DE2760203C2 - Antibiotically active oligosaccharide, process for its production and medicinal product containing it - Google Patents

Description

(a) an elemental analysis of:

C "= 43.84%; H = 6.41%, N = 1.08% and 0 = 49.67% (remainder);

(b) a specific optical rotation [a $ + 155 ° (c = 1 in water);

(c) no characteristic absorption peak in the UV spectrum;

(d) an IR absorption spectrum as a potassium bromide tablet, which corresponds to that shown in FIG. Ib of the drawing reproduced Spectrum, which is part of this claim;

(e) a nuclear magnetic resonance absorption spectrum (hydrogen) in deutero water corresponding to that in the F i g. 2b corresponds to the spectrum reproduced in the drawing, which is part of this claim; and

(f) a single spot with an R-raffinose value = 0.57 on paper chromatography with ethyl acetate / pyridine / water (10: 4: 3) as developing solvent and with an R-raffinose value = 033 paper chromatography with n-butanol / pyridine / EssigsätireAV-water (6: 4: 1: 3) as developer solvent, the R-raffinose values were calculated assuming that raffinose is a single spot at Rf ». Finds 1.00 on the same paper chromatography

2. A method for producing the antibiotic oligosaccharide SF-1130-x ₂ according to claim 1,

characterized in that Streptomyces myxogenes ATCC 31305 is grown in a culture medium, which contains nutrient swelling assimilable by common microorganisms, the fermentation broth or the filtrate obtained therefrom through a column of a strongly acidic cation exchange resin in the H + form, the resin column washes with water, then the resin column with 0.1 N aqueous ammonia

ak washes, the eluate is concentrated to dryness, the residue is dissolved in water, the aqueous solution obtained is adjusted to pH 3.5 by adding sulfuric acid or hydrochloric acid and then passed through a column of activated carbon, the activated carbon column is washed with water and then with an aqueous one The solution containing 30 to 35% ethanol is washed, the eluate is concentrated to dryness, the residue is dissolved in water, the solution thus obtained is passed through a column of a cation exchange resin in the NH ₄ + form, the resin column is washed with water and washed with 0 , 1 N aqueous ammonia is eluted, the eluate is concentrated to dryness, the residue is dissolved in water, the resulting aqueous solution is chromatographed on a cation exchange resin in the pyridinium salt form using an O ₁ IM pyridine-formic acid buffer (pH = 3.1) subjects as a developer solvent, the eluate is collected in fractions, the antibacterially active fractions are combined with one another and concentrated to dryness, the dabe The colorless powder obtained is taken up in water, the aqueous solution obtained is subjected to chromatography in a cellulose column using a mixed solvent of n-propanol / ethyl acetate / water (6: 1: 3 by volume) as the developer solvent, the eluate is collected in fractions and such fractions showing a single spot at an R-raffinose value of 0.57 (calculated assuming that the Rf value of raffinose = 1.00) on paper chromatography with development with ethyl acetate / pyridine / water (10: 4 : 3 in volume) results in

united with each other and constricted to dryness.

3. Medicament, characterized by a content of the oligosaccharide SF-1130-x ₂ as an active ingredient, optionally in combination with a pharmaceutically acceptable carrier for the active ingredient.

4. Medicament according to claim 3, characterized in that there is also at least one of the substances Contains maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose or maltoheptaose.

The invention relates to the subject matter of the claims.

Aminoglycoside antibiotics, for example kanamycins, are already known

A number of useful substances were found in the culture broth from various strains of Generated genus Streptomyces and isolated therefrom. It is known that Streptomyces myxogenes, SF-1130, the

% was identified by the depository number FERM-P 676, an antibiotic that forms as a substance

f | SK-1130, see Japanese Patent Publication No. 30393/73. From the applicant were

ig further investigations on the fermentation broth of the microorganism Streptomyces myxogenes SF-II3C

If carried out, it was found that new antibiotic substances, which against gram-negative

If bacteria are active, this microorganism is formed in the fermentation broth. It succeeded this

[• i isolate active substances from the broth, and one of the isolated substances was considered by the Arunelderin

ψ. Designated substance SF-1130-x ₂ .

ύ Furthermore, it was found that this active substance has an activity by which that in living

Ψ-. Animals and humans automatically existing immunity is protected from it, more or less than

■ As a result of the formation of tumors and / or as a result of the application of immunity-suppressing substances

l ·? Antitumor drugs to be reduced, and furthermore it has been found that this active substance therefore as

ils immuno-potentiating agents (immunopotentiators) for promoting the immune response in living animals

% and is beneficial in humans

The object of the invention is to provide a new, antibiotic substance that is used as an antibacterial

$ Rielles means and advanta- as an activator for promoting immunity in live animals and in humans

Another object of the invention is a method for producing this beneficial substance.

| S; This substance SF-1130-X2 was later found to have the following general formula:

CH ₂ OH OH CH ₃ CH ₂ OH

χ - · · ■ · ° ^0Η

OH OH

I OH OH OH OH

Culture medium growth Aerial mycelium soluble pigment Sucrose nitrate agar poor growth, low, white none colorless Glucose asparagine agar brown to none none greyish-brown that The back is matt colored in red color Clycerin Brown none none Asparagine agar Calcium malate agar thin growth, none none- pale brown Glycerin- Brown none none Calcium malate agar

20th 25th

D-glucose

The substance SF-1130-x ₂ is obtained by cultivating a strain of the genus Streptomyces which has been deposited with the American Type Culture Collection, Washington DC, USA under the depository number ATCC 31305. The cultivation takes place in the usual manner in an aqueous, liquid culture medium which contains sources of assimilable carbon and assimilable nitrogen, under aerobic conditions.

The strain SF-1130 with accession number ATCC 31305 has the following microbiological ones Characteristics:

Morphological observation

Substrate mycelia grow well on various culture media, while aerial mycelia formation in the general is bad. On starch agar and on starch yeast extract agar, where aerial mycelia develop, short, dense aerial mycelia are formed from the substrate mycelium. The mycelia form single-footed branches, no whorl-like branches are observed.

The aerial mycelia have spirals at their tips, most of which are of the compact, closed type and some of these are of the incomplete or open type. There will be no formation of sclerotium observed. Observation in the electron microscope shows that the surface structure of the spores is smooth. The spores are elliptical or cylindrical in shape and have a size of 0.6-0.7 0.9-1.0 Micron.

2. Culture characteristics on various culture media are compiled in Table I below

Table 1

55 60 65

continuation

Culture medium

growth

Aerial mycelium

soluble; pigment

5 Starch agar pale brown to powdery. Brown greyish-brown, development mainly on the periphery 10 the colony Oatmeal agar good, pale brown Gray Nutrient agar good growth none with wrinkles, brown on the 15th back Starch yeast extract agar pale brown to greyish brown Brown to gray Yeast extract good, reddish-brown none 20th Tyrosine agar dark brown none Potato piece high standing none Growth with Wrinkles, brown 25th Carrot piece greyish-brown none Growth with wrinkles Gelatin (20 ° C) cream colored none Skimmed milk (37 ° C) Growth on the ground, none 30th pale brown coagulated dark gray none Löffler Serum (37 ° Q Egg (37 ° C) dark gray none J5 to dark brown Czapek's glucose solution Growth on the ground, none cream colored Cellulose no growth

none

none brown

pale brown brown

blackish brown brown

pale brown

dark brown to black none

none

gray "pigment on the periphery of growth none

Note: The incubation temperature was 28 ° C, unless otherwise specified.

3. Physiological properties

Liquefaction of gelatin positive

Hydrolysis of starch positive

Formation of tyrosinase positive

Formation of hydrogen sulfide positive

Coloring power positive

Degradation of cellulose negative

Reduction of nitrate negative

Peptonization of skimmed milk negative

Negative coagulation of skimmed milk

Dissolution of coagulated Löffler serum negative

In addition to the above physiological properties, strain SF-1130 produces on agar medium and mucus in a liquid medium, this being an essential characteristic of the strain in question

On an agar medium such as a starch-yeast extract agar, starch agar, or glucose-asparagine agar medium the mucus was observed to form massively on the colony in about 10 days after incubation It was further observed that when the strain SF-1130 was shaken in a liquid Medium containing suitable carbon sources (glucose, starch, etc.) and nitrogen sources (yeast extract, soybean meal, Wheat germ, etc.), the culture broth gradually becomes viscous to form a slime.

4. Exploitation of carbon sources

(1) Utilized: xylose, glucose, galactose, maltose, sucrose, lactose, Raffmose, dextrin, starch, glycerine, Inositol, sodium acetate, sodium citrate, and mannose.

(2) doubtful: arabinose, fructose and salicin.

(3) not used: rhamnose, inulin, dulcitol, mannitol, sorbitol, sodium succinate and cellulose.

The aforementioned microbiological properties of the strain SF-1130 can be summarized as follows will:

(1) The Lufti.iycel forms closed spirals at its tip and the surface structure of the spores is smooth.

(2) The aerial mycelium, which is grayish-brown to gray in color, is only very slightly formed.

(3) The growth on synthetic culture media is brown to greyish-brown in color.
(4 J Coloring capacity (chromogenicity) is observed on organic culture media.

(5) Slime is formed on agar medium and in liquid media.

With regard to the microbiological properties described above, Streptomyces phaeoochromogenes, Streptomyces purpureochromogenes and Streptomyces noboritoensis as known, with the strain SI -'- 1130 related strains can be named. However, strain SF-1130 is not one of these known ones Stems as shown below:

Streptomyces phaeochromogenes produces large amounts of aerial mycei and forms a brown, soluble one Pigment on Czapek sucrose agar and calcium malate agar as culture media, while strain SF-1130 shows little formation of aerial mycelium and no formation of soluble pigment on these culture media.

Although Streptomyces purpureochromogenes grows orange to reddish-orange on potato tucibs shows, this strain does not form hydrogen sulfide and causes the coagulation of Skim milk, while strain SF-1130 shows brown growth on the same medium and the However, the formation of hydrogen sulfide does not cause coagulation of skimmed milk.

Streptomyces noboritoensis does not form spirals and produces a green, soluble pigment on starch agar cdium (The formation of the green, soluble pigment is not mentioned in the earlier documents, but is they actually observed appreciably for this type of strain), while strain SF-1130 However, Spirals does not form a soluble pigment on synthetic starch agar. In addition, it differs the strain SF-1130 of Streptomyces noboritoensis is also in the utilization or acceptance of mannitol and sucrose.

Hence, the strain SF-1130 differs markedly from each of the known, related species of Genus Sireptomyces. The strain SF-1130 is further distinct from any other known Species of Streptomyces in that the strain SF-1130 has the special property of the formation of mucus, Wn. previously described, shows, while the other species of Streptomyces, no mucus correspondingly the previous publications.

It follows that the strain SF-1130 was identified as a new species of the genus Streptomyces, where he was referred to as "Streptomyces myxogenes SF-1130".

The strain SF-1130 possesses properties that can vary, as is usually the case with others Streptomyces species is observed. For example, the strain SF-1130 can be variants or mutants form when exposed to various muiagenen such as ultraviolet rays, x-rays, high frequency electromagnetic Waves, radioactive radiation and chemicals are treated. Any natural or artificial Variant or mutant of the strain SF-1130 can be used in the method according to the invention, as long as it still has the ability to form the substance SF-113O-X2 ^ o

In the method according to the invention, the strain used can in a manner known per se in a Culture medium can be grown that contains nutrient sources that can be assimilated by common microorganisms are, contains. For this purpose, any known nutrient can be used, which are generally used in the Cultivation of known strains of Streptomyces was used. Examples of applicable nutrient sources include: glucose, starch, maltose syrup and molasses as carbon sources and soybean " flour, wheat germ, dried yeast, peptone, meat extract, corn steep liquor, ammonium sulfate and sodium nitrate as nitrogen sources. If necessary, inorganic salts such as calcium carbonate, sodium chloride, Potassium chloride, phosphates and the like can be added. In addition, such organic and inorganic Materials that support the growth of the trunk used and the formation of the substance Promote SF-II3O-X2 to be entered into the culture medium.

As a cultivation method which can be used according to the invention, liquid cultivation is preferred, in particular under aerobic immersion conditions, as is generally used in the production of antibiotics. The cultivation is especially frequently performed under aerobic conditions, advantageously at a temperature of 25 to 38 ⁰ C and at a temperature near 28 ° C. Under these circumstances, the formation of the substance SF-1130-X2 in the culture broth reaches a maximum after shake cultivation or tank cultivation for a period of 2 to 5 days.

The following procedure can be used to examine the substance SF-1130-x ₂ according to the invention: The culture medium used is a medium which contains 04% peptone, 03% meat extract and 1.5% agar (pH = 6X, known as mycin test agar medium) with 0.125% maltotriose contains Escherichia coli K-12R as the test microorganism. The examination can usually be carried out using the conventional paper disk method.

The substance SF-1130-X2 is an oligosaccharide of a weakly basic nature, which is given below Possessing physico-chemical properties and taking advantage of these physico-chemical properties of the substance SF-1130-X2, it can be obtained from the culture broth and then purified. For example, purification by adsorption on charcoal, chromatography and the use of Aqueous alcohol as the eluent or by reprecipitation from mixed solvents water / ethanol will.

For example, the substance SF-1130-X2 can be extracted from the fermentation broth according to the following procedure

cleaned and isolated: The fermentation broth containing the substance SF-1130-x ₂ is adjusted to pH = 3 by adding z. B. sulfuric acid or hydrochloric acid acidified uih! then filtered under acidic conditions / to remove the mycelia and insolubles, and the broth filtrate is passed through a column of activated charcoal for adsorption of the active substances. The activated carbon acid is then eluted with 50% acetone / water, ie a mixture of acetone and water in a ratio of 1: 1 by volume, in order to desorb the substance SF-1130-X2 from the activated carbon. The eluate is concentrated to dryness and the residue is dissolved in water. The aqueous solution obtained is in turn passed through a column of activated charcoal for adsorption, and the activated charcoal column is then successively mixed with aqueous solutions which contain ethanol in different concentrations in the range from 5 to 25% by volume. The eluate is collected into fractions, and those antibacterial active fractions containing the substance SF-1130-X2 are combined and concentrated. A volume of ethanol is added to the concentrated solution to _{precipitate the substance SF-1130-x 2} , which is easily recovered as a crude powder. The raw powder obtained in this way contains a substantial proportion of neutral oligosaccharides and in particular maltodextrin, which are also produced in the fermentation broth. Therefore, in order to obtain a highly pure product of the substance SF-1130-X2, it is more preferable to use the following extraction procedure:

The fermentation broth of the strain SF-1130, which contains the substance SF-1130-X2, or the broth filtrate obtained therefrom is passed through a column of a strongly acidic cation exchange resin in the H + form for adsorption of the substance SF-1130-X2. The resin column is washed well with water in order to remove traces of the neutral oligosaccharides such as maltodextrin, maltopentaose and maltohexaose, which may have been bound to the resin from the fermentation broth. After washing, the resin column is washed with O ₁ IN aqueous ammonia to desorb the substance SF-1130-X ₂ . The eluate is concentrated to dryness and the residue is dissolved in water. The aqueous solution obtained is adjusted to pH = 3.5 by adding sulfuric acid or hydrochloric acid and then passed through a column of activated carbon to adsorb the substance SF-1130-X2. The activated charcoal column is washed with water and then washed with an aqueous solution containing 30 to 35% ethanol. The eluate is concentrated to dryness, the residue is dissolved in water, and the solution thus obtained is again passed through a column of a cation exchange resin in the NH.4 ⁺ form. The resin column is washed with water and eluted with 0.1N aqueous ammonia. The eluate is concentrated to dryness, the residue is dissolved in water, and the aqueous solution thus obtained is subjected to chromatography on a cation exchange resin in the pyridinium salt form using a 0.1 M pyridine-formic acid buffer (pH = 3.1). subjected as a developer solvent. The eluate is collected in fractions, and the antibacterially active fractions are combined with one another and concentrated to dryness, a colorless powder being obtained which contains the substance SF-1130-x ₂ in the form of a mixture of the substance SF-1130-x ₂ with a contains another oligosaccharide. This raw powder can be used as such for carrying out the test of estimation of the physiological properties of the substance SF-1130-X ₂ . For the isolation of the substances SF-1130-X2, if necessary, the aforementioned colorless powder containing the mixture of these substances is taken up in water, and the resulting aqueous solution is then subjected to chromatography in a cellulose column using a mixed solvent of n -Propanol / ethyl acetate / water (6: 1: 3 by volume) as developer solvent. The eluate is collected into fractions, and those fractions which have a single spot at an R-raffinose value of 0.57 (calculated assuming that the Rf value of raffinose = 1.00) on paper chromatography under development with ethyl acetate / pyridine / water (10: 4: 3 by volume) are combined with one another and concentrated to dryness to give the substance SF-1130-x as a colorless powder

The substance SF-1130-x ₂ has the following characteristics:

(a) The elemental analysis found:

C "= 43.84%; H = 6.41%; N = 1.08% and O = 49.67% (remainder);

(b) a specific optical rotation of [a $ = + 155 ° fc = 1 in water);

(c) it has no characteristic absorption peak in the UV absorption spectrum;

(d) It has an IR absorption spectrum in the form of a potassium bromide tablet that corresponds to that in FIG The drawing corresponds to the spectrum shown;

(e) It has a nuclear magnetic resonance absorption spectrum on hydrogen in deutero water corresponding to the in FIG. 2b corresponds to the spectrum reproduced in the drawing; and

(f) it gives a single spot when R-raffinose = 0.57 in paper chromatography with ethyl acetate / pyridine / water (10: 4: 3) as developer solvent and when R-raffinose = 033 in paper chromatography with n-butanol / pyridine / Acetic acid / water (6: 4: 1: 3) as the developing solvent when the R-raffinose values are calculated assuming that raffinose gives a single stretch at Rf = 1.00 on the same paper chromatography
eo

The above-mentioned and the other physicochemical properties of the substances SF-1130-X ₂ according to the invention are shown in Table II below

Tables

properties

SF-1130-X ₂

Appearance Melting Point Elemental Analysis (%)

Molecular weight (determined by the vapor pressure method) specific optical rotation UV absorption spectrum IR absorption spectrum Hydrogen core absorption spectrum Solubility:

soluble in:

less soluble in:

insoluble in: stability:

Color reaction: positive for:

R-raffinose values in paper chromatography (assuming that Rf of raffinose = 1.00)

i) developed with

Ethyl acetate / pyridine / water

(10: 4: 3) ii) developed with n-butanol / pyridine / acetic acid / water (6: 4: 1: 3)

colorless, odorless, amorphous powder

195 ° C (dec.)

C = 43.84

H = 6.41

N = 1.08

900

[<χψ + 155Vc = 1, water)
no character. Absorption peak
in Fig. 1 b reproduced
in Fig. 2b reproduced

Water, dimethyl sulfoxide

Methanol, ethanol

Acetone, ethyl acetate, chloroform. benzene

stable under neutral conditions,

however somewhat unstable under acidic

and alkaline conditions

Silver nitrate, tetrazolium red, anthrone, ninhydrin
and Greig Leaback Reagent

0.57

0.33

In the case of carrying out the method according to the invention using the aforementioned Siammes SF-1130, the resulting culture broth contains, in addition to the substance SF-1130-x ₂ and a further oligosaccharide, the known antibiotic, substance SF-1130, see Japanese patent publication 30 393/73 as well as maltopentaose and maltohexaose produced by strain SF-1130; for these last two substances, reference is made to the applicant's German patent application filed on the same day, corresponding to Japanese patent application 99 756/76.

The substance SF-1130 is a neutral oligosaccharide, which is easily soluble in water, hardly soluble in methanol and ethanol and insoluble in acetone, while maltopentaose and maltohexaose are neutral oligosaccharides which are easily soluble in water but hardly soluble in methanol -are soluble in ethanoi and acetone. In contrast, the substance according to the invention SF-1130-x _{2 is} an oligosaccharide with a basic nature, which is easily soluble in water, is less soluble in methanol and ethanol, but is not soluble in acetone. By utilizing these differences in the properties of the aforementioned new substance and the known substances, the substance SF-1130-x _{2 can be separated} from the substance SF-1130 as well as from maltopentaose and maltohexaose. For example, the fermentation broth of strain SF-1130 can be acidified and filtered by adding the appropriate inorganic acid such as hydrochloric acid or sulfuric acid. When the acidified broth filtrate thus obtained is passed through a column of a strongly acidic cation exchange resin in the H + form, the substance SF-1130-x ₂ and the other oligosaccharide are adsorbed by the resin, while the neutral oligosaccharides such as maltopentaose and maltohexaose are adsorbed by the Pass the resin column through the resin without adsorption. When the resin containing the substance SF-1130-x ₂ adsorbed thereon is treated with 0.1N aqueous ammonia as an eluent, the substance SF-1130-x _{2 is} eluted from the resin

The substance SF-1130-X2 is an oligosaccharide of a weakly basic nature in the form of a colorless powder, which is soluble in water and dimethyl sulfoxide, less soluble in methanoi and ethanol, but in acetone, Ethyl acetate, chloroform and benzene is insoluble and the substance has a positive reaction with silver nitrate, Tetrazolium Red, Anthrone, Ninhydrin and Greig Leaback Reagent shows and taking the substance with acid Formation of glucose is hydrolyzable

In the drawing, the following spectra are shown in the figures:

F i g. 1 b shows the curve of the IR absorption spectrum of a sample of the substance according to the invention SF-1130-x ₂ as a pellet in potassium bromide;

2b shows the curve of the nuclear magnetic resonance absorption spectrum (hydrogen) of a sample of the substance SF-1130-x ₂ , determined in deutero water at 100 MHz.

The antibacterial activity of the substance SF-1130-x ₂ for inhibiting the growth of various bacteria was estimated by the conventional paper disk and plate method. For this purpose, the

Substance SF-1130-x _{2 dissolved} in distilled water at a concentration of 1 mg / ml or 2 mg / ml, and * y

with the test solutions prepared in this way, paper disks made from filter paper with a diameter of 8 mm were used

impregnated with knives, which were then air-dried. These paper discs were placed on an H

The upper plate layer of the known mycin test incubation medium, which contains 0.5% peptone, | 3

03% meat extract and 1.5% agar (pH = 6), and which comprised the test microorganisms to be incubated therein

nisms, this being placed over the lower layer of agar medium in the examination dish ρ

became. Incubation was carried out at a temperature of 37 ° C overnight, and then M

measured the diameter of the zones of inhibition formed around the paper letters. The ||

Test results are summarized in the following Table III, from which it can be seen that the substance ti

to SF-1130-X ₂ no antibacterial activity against the gram-positive bacteria and the acid-resistant | j

Owns bacteria. These tests were repeated in the same way as before, but with the addition of W _t 1.25 mg / ml of macotriosis to the top plate layer of the mycin test incubation medium- §5

The results obtained here are also shown in Table III, from which it can be seen that the %

additional presence of maltotriose improves the antibacterial activity of the substance SF-1130-x ₂ In I]

Tests of the same type have also found that the combined use of maltose or a K

Maltodextrin including maltotriose, maltotetraose, maltopentaose and maltohexaose with the substance β

₂ SF-1130-x, the first-mentioned substances of the erfindungsgemä- '■ /] Ben substance SF-1130-x ₂ generally improve the antibacterial activity, with the exception that an improvement of the antibacterial activity i

against Klebsieila pneumoniae is not observed. All results of the in this context carried out /

Tests carried out are listed in Table III. It should be noted that maltotriose and others: "

Maltodextrins show absolutely no antibacterial activity under the aforementioned test conditions. d

Table IH Ϊ3

^ 7..Λ

Tesunikroorganisimis diameter of the zone of inhibition (mm) with the substance SF-1130-xi? |

no additional '■': ._

Use of Maltotriose Use of Maltotriose £ Content of SF-1130-X ₂ ϊά

2 mg / ml 1 mg / ml 2 mg / ml 1 mg / ml. ·, '

Escherichiacoli IAM1239 0 0 14.8 14.2 '■:

Escherichiacoli K-12 13.0 low 18.8 153 V

Escherichiacoli 14.5 13.4 23.6 203; j

(resistant to chloramphenicol) C

Escherichia coli IAM1264 15.2 123 25.4 23.6 %

Shigella sun egg 13.0 0 193 17.8 γ-

Proteus vuigaris low Ö 15.4 12.8 '■ ?.

Salmonella typhi 13.4 11.4 19.0 17.6

Klebsieila pneumoniae 13.2 11.5 13.2 11.0

Bacillus subtilis 0 0 0 0 f-.

Staphylococcus aureus 0 0 0 0 f- \

Sarcinalutea 0 0 0 0 B

Mycobacterium smegmatis 607 0 0 0 0 -Ά

From the results of further investigations, it was found that the improvement of antibaktericl- Ά len activity of the substance SF-1130-xi which is attributed to the additional or combined use of maltose, maltotriose, maltotetraose, maltopentaose or maltohexaose, is significant when maltose . or Makodextrin, as mentioned above, is used in addition in a proportion of 0.1 to 20 ■

Parts by weight to 1 part by weight of the substance SF-1130-x ₂ is

To estimate the acute toxicity of the substance SF-1130-X ₂ , aqueous solutions of the substances SF-1130-x _{2 were} injected subcutaneously into mice, it being found that all the mice examined survived at dose levels of up to 400 mg / kg. From the results of further studies on acute toxicity it was found that the substance SF-1130-x _{2 has} an LD 50 value of not more than 500 mg / kg in the intravenous;

Injection in mice. From this it can be seen that the substance SF-1130-xj has a very low toxicity owns.

Tests for cell-mediated immunity were carried out according to the known technique of delayed hypersensitivity using mice vaccinated with an ascites tumor "Sarcoma 180", and the results obtained from these investigations were found to contain the substance SF-1130-x ₂ Has an immunopotentiating effect in tumor-bearing animals whose immunity is lowered. In addition, it was found from further tests that the aforementioned activity of the substance SF-1130-xj is also effective in order to reduce the immunity-suppressing effect of an antitumor agent such as cyclophosphamide (a Preparation of N, N-bis- (2-chloroethyl) -tetrahydro-2H-13,2-oxazophophorin-2-amine-2-oxide) and to completely restore the immune responsiveness of living animals. From this it can be seen that the substance SF-1130-x _{2 has} an immunomodulating activity in living animals. Results of further, similar tests showed that the additional use of a maltodextrin and in particular of maltopentaose and maltohexaose, the activity of the substance SF-I 130- x ₂ to increase the immune reactivity significantly improved.
The investigations were carried out according to the following method: groups of mice from the strain

ICR-JCL (6 male mice per group, average body weight 313 ± 3 g) were ^{inoculated with the ascites tumor "Sarcoma 180" (10 6} cells). 24 hours after inoculating the tumor cells, each of the drug samples to be examined was administered subcutaneously, and a known one Antitumor agent (cyclophosphamide) was administered intraperitoneally to the vaccinated mice. 2 days after the vaccination, an ethanolic solution of 5% picryl chloride was applied to the belly of the mice in which the hair had been shaved to induce sensitization. Each drug sample examined was then subcutaneously applied once applied on the day for the following 4 days. 4 days after vaccination, cyclophosphamide was reapplied. 9 days after vaccination, a solution of 1% picryl chloride in olive oil was applied to both sides of both ears of the mice to induce the second sensitization. 24 hours after the second sensitization, the extent (%) of the increase in the thickness of the ears in the test mice was calculated Ears are determined. The sample of the substance SF-1130-x used in this test was a mixture of the other oligosaccharides and the substance SF-1130-X2 in a weight ratio of 1: 2. The test results obtained are summarized in Table TV below

Table IV Dose (mg / kg) Extent of increase Percentage Applied drug sample the strength of the increase Ear (xlO- ³ cm) 100 9.2 ± 1.88 119 ^ SF-1130-x 100 + 75 10.4 ± 4 ^ 5 135.1 SF-1130-x + cyclophosphamide 100 + 200 10.4 ± 3.12 135.2 SF-1130-x + maltohexaose 100 93 ± 2.03 127.2 SF-1130-X ₂ 75 4.0 ± 2.60 51 ^ Cyclophosphamide 7.7 ± 2.06 100.0 untreated

Further tests using solid tumor of Sarcoma 180 were carried out to show that the substance _{according to the invention SF-1130-x 2 is active} in promoting the immune response in living animals. These tests were carried out according to the following method: Groups of mice from the ICR- Strain (5 male mice per group; average body weight 20 ± 2 g) were inoculated subcutaneously in the flank with a cell suspension of Sarcoma 180 (7 ΙΟ ⁶ cells / 0.05 ml) 6 days and 5 days before inoculating the tumor cells in these Mice were intraperitoneally administered with a mixture of SF-1130-x ₂ and maltopentaose in a weight ratio of 1: 9 as obtained in Example 2, 10 days after the inoculation, the formed tumor was removed from the body of the mice, and the size and the like The weight of the primary subcutaneous tumor was determined. The test results obtained are summarized in Table V below; from this it can be seen that a pretreatment with the substance _{according to the invention SF-1130-x 2} before the inoculation of sarcoma 180 brings about a significant inhibition of the growth of the solid tumor

In a separate experiment, male mice of the ICR strain (5 per group with an average body weight of 20 ± 2 g) were ^{inoculated in the flank with 7 10 6} living tumor cells of sarcoma 180 in 0.05 ml. After 24 hours, each group was inoculated the mice were treated by intraperitoneal injection of 0.1 ml of saline or sample dissolved in saline for 5 consecutive days. 7 days after vaccination, all mice were sacrificed and the primary subcutaneous tumor excised and weighed. The results are also summarized in Table V. The substance SF-1130-X ₂ in combination with maltodextrin or mitomycin C showed a significant effect on the inhibition of the growth of the tumor.

Tubelle V dosage Tumor weight Tumor size Extent (%) of Applied drug (mg / kg) (G) (cm ¹ ) Inhibition of Growth of Tumor 200 0.07 0.141 533 SF-1130-x ₂ + (Pre-dosing) Multopentaose 100 0.06 0.167 60.0 SF-1130-x ₂ + (Pre-dosing) Maltopentaose 20th 0.08 0.151 46.8 SF-1130-x (Pre-dosing) 0 0.15 0.326 - Control, no treatment 200 0.26 66 S F-1130-X ₂ + Maltopentaose 100 0.28 64 SF-1130-xj + Maltopentaose

continuation

Applied drug dosage Tumor weight Turaor size Extent (%) of (mg / kg) (G) (cm ³ ) Inhibition of Growth of Tumor

SF-1130-X ₂ + maltopentaose + mitomycin C mitomycin C control, no treatment

100 + 2

0.43 0.78

In the above table, the degree (%) of inhibition of growth of the tumor was determined as follows Calculated equation:

Escapement = ι 1 -

Weight of treated tumor ^ Weight of control tumor J.

xlOO.

In further investigations, the mice of the ICR strain were inoculated intraperitoneally with Sarcoma 180, to develop ascites tumor. After the tumor developed, the substance became SF-1130-x administered intraperitoneally to the mice affected by the tumor, and it was found that the substance SF-1130-x showed no beneficial influence at all in the treatment of the Sarcoma 180 tumor. this does not suggest any significant killing activity of the substance SF-1130-x against tumor cells. Hence became concluded that the substance SF-1130-x is able to increase the host's immune resistance to the tumor Activate or promote Sarcoma 180, so that an obvious therapeutic effect of the substance SF-1130-x can only be ascribed to the immunopotentiating effect on the tumor

More recently it has been found that patients infected by protozoan and patients with tumors have a considerably reduced immune response compared to normal individuals who have neither a protozoan infection still suffer from an attack of tumors, and that immunotherapy in the clinical practice must be begun in such a way that the restoration of the non-impaired, immunological, "response" favor the therapeutic effect of an applied drug can. The use of such immune activators as the bacterial cells of BCG vaccines as well as of various polysaccharides, all of which have a high molecular weight, is known for this application. There no report was made about this, it was surprising that the substance SF-1130-x, which is a mixture of Cugosaccharides with a relatively low molecular weight are used to activate the immunological system It is clear that the use of oligosaccharides of relatively low molecular weights is effective in practice is more favorable than the use of the polysaccharides, because the oligosaccharides are usually lighter are absorbed by and excreted from living animals (living bodies) and less so The cause of undesirable, allergic reactions and the risk of tuberculosis are not given is

Still further studies showed that the substance SF-1130-x has an activity for preventing bacterial infections in animals Substance SF-1130-x (i.e. a mixture of the substance SF-1130-X2 and the other oligosaccharide in one Weight ratio of 2: 1) was in phosphate buffered saline (pH = 7.2) in one concentration of 0.1 or 0.5% based on the substance SF-1130-x, and the resulting solution of the substance SF-1130-x was administered intraperitoneally to the test mice at a dose of 50 mg / kg / day, 0.2 ml or 10 mg / kg / Day / 0.2 ml applied three times at a time interval of 48 hours. The test mice used were mice from the JCL strain: ICR (8 male mice per group, average body weight 20 g). 72 hours after the final application, the mice were injected intraperitoneally with 0.5 ml / mouse of a cell suspension of Staphylococcus aureus, Smith-S-424 strain, which was obtained by diluting the incubated culture of this strain with physiological saline solution and subsequent addition of 5% mycin was. The number of surviving mice was determined for 5 days after vaccination, and the LD50 values of the Bacterial cells which killed 50% of the host animals were calculated in each case as control tests also carried out, in which the treatment with the substance SF-1130-x was omitted.

The test results obtained in this way are summarized in the following table VI, from which it can be seen that the application of the substance SF-1130-x increases the LD50 value of the bacterial cells by approximately 11.4 times at a dose of 50 mg / kg and by approximately 8.1 times at a dose of 10 mg / kg of the substance SF-1130-x compared to the control tests, which shows that the substance SF-1130-x has a preventive effect against bacterial infection despite the The fact that the substance SF-1130-x itself has no antibacterial activity against Staphylococcus aureus as shown in Table IM above.

10

Table VI Dose of the substance surviving mice at different doses. SF-1130-x from inoculated bacteria (cells / mouse) bacterial cells

9.3 χ 10 ⁷ 13 χ 10 ⁷ 3.7 χ ΙΟ ⁶ 7.4 χ ΙΟ ⁵

50 mg / kg 1/8 5/8 7/8 8/8 2.4 χ ΙΟ ⁷

10 mg / kg 1/8 3/8 7/8 7/8 1.7 χ ΙΟ ⁷

Control - 0/8 2/8 8/8 2.1 χ ΙΟ ⁶

It is known that some immune activators are sometimes used in the treatment of a disease as a result of a Autoimmunization are effective, e.g. in rheumatism. From the following tests it can be seen that the Substance SF-1130-x is significantly effective in preventing the development of experimental arthritis, this was believed to have some relation to rheumatism. The tests were carried out in carried out as follows: a 5.8 mg / ml of Mycobacteriumbutyricum containing cell suspension in physiological saline solution was introduced into the sole of the right leg of rats from SD strain (10 female Rats per group; average body weight 180 g) for the development of inflammation injected a Day after the injection of the bacteria, a solution of 27 mg / ml of the substance SF-1130-x (a mixture ία of the substances SF-II3O-X2 and the other oligosaccharide in a weight ratio of 2: 1) in with

• ig phosphate buffered saline solution 6pH = 7.2) subcutaneously in a dose of 150 mg / kg / day during the subsequent

Injected after 11 days 14 days after the inoculation with the bacteria, the inflammatory symptoms were

sj soft develops in the legs, ears, nose, eyes and waist of the treated advice «

ψϊ were estimated and numbered: 0 , 1, 2, and 3, and the number average values were determined

fil Furthermore, the volume of the sole of the left leg was determined in order to determine the anti-inflammatory activity of the

H to estimate substance SF-1130-x. For comparison, phenylbutazone was administered orally instead of the substance SF-1130-x in

at a dosage of 20 mg / kg / day applied during the following 11 days. Control

H tests carried out in the same way as before, with the application of the substance SF-1130-x or des

; &: Phenylbutazone has been omitted. The test results obtained show that the substance SF-1130-x for

|| Preventing experimental or artificially caused arthritis is significantly effective

| f The substance of the present invention SF-1130- :; ₂ can be made into injectable solutions by dissolving a suitable one

ψ amount in a physiological saline solution or other conventional, pharmaceutically acceptable,

f | liquid carriers such as aqueous carriers, e.g. B. Ringer's injection solution, dextrose injection solution, refined

f | th oils of vegetable origin or synthetic mono- and diglycerides in the form of fat emulsions

"; formulated, or by mixing with a pharmaceutically acceptable solid carrier such as suppository

; ■. '; ricn cocoa or formulated in a porous ethylene tube. For the application of the substance SF-

/> 1130-X ₂ , the injection solution prepared in this way can be administered as an intravenous drip, as a local injection, as an intramuscular

£ = injection, as an intrap! Eura! E injection or as an intraperitoneal injection or as a suppository.

The substance SF-1130-x ₂ can be used as an immune potentiator in a dose of approximately 10 to 400 mg / kg / day and

f | preferably from about 50 to 100 mg / kg / day every day or two or three times a week all at once

| .S mixture or in combination with antibacterial agents, antitumor agents or other the immunity

ig stimulants are given. As an antibacterial agent, a very high dose of over 400 mg / kg /

tis day required.

^ i The invention therefore further relates to ehien stimulator for host defense, d. H. a means to mainly

■; ::; promote immune response in live animals and humans, using this stimulator ah> active

Κ? Component of the substance SF-1130-x ₂ in combination with a known, pharmaceutically acceptable,

ü aqueous or non-aqueous carrier for the active ingredient includes Aqueous carriers include Rin-

p ger's injection solution and injectable nutrient solutions such as dextrose injection solutions and sodium chloride

IiJ injection solutions, and non-aqueous vehicles include water-in-oil emulsions made up of 65% sesame oil, 5%

;; of a surfactant and 30% aqueous solution of SF-1130-x, oil-in-water emulsions

s | sesame oil, 65% aqueous solution of SF-1130-x and 5% surfactant

Sf and water-in-oil emulsions, soft from 9% aqueous solution of SF-1130-x, 19.5% sesame oil, 1.5%

? i composed of surfactant, 65% distilled water and 5% nonionic polyether glycols. That

ύ medicament of the invention can effectively be the Immunresi- ■■ stenz used to prevent a reduction in the ng-Entwickh' normally: m \ formation of tumor or by application

* could be caused by the immunity-suppressing anti-tumor agents, whereby the medicament according to the invention

': neimittel additionally at least one of the substances maltose, maltotriose, m & itotetraose, maltopentaoae, MaI-

may contain tohexaose and maltoheptaose in sufficient quantities to maintain the activity of the substance.:,; SF-1130-x ₂ to improve.

j | The proportion of added maltose, maltotriose or the other added maltodextrin is 0.1

; ■}? up to 20 and preferably 0.5 to 10 parts by weight to 1 part by weight of the substance SF-1130-x ₂ .

The host defense stimulator according to the invention can optionally contain at least one of the known ones

antibacterial agent, anti-tumor center! and the other immunity potentiating agents together with or without a maltodextrin in addition to the substance SF-1130-x ₂ . It is clear from the statements made above that the substance SF-1130-x _{2 can be used} for prophylactic purposes against bacterial infections, against tumors and in rheumatic diseases.

The invention is illustrated in more detail by means of the following examples.

example 1

A seed culture of Streptomyces myxog; enes SF-1130 ■ = ATCC 31305 was in 200 l of a liquid culture medium inoculated which contains 5.01 corn syrup, 2.5% soybean meal, 1.0% wheat germ and 0.25% sodium chloride The inoculated medium was fermented in a container with aeration and stirring at 28 "C incubated for 64 hours.

At the end of this incubation, the culture broth obtained was adjusted to pH - 3 by adding hydrochloric acid and then filtered under acidic conditions to obtain 140 liters of broth filtrate. This filtrate was adsorbed through a column of 201 of a strongly acidic cation exchange resin in the H + form to the substances SF-1130-xi and -x? through the resin has been sent Mach which has been washed thoroughly with water the resin column was eluted with a total of 80 liters of 0.1 N aqueous ammonia. The eluate was in fractions collected, and those fractions showing antibacterial activity against Escherichia coli K-I2R were combined and evaporated to dryness, giving 250 g of a brown colored powder became.

is The exiting liquid (150 l), which comes directly from the aforementioned resin column when the Thereupon abandoned broth filtrate emerged, was acidified to pH - 3.0 with hydrochloric acid and then in a Column of 20 l of a cation exchange resin in the H + form for adsorption of the remaining active Substances given. The resin column was washed thoroughly with water and then with a total of 801 0.1 N aqueous ammonia eiuisrt The eluate was again collected in fractions, and the Fractions active against E. coli K-I2R were combined and concentrated to dryness, whereby 51 g of a brown colored powder were obtained as a second portion.

The brown-colored powder (47 g), which was received as the first portion, was dissolved in 250 ml of water dissolved was then adjusted to pH = 3.0 by adding hydrochloric acid. The acidic Solution was passed through a column (53 x 26 cm) of 600 ml of activated carbon to adsorb the active substances

sent. The charcoal column was washed with water and with an aqueous solution of 30% ethanol and then eluted with an aqueous solution of 35% ethanol. The eluate was collected in fractions, and the active fractions showing antibacterial activity against E. coli K-12R were compared with each other combined (4 l) and concentrated to dryness, with 5.6 g of a yellowish '·. brown powder.

This powder was again taken up in 100 ml of water, then it was adjusted to pH = 3 Addition of hydrochloric acid stopped. The acidic solution thus obtained was passed through a column of 600 ml of cation exchange resin sent through in the NH4 + form for adsorption of the active substances. This resin column was washed well with water and then eluted with 0.1 N aqueous ammonia. The eluate was in fractions collected and the fractions active against E. coli K12-R were combined with one another and used for Concentrated to dryness, 1.5 g of a yellowish-brown powder being obtained.

A portion (1 g) of this powder was dissolved in 70 ml of a buffer solution (0.1 M pyridine / formic acid solution, pH = 3.1), and the solution obtained was poured into a column (3.5 × 60 cm) of 50 ml Ion exchangers: given in the pyridinium form, grain size 0.074-0.037 mm for adsorption of the active substances. The resin column was then eluted with the same buffer solution as before and the eluate was collected in 10 ml fractions. The antibacterially active fractions 63-79, each having a single spot stained by the silver nitrate reagent with an R-alanine value: of 0.53 (calculated assuming that the Rf value of alanine = 1.00) for a Were combined with each other and concentrated to dryness, whereby 380 mg of a colorless powder was obtained which contained the substance SF-1130-x as a mixture of the substance SF-1130-xj with the other oligosaccharide dissolved in small volumes of water and the aqueous solution obtained was combined with a mixed solvent of n-propanol / ethyl acetate / water (6: 1: 3 by volume). This mixture was passed through a column (3 × 30 am) of 200 ml of cellulose to adsorb the active substances, and the cellulose column was eluted with the same mixed solvent as before. The eluate was collected in 7 ml fractions, and each fraction was examined by paper chromatography using a mixed solvent of ethyl acetate / pyridine / water (10: 4: 3 by volume) as the developing solvent. The antibacterial active fractions 351-445, each one single stain colored by the syllable nitrate reagent at an R-raffinose value of 0.57 (calculated assuming that the Rf value of raffinose = 1.00) in the aforementioned paper chromatography were combined with each other and concentrated to dryness, 150 mg of a colorless powder being obtained which contained the substance SF-1130-x ₂

Furthermore, the antibacterially active fractions 503-550, each of which was a single one by the Silver nitrate reagent colored stain with an R-raffinose value of 039 - corresponding to the previously given Definition - revealed on the same paper chromatography, combined and concentrated to dryness 70 mg of a colorless powder which contained the further oligosaccharide

The substance SF-1130-x ₂ containing, .colorless powder (150 mg) was dissolved in 6 ml of water and the resulting aqueous solution was in a column (2 × 5.5 cm) of 15 ml of activated carbon to adsorb the active Given substance. The activated carbon column was washed with water and then eluted with an aqueous solution of 30% ethanol and then with an aqueous solution of 35% ethanol. The eluate was collected in 3 ml fractions. The antibacterially active fractions 30-42 were combined with one another and used for Concentrated dry, a pure product of the substance SF-1130-X2 was obtained as a colorless powder with a melting point of Melting point = 195 ° C. (with decomposition). The yield is 120 mg.

The brown powder (51 g) obtained as the aforementioned second portion from the eluate directly ^{eluted from the second column of the ion exchange resin in the H +} form with 0.1 N aqueous ammonia was subjected to the same purification conditions and Isolation working methods as before

Throwing, 520 mg of a pure product of the substance SF-1130-x _{2 were} obtained.

Example 2

liinc inoculum of Streptomyces myxogenes SF-1130 (identified as FERM-P 676 or ATCC. 31305) was inoculated into 201 of the liquid culture medium having the same composition as that used in Example 1. The incubation was carried out in a Behälterfermentator at 28 ⁰ C during 66 hours under stirring and BclüHung.

At the end of the incubation, the fermentation broth was adjusted to pH = 3.0 by adding hydrochloric acid and filtered under the acidic conditions to give 15 L of broth filtrate. This filtrate was through a Column (6 χ 36 cm) of 1 1 activated carbon sent to adsorb the active substances. The activated carbon column was washed thoroughly with water and then with 5 l of an aqueous solution of 50% acetone at pH = 8 eluted, and the fractions active against E. coli K-12R (150 ml) were taken out with each other combined and concentrated to dryness, 67.5 g of a yellowish-brown powder being obtained.

A portion (45 g) of this powder was taken up in 200 ml of water and the aqueous solution obtained Solution was placed in a column (5.6 χ 42 cm) of t I activated carbon for adsorption of the active substances. The activated charcoal column was washed with water and then successively with aqueous solutions eluted containing 5%, 10%, 15%, 20% and 25% ethanol. The eluate was collected in 15 ml fractions. The antibacterially active fractions 366-510 were combined with one another and concentrated to dryness, whereby 5.7 g of a colorless powder were obtained.

This colorless powder (5 g) was dissolved in 15 ml of water and the resulting solution was filtered. to 180 ml of ethanol were slowly added to the filter to induce reprecipitation.

The precipitate was removed by filtration and dried in vacuo, leaving 4.6 g of a colorless Powder were obtained which was a mixture of 10% by weight of the substance SF-1130-X2 and 90% by weight Contained maltopentaose.

For this purpose 2 sheets of drawings

Claims (1)

Patent claims: 1. Antibiotic active oligosaccharide with the designation SF-1130-x _{2 of} the following (subsequently submitted) general formula: Η— ( OH [OH
/ CH ₃ > 0 CH ₂ OH - O] D-glucose \
OH /
OH \
OH / \
OH 4th ) - OH where the oligosaccharide SF-1130-x _{2 has} the further features: