.g. B. given intravenously to mice dissolved in water, then the LD ₀ value was approximately Table VI
Effectiveness of PA 105 against mycobacteria

y / ccm ranae ■
phlei smeg-
matis 607 25.0
12.5
6.25
3.12
1.56
0.78
o, 39
0.19 + - ■ I II + + + + + + I I + + + I

15 mg / 20 g mouse. If administered differently, the agent behaves similarly.

Finally, the antibiotic PA 105 also showed a high degree of effectiveness in vivo against various pathogenic agents. Organisms. Mice of approximately the same weight were injected intraperitoneally with certain strains of Strept. haemolyticus, D. pneumoniae and M. pyogenes var. aureus and ^{treated for 2 1} Z ₂ days by twice daily subcutaneous injections of 5 mg antibiotic each time. The effectiveness of the antibiotic PA 105 against these bacteria can be clearly seen in the mice treated in this way from Table VII, which compares the percentage ratio of the surviving treated and untreated mice.

Table VII Efficacy of the antibiotic P.A. 105 in vivo

organism

Treated

Untreated

Surviving,% rate, after

48 hours

96 hours

7 days

Strept. haem. C 203 Mv.
D. pneumoniae 1/230 ..

M. pyogenes var. Aureus
No. 235

Treated
Untreated

Treated
Untreated

Treated
Untreated

100
0

100
0

100
20th

100
0

100
ο

100
20th

100
0

100
ο

100
20th

The present invention also comprises a process for culturing the microorganism S. antibioticus, preferably working with aqueous nutrient solutions at about 24 to 30 ^° with submerged stirring and aeration. The nutrient solutions suitable for the process should be carbohydrates, such as sugar, starch, glycerine, corn flour, etc., a source of organic nitrogen, such as casein, soybean flour, peanut flour, wheat gluten, cottonseed flour, milk albumin, enzymatically dissolved casein, tryptone, a source of growth substances, such as soluble distillation residues, yeast extracts, molasses fermentation liquor, etc., as well as mineral salts such as sodium chloride, potassium phosphate, sodium nitrate, magnesium sulfate, etc., and trace metals such as copper, zinc and iron. If the nutrient media foam too much during fermentation, you can add antifoam agents to the fermentation solution, e.g. B. Vegetable oils, add. The _pH value of the fermentation usually remains pretty much unchanged; however, if changes occur, a buffer salt can be added to the nutrient solution, e.g. B. calcium carbonate.

The vaccine for the production of the antibiotic P.A. 105 by cultivating the S. antibioticus can be based on

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Nutrient solutions such as Emerson's agar or beef lactose can be obtained in cultures on inclined nutrient media. Shake flasks or larger containers can be used to develop submerged cultures

S inoculate this preculture, or you can later distribute the culture from the shake flask in the large container. The growth of the microorganism usually peaks in 2 to 3 days. However, you can Changes in the equipment used in the agitation and ventilation speed etc. the speed affect with which the highest effectiveness is achieved

■ will. In general, to produce the Antibiotic needed about 24 hours to 4 days.

The aeration rate of the nutrient solution in larger containers set up for submerged culture is kept at about ¹ ₂ to 2 parts by volume of free air per part of space of liquid per minute. The devices generally used in the fermentation industry can be used for stirring. Of course, aseptic conditions must be maintained throughout the transfer of the vaccine and cultures of the microorganism.

After the microorganism has grown, the mycelium, which is generally quite lush and vigorous, can be separated from the fermentation broth with various conventional devices, such as filter presses, centrifuges, etc. The antibiotic PA 105 can then be extracted from the fermentation broth in various ways. But you can also use the broth as it is, whole in a dried state. The antibiotic can also be further purified in various ways, e.g. As can the compound from the aqueous solution at neutral or slightly alkaline, preferably between about 6 and about 10 lying p _H values with a variety of water-immiscible organic solvents, which include the ethers, aromatic hydrocarbons, esters, ketones , lower alcohols and halogenated hydrocarbons, extract.

These include diethyl ether, benzene, toluene, ethyl acetate, butyl acetate, methyl isobutyl ketone, butanol and chloroform. Even with acidic p _H values, a certain amount of the antibiotic can be extended with some of these solvents. This applies in particular to alcohols that are immiscible with water, such as butanol, pentanols, etc. The antibiotic ■ can be made from most of them. Solvents are back extracted again with acidified water, preferably at _pH values below about 2.5.

However, it is expedient to concentrate the solvent extract before back-extraction with the acidified water. By adjusting the p _H -value in a neutral or alkaline reaction, the antibiotic of the above-mentioned solvent may be back extracted then again in a. After drying and concentrating the solution obtained in this way, the antibiotic crystallizes out in long white needles. It can be recrystallized by cooling a solution prepared with it in hot ethyl acetate, chloroform, methylene chloride or ethylene chloride. Further recovery processes that are obvious to the person skilled in the art are absorption on activated carbon with subsequent washing, treatment with ion exchange resins and chromatographic development from alumina columns.

The antibiotic PA 105 is a basic, white amorphous organic compound which is soluble in dilute aqueous acids and moderately soluble in water. It is very easily soluble in a number of organic solvents such as methanol, ethanol, acetone and butanol. It is insoluble in hexane, carbon tetrachloride and di-n-butyl ether. An aqueous solution of the compound is stable for several hours at room temperature within a wide p _H -area. However, when heated in acidic solution, it decomposes completely. In the dry state or dissolved in anhydrous solvents, however, it is resistant. The melting point of the crystalline anhydrous hydrochloride of the antibiotic is about 125 to 128 ⁰ . In the ultraviolet range, its absorption spectrum shows a broad peak of low intensity at around 286 to 289 χημ (ίο mg in 10 ecm of methanol). When the base of the antibiotic is dissolved in chloroform, it shows a number of characteristic absorption peaks in the infrared, of which the most notable frequencies are the following (in reciprocal centimeters): 3510, 2910, 2890, 2840, 2590, 1710, 1615, 1460, 1385, 1335-1305, 1280, 1190, 1160, 1145, mo, 1075, 1050, 1005, 985, 960, 935, 895, 885 and 860. The anhydrous hydrochloride also exhibits a number of characteristics when suspended in a potassium bromide bead Ultra-red peaks with the following more important frequencies: 3380, 2930, 2880, 1710, 1640, 1465, 1380, 1330, 1250, 1190-, 1160, 1115, 1075, 1055, 1010, 1000, 985 ', 935, 865 and 828. The infrared spectrum is shown in more detail in the drawings. The hydrochloride (0.1%) dissolved in methanol has an optical rotation of [a] ² | = - 80 °.

The molecular weight of the base of the antibiotic determined by the ebullioscopic determination The P.A. 105 was about 715.

A sample of the crystalline hydrochloride of PA 105 recrystallized from ethyl acetate was dried at 100 ° for 18 hours, after which a weight loss of 5 ° / _{0 was} observed. The dried fabric was analyzed and contained the following elements in the given weight ratios:

Coal 57.63%

Hydrogen 8.73%

Nitrogen 1.87%

Chlorine (ionic) 4.30 ° / ₀

Oxygen (the balance) 27.47 ° / ₀

This corresponds to the likely empirical formula Cg ₇ H ₆₇ NO ₁₃ · H Cl for the anhydrous hydrochloride. The hydrochloride also occurs in various hydrated forms, e.g. B. as a dihydrate according to the probable formula

C ₃₇ H ₆₇ NO ₁₃ -HCl ^ H ₂ O. ■■ ^iao

The antibiotic P. Ä. 105 differs how from the properties described above and on the basis of paper chromatographic Ti measurements This shows that its properties are distinct from other antibiotics. For the application

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Suitable salts of the antibiotic can by methods customary in the art, e.g. B. be prepared by treating the base with the corresponding acid in aqueous solution or under anhydrous conditions. For example, the hydrochloride can be prepared by dissolving the base in acetone and passing hydrogen chloride into the solution. Other acids, such as sulfuric and phosphoric acids, make the corresponding acidic salts of the antibiotic.

The invention is further illustrated, but not limited, by the following examples will.

Example I.
15th

On a slant culture of S. antibioticus ATCC11891

were developed under precisely controlled conditions on Emerson agar spores with which a Nutrient solution of the following composition has been inoculated:

Dextrose hydrate 10 g

Soybean meal 10 g

Sodium chloride 5 g

Soluble distillation residues 5 g

² S calcium carbonate ig

The nutrient mixture was diluted with water to 11, adjusted with calcium hydroxide to a _pH value of 7, and sterilized by heat. The nutrient solution was then cooled and the spores were added under aseptic conditions. The organism has now grown at about 25 ⁰ in shake flasks for 2 days. The resulting mixture of broth and mycelium was then placed in a sterile nutrient solution that was twenty times the volume of the first mixture and had the following composition:

Dextrose hydrate. ιό g / liter

Sodium chloride 5g

Digestate from alcohol production

through fermentation of grains 5 g

Corn starch ιό g

Soybean meal .. 10 g

This broth was first adjusted with calcium hydroxide to a _pH value of 7, with 1 g of calcium, per liter carbonate added, and then sterilized in the usual way, was added after which the broth and the mycelium from the shake flasks. After the latter were dispersed in the new nutrient solution, the mixture was stirred and ventilated under sterile conditions for 3 days. The concentration of the antibiotic in the broth was then 70 μ / cc. The mycelium was removed by filtration and ^{extracted twice with 1} J _{1 of} its volume of mexhyl isobutyl ketone. The combined solvent phases were concentrated to ¹ Z _{10 of} their volume in vacuo. The antibiotic was then extracted therefrom with water, which had been adjusted with sulfuric acid to a _pH value of about. 2 The aqueous phase was separated, washed with benzene to remove the methyl isobutyl ketone and adjusted to a _pH value of 6.5. The antibiotic was then extracted with ether several times and the extract obtained was dried over anhydrous sodium sulfate. When the ether was distilled off, the antibiotic remained in the form of long white needles; it was finally recrystallized from hot ethyl acetate. The recrystallized product thus obtained proved, as indicated in the experiments described above, to be highly effective against a number of different mycobacteria and gram-positive microorganisms.

Example II

Another fermentation solution was made from the following materials:

Soybean flour 15 g

Dextrose hydrate 20 g

Corn starch 10 g

Sodium chloride 5 g

Soluble distillation residues 5 g

Enzymatic protein hydrolyzate .... 5 g

After the addition of these substances to the water 11 of the p _H value was the resulting mixture with calcium hydroxide adjusted to 7 to 7.2. 5 g calcium carbonate was added to act as a buffer during fermentation. The nutrient solution was then treated in the autoclave and sprinkled under sterile conditions with S. antibioticus vaccine, which had been prepared according to the method described in Example I. After the inoculated nutrient solution was aerated for 2 days under sterile conditions at about 28 ⁰ and stirred for containing filtered broth 100 γ antibiotic PA 105 to 1 cc of solution.

Of course, modifications of this mode of operation are still possible which are within the spirit of the invention and fall within the scope of the following claim.

Claims (1)

PATENT CLAIM: Process for the production and extraction of the antibiotic PA 105, characterized in that Streptomyces antibioticus ATCC 11 891 or one of its biological variants or mutants is submerged under aerobic conditions in an aqueous nutrient solution, preferably at about 24 to about 30 ^° for 1 to 4 days, cultivates and optionally extracts the fermentation solution filtered from the mycelium with an organic, water-immiscible solvent in a neutral to alkaline reaction and isolates the antibiotic. Please refer to the sheet of drawings ι 509 656/174 1. 56