DE2017837A1 - Antibiotic AM 374 and its manufacture - Google Patents

Description

The invention relates to a new antibiotic, on its production by fermentation, on its extraction and enrichment from raw solutions, on its Purification and the manufacture of its salts.

The invention also relates to an improved one Animal feed, especially for use by young people Domestic animals, including poultry such as chickens, turkeys and Ducklings “It is also beneficial for young hinds, horses, pigs, dogs and sheep. '.

Accelerating the Growth of Young Pets' " and protection against disease is also an important one

Task. The economic importance of a rapid

Waiting speed is very fast, especially with this

rapidly growing domestic animals such as poultry, for example chickens, chicken and ducklings. The faster that Growth, the greater let the putter share, the

is converted into meat. It is also desirable to prevent disease in chicks. For these reasons, various narrow band ^ a / fcibiptioa, for example penicillin, bacitracin and streptomycin, and

009842/198 4 originally inspected

Broad spectrum antibiotics, such as tetracycline, oxytetracycline or chlortetracyeline, can be used. When these compounds are premixed in appropriate amounts with animal feeds, they actually promote weight gain, in some cases, and also help protect against infection. Despite the progress that has already been made, such means are still in need of improvement.

Surprisingly, an animal feed has now been found the usual nutritionally balanced putter ingredients and an effective amount of antibiotic AM 574 as contains new growth-promoting additive.

Within the scope of the invention are thinned forms, raw Concentrates and crystalline forms of the antibiotic. These new products are effective against numerous microorganisms, including gram-positive bacteria. The new antibiotic differs due to its effect on certain microorganisms in connection with its chemical and physical properties of antibiotics described so far. ■

The new antibiotic, named AM 374, is formed under controlled conditions during the cultivation of a new streptomycete isolated from a soil sample from Utah. A viable culture of the new microorganism was deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Divison, United States Department of Agriculture, Peoria-Illinois and included in their permanent culture collection .

2/1984 ^BATH

It is available from this depository under the deposit

3582 for everyone receives you ..

The -BG description and identification of this new microorganism, which is also used in the cultivation of the Lederle Laboratories, Pearl Eiver, Uew York by Dr. H. D. Iresner, the expert in these laboratories, carried out.

The culture merlanals, physiological merlanals and morphological characteristics of the culture were determined according to the methods described by Shirling et al., ^ / """Methods for Characterization of Streptomyces Species", Internat. Journ. Of. Syst. ^ Bacteriol. 16: 313- 340, (1966) J. The media used for the study were chosen from the media described by Pridham et al., ^ F ¹¹ A Selection of Media for Maintenance and 'Taxonomic Study of Streptomyces ", Antibiotics Annual (1956 - 1957), pp. 947 - 953 J for the breeding of Streptomycetes. Details are given in Tables I through IV and a general description of the culture is given below. Underlined Parb designations were taken from Jacobson et al "/""Color Harmony Manual" 3rd edition (T948) J.

: Growth

Moderate on most media, on potato dextrose agar Well; weak on Czapek solution agar. ■ * - "'.

IiUftraycel and / or spore colors en mass

Aerial mycelium whitish on most media; throws in sporulation areas ivory colored Ivory (2 dt) to light ivory colored Lt. Ivory (2 approx).

009842/1984

BATH ORIGINAL.

Loessl see P1.rrraente

Yellowish on different media. no pigin color formation on czapek solution, asparagine oextrose and inorganic Salts and starch agar.

? work on the liter side

On most media in yellowish tones. I) Various physiological responses

Nitrates are not reduced to nitrites in organic kitrate broth; complete liquefaction of gelatin in 7 days; no melanin formation on peptone-iron-agar. Utilization of carbon sources according to the method of • Pridham et al., / * J. Baot. 56: 107-TU, (1948) Vi average bio-good utilization of 1-arabinose, d-fructose, i7inositol, lactose, d-mannitol, d-melibiose, salicin, d-trehalose, d-xylooe; poor to no utilization of 1-rhamnose, adonit, d-meclezitose, d-raffinose and sucrose. '

ι Micromorphology

Aerial mycelium sparse, shows long intertwined and convoluted chains of elliptical to elongated spores with dimensions from 0.5 to 0.6ii. χ 1.0 to 1.3 αι .. spore surface • smooth (determined by electron microscopy).

Due to the general characteristics observed, the microorganism is a member of the genus Strepto myces. The WFD 3582 culture was compared to all accessible cultures of other Streptomycetes that share similar basic taxonomic characteristics. ”Two species

009842/1984

BAD OBIGiNAL

had several Merlanale with HEEL. 3532 common. However, as the literature descriptions of this related species were examined, some fundamental differences were found. These are given in Table Y. Besides these UEPJj 3582 generally shows a clear difference more limited growth on most media, and its sporulus is usually sparse.

From Table Y it can be seen that the spore morphology of UBEL 3582 belongs to the Rectus-3? Lexibils-Iyp according to Pridham et al., / "Appl. Microbiol. 6: 52-79, (1958 ) J / , while the spore morphology of the the other two species belong to the Spira type. This is a fundamental difference which alone suffices to distinguish between the organisms. If one also takes into account the other specified differences, the distance between the organism NRR 3582 and the other organisms is so great that that it is to be regarded as a species in its own right.

Due to the ivory color of the spores produced by the new microorganism are generated, the name Streptomyces eburosporeus n.s. as a more suitable descriptive Suggested nickname. .

009842/1984 b _{ad 0R (G1NAL}

Table I. Culture characteristics of Streptomyces eburosporeus n.s. NRRL 55S2

Incubation: 14 days

Temperature: 28

medium

growth

Aerial mycelium and / or spores

soluble
pigment

Color of
bottom

Remarks

Czapek * s solution agar

(O

s chwaeh

Aerial mycelium weii31ich, becomes ivory (Ivory (2 db)). ' to light ivory-colored (according to Ivory (2 ca)) in sporulation zones. Weak sporulation

none

pale melon yellow
(Lt. Melon
Yellow
(3 ea))

Limited growth

* ^ Asparagine-eex-

_ * trose agar ■ CD

moderate

Aerial mycelium
sparsej
rulatj on

whitish, no spo

none

■ bamboo

ben

(Bamboo

Growth limited, jagged colonies

Hickey and Tresner's Agar

. moderate

Aerial mycelium whitish, turns ivory in color (Ivory (2 db)) to .light ivory colored (Lt. Ivory (2 ca)) in Sporulation Zones. Sporulation schv / ach

yellowish;
schv / ach

hellborn- i surface-

s't coloring wax turn

. (According to Amor> wargenför- (3 ic)) j

'OO Ca »

Table I (cont.)

medium

growth

Luftmyeel and / or

Spurs

soluble
pigment

Color of the underside

Remarks

Yeast extract agar

moderate

LuftEycel Weißlieh, spjurenvreise Sporulation

yellowish;
weak

light amber stone color (Lt, Arnber

surface

.wrinkled

Küsters oat-like
flake agar

Aerial mycelium whitish, turns ivory (Ivory (2 db)) bia
light ivory color (It.Ivory (2 ca)) in sporulation zones
Bloating weakly yellowish brown;
schv; ach

helliaelonengclb. (Lt. Melon Yellov / (3 ea))

^ Starch hydrolysis

Tomato paste-eafer meal-.
Agar

moderate

Lyftmyeel whitish, becomes ivory colored (Ivory (2 db)) to light ivory colored (Lt. Ivory (2 ca)) in sporulation zones Sporulation pale orange-yellow;

moderate

light amber (Lt. Amber (3 ic))

Surface vfarsig

Kar-tof el-Dextrose-

Agar ■■. ■: ■■ ", · ■ ..

Well

Aerial mycelium whitish, sparse, becomes ivory (Ivory < 2 db).) To light ivory j-colored (Lt. Ivory (, 2 ca)) in j Spor.ulatiönszon.en. Spur j! Ation s chvrach f

orange yellow;

moderate

light amber (Lt. Amber (5 Ic))

Starch hydrolysis schv / ach.

Colony surface wrinkled to cracked

Table (Ports.)

medium Growth aerial mycelium and / or
] Spores Aerial mycelium whitish, sparse
lich, traces of sporu-
lation soluble
pigment "" ... -.
Color of
bottom Remarks Bennett'S-
Ag ar ¹
moderate •
Aerial mycelium is whitish
ivory-colored (Ivory
(2 db)) to light ivory.
colors (according to Ivory (2 ca))
in sporulation zones.
Weak sporulation yellowish;
is weak light amber
stone colored
(Lt., Amber
(3 ic)) surface
wrinkled
until
cracked.
■ inorganic
cal salts
Starch agar moderate none
^1
1
■ melon
yellow
(Lto Melon
Yellow
(3ea)) C.

Table II

Kicromorpliology of Streptomyces eburosporeus η. see NRRL 3582

medium

lift mycelium and / or spore chains " structure

Spore shape

Spore size

Spore surface

Küster's oatmeal agar

Iiuftmycel sparse, forming chains long ▼ erscblungene and corrugated spores

Elliptical
to elongated

Spurs
0.5-0.6 / rev

χ
1.0-1.3 / 1

smooth (through

Ilectronic

microslcopie

starred)

Table III

Various physiological reactions of Streptomyces eburosporeus n.s. NERL 3582

iEeHperatur: .28 ⁰ C

O CO OO

toc »

Kedium. Incubation
duration growth Physiological response organic Ni
kick broth 7 days Well no liitratreduk '; Lon organic ki-
kick broth 14 days Well no nitrate reduction gelatin 7 days Well complete liquefaction Peptone iron.
Agar 24 hours moderate no melanin produced

O, I.

κ: cd

OO

Table IV

C-source pre-consumption spectrum of Streptomyces

eburosporeus n.s. rTRRL 3582

C "sources

Adonit

1-arabinose · d-fructose i-inositol -.'.- ■ lactose

d-Mannlt d-Eelezitose d-melibiosis d-raffinose 1-thainnose Salicin

Sucrose d-trehalose "■ d-xylose dextrose negative control

5-good recovery 2-medium recovery

Incubation: 10 Tsge temperature; 28 days

Recovery

3
2

.3 ■

0.

: 1 . ■■■■ .'-

3;

0 ·

i.-bad waste 0-no recovery

009842/1984

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Table V

Comparison of Streptomyces NRRL 35 & 2 with S. albidoflavus and S. odorifer

ca DO 3D Q

w — J *

Streptomyces ITRRL

S. albidoflavus

S. odorifer

Aerial mycelium and / or j
spore-carrying ver
branches Long sporophores,
gev; ellt and ver
looped Sporophores short,]
form spirals \
: Long sporophores,
straight, branched,
form spirals Spore shape elliptical "to
elongated spherical spherical Gelatin ver
liquid complete liquefaction
approval rapid ver
liquid slow ver
liquid soluble pigments ■ mainly yellowish mainly
yellowish mainly
brownish Growth on as-
paragin-dextrose-
Agar Growth yellowish; Air-
mycelium whitish, sparse
lich Growth brown;
Aerial mycelium will
whitish-yellow Cream-colored growth
to brownish.
Aerial mycelium abundant,
cream colored

OO CO

1 I I

- 13 -

It is emphasized that the invention for the production of the new antibiotic does not target this particular microorganism or limited to microorganisms that have the growth characteristics given above for illustrative purposes and fully meet microscopic characteristics .. Rather, within the scope of the invention is also the use of Hutantes resulting from the described Organism can be obtained by various measures such as x-ray irradiation, ultraviolet irradiation, treatment with nitrogen dissolves, phage exposure and the like,

Pas permentation process

The cultivation of the organism S. eburosporeus can be carried out in a variety of liquid culture media. Media which are suitable for the production of the new antibiotic contain an assimilable carbon source, for example starch, sugar, molasses, glycerol and the like, an assimilable nitrogen source, for example protein, protein hydrolysates, polypeptides, amino acids, corn swell and the like, and inorganic anions and cations , for example, potassium, sodium, calcium, sulfate, phosphate, chloride and the like. Trace elements such as boron, molybdenum, copper and the like are added to the media as impurities in other constituent parts. Ventilation in tanks and bottles is provided by passing sterile air through the fermentation medium or on its surface. Mechanical stirring devices are used to continue through the storage in tanks. If necessary, a de-haze agent, for example 1 ^ octa decanol in lard oil, can be added.

- 14 Inoculum preparation

A shake bottle inoculum of S. eburosporeus v / ird by inoculating 100 ml of sterile liquid medium into ml 'flask with spores scraped off or washed off from an agar slant culture of the strain. Usually the following will occur -Medium used.

ι.

Molasses. .20 g

Glucose ..... 10 g

Bactopeptone 5 g

Water to 1 000 ml

The flasks are kept at a temperature of 25 to 29 ° C, preferably 28 ° C, incubated and on an eotation shaker Intensive exercise for 30 to 48 hours. These 100 ml Inokua are used to inoculate 1 1- and 12 1 batches of the same medium in 2 1 and 20 1 glass fermenters used. The inoculum is allowed to grow for 30 to 48 hours while aerating with sterile air. These Inoculum approaches are used to inoculate tank fermenters.

' Tank fermentation

Purely the production of the antibiotic in tank fermenters. the following fermentation medium is preferably used.

Soybean meal ......... 10g

Gerelose 10 g '

Sodium Chloride ................. 5g

Calcium carbonate. * .. 1g

Distillers' Solubles from Corn. ** 5 g

Water to 1000 ml

Jader Tank comes with $ 3 to $ 10 of the above prepared inoculum. The ventilation takes place at a rate of 0.5 to 1.0 1 sterile air per liter of broth and per minute, and the fermentation mix is stirred by a stirrer which is operated at 200 to 400 revolutions per minute. The temperature is maintained at 25 to 29 ° C, usually 28 ° C » The fermentation is usually carried out for 65 to 90 hours, after which the mash is harvested.

Cleaning process

After the fermentation has ended, the fermented mash, which contains the antibiotic according to the invention, is filtered to remove the kyeel, preferably at pH 6. Diatomaceous earth or another customary filter aid can be used to aid the filtration. Usually the mycelial cake is washed with water and the wash water combined with the filtrate. The antibiotic activity is adsorbed on activated charcoal Darco G-60 or other suitable adsorbent charcoal, for which about $ 0.5 (weight / volume) of the adsorbent is used. The antibiotic activity retained on the activated carbon can be eluted by stirring the activated carbon for about half an hour with 40% aqueous acetone adjusted to pH 2 with concentrated sulfuric acid, using a volume of eluent that is about one Corresponds to quarter of the original mash volume. The eluate is concentrated under reduced pressure to an aqueous phase which corresponds to about one twentieth of its original volume. The pH of this phase is adjusted to about 5-5 with barium hydroxide, and the barium sulfate precipitate that forms is filtered off. The set solution (Filtrat) is continued except for

009842 / 198Λ

BADORiOiNAL

concentrated about 400 to 800 ml. This concentrate is then slurried with acidified alumina, (using about 1/5 the amount of alumina in G-rasim compared to the volume of the concentrate), and the slurry is transferred to a suitable column of acidified alumina (about ten times the amount of aluminum oxide used in the slurry), which was wet-filled into methanol. The antibiotic activity is eluted from the column with aqueous methanol (usually 25 to 50%) with appropriate active fractions being collected. The active fractions are combined and concentrated to a small volume (1 liter or less) under reduced pressure. The pH of this concentrate is adjusted to about 6.0 to 6.5 with barium hydroxide. The barium sulfate precipitate formed is filtered off again and the clear filtrate is lyophilized, whereby the crude antibiotic AM 374 is obtained. This lyophilized product is then further purified by column chromatography on a suitable ion exchange resin, for example on CM Sephadex C-25 (H -form). The column will be eluted with dilute sulfuric acid solutions. The eluate, which contains the antibiotic activity, which is shown by absorption at 280 nm, is adjusted to about pH 6.3 with barium hydroxide. The resulting barium sulfate precipitate is filtered off and the filtrate is concentrated to a volume of about 30 to 80 ml. The pH of this solution is adjusted to about 8.0 with ion exchange resin IR45 (OH * "). The resin is filtered off and the filtrate is added to a larger amount of acetone with stirring. The resulting precipitate is filtered off with Acetone washed and dried at moderate temperature under reduced pressure, -which antibiotic AM 374 is obtained in the base form.

009842/1984

BATH ORIGINAL

Physical Properties

A microanalysis sample can be obtained by babbling AM 374-Base from methanolic and / or ethanolic acetone and 2 days long drying of the precipitate in a high vacuum (10 mm) "Made at 100 ° C · Made in this way Antibiotic AM 374-Base contains the elements carbon, Hydrogen, oxygen, nitrogen and chlorine in handy the following weight percentages

Carbon 54.54

Hydrogen .................... 6.10

• Oxygen ... 29.01

Nitrogen 7.60

Chlorine ......... 2.47

The product does not have a defined melting point and slowly decomposes above 250 ° C. The optical rotation value is / "a J ^ ⁵ = 102 ° (+ 2.9 °) (C = 1.045 · in Waaser).

Ultraviolet maxima occur

282 mn (E * cm «42) in acidic solutions 282 mu (e3 "" = 42.5) in neutral solution

f Ί CIu

¹ -

305 mu (E, _ * 51 »5) in basic solutions 3h260 mn ( ^Ε ] οη, ■ 92)" in basic solutions

An infrared absorption spectrum of AM 374 base in a like Commonly produced KBr molding has oharacteriotic Absorptions at the following wavelengths, expressed in microns on: 3.0, 3.45, 6.0, 6.20, 6.30, 6.67, 6.88, 7.05, 7.22, 7.35eh, 7.52, 7.67, 8.2, 8.65sh, 8.85, 9.45, 9.75, 9.90, 10.45ih, 11/07, 11.5, 12.0, 12.35, 13.35,

The infrared absorption curve is shown in Figure 1 of the "accompanying" Drawing shown.

Antibiotic AM 374 has the specified below Solvent systems using the following Bacillus subtilis pH 6.0 or Corynebaeterium xerosis as detection organismeri determined R ^ values on:

Rp value. Iib'sungsEulelsystem

0.90 5 # aqueous NE.Cl solution

0.20 pyridine 2 parts,

s-collidine 2 parts, '' sec-butanol 1 part, water 1 part.

Antibiotic AM 374 base is in water and dimethyl sulfoxide Easily soluble, moderately soluble in methanol and to a lesser extent soluble in ethanol.

Antibiotic AM 374 clearly differs from other antibiotics in the characterization data given above and by its anti-microbial activity. The in vitro antimicrobial activity of this new antibiotic and its hydrolysis product is given in the table below, which is the minimum inhibitory concentration shows which is required to inhibit the growth of representative microorganisms in a nutrient medium.

Q4i * 4 * / 1M4

Tabο11ο VI

In Vitro antiraicrobial activity of the antibiotic AM 374 and its hydrolysis product *

Minimal inhibitory concentration (mc.g / ml)

-. - - Aa ΐ ibi ο 11cum
M374 , Hydrolysis
product t Staphylococcus aureus]
ATCC 6538Ϊ "1
■ ■ i 3.1 ■ 6.2. Staphylococcus aureus STo. 69 I.
"'""· ■ ■" ι 3.1 Staphylococcus aureus ■
ATCC trousers H 1.54 ■ ;. 6.2 y 6.2 . . i
Staphylococcus aureus
Smith ATCC 13709
■! • 6.2 6.2 .- ■■ '■ ■. ■ ■ '·
Streptococcus pyogenes C2O3 0.62 1.25 -
Streptococcus' faecalis
ATCC 8043
- ■ -: ■■ - 3.1
■ 6.2. A. ■
Streptococcus sp ,, non-
hemolytic No. 11 -
■ ■
6.2 6.2 Streptococcus sp., Ss-hemo-
Iytisch JTo. 80 6.2
■ 6.2 ■ Mycobacterium smegmatis
ATCC 607 .. ■■■■ -... ■
> 250 '. .125 ■
Salmonella thyphosa
ATCC 6539 •
> 250 > 250 Proteus vulgaris ATCC 9484 > 250 > 250 '. Escherichia coli TJ311 ' > 250 > 250 ■ Escherichia coli DY > 250 > 250 " Klebsieila pneumoniae, strain AD > 2.50 > 250 Enterobacter aerogenes Ko. 75
PseudoBionas aeruginosa ATCC 10145 > 250
> 250 > 25O
> 250

* determined using the agar dilution method

0 0 9842 / 198A

BATH ORIGINAL

13. 374 is also active in vivo against numerous gram-positive microorganisms, for example staphylococci, pneumococci and streptococci. Thus, the new antibiotic comes into consideration as a therapeutic agent for the treatment of bacterial infections in mammals caused by such microorganisms. It can be expected that the new antibiotic can be used to advantage in the treatment or control of such infections by topical or parenteral administration.

ψ The usefulness of the new antibiotic can be demonstrated by its ability to combat lethal systemic infections in mice. The new antibiotic shows high in vivo antibacterial activity in mice against Staphylococcus aureus, strain Smith; Staphylococcus aureus, trunk pants; Streptococcus pyogenes, 0203; and Diplococcus pneumoniae, SVl, when administered as a single subcutaneous dose to groups of female mice (Carworth Farms CF-1) weighing about 20 g, the intraperitoneally ■ with a lethal dose of these bacteria in 10, 10 °, 10 or 10 "trypticase soy broth (TSP) dilutions of a 5-layer TSP blood culture were infected.

> ■■■ "-. ■

The following table VII explains the in vivo anti

bacterial activity of AM 374 »during the subsequent Table VIII shows the in vivo anti-bacterial activity of the hydrolysis product.

0098 42/198 4 baths

Table VII

φ co

= 5 g

co

rn

in vivo antibacterial activity of antibiotic AH374 live mice / all mice: 14 days after infection

dose Staphylopoccus Staphylococcus Streptococcus Diplococcus ■. . 20/20 body weight aureus
Tribe of smith aureus
Trunk pants pyogenes
C203 pneumoniae
SVl • 20/20 - ₁ 320 20/20 15/20 80 18/20 0/20 20th 20/20 4/20 20/20 5 20/20 0/20 20/20 t, 25 1/20 3/20 0.32 2/20 0/20

▼ on. the infected, untreated control mice died $ 95-100 within 5 days of infection.

ax co

dose Table VIII in vivo antibacterial activity of AM 374 20th Hydrolysis product 5 live mice / all mice 1.25 14 days after infection 0.32 Staphylococcus aureus 0.08 Tribe of smith 5/5 . 3/5 - 0/5 1/5 . 0/5

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

example 1

I · ■

Inoculum therapy

A typical medium for propagating the first inoculum is prepared according to the following recipe:

Molasses 20 g

Glucose 10g

Bactopeptone 5 g Water .............. ad 1000 ml

00

39 8 4 27 1 S U, ° «ginal / nspected:

Those washed off from an agar shred culture of S. eburosporeus or scraped-off spores are used for inoculation-of • Two 500 ml flasks are used, each 100 ml of the above specified medium. The pistons are on a Put a rotary shaker device and agitated intensively for 48 hours at 28 ° C. The piston inoculum obtained is transferred to a 19 liter (5 gallon) glass fermenter containing 12 sterile media. The glass fermenter is during a cultivation time of about 48 hours with sterile Mift ventilated, whereupon its contents for inoculating a 300 1 tank fermenter is used. . ■

B e i s ρ i e 1 2

fermentation

A fermentation medium is made according to the following recipe prepares:

Soybean flour 10 g

Gerelose 10g

Sodium chloride ...................... 5g

Calcium carbonate 1g

Distillers' S, olubles of corn, ..5 g

Water ........ ................ to 1000 ml

The permeation medium is at 120 ° C for 45 to 60 minutes sterilized for a long time with steam at 1 atu (15 psi) pressure. After sterilization, the pH value of the medium is 6.6. 300 l sterile medium in a 400 l tank fermenter are loaded with 12 l of the inoculum described in Example 1 inoculates, and -permentation .-- is using at 28 ° C carried out by Hodag LG-8-Ö1 as a defoaming agent. the Aeration occurs at a rate of 0.5

009842/198 A

ί "

sterilizing air per liter of medium and per minute. The medium is' stirred with a stirrer that. with 500 revolutions operated per minute. After a fermentation period of around 70 hours, the maize is harvested.

B eis ρ ie 1 3 Isolation and purification

fc 300 liters of fermented mash are filtered with about 2 <fo (weight / volume) filter aid (diatomaceous earth), and the filter cake is washed with about 30 liters of water. The fermented mash is previously adjusted to pH 6.0 with sodium hydroxide. The antibiotic activity in the combined filtrate and wash water is adsorbed on 900 g activated carbon (D, arco G-60), and colored impurities are removed from the activated carbon suspension by stirring with about 30 1 40% aqueous acetone. The activity is eluted from the activated charcoal by stirring with 75% aqueous acetone which has been adjusted to pH 2.0 with concentrated sulfuric acid. The suspension is filtered, the eluate is reduced under reduced pressure

* Concentrate about 4 liters of aqueous phase and the pH is adjusted adjusted to 5.2 with barium hydroxide. The barium sulfate precipitate is filtered off and the filtrate is further concentrated to about 700 ml. This concentrate is slurried with ■ 100 g of acid-treated aluminum oxide and applied to a column containing 1 kg of acid-treated Alumina was filled in methanol * Daa 'acid treated Aluminum oxide is made by acidifying an aqueous slurry of aluminum oxide (Farbikat Merck) with concentrated sulfuric acid to a constant Prepare pH 3.0, filter, wash with water and methanol and dry on the IiUft. Then it will be

009842M984 _BAD original

the column was eluted with 5 l of methanol, gut, with 60 l of 50% aqueous methanol and then with 10 l of 25% of aqueous methanol. Suitable fractions containing the antibiotic AM 374, such as by bioassays against C. xerosis is found, are combined, concentrated under reduced pressure to about 500 ml and adjusted to pH 6.2 with barium hydroxide. The barium sulfate precipitate is filtered off and the filtrate is lyophilized, yielding 5.1 g of crude AM 374 (purity about $ 30 to $ 50>) can be obtained.

The crude AM 374 obtained in this way in an amount of 5.1 g is dissolved in 30 ml of water and applied to a column with ion exchange resin CM Sephadex C-25 (H form). To prepare the Sephadex resin3 for use, 250 g of it are slurried in about 3 liters of water, and the slurry is _{adjusted to pH 2.0 with concentrated H 2} SO. The excess water is decanted off and that Resin is washed several times with water, the slurry is poured into a column with an internal diameter of 7.6 cm and washed with an additional 5 liters of water to remove the excess acid.

The column is with a gradient between 4 liters of water and 4 1 water that with concentrated sulfuric acid to pH 1.4 is set, and then with a further 4 1 to pH 1.4 adjusted water eluted; Fractions of about 100 ml each are collected and the bioactivity (C. xerosis) and the ultraviolet absorption (290 mu) are from 1- to 100-fold dilutions of these fractions were measured.

As can be seen, fractions 58 to 80 contain the majority of the desired antibiotic, and these fractions are combined. The pH of these combined erections is adjusted to 6.3 with barium hydroxide, and

- "26 -

the deposited barium sulfate is filtered off with egg from diatomaceous earth. The piltrate is concentrated to about 200 ml and. lyophilized to give about 1.5 g of crude antibiotic. The raw antibiotic is dissolved in 50 ml of water and the pH of the solution is adjusted to about 8.2 with ion exchange resin IR45 (OH ~ form). The suspension is filtered and the filtrate is lyophilized. The lyophilized product is dissolved in 40 ml of water and the antibiotic is precipitated by adding 400 ml of acetone. The crystalline precipitate is filtered off and dissolved in a small amount of methanol. By adding acetone, the purified AM 374 antibiotic separates and is filtered off. The yield is 1.18 g. '

A micro analytical sample is prepared by precipitation of long as obtained above AM 374 from ethanolic acetone and 2 days drying the precipitate in a high vacuum (10 mm) at 100 G ^0. The results of the chemical analysis of this product and the other physical and biological properties of the new antibiotic have already been given.

'. Example " 1" 4.

Conversion of AM 374 base to AM 374 sulfate

150 mg of AM 374 base are dissolved in 60 ml of warm methanol, whereupon 1 drop of a 1; 1 solution of methanol and more concentrated Sulfuric acid is added. The resulting precipitate is filtered off and mixed with a little methanol and acetone washed to give 98 mg of AM 374 sulfate.

Alternatively, AM 374 sulfate is made by adjusting an aqueous solution of AM 374 base with sulfuric acid to about pH 6.3

and subsequent precipitation with acetone.

Microanalysis of sample (mm. 10) by deposition, long from aqueous acetone and 2 days drying the precipitate in a high vacuum: receive at 10Q ⁰ C, In.dieser manner hergestelttes AM contains 374-sulfate, the Eemente carbon, hydrogen, oxygen, nitrogen, Sulfur and chlorine in practically the following weight percentages:.

■ carbon 51, 68

Hydrogen ................ 5.85

Oxygen 30.25

Nitrogen, 7.02

Sulfur 1.36

The optical rotation value is £ & J ^ = 104 ° (- 1.8 °). (C? S-1.1075 in water). Ultraviolet maxima occur

282 mn (E ₁ = 44) in acidic solutions 282 mn (E ₁ ■ „. = 43) in neutral solutions

305 mn (E ₁ ' ⁰ - = 48) in basic solutions

/ · CHi. _t

sh260 mu (E ₁ _ = 85) in basic solutions

j · CHI ·

An infrared absorption spectrum of AM 374 sulfate in one Commonly produced KBr compact has characteristics. Absorptions at the following wavelengths, given in microns: 3.0, 3.45, 5.75sh, 5.9Osh, 5.99, 6.03, 6.20, · 6.32, 6.67, 6.78sh, 6.87, 7.05, 7.20, 7.53, 7.65, 8.25, 8.65sh, 8.87, 9.45, 9.73, 9 * 88sh, 1O, 45sh, 11.05, x 12.0, 12.35, 13.3, H, 4. ^

009842/198 4 ^{BAD ORSGLI4AL} |

- 28 example ......

Conversion of AM 574 base into AK 574 chloride

200 mg AM 374 base are dissolved in 10 ml 0.1 η hydrochloric acid, and the solution is concentrated under reduced pressure to about 2 ml, whereby a partially microcrystalline Precipitation forms. This precipitate is filtered off, redissolved in a little water and again with acetone pleases. After filtering off and washing with a little more acetone, 116 mg of AM 374 chloride are obtained.

A micro analytical sample is obtained by deposition from aqueous acetone and 2 days long drying the precipitate in a high vacuum (10 mm) at 100 ^u C. AM 374 chloride produced in this way contains the elements carbon, hydrogen, oxygen, nitrogen and chlorine in practically the following percentages by weight:

Carbon 52.20

Hydrogen 6.22

Oxygen, 28.03

Nitrogen. 7.26

Chlorine 6.34

The optical rotation value is £ & J jp = 106 ° (+ 2.8) (G = 1.057: in water). Ultraviolet maxima occurred

at w

282 mu (E. / = 42) in acidic solutions
/ ι cm

282 mu (E ^ _m = 39t5) in neutral solutions. 305 mu (E ₁ ^ _n = 52) in basic solutions

sh260 mu (E ₁ ^ _1n »92) in basic solutions

00 9 842/1984

; - ■ - BATHROOM

An infrared absorption spectrum of AK 574-Clilorid in a KBr compact produced as usual has characteristic absorptions at the following wavelengths, given in microns: 3.0, 3.27, 3.40, 5.75sh, 5.9Osh, 5 , 98, 6.15, 6.28, 6.65, 6.82sh, 7.03, 7.15, 7.50i 7.65, 8.25, 8.60, 8.83, 9.42, 9.70, 9.88, 10.45sh, ".11.05, 11.9, 12.3, 13.3, U, 37.

Examples of acid hydrolysis product of AM 374 base

1 g of antibiotic AM 374 base is dissolved in 7.5 ml of boiling water. After adding 1.25 ml of 5 η hydrochloric acid , the resulting solution is boiled for 2 to 3 minutes. The solution is mixed with a further 2 ml of 5N hydrochloric acid, cooled and filtered. The precipitate is washed with a total of 3 ml of 5 η hydrochloric acid, redissolved in water and concentrated to a residue. This procedure is repeated twice, whereupon the residue is precipitated from aqueous acetone. 516 mg of acid hydrolysis product are obtained.

A microanalysis sample is obtained by separating out aqueous acetone and drying the precipitate for 2 layers in a high vacuum (10 "* ^ mm) at IQO ⁰ C. The AM 374 hydrolysis product produced in this way contains the elements carbon, hydrogen, oxygen, nitrogen, and chlorine in practically the following percentages by weight:

Carbon 52.54 *

Hydrogen 5.81

Oxygen -25.52

Nitrogen 8.32

Chlorine 7.11

The optical rotation value is / ~ a_ / ^ - '= -69 ° (1 3.6 °) (C = 0.839 in water)

Ultraviolet maxima occur at:

mu (E ₁ ^ = 46) in acidic solutions

280 lu (eJ "_ = 51.5) in neutral solutions

/ f CIu

mu (E ₁ ^ V = 86) in basic solutions j ι cm

sh260 nm (E _1. = H3) in basic solutions

An infrared absorption spectrum of AM 374 hydrolysis product in a KBr compact produced as usual has characteristic absorptions at the following
Wavelengths, given in microns, on: 3.0, 3.3Osh, 3.43, 5.75sh, 5.92sh, 6.0, 6.15sh, 6.25, 6.48sh, 6.68, 6, 85, 7.03, 7.18, 7.5, 7.67, 7.95sh, 8.30, 8.55sh, 8.86, 9.15, 9.45, 9.9, TQ, 45sii _r 11.1, 11.55sh, 11.9, 13.3, 14.4. ...

INSPECTED

JIIIIiIIMlI

B e i s ρ i e 1 7

Component g / kg

ground yellow corn 514

Soybean Oil Oil (44 fi) 300

Kaisgluten flour. 50

Menhaden-PischEehl (60fo) "50

Pett ■ 40

dehydrated alfalfa flour ($ 17) 20

ground limestone 5

Dicalcium phosphate 12

Sodium chloride 3

Trace Miriers - 1

Vitamin premix 5

Trace minerals are manganese (6, CfO, iodine ($ 0.12),

Iron (2, Of ₉ ), copper (0.2%), zinc (2.0 / 0. Cobalt (0.02 ^c / o)

and calcium

2
Vitamin premix per kg putter contains 125 mg butylated hydroxytoluene, 500 ml DL-methionine, 3300 IU vitamin A, 1100 IU vitamin D ₅ , 2.2 IU vitamin E, 11 meg. Vitamin Bp »4.4 mg riboflavin, 27.5 mg niacin, 8.8 mg pantothenic acid, 500 mg choline chloride, 1.43 mg poly acid and 1.1 mg menadione sodium bisulfite on 5 g ground yellow corn. ·

From a merchant-related day-old chicks (6 male and 6 female animals per group) are housed in heated brooders and held in a chick space which is maintained at about 24 ⁰ C. All groups of chicks are weighed at the start of the tests and at the end after 20 days. Putter and water are available ad libitum

0098Ä2 / 198A

BATH ORIGINAL

posed. The forage described above is used for all tests. The results are determined for (a) untreated controls, (b) 10 ppm AM 374, (c) 2 ppm AM 374. The results obtained are in the the following table, from which it can be seen that with both treatment concentrations of AM 374 the Growth of the treated animals is considerably improved.

009842,198 *

Table IX

Growth and putter conversion of chicks when given feed containing AM 374H

Feed additive

Weight putter / ratio

Amount of gain (g), weight survivors (ppm) look for 0-20 «day would take

Improvement % food weight / gain weight gain

none

AM 374H none AM 374H

40

266 1.70 29/30 284 1.62 30/30 288 1.75 39/40 302 1.63 18/20

6.8

4.9

4.9

7.4

2.

CO

ι Ο

- 34 - '
Examples

Composition of the putter

Constituent concentration (° / o)

ground yellow corn '"51.4

'Soybean Oil Flour ($ 44) 30.0>

Corn gluten flour 5.0

Menhaden fish meal (60%) 5.0

Fat (NRG-50) "4.0

■ dehydrated ilfalfa meal ($ 17). ' 2.0

ground limestone ($ 33 Ca) 0.5

Dicalcium phosphate '1.2

Sodium chloride '· .0.3

Trace minerals ^ 0.1

ρ
Vitamin premix 0.5

! Lime Crest Z-2 Delamix; contains 25.5 $ calcium, $ 6.0 manganese, $ 2.0 iron, $ 2.0 zinc * $ 0.12 iodine and $ 0.02 cobalt.

^ Supplies 3300 IUVitamin A, 1100 IUVitamin D ₃₁ 2.2 I, U.Vitamin B, 500 mg choline chloride, 500 mg; DI-methionine, 125 mg ethoxyquine, 27.5 mg niacin, 8.8 mg panto-theanic acid, 4.4 mg riboflavin, 1.43 mg folic acid, 1.1 mg menadione, 0.011 mg vitamin B ₁₂ P. ¹¹ O kg of feed.

009342/1984

growth 1
Putter··· • 2
lot 10 10 and feed conversion_ - 1563 4th
Weight Table X 6th
relationship 29/30 Lining with different 8th
eration NJ
O additive (ppm) 10 2 , increase (g)
0-2O day weight gain survivors 28/30 Lining / Ge
weight gain none ... ίο 2 3
Ver 266 of chicks when administered with 1.70 30/30 .7
ia_ improve _{I1) L u} , ..., Penicillin 200 ., -. search 315 Additives 1, 50 30/30 : Gev; nothing to
would take 13.3 Payzone Penicillin 200 1559 298 ⁵ /
Lining / Ge 1.58 28/30 7.6 AK374H Payzone 284 1.62 39/40 18.4 4.9 · AV290D AM374H 282 1.59 20/20. 12.0 6.9 none AV29OD 288 1.75 20/20 6.8 ■ —- O 317 1.70 18/20 6.0 Φ
OO 289 1.75 20/20 0.0 S. 302 1.63 10.0 7.4 312 . 1.70 0.3 2.9 CQ ■ 4.9 03 8.3

Footnotes to table X;

Column 4: "Weight gain" (g), 0th to 20th day, gives the total weight gain during the test period from 20 days on.

Column 5i "feed / weight gain" gives the ratio of food consumed in g to increase in body weight in g.

Column 7: "Percent improvement (weight gain)" gives the improvement in weight gain in percent.

on, for example

315 x 100

Oj-

100 = 18.4 ..

Column 8 "Percent improvement (feed / weight gain)" gives the percentage improvement in the ratio of Grams of food per gram of weight gain, for example

1.7 x 100 -100 = 13.3.

1.5

009842/1984

ORlGiNAL INSPECTED

• · Be is ρ i e. 19 Manufacture of tablets containing AM 374

Antibiotic AM 374 is processed into common pharmaceutical tablets according to the following recipe:

component mff per tablet AM 374 '■■■' ■ ·. 50 Lactose 225 Corn starch (to mix) 50 Corn starch (for paste) 25- Magnesium stearate 5 -

355

The active ingredient AM 374, the lactose and the corn starch for mixing are mixed together. The corn starch for paste is suspended in water in a ratio of 100 g corn starch per 800 ml water and made into a paste by heating with stirring. "Then the resulting paste is used to granulate the mixed powder. If necessary, additional water is used . The wet granulation is passed through a sieve having a mesh width of 2.38 mm (sieve No., 8) and dried at 49 ° C (120 ⁰ P) · The dry granulate is passed through a. sieve with an Maeohenweite of 1 19 mm (sieve no. 16) and made slippery with the "magnesium stearate." Then the granules are compressed into tablets in a suitable tabletting machine (tablet weight 355 ng). Each tablet contains 50 mg of active ingredient.

ι 1

Example 10 Γ

"'V Manufacture of AM 3-74 containing hard-

Are • The active ingredient AM 374 can be administered in capsules hartachaligen enough '. The following recipe is for making them

·

ia

\ - ■ / ^ ■ ^ ■ ..iiJ.y ^

Capsules suitable:

Component per capsule for 1000 capsules,

mg g

AM 374 50 50

Lactose 90 90

Magnesium te ar at. 1 '1

141 · 141

-ν

"The active ingredient, the lactose and the magnesium stearate

are mixed together. With the mixture become hard. ' shell capsules of suitable size with a filling weight of 141 mg per capsule.

Example 11 \

Preparation of an oral solution of AM 374

component Amount, % C weight / WoI) AM 374 1.00 Magnesium aluminum silicate gel 0.50 Sucrose 60.00 Methyl paraben 0.08 ^ Polyparaben ''. 0.02. Flavoring agents upon need distilled water ad - 100.00

The parabens and sucrose are stirred in about 2/3 of the final volume of distilled water at 80 ⁰ G »and daa gel (Veegum) is added with stirring. After cooling the solution to ^4Ο υ Ο the active ingredient and daa flavoring agent are added under stirring. The cooled solution is adjusted to the final volume with distilled water.

The solution delivers 50 mg of active ingredient per 5 ml of solution as Doöio.

0Q9842 / 1984

-.39 - example 12

With the active ingredient AM 374, an injection solution can be used the following recipe "must be prepared:

Component quantity fo (Wt./YoI.)

AM 574 ■ V '5.0

Polyethylene glycol 4000 U.S.P. 4.0

Sodium chloride π.S.P. 0.90

Benzyl alcohol, reagent grade 0.90

Water for injections. ad 100.00

The carrier is prepared by mixing all of the above ingredients with the exception of AM 574 ". The carrier and the finely ground active ingredient are made by, suitable Measures sterilized. A solution of the active ingredient in the Carrier is made by slurrying the active ingredient with part of the carrier and then adding the remainder Part of the support prepared with stirring.

, A dose of this solution of T ml provides 50 mg of active ingredient.

ORIGINAL

Claims (7)

Claims Antibiotic AM 374, characterized in that it (a) in practically pure crystalline form or in farms of a salt, the growth of gram-positive Inhibits bacteria, (b) Easily soluble in water and dimethyl sulfoxide, Slightly soluble in methanol and ethanol and proportionally in other common organic solvents ^ is insoluble, (c) has the following values of the elemental analysis: C 54.54; H 6.10; 0.29.01; N 7.60; Cl 2.47; (d) tlltraviolet maxima at 282 DIu (E ₁ ^ = 42) in acidic solutions, 282 mu (El ^ = 42.5) in neutral solutions, / 1 cm • 315 wül (t ^^ ° = 51.5) in basic solutions / 1 cm sh260 ¹ ^ - (E ₁ Q _1n = 92) in basic solutions having; (e) an optical rotation value of / ~ a _7jj = -102 ° C ² »9 °) (C = 1.045 in water) and (f) has a characteristic infrared absorption spectrum as shown in FIG. 2. A method for the production of antibiotic AM 374, characterized in that an antibiotic AM 374 producing strain of Streptomyces eburo- 009842/1984 sporeus nov. sp. cultured in an aqueous nutrient medium under aerobic conditions until the medium exhibits considerable antibacterial activity as a result of the production of antibiotic AM 374. 3. The method according to claim 2, characterized in that the antibiotic produced AM 374 isolated from the medium and optionally converted into a salt or hydrolyzate. · In that it contains 4 or animal feed -tränkmittel, characterized in that as an inhibitor for the growth of gram-positive bacteria such as identified in claim 1. A compound AM 374 or derivatives thereof. 5. Animal feed balanced in terms of nutrients, characterized in that it is as identified in claim 1 Antibiotic AM 374 or derivatives thereof. 6. Animal feed or animal drinker, characterized in that it contains the antibiotic produced according to the method of claim 2 and / or 3 as an inhibitor for the growth of gram-positive bacteria. 7. Nutritionally balanced animal feed, characterized in that it contains antibiotic AM 374 or a derivative thereof produced by the method of claim 2 and / or 3. ORIGINAL «Λ Blank page