DE2009116A1 - - Google Patents

Description

VEB Jenapharm

Process for obtaining a trypanocidal antibiotic

The invention relates to a method for obtaining a pigment antibiotic with new and surprising effectiveness against pathogens of certain protozoan diseases in animals and humans.

Since it hitherto against those caused by certain Trj ^ anosoma-kinds Diseases no or insufficient. " Cure there, is that the aforesaid methods of great value in chemotherapy of protozoal diseases,

In the antibiotics documentation center of the Institute for Microbiology and experimental therapy, Jena, the German Academy the visa senses in Berlin have approved more than 120 antibiotics,

which are formed by streptomyoetes and dit against various Types of protozoa are effective. Four of these antibiotics provide red pigments with indicator properties. In none of the different natural substances, however, has so far been trypanooids effective ffststellfn. - -

The invention aims to produce an »antibiotic with Effect against certain Trypanoeoma species,

The task of discovery b "" is in, "in proceedings" ur Herete! " lung elnfi trypanocidal natural product by cultivating fines Streptoaycft * n strains and anachlifflendf isolation df » kuas from 4 # r KulturbrUhe b »rtitaust · ll * n,

It was found that in the training sessions df

3A 10081 fin for 4if fherapif from Proto * o «n-Krenkheit« n fe ~ Äatifeiotiku »» ii auiÄt * borrowed * r Ktsmffektuni on ά & έ

of tumor cells in vitro, on microorganisms and on the multiplication of bacterial and animal viruses, is formed in good yield and can be isolated under the process conditions described below.

The microorganism used to obtain this substance is similar to the type of the species Streptomyces diastatochromogenes (Krainsky) Waksman et Henrici described by Waksman (1961) in 1948 Strain 207 (ATCC 12309) and is in the strain collection of the Institute for Microbiology and Experimental Therapy, Jena, der Deutschen Academy of Sciences in Berlin deposited under the number JA 10081. The IMET JA 10081 strain was obtained from an Italian soil sample isolated. The sample comes from a vineyard on the island of Capri of the village of Anacapri and has the morphological, macroscopic, microscopic and bioohtvisohtn properties.

The method that has been found "consists" in that under aerobic culture conditions in ink-liquid nutrient diua "with tntsprtohttn carbon and S-ticketoffqutlltn eowit Kintralsalstn in Streptomyoee strain is given, bti dt" tsoft dtsioh at the beastechptneyots «An 19Ö1) IhnUtatn 8t ·» »IMST JA 10011 odtr stift · HuUntt * tdtr Variants handles. The Zuthtuag is i »gttifnttta tfahrfttalen, inebtttwUrt under the ingredient * of Sehwermttallsali & tn, bti tiatr Ttsptratur before 25 to 3? ⁰ C for 2 to 6 days of unitary warming and clocking ftfllt. The AaUbiotiku »becomes aath Abts ^ nntn dts Myttls Ijitrakiion Kit organ is eh tn iiBsu »gsaiti» Xn both from the culture filtrate and from the dt »Myetl, through conversion into the vMttrigt Phast, ansthiitötndt RttÄtraktitn from dtn wuerttn iohkonitntrattn in the organisoht Phast «owit Auefallen from the raw material with

0098A0 / 2083

obtained from a fraction of low-boiling hydrocarbons. That so ioodized antibiotic is known as trypanomycin.

The IHET JA 10081 strain and its variants and mutants are cultivated under aerobic conditions. Starting with spore material dried lyophilically on soil, suitable agar nutrients and then selected liquid nutrient media are inoculated. The production of the antibiotic is carried out by the customary methods known per se and consists essentially in that the strain IMET YES 10081 in a liquid, previously sterilized culture medium preferably under aerobic conditions at a temperature of 25-37 ⁰ C (28 ⁰ C ) is grown over a period of 2 to 6 days (preferably Λ days) at an acidity which is initially pH 6.3 to pH 7.0 and at the end of the fermentation process pH 7.5 to pH 8.3. The culture medium consists of a source of carbon and nitrogen as well as inorganic salts. As the carbon source, starch, glucose, glycerin, mannitol, maltose, dextrin, soybean oil and soybean meal can be used. As a nitrogen source, in addition to some of the nitrogen-containing substances mentioned above, dry yeast, meat peptone and casein can also be used.

Good fermentation results can be obtained using the following Achieve ammonium salts: ammonium nitrate, ammonium sulfate, diammonium phosphate.

The addition of mineral salts, which favors the progress dos fermentation process may change depending on your soil. Additions of calcium carbonate, sodium phosphate and potassium phosphate have proven to be beneficial in a nutrient medium that contains complex substances such as various flours. In one containing glucose and yeast or ammonium salts

00984a / 2Ö83 ~ 4-

The nutrient medium is the addition of mineral salts such as potassium, magnesium, Iron, zinc, manganese and copper salts to increase the yield Antibiotic necessary. In particular, the addition of iron salt to the fermentation medium stimulates the biosynthesis of the antibiotic. The fermentation of the creator can take place in steep chest bottles and round bottom flasks with different contents, in the glass fermenter as well as in the V2A tank be performed.

The inventive method for isolating the pigment antibiotic Trypanomycin from the strain IMET JA 10081 consists in the fact that the antibiotic trypanomycin from the culture filtrate met organic, not or only slightly miscible with water, and from the mycelium with organic, extracted with water-miscible solvents, transferred to the aqueous phase after concentration of the extracts, from the aqueous phase at pH 3.0 to pH 7.0 (preferably at pH 6.8) with organic, water-immiscible solvents and re-extracted from the re-extract concentrates by adding petroleum ether or gasoline is precipitated with stirring, freed from the solvent by filtration and dried.

The antibiotic trypanomycin is a complex of amorphous red-brown substances that dissolves well in chloroform, acetone, dimethylformamide, Little in alcohols, esters, benzene, on the other hand dissolves poorly in water and has indicator properties.

Both fresh fermentation broths of the strain JA 10081 and that from it isolated antibiotic trypanomycin inhibit the growth of non-pathogenic and pathogenic protozoa in vitro and in vivo, of Nocardia, aotinomycetes, gram-positive bacteria and stable L-forms as well as sparoplasts of gram-negative bacteria. Yeasts, Filamentous fungi and basidiomycetes is the inhibitory effect of the antibiotic low or absent,

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The antibiotic trypanomycin and its derivatives, the products of its hydrolytic degradation and the mixtures thereof can be used as a hemotherapeutic against protozoal diseases. "

Investigation of the antiprotozoal effect of the antibiotic Trypanomycin.

The study of the antiprotective effect of the antibiotic Trypanomycin was seen by means of a "spectrum" of saprophytic and pathogenic protozoa in vitro and in vivo on the mouse.

1. Sensitivity to different types of protozoa in vitro the trypanomycin.

Table I shows that non-pathogenic ciliatan, pathogenic entamoebs and especially Tiypanosoma-Aften * eh » are sensitive to trypanomyols. ".,

Test Organisiaen Smallest Inhibition Factors

ug tvypapom $ eiick / ni ' (death after 6 h)

apathogenic Euglena gracilis Polytema avella ParMUOou »oaadatum Tetrahyaena vorax

100.00 1.25

5.00

pathogenic Entamoeba invadens

Entamoeba histolytica Trichomonas vaginalis Leishmania brasilieneis Trypanosoma equiperdum Ti-ypanosoma gembiense Trypanosoma crÄzi

50.00 10.00 50 »00 100.00

0.05

0.05 25.00

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2, sensitivity of Trypanosoma gambiense (causative agent of sleeping sickness) in vivo (mouse) versus trypanomycin. Female mice of the AB-KoI strain were aged 7 to 9 Weeks, see above, infected with Trypanosoma gambiense. From the 2nd day of the experiment, the animals were given 4 consecutive days Days of trypanomycin in various concentrations i.v. applied. Table II gives the results. It can be seen) that the examined antibiotic in the dose range of 1.0 to 2.0 mg / 20 g mouse (daily single dose) a significant prolongation the survival time of the treated animals. This effect of red pigment antibiotics with indicator properties is surprising and not yet known.

Table II Binseldose
in mg for
20 s Mau " Survival time
(Day* extended"
Survival
duration % Animal-
number substance ■■ M * ■ * ■ * 14th Controls 2.0 19.5 217.4 6th Trrpano »yoi» 1.0 12.8 108.4 5 η ■ 0.5 10.5 70.9 6th η 0.25 6.3 3.1 6th Il

As Table III shows, the acute LD ₅₀ of trypanomycin depends on the type of application.

Method of administration (mouse)

(ag / kg)

Injection! intravenous 60.0
intraperitfneal 31.0

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Comparison of the trypanomycin with the known antiprotozoal

v; effective strep to myces antibiotics

The known natural substances used for comparison are they are also pigments that have indicator properties. These are the antibiotics cinerubin, granaticin, danubomycin and Hedamycin.

ir those described for the granaticin, danubomycin and hedamycin Formers such as Streptomyces olivaceufi (VJaksman), Stretomyces griseus (or similar) and Streptomyces griseoruber disagree with the taxonomic criteria of the trypanomycin producer Streptomyces diastatochromogenes, IMET JA 10081. That of the latter || Synonyma to be assigned to type (based on a suggestion by Hütter) are from a compilation of

Hütter, Ralf "Systematics of the Streptomycetes under particular Consideration of the antibiotics they produce "

Bibliotheca Microbiologica, Fase. 6 Basel-S. Karger New York 196?

refer to.

In contrast, cinerubin formers from strains of Streptomyces galilaeus of the type species Streptomyces diastatochromogenes are assigned to μ , so that no taxonomic differences need exist between known cinerubin formers and the trypanomycin former characterized in the invention, IMET JA 10081.

2. The im. Trypanomycin described in the patent differs in its biological activity from all 4 comparable antibiotics. In contrast to trypanomycin, cinerubine inhibits the growth of yeasts and has only a weak effect against Entamoeba histolytica

-8th-

009840/2083

Granaticin also has only weak effects on Trichoraonas vaginalis and Entamoeba histolytica (100 mcg / ml).
Unlike trypanomycin, danubomycin inhibits fungi and Trichomonas fetus.

Like cinerubin, hedamycin inhibits yeasts for which no sensitivity to trypanomycin has been proven. Hedamycin inhibits Tetrahymena pyriformis.

A trypanocidal activity has not been reported for any of the previously described red pigment antibiotics with indicator properties been. It is therefore surprising that in the case of the trypanomycin from the strain IMET JA 10081 described in the w patent Antibiotic with a strong trypanocidal effect is present in vitro and in vivo.

Based on the state of the art, this effect can be described as novel.

3 »With the exception of the cenerubins, the compared natural substances Granaticin, Danubomycin and Hedamycin as distinctly different in their physicochemical properties from those of Trypanomycin be considered. Trypanomycin belongs chemically to the same substance— "great like the cinerubine (anthracycline). An identity with However, cinerubine has not yet been detected. A trypanocidal effect has not been described for cinerubine.

The following examples serve to illustrate the invention without to limit them to this.

example 1 Two 500 ml steep brewing surfaces each contain 80 ml of the following

Vorzuoht nutrient medium

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Glucose 1.5%

Soy flour 1.5%

Calcium carbonate 0.1% Sodium chloride 0.5% ■ '■■ · .-

primary potassium phosphate 0.03%

Fractional sterilization »3 times 30 min. At 100 ^° C After sterilization, the acidity is pH 6.3.

Each steep chest bottle is filled with a tenth of a spore and Mycelium suspension is inoculated, which is inoculated with sterile isotonic Koohsalζlösung from a 14 ΐage old culture of the strain IMET JA 1008; manufactured will:

Sucrose 0.30% '

Dextrin 1.50%

Urea 0.01%: ^

Book extract Ö _# 10 % ;

Ptpton (bakt _f ) 0.50% Sodium chloride '0.05%' / Beech Sulphate 0.001%. Primary potassium phosphate 0.05% Agar (wash) 2 * 0% _s i Aqua dtstillata Steriliefttioni 40 minutes at 116 ⁰ C

After sterilization, the aoidity is between pH 6.5 and pH 7.0,

One incubates the btiapften Vorzuchtkulturen ήβ hours at 28 ⁰ C on a "SchUtt.lgerät with 180 U / rain, 8 “1 such!” “Breeding cultures are used for inoculation of 500 al-steep chest bottles, each 80 ml of the following

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Production culture medium included *

glucose 3.0 fa Soy flour 2.0 % calcium carbonate 0.3 % Sodium chloride 0.5 % in tap water

Sterilization »35 minutes at 110 ^° C. The acidity after sterilization is pH 6.3. It is incubated at 28 ^° C. at a shaking frequency of 180 rpm. After 96 hours of fermentation, the highest content of Trypano- ™ mycin is reached (150jug / ml). The activity is determined by a known microbiological agar diffusion test method in which the strength of the inhibitory effect on the proliferation of temperate viruses in bacteria is used as a measure of activity of the antibiotic applies (BIP-TtBt ₅ Fleck, W., 1968).

Betaplel 2

One seduces as in the example with the difference that the Production-nutrient medium has the following composition

Gfluoose 3 % Soy flour <
3 % Ial * iu * carbonate 0.3 % ] f »triu» efalerid 0.3 % lisene chloride hydrate 0.025 S * in outlet water

Sterilization * 35 minutes at 1X0 ⁰ C The aoidity is ηaoh the sterilization at pH 6.5. The highest content of trypanomyoin is reached after 100 hours

(170 JOf / el).

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Example 3

Proceed as in Example 2 with the difference that the pre-cultivation and production media have the following compositions:

Pre-growth medium

Peptone 0.6%

Dry yeast 0.3 %

Calcium nitrate hydrate 0.05 % tap water

Sterilization: 20 minutes at 120 ^° C., pH 7.2.

Production culture medium

Glucose. 3 % - ■

Dry yeast 1.5 %

Sodium chloride 0.3 %

sec. Potassium phosphate 0.1 %

Calcium carbonate 0.15 %

Magnesium sulfate 0.01 %

Iron sulfate 0.002 %

Zinc sulfate "0.002 % -

Copper sulphate 0.001 %

in tap water

pH 7.0. ■ '_ The sterilization is carried out in the usual way. The highest content of trypanomycin is reached after fermentation for 120 hours (160 g / ml).

Example 4

With a culture of the IMET JA 10081 strain on a solid medium, as described in example ^, inoculate A-OO ml of the im Example 1 described liquid preculture medium, which in a 2000 ml glass flask is included.

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Incubation is carried out for 24 to 48 hours at 28 ^° C. on a shaker at 180 rpm. The 400 ml culture broth thus obtained are used to inoculate 20 liters of the product nutrient medium described in Example 1, which is contained in a 32 liter glass fermenter. The fermentation takes place at 28 ⁰ C with an air supply of 20 1 per minute and a stirring speed of 350 rev / minute. The foam formation that occurs is controlled by adding small amounts of soybean oil.

The maximum production obtained in 96 hours of fermentation Trypanomycin corresponded to a concentration of 100jag / ml,

Example 5

The method differs from that of Example 4 in that the production nutrient medium has the composition given in Example 2. From 20 1 of a culture broth obtained according to Example 4, 18 1 of culture filtrate is obtained after separation of the mycelium with unchanged acidity, which is extracted with 4.5% of butanol. The mycelium (1 | 8 kg fresh weight) is extracted three times with 3.6 l of methanol. The extracts are concentrated to one fifteenth of the initial volume, combined and extracted twice with half the volume of chloroform through 1.5 l aqueous suspension, the chloroform extract is concentrated to one tenth of its volume and the antibiotic is precipitated with the same volume of petroleum ether.
The yield is 2 g of trypanomycin.

Example 6

The procedure is as in Example 4, with the difference that the 20 l of culture broth in the 32 l glass flask is already after a 24-hour fermentation used in the production nutrient medium described in Example to inoculate 400 l of the same medium in a 700 l V2A tank.

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The fermentation takes place at 28 ^° C. with an air supply of 450 l / minute and a stirring speed of 270 rpm. The yield was 30 S 'Trypanomycin,

Example 7

The procedure differs from that described in Example 6 by exchanging the production medium described in Example 1 by the medium described in Example 2. The yield was 60 g of trypanomycin.

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Claims (3)

Claims 1. Process for obtaining a trypanocidally active antibiotic by breeding a Streptomyces strain, characterized in that that as a strain Streptomyces diastatoohromogenes IMET JA 10081, its mutants or variants can be used. 2. The method according to claim 1, characterized in that the Cultivation under aerobic conditions in a nutrient medium containing essentially carbon and nitrogen sources is carried out. ™ 3. The method according to claim 1 and 3t, characterized in that the antibiotic by extraction with organic solvents, subsequent transfer to the aqueous phase and re-extraction is obtained with subsequent precipitation using low-boiling hydrocarbons. 009840/2083