DK143036B - METHOD OF PREPARING METABOLIT A-27106 OR THE ACID, AMMONIUM, LITHIUM, POTASSIUM, RUBIDIUM OR CAESIUM FORMS THEREOF - Google Patents

Description

(11) PUBLICATION 143036 C 12 P 19/58 DENMARK (51) int. ci.3 c 07 G 3/00 C 07 G 11/00 C 07 H 15/26 // A 23 K 1/18 (21) Application No. 51 ^ / 7 ^ (22) Filed on J1. Jan. 197 ^ · vSfctS «(24) prayer day 51 · jan. 197 ^ ν '(44) The application submitted and the petition published on 1 6th March. 1 98l

DIRECTORATE OF

PATENT AND TRADEMARKET SYSTEM (30) Priority requested from it

Feb 1 1973, 328586, US

<71> ELI LILLY AND COMPANY, 7 East McCarty Street, Indianapolis,

Indiana, US.

(72) Inventor: Donald Ray Brannon, R.R. 1 Box 201B, Pittsboro, Indiana, US: Donald Roy Horton, R.R. 2 Box 138M, Brownsburg, Indiana, US · (74) Plenipotentiary:

Hofman-Bang & Boutard Engineering Company.

(54) Process for the preparation of metabolite A-27106 or the acid, ammonium, lithium, potassium, rubidium or cesium forms thereof.

The present invention relates to a method for preparing the novel metabolite A-27106 having the physical data or the acid, ammonium, lithium, potassium, rubidium or cesium form thereof as claimed in the preamble of claim 1, of the characterizing part of claim 1.

The present compounds can be used to prevent or treat coccidiosis in poultry by administering to the animals an effective amount of a mixture consisting of metabolite A-27106 or one of the aforementioned derivatives thereof and a physiologically acceptable carrier. They may further be used to increase the efficiency of food utilization in ruminants having a developed ruminant function by oral administration of a propionate increasing amount of metabolite A-27106 or one of the aforementioned derivatives thereof to such animals.

Coccidiosis is a well-known protozoal disease that results from infection with one or more species of Eimeria or Isospora (see Lund and Farr in "Diseases of Poultry," 5th ed., Biester and Schwarte, eds., Iowa State University Press, Ames, la., Pages 1056-1096).

Given the large financial losses from coccidiosis and the difficulties in using some of the known coccidiostats, the investigation for better coccidio states continues.

Since ruminants are animals of economic importance, an increase in ruminants' food utilization is a very desirable thing. The mechanism for the utilization of the nutritional portion (carbohydrates) in the ruminant's feed is well known. Microorganisms in the animal's stomach turn carbohydrates into monosaccharides, and then these monosaccharides are broken down into pyruvate compounds. These are metabolized by microbiological processes to form acetates, butyrates or propionates, collectively known as volatile fatty acids (VEA).

The relative effectiveness of utilizing the VEAs is discussed in Feedstuffs, June 19, 1971, page 19; Eskeland et al., J. An.

Sci. 33 »282 (1971)» and Church et al., "Digestive Physiology and Nutrition of Ruminants", Vol. 2, 1971 »pages 622 and 625. Although acetates and butyrates are utilized, propionates are used with a relatively better efficiency. When too little propionate is available, the animals can develop ketosis. A beneficial agent increases the production of propionate from carbohydrates, thereby increasing the carbohydrate utilization efficiency and also reducing the incidence of ketosis.

Monensin from which metabolite A-27106 is prepared is described in U.S. Patent No. 3,501,568 as a factor A of the antibiotic A 3823 complex. Monensin is also an anticoccidial agent.

The biologically active agent produced by the process of the invention is called arbitrary metabolite A-27106. The term

3 H303G

MA-27106 "herein refers to the sodium salt.

One of the starting materials for the preparation of metabolite A-27106 is monensin, whose sodium salt has the formula: CHa

in CHa sK

vi a * · ........ (i E y I CHa

HaCO-CH H O ”

c * C

I No CHa

Monensin in its sodium form is produced by Streptomyces cinnamo- nensis as described in U.S. Patent No. 3,501,568.

Metabolite A-27106 is prepared as claimed in claim 1, characterized by monensin in the presence of glucose by an enzyme or enzymes developed by a new strain of Streptomyces candidus.

A culture of the new strain has been deposited with no restrictions on availability in the permanent culture collection at the Northern Utilization Research and Development Division of Agriculture Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois 66060 assigned the accession number NRRL 5449.

Due to the uncertainty of taxonomic studies of the Streptomyces group of organisms, there is always a question of doubt blinded to the classification of newly discovered organisms. However, in its most important characteristics, the organism NRRL 5449, which converts monensin to metabolite A-27106, is most similar to Streptomyces candidus (Krassil-nikov) Waksman 1953. Waksman in "The Actinomycetes," vol. II, Williams and Wilkins, Baltimore, 1961, page 187. The type organism is also deposited with the Institute of Microbiology of Rutgers University, New Brunswik, NJ, under the accession number IMRU 3416. The present organism is considered as a new strain of the described organism. Although S. candidus NRRL 5449 and the known strain are similar in general mycelial morphology and color and in trace appearance, there are sufficient differences between them to claim that the organism described herein is a new strain. For example, the previously described organism does not coagulate milk and makes gelatin slowly flow, while the new strain forms whey after 14 days and does not make gelatin liquid in 21 days. What is more important is that a sample culture propagated from strain IMRU 3416 does not convert monensin to metabolite A-27106.

The organism converting monensin to metabolite A-27106 was isolated from a soil sample collected on Mount Ararat, Turkey, by suspending parts of the soil in sterile distilled water and removing the suspension on nutrient agar. The sown agar plates were incubated at 25 - 35 ° C until visible colonies were observed. At the end of the incubation period, colonies of selected organisms were transferred using a sterile platinum needle to agar slant substrates.

One of the oblique substrates was then incubated to obtain a suitable amount of seed material of the organism NRRL 5449.

Taxonomic studies of S. candidus NRRL 5449 were performed using methods recommended for the International Cooperative Project for the Description and Deposition of Streptomycetes in accordance with procedures described by Shirling and Gottlieb, "Methods for Characterization of Streptomyces Species", International Bulletin of Systematic Bacteriology 16 313-340 (1966), as well as other supplementary tests. The prefix ICP refers to media described by Shirling and Gottlieb. The remaining media is described by Waksman, cited above. The color names are listed according to Kelly and Judd in "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names", U.S. Department of Commerce circular 553, 1955. The letters in brackets refer to the color series described in "System of Color" Wheels for Streptomyces Taxonomy ", Appl. Microbiol.

11, 335 (1963). Color label designations are underlined, and Maerz and Paul ("Dictionary of Color", McGraw-Hill, N.Y., 1950) color blocks are indicated in angled brackets.

Streptomyces NRRL 5449 is characterized by straight to corrugated sporophores and oval to slightly cylindrical spores, with an average dimension of 1.165 µm x 0.57 µm, in chains of 10-50. The traces 5 143036 are smooth when observed by means of an electron microscope. Air mycelia are usually white. At 26 ° C only vegetative growth occurs and at 45 ° C no growth occurs. The maximum growth and sporulation occurs at 30 - 27 ° C.

According to standard methods, the growth of the microorganism NRRL 5449 was studied on a wide variety of media accepted by the study of Actinomyceter. The cultivation methods were uniform and standardized. The media used and the cultural characteristics observed are listed below: IGP 1: Reasonable growth; pale yellow back [1101]; no aerial mycelium or spores; no soluble pigment.

IOP 2: Abundant growth; reverse light yellow [10J3]; ample air mycelium or spores; (W) white a; no soluble pigment.

IOP 5: Good growth; reverse light yellow-green [1001]; good aerial mycelium and spores; (W) white a; no soluble pigment.

TCP 4: Good for ample growth; reverse moderately yellow [10H4]; good for abundant aerial mycelium and spores; (W) white a; brown soluble pigment.

IPO 5 ·· Abundant growth; reverse moderate orange-yellow [1016]; abundant aerial mycelium and spores; (Y) pale yellow 2db; light brown soluble pigment.

ICP 1: Good growth; reverse light yellow-green [10B1]; good growth mycelium and spores; (W) white a; no soluble pigment.

Glyoerol-glycine:

Abundant growth; medium brown reverse [11J4]; abundant sporulation and aerial mycelium; (W) white a; (Y) pale yellow 2db; a bit of light brown soluble pigment.

Emerson's:

Abundant growth; greyish-yellow back [12H3]; no aerial mycelium or spores; no soluble pigment.

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Bennett1 s:

Good growth; light yellow tag page [1112]; sparse aerial mycelium and spores; (W) white a; no soluble pigment.

Ozapek's:

Abundant growth; moderate orange-yellow back [HJ7] j abundant aerial mycelium and spores; (W) white a; no soluble pigment.

Glucose-Asnaragin:

Reasonable for good growth; hawg yellowish green back [10B1]; no aerial mycelium or spores; no soluble pigment.

Calcium:

Abundant growth; reverse grayish yellow [11E4]; abundant aerial mycelium and spores; (Y) pale yellow 2ba; a small amount of brown soluble pigment.

nutrient;

Good growth; reverse pale yellowish-green [10C1]; no aerial mycelium or spores; no soluble pigment.

The organism was studied for selected physiological properties according to standard methods. The properties and characteristics observed are as follows:

Observed property Characteristics

Effect on skimmed milk Clearance; curd after 14 days.

Nitrate reduction Positive

Melanin pro ducti on

Trypton yeast extract Rings

Tyrosine Agar None

Gelatin transfer None after 21 days

Temperature requirements 26 ° C: Vegetative growth only 30-37 ° C: Good vegetative growth; good air mycelium and spores 43-55 ° C: No growth.

7 U303G

The results of carbon utilization tests performed with the organism NRRL 5449 are given below. The symbols used to indicate growth are the following: + = Exploitation - good growth [+] = Probable utilization - poor growth - [-] = Possible utilization - little or no growth.

= No utilization - no growth.

Carbon Source Response raffinose + D-fructose [+] cellobiose [+] 1-arabinose [+] D-mannitol [+] rhamnose [+] cellulose dextrose [+] to [-] D-xylose [+] inositol + -C ( not carbohydrate) [-] to [+]

The culture medium used to grow S. candidus NRR1 5449 can be any of a number of media. For the sake of economic production, optimal yield and easy isolation of the product, certain culture media are preferred. Thus, among the preferred carbohydrate sources for large scale fermentation are invert sugar or corn syrup, although glucose, fructose, maltose, starch, inositol and the like can also be used. For monensin to be converted to metabolic A-27106, the medium must contain a glucose source to achieve efficient conversion. When S. candidus NRRL 5449 is grown to produce its enzyme system for later use in converting monensin to metabolite A-27106, glucose may be present during the fermentation if desired. Preferred sources of nitrogen are peptone, soybean meal, amino acid mixtures and the like. Among inorganic nutrient salts that can be incorporated into the culture media are usually soluble salts which can provide ions such as iron, sodium, potassium, ammonium, calcium, phosphate, chloride, carbonate and the like.

Essential trace metals needed for the growth and evolution of the organism must also be present in the culture medium.

Such trace metals are usually present as impurities in the other constituents of the medium in amounts sufficient to meet the organism's growth requirements.

The initial pH of the culture medium can be varied. However, before inoculation with the organism, it is desirable to adjust the pH of the culture medium to ca. 5 »7-7.5, depending on the medium used. As with other Actinomycetes, the medium gradually becomes more alkaline as the fermentation progresses, and can rise from an initial pH of approx. 5 * 9 to pH 6.9 or higher during the growth period of the organism. The final pH is determined at least in part by the initial pH of the medium, the buffers present in the medium and the time allowed for the organism to grow.

Although pH can be regulated by the addition of either acid or base, good results have been obtained without any pH adjustment.

Like other Streptomyces species, the organism requires NKR1 5449 aerobic growth conditions. Small volume propagation is conveniently carried out on agar slant substrates or plates, in shake flasks or in flakes. For large scale production, submers aerobic cultivation in large containers is preferred.

The fermentation medium in a sterile container can be seeded with a sporulated suspension to start fermentation. However, as grafting with a sporulated suspension causes a growth delay, a vegetative graft material is preferred. This is accomplished by inoculating a small volume of culture medium with spores from mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative grafting medium is then transferred to a large container. The medium used for growing the vegetative seed material may be the same as that used for large scale production, but other media can of course be used.

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However, the organism S. candidus NRRL 5449 can be grown in a temperature range between 26 and 40 ° C.

As is customary with aerobic submergence, sterile air can be blown through the culture medium during fermentation. In order to achieve efficient growth of the organism and production of metabolite A-27106, the volume of air used in the container must be above approx.

0.1 volume of air per volume of culture medium per minute. Optimal yields are obtained when the volume of air used is at least one-third to one-half the volume of the culture medium per liter. minute.

The fermentation time required to convert monensin to metabolite A-27106 varies. The presence of a sufficient amount of glucose is essential for the conversion of monenslon to metabolite A-27106. Generally, when glucose is present in sufficient amounts and monensin is present at a concentration of 0.1 - 1.0 gram per gram. liter of the medium, the conversion of monensin to metabolite A-27106 is substantially complete in 36-72 hours. The optimal conversion occurs when monensin is present at a concentration of 0.5 - 0.7 grams per minute. liter of medium. A sufficient amount of glucose is 2.0 to 2.5% by weight of the medium. A glucose sensitive paper can be used to analyze the concentration levels.

When the glucose content falls below 2 #, glucose must be added to keep the concentration at the optimum value.

It is particularly advantageous according to the invention that the source of monensin is a sterilized culture medium in which monensin is produced by the cultivation of Streptomyces cinnamonensis under submerse aerobic conditions. Since monensin must in all cases be produced by such a fermentation process, it is much faster and cheaper to simply sterilize the culture medium in which the monensin is produced, to inactivate the original organism and then add more stirring agents to the sterilized medium and use it as a culture medium. for the new cultivation of Streptomyces candidus NRRL 5449.

When the S. candidus enzyme system is used to convert the monan to metabolite A-27106, it is essential that the enzyme preparation is extremely pure. For example, the filtered fermentation liquid can be lyophilized and stored for up to 2 weeks prior to reconstitution with an aqueous buffer, using approx. one-sixth of the volume of the original medium. Effective conversion will be achieved after 72 hours when 2.5 g of such lyophilised preparation is reconstituted in the presence of 25 mg of monensin and a sufficient amount of glucose

That active enzyme system is present in "both the filtered liquid and the cells. An enzyme preparation of greater purity can be obtained from the separated yeast cells. The cells can be frozen and stored for periods, at least three months. The thawed cells can then The buffer suspension is further purified by sonication and centrifugation. The purified cell walls are resuspended in buffer and dialyzed. 25 mg monensin for metaholite A-27106.

The conversion process can be followed by thin layer chromatography. On silica gel (sold under the designation "F-254" by EN Laboratories, Inc. Elms-ford. NY) in benzene / methanol (7-3), the Rf value for monensin is 0.62, while the value for metaholite A -27106 is 0.49. A vaniline spray reagent can be used for detection. This reagent is prepared by adding fuming sulfuric acid (2 ml) to a solution of vanillin (3 g) in absolute ethanol (100 ml).

Metaholit A-27106 is present in both the culture fluid and the mycelium.

Therefore, the technique used to isolate metaholite A-27106 is designed to allow maximum recovery of the product from one or both sources. For example, the fermentation medium is filtered and both the filtrate and the mycelial cake are extracted with appropriate solvents to give metaholite A-27106. The product is recovered from the extraction solvents by known methods.

Alternatively, the solids from the culture medium comprising media constituents and mycelium may be used without extraction or separation, but preferably with the removal of water from the mycelium and the culture medium as a source of metaholite A-27106. For example, the culture medium can be dried by lyophilization and mixed in the feed. Likewise, the solids can be converted without total removal of water into a thin slurry suitable for addition to wet mask and similar feed materials.

No specific extraction / isolation method is required. In a satisfactory manner, the final culture medium is filtered using a filtration aid. The filter cake is extracted with a polar solvent such as methanol. The methanol extract is concentrated and added to the original aqueous filtrate. This combined solution is extracted twice with half the volume of chloroform. The chloroform extracts are evaporated in vacuo to give a dark oil.

This is decolorized over a column of activated charcoal using chloroform and ca. 20 g of activated carbon per grams of oil. The eluate is concentrated again in vacuo to give a pale yellow to colorless oil. This is dissolved in a minimal amount of chloroform, chromatographed on a silica gel column using ethyl acetate as the solvent. The elution is followed by thin layer chromatography. Impurities are eluted with ethyl acetate. Elution with ethyl acetate / methanol mixtures yields metabolite A-27106.

Metabolite A-27106 (sodium salt) is a white crystalline solid, with a melting point under bubbling at 170 - 175 ° C. Metabolite A-27106 appears to form a hydrate or other solvate very easily. When A-27106 is hydrated or solvated, its melting point varies, generally melting a few degrees lower than the stated value.

Elemental analysis of metabolite A-27106 gave the following percentage composition: 58.78% carbon, 0.51% hydrogen, 27.85% oxygen and 5.65% sodium. These values are correlated with the empirical formula C ^ g ^ iOigNa having the following theoretical composition: 59.00% carbon, 8.37 # hydrogen, 29.94 4> oxygen and 2.42 $ sodium.

Metabolite A-27106 has virtually no UV absorption above 235 µm.

The infrared absorption spectrum of metabolite A-27106 in chloroform is shown in the drawing. The discernable absorption maxima in the spectrum are as follows: 3.1, 3.36, 6.39, 6.82, 7.1, 7.25, 7.9 (shoulder), 8.1, 8.3, 8 , 67, 8.8 (shoulder), 9.04, 9.22, 9.51, 9.66, 10.03, 10.26, 10.66, 11.23, 11.47, 11.83 and 12.15.

The mass spectrum of metabolite A-27106 shows a molecular ionic peak and other characteristic peaks as listed below: —___ g / e_

Calculated Observed Fragment 854,46230 854,44630 C42H? 10l6Na (M +) 836.45229 836.44129 ° 42H69 ° l5Na (m + -h2o) 779.45459 779.4690 C4 () H68013Na (m + - [ch3o + co2]) 44540 761.4740 C40H66 ° 12Na (m + [ch5o + co2 + h2o])

These findings confirm the assumed empirical formula and a molecular weight of 854 for metabolite A-27106.

In general, metabolite A-27106 is readily soluble in highly polar solvents, is insoluble in non-polar solvents, and varies in solubility in solvents with polarity therebetween. By way of illustration, A-27106 is soluble in lower aliphatic alcohols, is partially soluble in phenol, diethyl ether and acetone and is relatively insoluble in liquid lower alkanes.

Electrometric titration of metabolite A-27106 in acid form in water at an initial pH of 8 indicates the presence of a titratable group having a p value of 7.2.

Bones described above monosodium form are generally the natural form of metabolite A-27106. The sodium salt can easily be prepared from the acid. From the acid, the ammonium and other alkali metal salts of this invention can be prepared. The various metal salt forms behave somewhat similar to alkali metal carboxyl species and somewhat similar to chelates.

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In preparing another form, metabolite A-27106 (in sodium form) is dissolved in an aqueous solvent such as methanol / water; an acid such as hydrochloric acid 'is added to lower the pH to 5 or lower. The methanol is removed under vacuum and the resulting aqueous acid is extracted with chloroform. The chloroform extract is dried and evaporated to give the free acid form. Dennp may be used as such or may be further modified by titration with an aqueous alkali metal hydroxide or aqueous ammonia to obtain the corresponding lithium, potassium, rubidium, cesium or ammonium forms. The acid, ammonium and alkali metal forms are all biologically active.

The exact structure of metabolite A-27106 is not known. It has been found that glucose is necessary for the conversion of monensin to metabolite A-27106 and is consumed even in the absence of metabolizing S. can-didus cells. The molecular weight of A-27106 corresponds to the molecular weight of a glucosylmonensin.

in addition to the highly sterically hindered tertiary hydroxy group on the E-ring, there are five hydroxy sites on a glucosylmonensin, all of which can react to form a simple ester such as eb acetate. The presence of all five such reactive sites is demonstrated by esterification experiments.

Based on the physical characteristics listed above, the following structure can be proposed for metabolite A-27106: CH a. CH 3 / S? · * 8 \ C / \ D / CHa)

£ ® \) - (Formula II

, Na: ........ E y kAJ-OH a / y un a

HaC-CH I / H

in CHa /

HaCO-CH H o “0 (glucose) ^ C - οζ I ^ 0 CHa

Since the structure is only postulated, it is clear that the structure stated above is only a working hypothesis.

14- 143036

Metabolite A-27106 is less toxic than monensin. In trials in which metabolite A-27106 was administered intraperitoneally to groups of every 6 mice, one in six died at a dose of 50 mg / kg and 3 in six at a dose of 100 mg / kg. In similar trials where monensin was administered intraperitoneally to groups of every 6 mice, one in six died at a dose of 10 mg / kg and 3 in 6 died at a dose of 20 mg / kg.

To prevent or treat coccidiosis in poultry, a nontoxic, coccidiostatic amount of metabolite A-27106 'is administered daily to the birds, preferably orally. Although a wide variety of factors must be taken into account when determining a suitable concentration of A-27106, the amount administered will generally be in the range of 0.005 to 0.05% by weight of the feed and preferably in the range of 0.01 - 0.04 %. Metabolite A-27106 can be added in many ways, but is most conveniently added together with a physiologically acceptable carrier, preferably to the feed eaten by the birds.

Metabolite A-27106 also improves the food utilization of ruminants who have a developed ruminant function. Young ruminants, especially those who are not accustomed, act as monolingual animals. As young ruminants start eating solid food, ruminant function begins to develop and the microbiological population in the stomach begins to increase. After the animal has eaten solid food for a period of time, its stomach function reaches full development and continues to function throughout the animal's life. Some economically important ruminants are cattle, sheep and goats.

Metabolite A-27106 is typically effective in increasing the efficiency of food utilization when administered orally to ruminants in an amount of from 0.05 mg / kg / day to 2.5 mg / kg / day. Most favorable results are obtained in an amount of 0.1 - 1.5 mg / kg / day. A preferred method of administering metabolite A 27106 is to then mix with the animal's feed. However, it can also be administered in other ways, e.g. in the form of tablets, capsules as cattle medicine or as large pills. The composition of these various dosage forms can be carried out by well known methods. Each dose unit should contain an amount of metabolite A-27106 which is directly proportional to the appropriate daily dose for the animal to be treated.

The following examples illustrate the method according to the invention.

EXAMPLE 1

Preparation of A-27106 from Monensin by S. candidus NRRL_5449________________________________________________________ S. candidus NRRL 5449 was grown on an agar substrate made from Bennett's medium to obtain a well-defined colony. This was removed and slurried with sterile water (10 ml).

This slurry was divided into four 500 ml shake flasks, each containing 100 ml of medium of the following composition:

Ingredient Amount of "Distillers' solubles" from distillation of fermented corn * 25.0 g

Lactose 10.0 g

Maltose 10.0 g

FeSO4.7H2 O 0.01 g

MgSO4.7H2O 2.0 g KH2PO4 2.0 g

CaCO 2 2.0 g

Ion-exchanged water for 1, 1 liter H Available under the name "Nadrisol" by National Distiller's Products Company, U.S.A.

The four seeded flasks were incubated at 30 ° C in a rotary shaker at a speed of 250 rpm for 24 hours. The vegetative medium (10 ml portions) was used to seed 15 shake flasks (500 ml), each containing 100 ml sterile fermentation medium of the following composition:

Quantity of Ingredients

Beef echo extract 5 g

Casein panereatic hydrolyzate peptone 5 g

NaCl 5 g

Glycerol 15 g

CaCO 3 2 g

Ion-exchanged water to 1 liter

The medium had a pH of 7.0 which was not controlled. The seeded medium was incubated for 72 hours as described above.

In a 40 liter fennentor, a production medium of the following composition was prepared:

Quantity of Ingredients

Polysiloxane oil antifoaming agent 5 g

Glycerol 375 g

Glucose 625 g

Casein pancreatic hydrolyzate peptone 125 g

Beef extract 125 g

NaCl 125 g

CaCO 50 g

Ion-exchanged water for 24 liters

The initial pH of the medium was 7.0. The medium was sterilized by autoclaving at 120 ° C for 30 minutes at a pressure of 103 - 138 kPa.

After sterilization, the pH of the medium was 7.6.

25 g of purified monensin was dissolved in 200 ml of ethanol and added to the sterilized medium and the second stage vegetative inoculum (700 ml) prepared as described above was introduced.

The fermentation medium was aerated with sterile air at a rate of approx. 10 liters per and stirred with a conventional stirrer at a rate of 420 rpm. minute. The seeded medium was incubated at 30 ° C for 114.5 hours.

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The fermentation process was followed by thin layer chromatography on silica gel as described above. Early in the fermentation, only monensin was present. Gradually, another stain indicated the presence of metabolite A-27106, and finally only the stain of metabolite A-27106 was present.

Isolation and purification of metabolite A-27106

The fermentation liquid prepared as described above was filtered using filtration aids. The mycelial cake was extracted with methanol (about 5 liters) at room temperature. The methanol extract was filtered and the filtrate was concentrated under vacuum to remove methanol and to give an aqueous concentrate.

This was combined with the original filtrate. The combined solution (about 22 liters) was extracted twice with half the volume of chloroform. The chloroform extracts were combined and concentrated in vacuo to give 500 ml of a dark amber oil.

This was dissolved in chloroform and decolorized over a 10 kg carbon column 30.5 x 101.6 cm (the carbon sold by Pittsburg Activity Co., Division of Calgan Corp.) eluting with chloroform (5 liters). The chloroform eluate was evaporated in vacuo to give 500 ml of a colorless to pale yellow oil.

The decolorized oil was chromatographed in a minimal amount of chloroform over a 25 kg column of silica gel (sold under the designation "Grade 62" by W.R. Grace & Company) in ethyl acetate. The elution was followed by thin layer chromatography as described previously. After the impurities were removed with ethyl acetate, metabolite A-27106 was eluted from the column using ethyl acetate / methanol (19: 1). The fractions containing metabolite A-27106 were combined and evaporated to dryness in vacuo to give an amorphous, almost white, substance. This was washed with hexane and dried to give 12.84 g of metabolite A-27106 (one spot by thin layer chromatography).

EXAMPLE 2

Preparation of Metabolite A-27106 by S. cinnamonensis and S. candidus NRRL 5449

Another method for preparing metabolite A-27106 in fermentor culture is illustrated by the following procedure:

Streptomyces cinnamonensis ATCC 15413 was conventionally grown (see U.S. Patent No. 3,501,568) in 55 ml of medium in a 250 ml shake flask during incubation for 47 hours to obtain a vegetative inoculum. This was added to 220 ml of medium in a 1 liter flask and incubated for 21 hours to obtain an inoculum for inoculation of the fermentor.

The inoculum thus prepared was used to seed a 40 liter fermentor containing a heat sterilized medium of the following composition:

Quantity of Ingredients

Glucose 750.0 g

Soybean meal 625.0 g

Soybean oil 500.0 g

Methyl oleate 500.0 g

Eolysilox oil anti-foaming agent 5.0 g

Potassium chloride 2.5 g

Dicalium hydrogen phosphate 2.5 g

Manganochloride, tetrahydrate 15.0 g

Hydrated ferric sulfate 7.5 g

Calcium carbonate 25.0 g

Ion-exchanged water for 24 liters

The resulting medium had a pH of 5.5 which was adjusted to pH 8.0 by the addition of 10 N potassium hydroxide o solution (15 ml). The seeded medium was incubated at 32 ° C for 234 hours.

19 1Λ 3 O 3 6

After 42 hours the aeration increased from approx. 10 to approx. 23 liters per and stirring increased from 500 to 700 rpm. minute. Monensin production was substantially complete after 210 hours as determined by thin-layer bioassays with Bacillus subtilis ATCC as a detection organism.

After 234 hours, the fermentor content was pasteurized to inactivate S. cinnamonensis. Wavy nutrients were added to the fermented impure:

Quantity of Ingredients

Glucose 750 g

Soybean meal 625 g

Manganochloride, tetrahydrate 155 g

Calcium carbonate 12.5 g

Eerrisulfate, hexahydrate 7.5 g

Potassium chloride 2.5 g

Eicalium hydrogen phosphate 2.5 g

Methyl oleate 250 ml

Soybean oil 250 ml

Ion-exchanged water to 24 L of pH was adjusted to 8.9 with 215 ml of 5 N sodium hydroxide solution and the medium sterilized. The medium was seeded with a fast growing vegetative culture of Streptomyces candidus NRRL 5449 and incubated for 137 hours at 30 ° C. The fermentation was aerated with sterile air at a rate of 10 liters per minute. The fermentation medium was stirred with a conventional stirrer, first at 120 rpm. increasing after 16 hours to 420 rpm. and after 40 hours to 500 rpm. minute. 400 g of glucose was added after 44, 66.5, 89, 97, 113 and 127 hours. 175 g of calcium carbonate was added after 72 and 99 hours.

Thin layer chromatography as described above was used to follow the fermentation. After 137 hours of production of metabolite A-27106, this was substantially completed. Work-up and purification of metabolite A-27106 followed the method described in Example 1.

EXAMPLE 3

Preparation of metabolite A-27106 by a special S. can-didus NRRL 5449 enzyme

Preparation S. candidus NRR1 5449 was grown as described in Example 1 on a 100 liter scale. The cells were separated from the fermentation medium by vacuum filtration and divided into 200 g of samples stored by freezing.

Two of these samples were thawed at room temperature and suspended in 0.05 N phosphate buffer (pH 5.8) to a volume of 600 ml. This cell suspension was subjected to ultrasound for 30 minutes and centrifuged at 10,000 rpm. 30 minutes. The resulting cell residue was suspended in 100 ml of the above buffer, and the suspension was dialyzed for 18 hours with 5 liters of cooled buffer. To 50 ml of the particular dialysate was added D-glucose (120 mg) and 25 mg of monensin in 2 ml of ethanol. The reaction mixture was stirred for 72 hours at 30 ° C and filtered. The filtrate was extracted with chloroform (100 ml). The chloroform extract, after concentration in vacuo, was chromatographed on a silica gel column (10 g). Elution with ethyl acetate gave only a trace of monensin. Elution with ethyl acetate / methanol (19: 1) gave 17 mg of metabolite A-27106, which was identical to that obtained in Example 1.

EXAMPLE 4

Acid form of metabolite A-27106 100 mg of metabolite A-27106 prepared in sodium form by the procedure described in Example 1 was dissolved in 100 ml of methanol / water (1: 1).

The resulting solution was titrated to pH 3 by dropwise addition of 1N hydrochloric acid. The methanol was removed under vacuum.

The resulting solution was extracted twice with chloroform (200 ml each time). The chloroform extract was dried over magnesium sulfate and evaporated in vacuo to give 85 mg of the acid form of metabolite A-27106.

21 143036

The acid form of A-27106 is a white, amorphous substance, with a molecular weight of approx. 832. The biological activity of the acid form is almost the same as that of the sodium form.

EXAMPLE p

Control of Ooccidiosis by Metabolite A-27106 ------- 1 -------------- f -------- ~ ----------- -

A group of 75 one week old, healthy, incubator and battery-raised males of a severe broiler type were used. The birds are divided into groups of 15 birds with three repeated 5-bird subgroups. Each subgroup was kept out of touch with the other subgroups. A first group was kept as untreated healthy controls under favorable conditions. Another group was treated as the first group but inoculated with poccidiosis by oral administration of a dose containing 10 ^ sporulated oocysts of Eimeria tenella. The third, fourth and fifth groups were treated as the second group with the exception that 24 hours before this inoculation, their feed was altered by the addition of metabolite A-27106 at concentrations of 100, 150 and 200 ppm.

Seven days after inoculation, the birds were weighed, slaughtered and examined for the presence of coccipial lesions. The coccidial damage is expressed on an arbitrary scale from 0 to 4. The lesion score indicates the number of birds at each level. 0 indicates no evidence of ooccidiosis. 1 denotes the smallest coccidial damage that can be observed. 2 denotes moderate damage with little or no bleeding and no damage to the tissue. 3 indicates bleeding, a swelling of the appendix and a widespread destruction of the tissue. 4 indicates hemorrhage, a cochlear appendage of clotted blood and damaged epithelial cells. Birds with a grade of 4 or less are usually cured if they are not further infected with coccidia.

Using the above method, the following results were obtained: 22 U3036

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The treated lesions with the high lesion characteristics had little or no fecal blood and the birds appeared to be in good condition. Their good weight gain is considered more meaningful than the lesion characters.

Since studies indicate that metabolite A-27106 usually kills E. tenella when first trying to establish itself in the host cell, a prophylactic treatment method is preferred.

In other studies, metabolite A-27106 was found to be more palatable to birds and less toxic than monensin.

Several other studies were performed with metabolite A-27106 both alone and in combination with monensin. These studies showed that although metabolite A-27106 did not give any greater weight gain at 0.04% than at 0.01%, the greater amount resulted in fewer appendicitis lesions. There was no trace of toxicity at the highest dose.

Within the generally used range, monensin and metabolite A-27106 combined in the diet show at least one additive effect, but not in toxicity. The toxicity of the combination is lower than the toxicity of monensin alone in the amount corresponding to the combined amount.

EXAMPLE 6

Improved reuse utilization by metabolite A-27106

Stomach juice is obtained from a stud with a surgically installed fistulQ opening into the stomach. The stud is kept on a feed ration, the composition of which is as follows; 69.95% coarsely ground corn 10% ground corn cobs 8% soybean meal (50% protein) 5% alfalfa flour 5% molasses 0.6% urea 0.5% dicalcium phosphate 24 1 / 4303b 0.5 f calcium carbonate 0.3 $ Salt 0.07 $ Vitamin A and D2 "Mixture * 0.05 $ Vitamin E Mixture ** 0.03" $ Trace Mineral Mixture *** * Containing Per Kg: 4,409,000 Int Unit Of Vitamin A, 500 goo per unit of vitamin D2 and 850 g of soybean meal with 1% oil.

xx Distillation residue of fermented maize containing 44 000 int. U. α-tocopheryl acetate per kg.

xxx Containing manganese (II) oxide, potassium iodide, cobalt carbonate, copper oxide and zinc sulfate.

A sample of the gastric juice is pressed through a cheese cloth filter layer and the filtrate is collected. The particulate material remaining on the cheesecloth is resuspended in sufficient physiological buffer to obtain the original volume of gastric juice and this suspension is filtered again. The buffer used has the following composition: g / liter Ingredient 0.316 Na2 HPO4 0.152 kh2po4 2.260 NaH003 0.375 EC1 0.375 NaCl 0.112 MgSO ^ 0.038 CaCl

0.002 CUSO4.5H2O

0.001 CaCl2 as described by Cheng et al. 1 J. Dairy Sci. 38, 1225 (1955).

The two filtrates are combined and allowed to stand until particulate matter is deposited on top. The clear phase is separated, diluted with the same buffer (1: 1) and taken to pH 7.0.

The diluted gastric juice (10 ml) is placed in a 25 ml flask with 40 mg of the above feed and an additional 5 mg of soy protein and the compound tested. Four sets of flasks are used per treatment.

Two sets of four control flasks are also used. A zero-time check and a post-incubation check for 16 hours are used. All sample flasks are incubated for 16 hours at 38 ° C. After incubation, pH is measured and 25% metaphosphoric acid (2 ml) is added to each flask. The samples are allowed to settle and the supernatant is analyzed by gas chromatography for propionate, acetate and butyrate components. The active compound significantly increases propionate production relative to the control samples.

The test compound results are statistically compared with the control results. The table below shows the ratio of volatile fatty acids in metabolite A-27106-treated flasks to the concentration in the control flasks.

yug metabolite A-27106 / ml diluted gastric juice Propionate Butyrate Total volatile fatty acids 5 2.15 0.73 1.03 1 1.42 0.96 0.98 0.2 1.02 0.91 1.07

Claims (1)

26143038 Patent Claims: 1. A process for the preparation of metal o A-27106 or the acid, ammonium, lithium, potassium, rubidium or cesium form thereof, which metabolite is a white crystalline solid which is relatively soluble in lower alkanols, but generally insoluble in lower alkanes, having (a) a molecular weight of 854 °, as determined by mass spectrometry; (b) an approximate elemental composition of 58.78 fo carbon, 8.51 fo hydrogen, 27.85 fo oxygen and 5.63 fo sodium; (c) an empirical formula of Ο ^ Ηγ ^ ° 16Na; (d) a chloroform infrared absorption spectrum as shown in the attached drawing; (e) a mass spectrum showing a molecular ion peak at m / e 854.44630 and characteristic peaks at m / e 836.444129, 779.44690 and 761.44440; and (f) an Rf value of 0.49 by silica gel thin layer chromatography in benzene / methanol (7: 3); and wherein the acid form is a white solid having a molecular weight of 832 and a titratable group having a ρκ & value of 7.2, characterized. by culturing Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts, including a sodium salt, under submerged aerobic conditions, adding glucose and monensin to the culture medium before, during or after culture, or to filtered culture fluid or to cells isolated from the culture fluid after culture, to convert monensin to a substantial amount of metabolite A-27106 by the enzymes produced by the microorganism, and, if desired, the acid form of metabolite A-27106 is released by the addition of an acid and the acid , if desired, is converted into one of the salt forms specified in the preamble of the claim by reaction with a corresponding alkali metal hydroxide or with ammonia water.