CS207694B2 - Method of making the antibiotic a-40104 factor a - Google Patents

Description

The present invention relates to a process for the production of the A-40104 factor A antibiotic.

This factor is a derivative of pleuromutilin and D-xylose and my form of acetal.

The antibiotic pleuromutillin was isolated in 1951 and is described in Kvanagh et al. [Proc. Nati. Acad. Soc. 37, 570-574 (1951)]. Pleuromutillin can be represented by the following formula

Alkaline hydrolysis of pleuromutilin produces a compound known as mutllin, which can be represented by the following formula

OH

A number of pleuromutilin derivatives have been prepared (Swiss Patent No. 572,894 (Derwent No. 26553X), the Dutch patent δ). No. 69,11083 (Derwent No. 40,642), Knauseder et al., U.S. Patent No. 3,919,290, Brandl et al., U.S. Patent δ. No. 3,949,079, Riedl, U.S. Pat.

No. 979,423 to Baughn et al., U.S. Patent No. 3,987,194 to Egger et al., U.S. Pat. 4,032,530,

K. Riedl, Studies on Pleuromutilin and Some of Its Derivatives, J. Antibiotics, 29, 32-139, (1976), H. Egger and H. Relnshagen, New Pleuromutilin Derivatives, including Enhanced Antimicrobial Aetivity. I. Synthesis, J. Antibiotics 22: 915-922 (1976) and II. Struoture-Activity Correlations, ibid., 923-927 ¢ 1976), F. Knauseder and E. Brandl, Pleuromutilin Fermentation, Structure and Biosynthesis, J. Antibiotics 22, 25-131 (1976), J. Drews. et al., Antimicrobial Activitcs of 81,723 hfu, and New Pleuromutilin De rivative, Antimierob. Agents and Chemitherapy 2, 507-516 (1975) 1.

It has now been found that there are two other new antibiotics which also belong to the pleuromutilin family. In addition, a new process for the production of pleuromutilin could be proposed.

SUMMARY OF THE INVENTION The present invention provides a process for the production of antibiotic A-40104 factor A, characterized in that Clitopilus pseudopinsitus NRKL 11179 is grown in a culture medium containing usable sources of carbohydrates, nitrogen and inorganic salts in submerged culture under aerobic conditions for at least two days. After fermentation, the broth is extracted with chloroform and the A-40104 factor A antibiotic is isolated from the extract.

Physical properties of factor A antibiotic A-40104

Factor A of the A-40104 antibiotic crystallizes from chloroform and has a melting point of 17-120 ° C. The empirical formula of factor A of A-40104 is ¢ 27 ^ 42 ^ 9 '^ ^elc W ³ · ^{0 as} determined by elemental analysis, molecular weight 510 as determined by mass spectrometry.

The absorption spectrum of the A-40104 antibiotic factor A in ultraviolet light in trifluoroethanol has an absorption maximum at 284 nm (e 32).

The spectrum of factor A antibiotic A-40.40 in circular dichroism in trifluoroethanol has the following maxima:

Δε = +2.84 at 200 nm Δε = +172 at 295 nm.

Factor A of A-40104 has the following specific rotation:

[α] 25 _{D 4} ° ( _c = 1.0, C ₂ H ₅ OH).

The absence of titratable groups can be detected by electrometry by titration of Factor A with A-40140 in 66% aqueous dimethylformamide.

The absorption spectrum of KBr factor A antibiotic A-40.04 is shown in Figure 1 in the accompanying drawings. Absorption maxima can be observed at the following frequencies:

(cm ^-1 )

3,590, 3,500, 3,450 (wide), 3 280 (wide) , 2,990, 2,980, 2,960, 2,940, 2,920, 2,890, 2,860, 1,755, 1,735, 1 645, 1 465, 1 447, 1 420, 1,380, 1,330, 1,310, 1,274, 1,240, 1,220, 1,160, 1,117, 1,093, 1,070, 1 060, 1 1 040, 1 014 984 955 940 921 905 865 760 742

698, 674, 603, 530 and 442.

A-40104 is soluble in alcohols such as methanol or ethanol, partially soluble in chloroform, and insoluble in water.

According to its physical and chemical properties, the A-40104 factor A is probably a beta, D-xylopyranosylaoetone derivative of pleuromutilin with the following formula:

A strain of Clitopilus pseudopinsitus suitable for the production of the A-40104 factor A antibiotic was obtained from Centraalbures voor Schimmelcultures (CBS) in the Netherlands. CBS culture (CBS 270.36) was classified as Octojuga pseudopinsite. The generic name Clitopilus, which is synonymous with Octojuga, will be used to refer to the microorganism used. The ability of this culture to produce an antibiotic has not been known to date. This ability to produce the A-40104 antibiotic has been demonstrated in broad-based assays with various types of microorganisms.

The strain Clitopilus pseudopinsitus was deposited in the public collection of Cultures of Northern Marketing and Nutrition Research Division, US Papartmen of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, from which it can be obtained under NRRL 11179.

As with other microorganisms, the characteristics of the culture of Clitopilus pseudopinsitus NRRL 11179, which produces the A-40104 factor A antibiotic, are subject to variations. For example, various artificial variants and mutants of the NRRL 11.79 strain can be obtained by the action of various known mutagens, such as ultraviolet rays, X rays, radio waves, radioactive rays, and chemicals. All natural and artificial variants and mutants having substantially the same properties as Clitopilus pseudopinsitus NRRL, 1.79 and producing the A-40140 antibiotic can be used in the method of the invention.

however, the culture medium used for the cultivation of Clitopilus pseudopinsitus NRRL 11179 may vary widely. However, for specific reasons and in order to obtain optimum yield and for easy isolation, some particular media are preferred. For large-scale fermentation, for example, glucose, tapioca dextrin, starch and corn oil are preferred carbohydrate sources, although glycerol, maltose, fructose and other sources may also be used. Other preferred sources of carbon are oleic and lauric acid salts and lecithin. Soya flour or yeast extract are preferred nitrogen sources.

Other suitable sources of nitrogen are yeast, cottonseed flour, peanut flour, meat peptone, dried dog food. Although inorganic salts are not essential for the growth and production of the antibiotic, soluble salts containing sodium, magnesium, calcium, ammonium ion, chloride ion, carbonate ion, sulfate ion, nitrate ion, and the like can be added. 05% MgSO 4 .7H ₂ O and 0.2% CaCO 3 to the culture medium.

The essential trace elements necessary for the growth and development of the micro-organism should also be added to the culture medium. These trace elements are commonly found as impurities in other nutrients in the environment in an amount sufficient to meet the growth requirements of the microorganism.

Sometimes it may be necessary to add a small amount, i.e. about 0.2 ml / liter, of an antifoaming agent, for example polypropylene glycol, since foaming becomes a problem especially in large-scale fermentation.

To produce large amounts of the A-40140 Factor A antibiotic, it is convenient to use fermentation in submerged culture under aerobic conditions in large tanks. A small amount of the A-40104 factor A antibiotic can be obtained in shake flasks. Since the lag period is usually very long in the production of an antibiotic in large tanks using a small amount of culture as the inoculum, it is preferable to use a vegetative inoculum that contains more cells in the active growth stage.

The vegetative inoculum is prepared by inoculating a small volume of culture medium with mycelium fragments to obtain a fresh, active, growing culture of the microorganism. The vegetative inoculum is then transferred to a larger tank. The medium used for the growth of the vegetative inoculum material may be the same as for larger scale cultivation, but a different medium may also be used.

The microorganism Clitopilus pseudopinsituš which produces the A-40104 factor A antibiotic can be grown in the temperature range of 20 to 33 ° C. Optimal production of A-40104 occurs at temperatures close to 30 ° C.

As is common in aerobic fermentation in submerged cultures, sterile air is injected into the culture medium while stirring. For efficient growth of the micro-organism, it is necessary to sufficiently aerate and mix the environment so that the dissolved oxygen level is kept as close as possible to 80%. Lower dissolved oxygen levels reduce factor A production.

A suitable incubation period for the production of factor A of the A-40140 antibiotic is about 12 days. The addition of corn oil to the culture medium promotes the conversion of factor C to factor A during fermentation. Under conditions that are optimal for the production of antibiotic factor A-40104, factor A accounts for 70 to 90% of all antibiotic produced.

The filtered broth is extracted with chloroform and the A-40104 antibiotic factor A is isolated from the chloroform extract.

Efficacy of the A-40104 factor A antibiotic

Antibiotic A-40104 factor A inhibits the growth of various pathogenic microorganisms, particularly gram-positive bacteria. The minimum inhibitory concentrations (MIC) for factor A and for various bacteria are shown in the agar dilution in Table III below

Table III

MIC Qug / ml)

Microorganism A-40104 factor A

Staphylococus aureus 3055 <0.5

Staphylococcus aureus 3074 <0.5

Streptococeus faecalis <0.5

Bordetella bronehiseptica <0.5

Antibiotic A-40104 factor A is also effective against microorganisms that are resistant to other antibiotics. Table IV below summarizes the results obtained on agar plates using A-40104 factor A antibiotic discs using various microorganisms. The potency was measured as the diameter of the zone of inhibition in mm. The diameter of the disc was in any case 6.35 mm ·

IV

Zone diameter (mm)

Mikroorgani smus Concentration (jug / disk) A-40104 factor A Staphylococcus aureus 3055 ¹ 100 ALIGN! 31.2 Staphylococcus aureus 3055 ¹ 10 27.2 Staphylococcus aureus 3074 ² 100 ALIGN! 29.2 Staphylococcus aureus 3074 ² 10 25.4 Staphylococcus aureus 3130 ^ 1 00 34.0 Staphylococcus aureus 3130 ^ 10 29.2 AND, Streptococcus pyogenes 100 ALIGN! 27.0 Streptococcus pyogenes 10 21, 0 Streptococcus sp. 9960 ^ 100 ALIGN! 8.7 Streptococcus sp. 9960 ^ 10 0 Diplococcus pneumoniae 100 ALIGN! 20.0 Diplococcus pneumoniae 10 14.0

penicillin-sensitive G penicillin-G resistant methicillin resistant group A group D

It can be seen from Table V that factor A of A-40104 can be compared to a favorable result with ampicillin in a number of in vitro experiments performed using a dilution series on agar plates.

Table

k and V BALL ((Ug / ml) Microorganism A-40104 factor A Ampicillin Streptococcus pyogenes <0.01 <0.01 Streptococcus sp. X-66 (Group D) 0.5 0.8 Streptococcus sp. 9960 (Group D) 70 0.6 Neisseria gonorrhea 0.5 0.5 Neisseria gonorrhea Sand. 0.07 0.07 Neisseria gonorrhea Lind. 0.5 0.5 Hemophilus influenzae Brun ’ 2 0.5 Haemophilus influenzae Dick. ¹ 2 0.5 Hemophilus influenzae Laiv. ¹ 4 0.5 Hemophilus influenzae 251 ² 0.7 70 Hemophilus influenzae 259 ² 0.7 70 Hemophilus influenzae 260 ² 0.7 70

Antibiotic factor A-40104 has antimicrobial activity in vivo against experimental bacterial infections. Two doses were administered to mice, the observed efficacy was measured as the ^ED 50 'dose in mg / kg, which protects 50% of the test animals as described by Darren tick et al., J. Bacteriol. 81, 233-235 (1961). The values were summarized in Table VI below.

Table VI

The efficacy of factor A antibiotic A-40104

Microorganism

Staphyloeocous aureus 3055 Streptococcus pyogenes C202 Streptoooocus pneumoniae Park I

Route of administration ^ 50 * Method of infection subcutaneously 15.7 1,260 x intraperitoneally subcutaneously 3.6 195 x LDjq intraperitoneally subcutaneously 21.5 1,280 x LD ^ q intraperitoneally

A-40104 also inhibits the growth of various anaerobic bacteria.

The following Table VIII summarizes the efficacy of factor A antibiotic A-40104 as determined by a standard dilution series on an agar medium.

and b u 1 k a VIII Microgranism MIC (jug / ml) * Actinomyces israelli * 0.5 Clostridium perfringens 8 Clostridium septicum 8 Eubacterium aerofaciens 16 Peptococcus prevoti * 0.5 Peptococcus asaccharolytious * 0.5 Peptostreptococcus anaerobius 2 Peptostreptoeoccus intermedius * 0.5 Bacteroides fragilis 111 8 Bacteroides fragilis 1877 8 Bacteroides fragilis 1936B 8 Bacteroides thetaiotaomicron 2 Bacteroides melaninogenicus 1856/28 4 Bacteroides melaninogenicus 2736 4 Bacteroides vulgaris 4 Bacteroides corrodens 8 Fusobacterium symbiosum 4 Fusobacterium necrophorum * 0.5 BALL established agar dilution method. Endpoints were read after 24 hours incubation.

The LD 50 for factor A when administered intraperltoneally in mice is greater than 300 mg / kg.

Efficacy against mycoplasmas is a particularly important aspect of the antimicrobial efficacy of the A-40104 factor A antibiotic. Mycoplasmas are pathogenic to humans and various animals. There is therefore a need for active substances for the prevention and treatment of these diseases in poultry, pigs and cattle.

Table IX below shows the minimum inhibitory concentrations of Factor A, the A-40104 antibiotic against various mycoplasmas as determined by the in vitro dilution series.

Table IX

Minimum inhibitory concentration

Microorganism

A-40104 factor A

Mycoplasma galiisepticum Mycoplasma synoviae Myooplasma hyorhinia Mycoplasma hyopneumoniae <0.78 <0.78

1.56 0.195

An important aspect is the use of the A-40104 factor A antibiotic to treat swine dysentery. As explained by W. E. Brown in U.S. Patent No. 4,041,175, pleuromutilin is effective in treating swine dysentery. It has now been found that the A-40104 factor A antibiotic produced by the method of the invention is also effective against Treponema hyodysenteriae, i.e. against a microorganism that usually causes swine dysenteria. Efficacy was determined in vitro using doses of 50, 5.0, 0.5 and 0.05µg / ml on trypticesis and soybean agar plates containing 5% bovine defibrinated blood. The agar surface was inoculated with 0.1 ml of a 10 'dilution of T. hyodysenteriae suspension. Plates were incubated under anaerobic conditions for four days and then evaluated for the presence or growth of hemolytic treponematos. In this experiment, A-40104 antibiotic factor A was shown to inhibit the growth of T. hyodysenteriae at concentrations of 50, 5.0, and 0.5 µg / ml.

When used to treat swine dysentery, the A-40104 factor A antibiotic may be administered to pigs orally in the form of tablets, capsules, powder, and the like.

However, a preferred route of administration is to incorporate the A-40104 factor A antibiotic into pig feed.

In addition, the A-40104 factor A antibiotic produced by the method of the invention can be used against Pasteurella. For example, P. multocida is a transverse airway infection in cattle, poultry and pigs. P. hemolytica is the most common cause of respiratory disease in cattle. The efficacy of factor A against Pasteurella was determined by a standard in vitro method. The results are shown in Table X.

Table X

MIC Qug / ml)

Mikroorgani smus

Factor A - A-40104

Pasteurella multocida (Cattle) Pasteurella multocida (Crocodile) Pasteurella hemolytica

6.25

12.5

25.0

It is also possible to produce a pharmaceutical composition which comprises an inert carrier and as an active ingredient the antibiotic A-40104 and which is effective against pathogenic microorganisms in humans.

Another important aspect is the efficacy of the A-40104 antibiotic against Spiroplasma citri. Spiroplasma citri is the cause of diseases of citrus fruits, other epiroplasms adversely affect the growth of maize. In vitro experiments with Spiroplasma citri showed that factor A of A-40104 inhibited S. citri at doses of 0.01 ppm.

Antibiotic A-40104 factor A is also effective against organisms that cause plant diseases. For example, when applied to foliar or soil, factor A of the A-40104 antibiotic is effective against fungi in both beans and barley, especially when applied to soil where the factor A

And A-40104 antibiotics effective at a dose of 100 ppm, which means that the compounds appear to have eystemic activity. The systemic effectiveness of antibiotics is particularly advantageous in controlling organisms, plant pathogenic or Spiroplasma.

The invention will be illustrated by the following examples.

Example 1

A. Fermentation of A-40104 in shake flasks

The mature culture of Clitopilus pseudopinsitus NRRL 11179 on slant agar is collected with a sterile instrument and aseptically transferred to slant agar of the following composition:

Component Amount sucrose 25 g molasses 36 g corn extract 6 g malt extract 10 g k ₂ hpo ₄ 2 g enzymatically hydrolyzed casein * 10 g washed agar 25 g deionized water to 1 liter

* N-Z Time, Humko Sheffield, Norwich, N.Y.

The inoculated slant agar is incubated for 14 days at 30 ° C. Mature culture on a sloping p

The agar is collected with a sterile instrument and the mycelium from an area of approximately 5 cm is used to inoculate 50 ml of a vegetative medium of the following composition:

Ingredient Amount of glucose 1 0 g tapioca dextrin * ¹ 0 g powdered soy hydrolyzate ** 10 g brewer's yeast fraction *** 5 g tap water up to 1 liter pH of the environment is 5.3 and needs to be adjusted to 6.9 by adding Sodium hydroxide * Stadex 11, AE Staley, Decatur, 111.

** HySoy T. Humko Sheffield, Nerwich, N.Y.

*** Amber BYF300, Amber Laboratories, Juneau, Wisc.

The inoculated vegetative medium is incubated in 250 ml Erlenmeyer flasks at 25 ° C for 96 hours on a rotary shaker at a 5 cm pivot and 250 rpm.

The incubated vegetative medium can be used directly to incubate the vegetative medium in the second stage. Preferably, the medium may be stored for further use by storing the culture above liquid nitrogen. For this purpose, the culture is divided into small vials by placing in each vial 4 ml of the incubated vegetative medium and 2 ml of the glycerin-lactose solution of the following composition:

Component

Quantity glycerine 100 g lactose 150 g deionized water to 1 liter

The prepared suspensions are stored over liquid nitrogen until use.

ml of the thus prepared stored suspension is used to inoculate 50 ml of the vegetative culture medium as described above for the vegetative medium. The culture is then incubated in 250 ml wide-neck Erlenmeyer flasks for 4 days at 25 ° C on a rotary shaker with a 5 cm pivot at 250 rpm.

B. Fermentation in a tank

In order to obtain a larger volume of the inoculum material, 10 ml of the above-mentioned first stage vegetative medium is used to inoculate 400 ml of the second stage vegetative medium. My environment has the same composition as mentioned above for the vegetative culture medium. This medium is incubated in 2-liter wide-necked Erlenmeyer flasks at 25 ° C for 48 hours on a rotary shaker at a 5 cm swing at 250 rpm.

800 ml of the second stage vegetative medium thus incubated is used to inoculate 100 liters of sterile production medium of the following composition:

Component Quantity g / liter glucose 15 Dec glycerine 5 Tapioca dextrin 30 dried dog food ^x 25 yeast extract 5 KC1 1 MgSO ₄ .7H ₂ 0 0.5 FeSO ₄ .7H ₂ 0 0.1 MnCl ₂ .4H ₂ Q 0.05 ZnSO ₂ .7H ₂ O 0.05 CaCO2 0.1 deionized water to 1 liter

The pH was adjusted to 5.0 with 4N hydrochloric acid.

* Purina Dog Chow, Talston Louis, Mo. 63188

The inoculated production medium is fermented in a 165 liter fermentation tank at 30 ° C for approximately 12 days. The fermentation medium is aerated with sterile air, maintaining the soluble oxygen level at 80%.

Example 2

The fermentation is carried out in the same manner as in Example 1, except that a vegetative medium of the following composition is used:

Component

Quantity g / liter dextrose 5 galactose 5 glycerin 5 soluble starch 5 yeast extract 2 dried dog food ^x 5 soybean oil 2 corn oil 2

Component

ΚΗ ₂ Ρ0 ₄

MgSO ₄ .7H ₂ O

PaSO ₄ .7H ₂ O

CaCOj

MgCl ₂ .4H ₂ O ZnSO ₄ .7H ₂ 0 deionized water to

The medium has a pH of 5.7 and needs to be adjusted.

* Purina Dog Chow

Example 3

Quantity g / liter

0,5 0,1 1,0 0,05 0,05 l to pH 5,0 by addition of 4N hydrochloric acid The fermentation is carried out in the same manner as in Example 1, except that a fermentation medium of the following composition is used:

Component

Quantity g / liter glucose pulp from soya beans

MgSO ₄ .7H ₂ 0

Deionized water to

7,5 1, 0 2,0 liter

The medium has a pH of 7.4 and must be adjusted to 6.5 by adding 4N hydrochloric acid.

Example 4

Isolation of factor A antibiotic A-40104

500 liters of the fermentation broth obtained in Example 3 were filtered using diatomaceous earth as the filter aid (Hyflo Suppercel Johns-Manville Products Corp.) to give 3,640 liters of filtrate. The mycelial cake was washed with deionized water to give an additional 1,820 liters of filtrate. The filtrates were combined and the total amount of filtrate of 5,460 liters was extracted without adjusting the pH with half the volume of toluene, yielding 2,500 liters of toluene extract after an hour of extraction. This toluene extract contains the predominant factor C of the antibiotic A-40104, i.e. pleuromutilin.

The aqueous solution remaining after the toluene extraction was then extracted for one hour with half the volume of chloroform. The chloroform extract thus obtained is concentrated to a volume of 10 liters and then cooled to 4 DEG C. for 72 hours. The crystals were collected by filtration to obtain 588 g of factor A antibiotic A-40104.

Claims (1)

SUBJECT OF THE INVENTION A method for the production of antibiotic A-40104 factor A, characterized in that Clitopilus pseudopinsitus NEEL 11179 is grown in a culture medium containing useful sources of carbohydrates, nitrogen and inorganic salts in submerged culture under aerobic conditions for at least 2 days at 20 to 33 ° C; after the fermentation is complete, the broth is extracted with chloroform and the A-40104 factor A antibiotic is isolated from the extract.