DE1203423B - Process for the preparation of the antibiotic ZN-6 (fusidic acid) - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

Int. α .:

C12d

GERMAN

PATENT OFFICE

EDITORIAL

German class: 30h -6

Number:
File number:
Registration date:
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1203 423
L42612IV a / 30h
2nd August 1962
October 21, 1965

The patent application L 40063 IVa / 30h has a process for the production of the new antibiotic ZN-6, which in the meantime has received the name "fusidic acid", is the subject of this is characterized in that Fusidium coccineum Fuck (K. T u b a k i), strain ATCC 14700 on the usual biologically grown and the fusidic acid formed isolated from the culture medium.

In the course of the further development of this process it was found that when it is carried out mutants of the fungus Fusidium coccineum Fuck (K. T u b a k i), strain ATCC 14700 can also be used.

According to the invention is therefore a method for producing the antibiotic ZN-6, which has meanwhile been has received the designation "fusidic acid", according to patent application L 40063 IVa / 30 h by cultivation of Fusidium coccineum Fuck (K. T u b a k i), strain ATCC 14700 and isolation of the formed Fusidic acid from the culture medium, characterized in that one obtained in a known manner, Fusidic acid producing mutants of Fusidium coccineum Fuck (K. T u b a k i), strain ATCC 14700 breeds in the usual biological way.

Fusidic acid is an antibiotic based on a completely new process for the production of the antibiotic
ZN-6 (fusidic acid)

Addition to registration: L 40063IV a / 30 h interpretation document 1192 785

Applicant:

L0vens Kemiske Fabrik Ved. A. Kongsted,

Ballerup (Denmark)

Representative:

Dr.-Ing. F. Wuesthoff, Dipl.-Ing. G. Pulse and

Dipl.-Chem. Dr. rer. nat. E. Frhr. v. Bad luck man,

Patent Attorneys, Munich 9, Schweigerstr. 2

Named as inventor:
Inger Kirk, Copenhagen

Claimed priority:

Great Britain August 2, 1961 (28109)

Structure, namely a Cycllpentanoperhydrophenanthrenderivat of the likely formula:

, CH,

CH ₂ CH ₂ CH = C:

HO C-COOH! H

- OCOCH ₃

HO

CH ₃

Fusidic acid is sparingly soluble in water. It is a weak acid with a pK _a of about 5.3 and can form salts with a large number of inorganic or organic bases.

Extensive clinical trials have shown that fusidic acid and certain of its salts are pharmaceutically acceptable Acceptable antibiotics meet requirements and are valuable agents for treatment of infectious diseases, as shown, for example, in the journal "The Lancet", Vol. I, 1962, pp. 928-937.

According to the invention, a fusidic acid-producing mutant of the fungus Fusidium coccineum is cultivated Fuck (K. Tubaki), strain ATCC 14700 under aerobic conditions in an aqueous nutrient solution, the carbon and nitrogen sources as well as inorganic

509 718/415

contains niche salts until this solution has achieved substantial antibiotic activity.

Media are preferably used which have one or more of the substances as nitrogen sources Corn steeple, soybean meal, casein or other protein-containing dairy products, meat and bone meal contain.

The amount of available nitrogen in the culture medium is e.g. About 0.01 to 1.5%, preferably about 0.15%> based on the culture medium.

Sucrose and glucose in particular can be present as assimilable carbon sources. It is also advantageous to supplement the nutrient medium by adding fats or fatty acids, which form part of the carbon and energy sources, as these substances are in quantities of about 0.1 to 5.0% of the culture medium appears to promote the formation of fusidic acid. For this purpose fatty acid glycerides and the free fatty acids contained in them or syn- ao are preferably used thetic esters of these fatty acids, in particular fatty acids having 12 to 20 carbon atoms or derivatives such fatty acids.

When the formation of fusidic acid in the fermentation process stops or a satisfactory one Yield is achieved, this can be isolated by various methods.

Preferably, the mycelium is first separated from the culture fluid, e.g. B. by filtration; afterward the fusidic acid can be concentrated by treating the solution freed from the mycelium with a brings together solid adsorbent and then the antibiotic is eluted from the adsorbent. Fusidic acid can also be added to the nutrient solution by adding an amine or an acid Amounts that substantial amounts of the fusidic acid present in the culture medium are converted into an organic Solvent-soluble form are converted and subsequent extraction of the antibiotic with an essentially water-immiscible organic solvent, e.g. B. a Esters, a ketone or a halogenated hydrocarbon as such or in the form of one of their salts. This gives an organic layer, the fusidic acid as such or in the form of an amine salt contains in relatively high concentrations. Then the desired connection can be made known processes can be isolated in a degree of purity that is suitable for the preparation of the compound in of pharmaceutically acceptable quality is sufficient.

To obtain a mutant of Fusidium coccineum Fuck which can be used according to the invention (K. T u b a k i), strain ATCC 14700, one of the known mutation methods in which the spores of the fungus experienced an effect, can be applied. For example, a spore suspension irradiated with ionizing or non-ionizing rays or the spores can be exposed to mutagenic chemicals such as diethyl sulfate or mustard gas.

It has been found, however, that the mutation of the fungus can be brought about in a very convenient manner by irradiating a spore suspension with UV light for a period of time which ensures a survival percentage, preferably from about 0.05 to about 5.0%. The required irradiation intensity can be found in the experiments below. In these experiments suspensions of spores of the fungus, with 2 x 10 ⁶ spores / ml, which were enclosed in quartz tubes, with the light of a Hanovia Steri lamp (85% of this light had a wavelength of 2537 Å) from a distance of 18.5 cm irradiated for the periods listed in column A of the table below; the numbers in column B of the table indicate the percentage of surviving spores:

A. ] Experiment II Time B. 36.7 in seconds % surviving spores 4.3 15th Attempt I. 1.2 30th 27 0.3 45 5.8 0.04 60 1.0 75 0.3 0.06

The irradiated spores were then placed on agar plates and the colonies formed from individual spores were examined by cultivating them in shake flasks and determining both their ability to form fusidic acid and their need for special nutrients. The agar plates had the following composition:
Substrate 25 A:

Glycerin 7.5 g / l

Corn steep water 2.5 g / l

MgSO ₄ -7H ₈ O 0.05 g / l

KH ₂ PO ₄ 0.06 g / l

Perton 5.0 g / l

NaCl 4.0 g / l

FeSO ₄ - 7H ₂ O 0.005 g / l

CuSO ₄ -5H ₂ O 0.004 g / l

Glucose 10.0 g / l

Casein hydrolyzate 1.5 g / l

Agar 30.0 g / l

Vitamin solution *) 10 ml

The pH of the agar plate was 7.0.

*) The composition of the added vitamin solution was as follows:

Thiamine 100 mg / l

Riboflavin 50 mg / 1

Pyridoxine ■ HCl 61 mg / 1

Calcium salt of pantothenic acid 220 mg / 1

p-aminobenzoic acid 50 mg / l

Nicotinic acid amide 200 mg / 1

Choline · HCl 129 mg / 1

Inositol 400 mg / 1

Following this procedure, three mutants of particular interest were isolated. One of these Mutants, hereinafter referred to as Leo-U42, were produced from a 45-second irradiated spore suspension and two mutants, hereinafter referred to as Leo-U 79 and Fc-U 404, respectively, from one 60 seconds irradiated spore suspension isolated.

The rate of growth, the color of the colonies, the rate of sporulation and The morphology of the mutant Leo-U 42 was unchanged compared to the parent strain, but required he for its growth the presence of methionine or cysteine in the culture medium, for Difference from maternal line.

Compared to the parent strain, the mutant Leo-U 79 showed reduced spore formation and

The rate of growth and a change in the color of its colonies from white to pink; even its colonies showed an extraordinary one on agar plates of the above composition strongly ruffled surface.

The mutant designated as Fc-U 404 differed from the parent strain in that it was moderately smaller Growth rate, but gave much higher yields of fusidic acid.

Of these mutants, in particular, the strain Leo-U 42, which requires the addition of amino acids, and the strain Fc-U 404 for industrial Fermentation processes of interest.

However, the invention is in no way restricted to a use of the mutants mentioned above, since a large number of other fusidic acid producing mutants could also be obtained. This for Production of fusidic acid usable mutants can be derived from the parent strain by their growth rate in liquid culture media, by the Amount of spores formed and differentiated by the shape and color of their colonies on agar plates and may need the addition of special nutrients for optimal growth. at microscopic examination reveals no differences compared to the parent strain.

The dam line was obtained from K. T u b a k i from Bureau voor Schimmelcultures, Baarn, Holland and in the "Mycological Journal of Nagao Institute", September 1954, No. 4, described.

The mycelium is hyaline and has a diameter of about 1 to 1.6 μ, the conidia are spindle-shaped, and the conidia carriers develop into individual hyaline cells that form a long chain from which the outermost link is repelled when mature.

The sizes of the conidia of the mutants are about 4.6 to 6.8 x 1.4 to 2.0 µ, similar to that Mother line.

The sizes of the conidia isolated from the mutants mentioned above are given below:

Fc-U 404

6.4-1.6 µ
6.4 x 2.0 µ

6.7 -2.0 μ

4.8 x 1.6 µ

Leo-U 42

6.4 x 1.6 µ
4.8 x 1.8 µ

Leo-U 79

4.8-1.4 µ
4.8 x 1.6 µ
5.6-1.6 µ
6.4-1.6 µ

The mutants which can be used, like the parent strain, can furthermore not assimilate lactose and require biotin and therefore do not develop on Czapek agar plates.

Some of these mutants produce fusidic acid in higher yields than the parent strain.

The mutants Leo-U 42, Leo-U 79 and Fc-U 404 are in the "Commonwealth Mycological Institute" in Great Britain kept under the names mentioned and can be used from there for the purpose of a Identification can be obtained.

The invention is explained in more detail using the following examples:

example 1

Spores from 14-day-old colonies of Leo-U 42 developed in a flask on substrate 25 A were used as inoculum for a shake flask with a capacity of 500 ml containing 50 ml of a sterile culture medium the following composition was used:

Glycerin 7.5 g / l

Corn steep water 2.5 g / l

Meat and bone meal 15.0 g / l

Glucose 20.0 g / l

MgSO ₄ -7H ₂ O 0.05 g / l

NaCl 4.0 g / l

pH = 7.0

The shake flask was incubated on a reciprocating shaker for 72 hours at 26 ^° C. in order to achieve vegetative growth.

A portion of this culture was used as an inoculum for 125 ml of a sterile culture medium of the following Composition used, which was in a flask of 1000 ml content:

Corn steep water 20.0 g / l

KH ₂ PO ₄ 10.0 g / l

MgSO ₄ -7H ₂ O 0.5 g / l

Sucrose 50.0 g / l

pH of the medium = 6.7

After the inoculation, the culture medium was ^{incubated for 120 hours at 25 °} C. on a rotating shaking table. A fusidic acid concentration of 116 mg / l was then found in the culture filtrate in the agar dish test against Staphylococcus aureus as the test substance. The identity of the compound obtained with fusidic acid was demonstrated chemically.

Example 2

A glass fermentation vessel with a working volume of 51 was filled with a nutrient medium from the following composition:

Corn steep water 40.0 g

Sucrose 55.0 g

KH ₂ PO ₄ 5.0 g

MgSO ₄ -7H ₂ O 0.5 g

Tap water to 1000 ml

After sterilization, the pH was adjusted to 7.2 and the medium with 5 percent by volume 48 hour old vegetative culture of Fc-U 404 grown in a liquid medium of the same Composition like Substrate 25 A, which still contains 1.5% of the under the trade name »Staley's Special Nutrient «well-known nutrient medium had been supplemented, inoculated.

The fermentation was carried out at 25 ^° C. with stirring at 470 rpm and aeration with 0.2 volumes of sterile air per volume of culture liquid per minute. To prevent foaming, lard oil was added, if necessary, in amounts of 0.25 ml each. The total amount of lard oil added during fermentation was 40 ml.

After 40, 64, 88 and 112 hours, samples were taken and in these the amount of the resulting Fusidic acid determined. 10, 85, 185 and 335 mg of fusidic acid were found per liter of culture medium. Of the The last-mentioned value was confirmed by the recovery of fusidic acid in its pure form.

When carrying out a fermentation with the mother strain took place under the same conditions one after 112 hours in the culture liquid only 240 mg / 1 fusidic acid.

Claims (1)

9 10 Claim: dung L 40063 IVa / 30 h, "characterized by this Process for the production of antibiotic zei chn et that one obtains in a known manner ZN-6 (fusidic acid) by breeding fusidic acid, fusidic acid producing mutants of fusidic dium coccineum Fuck (K. T u b a k i), strain dium coccineum Fuck (K. T u b a k i), strain ATCC14700 and isolation of the fusidin-5 ATCC 14700 formed by the usual biological route acid from the culture medium according to patent application. 509 718/415 10.65 © Bundesdruckerei Berlin