DD282710A5 - PROCESS FOR THE PRODUCTION OF BESTATIN - Google Patents

Abstract

The invention relates to a process for the preparation of the immunomodulatory effective inhibitor of aminopeptidase B bestatin on a microbiological route. Its aim is to produce this dipeptide, which is important in human medicine and as a research tool in immunology, in an effective manner. The technical problem associated with this goal is achieved by aerobic, sterile submerged fermentation of the new strain Streptomyces spp. Am 33378 dissolved in media with carbon and nitrogen sources and mineral salts, wherein the target product with extractive and adsorptive steps from the culture filtrate is obtained. The microorganism Streptomyces sp. AM 33378 is deposited in the depository for microorganisms of the GDR under the number IMET 43894. {Enzyme inhibitor; Aminopeptidase B; Leucine aminopeptidase; bestatin; dipeptide; biosynthetic production; Immune drugs; Krebschemotherapeuticum; Microorganism Streptomyces spec. AM 33378; Deposit number IMET 43894; culture}

Description

2. The method according to claim 1, characterized in that

- The washed crude extract is diluted with water until the alcohol content is below 10%, and

- The active substance is bound from this aqueous solution to a porous polystyrene resin with a large inner surface.

Field of application of the invention

The invention relates to a microbial process for the preparation of a dipeptide, bestatin, which is of potential use as an immunomodulator and cancer chemotherapeutic in the treatment of infectious, tumor and immune diseases in humans. The field of application of the invention is thus in the microbiological and pharmaceutical industry, the field of application of the product according to the method, however, primarily in human medicine

Characteristic of the known state of the art

It is known that bestatin, a dipeptide with enzyme inhibitory activity on aminopeptidase B and leucine peptidase, is being clinically tested as an immunopharmaceutical in combination with anti-infective agents or antineoplastic agents. Important test results with the immunostimulant are recorded in the following monographs:

Small Molecular Immunomodulators of Microbial Origin. Fundamental and Clinical Studies of Bestatin (Edited by Hamao Umezawa, Japan Scientific Societies Press, 1981);

- Immunostimulants Now And Tomorrow (Edited by I. Azuma and G. Jolles, Japan Scientific Societies Press, Springer-Verlag, 1987).

It is also known that bestatin can hitherto only be formed by a single type of microorganism, Streptoverticillium olivoreticuli. Using a strain of this species, the microbiological production of bestatin in 1974 has been described for the first time (see patents DE 2528984 and DE 2628354). However, these process solutions are characterized by a low product yield, which is mainly due to the use of elaborate treatment procedures.

Object of the invention

The aim of the invention is to produce the dipeptide bestatin, which is important in human medicine as a research tool in immunology, in an effective manner.

Explanation of the essence of the invention

The invention has the object lugrunde to describe a technologically simple process by means of which the enzyme inhibitor and immunomodulator bestatin can be obtained by resorting to a new microorganism by fermentation using readily available nutrient substrates.

According to the invention this object is achieved in that a strain Streptomyces spec. AM 33378 designated microorganism under aerobic and sterile culture conditions in a 'üssigen medium with per se known carbon and nitrogen sources, such as glucose, starch or soybean meal, and is submerged fermented with known mineral salt additives. The microorganism AM 33378 has been deposited in the depository for microorganisms of the GDR, Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences, DDR - Jena, 6900, Beutenbergstrasse 11, under the registration number IMET43984. This microorganism, which makes it possible to produce bestatin with readily available fermentation feeds in good yield, has been isolated during mutagenesis and selection work from the population of a Streptomycete strain known to form a polyether antibiotic as well as a cAMP phosphodiesterase inhibitor , By comparison of its properties with those given in the literature (in particular those of the "International Streptomyces Project" by Shirling and Gottlieb 1972), the microorganism AM 33378 on the one hand could not be assigned to any of the known Streptomycetes species

Bestatin-Former Streptovertllllllum ollvoretleull ATCC 31159 differs from the other microorganism AM 53378 on the other hand in its morphological (described by Arai et al., In: Antibiotics and Chemotherapy 7,435-442,1957) as well as in its physiological, biochemical and genetic properties. Differences exist, for example

- in the educational capacity for penicillin-inactivating β-lactamases,

- in the educational capacity for a polyether antibiotic (26-hydroxylaidlomycin),

- in the educational capacity for cinnamamide,

- in the production capacity for proteinases (alkal., neutral),

- in educational capacity for a hexa-antibiotic,

- in the resistance or sensitivity pattern to different active substances (antibiotics). The differences were determined by direct comparison with the ATCC 31159 strain using the Adamczyk granular dispenser technique; Thereafter, the bestatin generator used according to the method is resistant to a number of antibiotics in comparison with the ATCC 31159 strain and thus has genetically and biochemically deviating properties as well

- in the formation capacity of a cyclic AMP phosphodiesterase inhibiting metabolite (media dependent). The cultivation of the strain AM 33378 takes place under aerobic and sterile conditions. Lyophilized Mycelfragfmente are inoculated onto appropriate nutrient agar and subsequently in liquid, previously sterilized culture media, and the mycelium is in a known manner at a temperature between 20 ° C and 35 ⁰ C, preferably at 28 ° C, over a period of 2 days to 6 Days, preferably 4 days, cultivated at an acidity which is between pH 6.2 and pH 6.5 at the beginning of the fermentation process and between pH 6.8 and pH 7.0 at the end of the process.

The nutrient medium consists of carbon and nitrogen sources as well as inorganic salts. Glucose, starch, maltose, glycerol and as a source of nitrogen soybean meal, peptones, yeast extracts can be used advantageously as the carbon source. Good results can be achieved with the addition of mineral salts. The latter favor the course of biosynthesis during fermentation, depending on the medium used. In complex media containing various flours, additions of calcium carbonate, sodium phosphate and potassium phosphate have been found to be beneficial. In addition, all substrate additions containing L-leucine and L-phenylalanine have a favorable influence. The fermentation of the bestatin formulator AM 33378 can be carried out in steep breast bottles and round-bottomed glass of various contents, in glass fermenters and in V2A steel tanks.

The isolation of the bestatin from fermentation solutions of the strain Streptomyces spec. According to the method, AM 33378 is carried out in such a way that the active substance, due to its enzyme inhibitory activity against leucine aminopeptidase (EC 3.4.11.1) at pH 3.0 to pH 4.0, is freed from the mycelium-free culture filtrate with n-butanol or another Extracted water immiscible alcohol, the alcohol extract (crude extract) by washing with aqueous alkali or by washing with water freed of dark pigments and becomes detectable. The butanol extract is concentrated in vacuo and the enzyme inhibitor precipitated with petroleum ether or another gasoline fraction. The inhibitor-containing crude product is dissolved in a small amount of methanol, diluted with water to 8 to 12 times the volume (until the alcohol content is below 10%) and adsorbed on a porous polystyrene resin with the largest possible inner surface. The elution takes place in methanol-water mixtures, the methanol content being at least 70%. The further purification is carried out in a conventional manner by column chromatography on silica gel using chloroform-methanol mixtures or butyl acetate-n-butanol-glacial acetic acid-water mixtures, on crosslinked dextrangeles using methanol and / or on cation exchange resins using aqueous buffer mixtures. By combining these purification steps, further secondary metabolites formed from strain AM 33378, including pigments, are separated. The thus obtained leucine aminopeptidase (LAP) inhibitor assay active fractions are concentrated and, after being dissolved in a low-boiling alcohol at elevated temperature, purified with charcoal. The colorless substance obtained after concentrating the alcoholic solution is finally recrystallized in alcohol, preferably in methanol, or in alcohol-ethyl acetate mixtures.

The biological activity of the culture filtrates, the differently enriched crude products and the pure substance is carried out by means of the LAP test, in which the inhibition effect on the leucine aminopeptidase is taken as a measure of the concentration of the bestatins. The test principle is based on the inhibition of a leucine aminopeptidase isolated from bovine eye lenses, which cleaves leucine hydrazide, so that the resulting hydrazine is reacted with 4-dimethylbenzaldehyde to give an orange compound which can be photometrically quantified at 450 nm. The LAP inhibitor isolated from strain AM 33378 inhibits LAP activity by 50% at a concentration of 80 ng / ml under the chosen test conditions.

The melting point of the purified inhibitor is between 231 ° C. and 235 ° C. The inhibitor is optically active, the value Ia) S ³ being -29.5 ° C. in a 0.4% strength methanol solution.

The elemental analysis gives C = 60.72%, H = 7.95% and N = 8.54%. In addition, the inhibitor from the strain AM 33371} shows a positive ninhydrin and Rydon-Smith reaction. The physico-chemical properties, the inhibitory effect on leucine aminopeptidase and the direct TLC comparison with reference bestatin show that the inhibitor 33378 is identical to the bestatin described by the Umezawa group in 1976 (Suda et al., J.A. tibotics 29 (1), 100-101)

 The Vo parts of the invention are seen as follows:

- Since only one type of microorganism has ever been known as the bestatin formator, it is an advantage if the range of bestatin formers could also be extended with the aid of the traditional method.

In the principle solution shown in the method according to the invention, the biosynthesis capacity is 30 mg / l of culture fluid, i. H. by a factor of 10 over the literature known prior art.

embodiments By the following embodiments, the invention will be explained, but not limited. example 1

Two 500 ml steep breast bottles each contain 80 ml of the following pre-breeding medium:

Strength 2.5%

Soybean meal 1.6%

Sodium chloride 0.5%

Calcium carbonate 0.1%

Potassium hydrogen phosphate 0.3%

in tap water

Sterilization: 35 minutes at 12O ⁰ C. After sterilization, the acidity is at pH 6.3.

Each steep-breast bottle is inoculated with a mycelial suspension which is filled with sterile isotonic saline from a 10-day to 14-day culture of the strain Streptomyces spp. Cultivated in the test tube on the following seed medium. AM 33378 is manufactured:

Saccarose 0.3%

Dextrin 1.5%

Urea 0.01%

NaCl 0.05%

K ₂ HPO ₄ 0.05%

MgSO ₄ 0.05%

Peptone (bacteriol.) 0.5%

Meat extract 0,1%

Fe sulfate 0.001%

Agar 2.5%

Sterilization: 40 minutes at 12O ⁰ C. After sterilization, the acidity is adjusted to pH 7.0.

The inoculated Vorzucht-cultures are incubated on a shaker table with a Schwingfrequ & nz of 180 U / min for 48 hours at 2O ⁰ C. 8ml of a pre-culture incubated in this way are used to inoculate 500 ml steep-breasted bottles, each containing 80 ml of the following production medium:

Glucose 2%

Soybean meal 2%

Potato starch 2%

NaCl 0.25%

KH ₂ PO ₄ 0.025%

CaCO ₃ 0.5%

MgSO ₄ 0.05%

tap water

Sterilization: 35 minutes at 120 ° C. Acidity is 6.2 after sterilization. The temperature during the fermentation is 28 ⁰ C, the shaking frequency, however, 180 U / min. After 80 to 96 hours of fermentation, the highest yield of the LAP inhibitor 33378 is achieved.

Example 2

With a culture of the strain AM 33378 from a semi-solid medium, as described in Example 1, 400 ml of the liquid pre-emergence medium cited in Example 1 are seeded, which is contained in a 2 l glass flask. It is incubated for 48 hours at 28 ⁰ C on a shaker with a frequency of 180U / min. The 400 ml culture broth thus obtained was used to inoculate 2Ol of the production medium described in Example 1, which is contained in a 32 l glass fermenter.

During the fermentation, which is carried out with a stirring speed of 400 rpm and an air supply of 15 to 20 l / min, foaming is controlled by the addition of small amounts of sunflower oil or silicone-containing antifoaming agent.

Example 3

With the media described in Example 1, the Vorzucht is first carried out for 24 hours in 500 ml Steil breast bottles with 80 ml and then for another 24 hours in the same medium in 2-liter glass flask with 400 ml content before a 450 1 liter can be inoculated from 1% to 2% of the pre-culture culture containing the production medium described in Example 1. The stirring speed is 280 rpm and the air supply is up to 400 lpm minute. After 96 hours to 120 hours, the fermentation is stopped.

3501 of the obtained culture solution are acidified to pH 3.0 with sulfuric acid and extracted with 120 l of n-butanol. The butanol extract (95I) is concentrated in vacuo to a volume of 91 and stirred with 11 of a 3% sodium hydroxide solution to remove dark pigments, wherein in the aqueous phase pH 7.0 to pH 7.2 are present. Then it is washed twice with 11 water each. After further concentration of the butanol extract under vacuum up to a volume of 200 ml is added 6 times the amount of petroleum ether, with 34.1 g of brown crude I precipitate (0.7% active substance). The crude products I from several fermentation mixtures are combined and in each case 50 g of crude product I dissolved in 400 ml of methanol with heating and poured into 4.61 water with stirring. The solution clarified by filtration is passed through a 2 cm diameter 2I column containing a porous polystyrene-based adsorbent resin. It is then washed with methanol-water mixture (1: 9) and the active substance is eluted with methanol-water (8: 2). The active fractions are combined and concentrated in vacuo to the aqueous solution, from which 10.2 g of light brown crude product II having an activity of 0.8% are obtained after lyophilization.

50 g of crude product II are further purified by column chromatography on silica gel. By dissolving in 200 ml of methanol and concentrating the solution under vacuum together with 50 g of Kiesefgel, the silica gel loaded with the substance is poured over 850 g of silica gel (0.063-0.2 mm) in a column (diameter: 8 cm) with the solvent mixture chloroform-methanol = 9 : 1 is equilibrated, stored and then eluted with chloroform-methanol mixtures. The composition

of the mixture is gradually changed from 9: 1 to 2: 8, the main activity being obtained at a ratio of 6: 4. Freeze-drying of the pooled active fractions gives 5.1 g of crude product III (10.2% activity). 10.2 g of crude product III with a light brown color are chromatographically separated again on a silica gel column in the system butyl acetate-butanol-acetic acid-water after dissolving in methanol, adsorption on 70 g of silica gel and applying to a column with 900 g silica gel (0.04 to 0.06 mm ) slurried with butyl acetate-butanol-glacial acetic acid-water = 10: 6: 1: 1. The eluent mixture is varied from 10: 6: 1: 1 to 4: 4: 1: 1 with the major activity being eluted at a ratio of 6: 6: 1: 1. The combined, active fractions give, after freeze-drying, 3.62 g of crude product IV (activity: 22%). 3.6 g of crude product IV are subjected to a final purification over a column packed with 1.91 g of o-alkyl-crosslinked dextran gel filtration support by dissolving this crude product IV amount in 10 ml of methanol, adding and eluting with methanol. After concentrating the active fractions in vacuo, 0.95 g of pale yellow powder is obtained with an ID _M value of 0.08 μg against leucine aminopeptidase using leucine hydrazide as substrate. After dissolving the pale yellow powder in 20 ml of methanol and addition of 100 mg of activated carbon, after warming to 6O ⁰ C, filtration and addition of ethyl acetate to turbidity, finally 0.6 g of pure bestatin are obtained as a white powder after cooling.

Table 1

Overview of known microorganisms with biosynthetic capacity for the immunomodulator Bestatin

Illustrator reference

1. Streptomyces olivoretlculi MD 976-C 7

Described in: DE 2528984

Isolated from a soil sample

from Kaitamura-Nishino,

Kisogun, Nagano / Japan

Deposited on 18.05.1974 in

Kogyo Gijutsuin Hakko Kenkyusho

under No. 2 590.

Further, deposited under No. ATCC 31159

in the American Type Culture Collection

The bestatin image generator MD 976-C 7 was characterized in its properties with those of Arai et al. Antibiotics and Chemotherapy 7,435-442 (1957) and directly compared with the properties of strain ISP-type culture 5105 (ISP = International Strepvomyces Project). Since no significant differences were detectable, the bestatin image generator MD 976-C7 was assigned to the species Streptomyces olivoretlculi (DE 2528984). Other microorganisms with biosynthetic properties for Bestatin si <.d have not been identified in patent or literature research. In "Applied Libraries of bacterial names" (Int. J. Syst. Bacterioi., Vol. 30, 1980, p. 410), the bestatin formner is assigned as Streptovertlcillium olivoreticulum to another genus.

Table 2

Selected process and substance protection patents for the immunomodulatory inhibitor of the leucine aminopeptidase bestatin or its analogues

Ser. No. Appl. date Patent no. applicant " protected object 1 28/6/74 DE 2528984 Zaidan Bestatin and procedure 2 24/6/76 DE 2628354 Zaidan Bestatin and procedure 3 21/7/76 CH 630892 Zaidan Bestatin analogs 4 26/3/76 Japan Kokai Foundation Bestatin analogs 77116435 5 26/3/76 Japan Kokai Foundation Bestatin-bleomycin 77117435 combination preparation 6 19/9/78 DE 2840636A1 Zaidan Bestatin analogs 7 07/07/80 FR 24859 ^O 4 Foundation Bestatin analogs 8th 10/6/81 US 4473554 Foundation Bestatin analogs 9 24/12/81 DE 3246135 Foundation Crystalline Bestatin 10 09/01/82 EP 83931 Zaidan Bestatin analogs 11 26.03.84 EP 156279 Foundation Bestatin analogs

¹¹ Abbreviations used:

Zaidan = Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai, Tokyo / Japan Foundation = Microbiochemical Research Foundation, Tokyo / Japan

Table 3

Overview of the differences between the inventive and known bestatin production method and the advantages derived therefrom

1. Origin of the Bestatln Bldner:

Dor known bestatin formers was originally isolated from a Japanese soil sample, assigned to the known species Streptomyces ollvoretlcull and deposited as strain MD 976-C7 in an international strain collection under the number ATCC 31159 (see Table 1 and Umeiawa et al., 1976, J .Antibiot., 29 [1], 97-99).

In contrast, in the bestatin production process of the present invention, a microorganism originally isolated from a soil sample from the Schwellenburg near Erfurt (GDR) was used, and due to its morphological properties, it could be assigned to the genus Streptomyces and as strain AM 33378 in an internationally recognized strain collection deposited under the name IMET 43894.

2. Characteristics of the Bestatln Bldner:

The bestatin image generator Streptomyces spec. IMET 43894 differs in the morphological properties typical of Streptomyces ollvoreticuli ATCC 31159 (described by Aral et al., In Antibiotics and Chemotherapy 7, 435-442, 1957).

The species Streptomyces ollvoreticuli was included in the Approved lists of bacterial names in 1980 (Int.J.Syst.Bacteriol., 30, 368ff.) The species feature "sporophore morphology" is so slight in almost all culture media in the bestatin formers of the invention trained that no clear type assignment made and the strain AM 33378 as Streptomyces spec., Is designated. The inventively 3 bestatin formers also differs from the known bestatin formers in its physiological, biochemical and genetic properties z. B.

- in the educational capacity for penicillin-inactivating β-lactamases,

- in the educational capacity for a polyether antibiotic (26-hydroxylaidlomycin),

- in the educational capacity for cinnamamide,

- in the production capacity for proteinases (alkal., neutral),

- in educational capacity for a hexa-antibiotic,

- in the resistance or sensitivity pattern to different drugs (antibiotics): the differences were determined by direct comparison with the ATCC 31159 strain using the Adamczyk granular dispenser technique. Thereafter, the bestatin generator used according to the method is resistant to a number of antibiotics in comparison with the ATCC 31159 strain and thus has genetically and biochemically deviating properties,

- in the formation capacity of a cyclic AMP phosphodiesterase inhibiting metabolite (media dependent).

3. Advantages of the invention

Since only one type of microorganism has hitherto been known as the bestatin formers, it is an advantage if the range of bestatin formers is expanded with the bestatin production method according to the invention.

In the proposed inventive principle solution according to the invention, the biosynthetic performance is 30 mg / l culture liquid and thus by a factor of 10 than higher in the principle solution according to patent DE 2528984.

Claims (2)

1. A process for the production of Bestatin by fermentation under aerobic and sterile conditions in liquid nutrient media containing a carbon and nitrogen source and mineral salts, at a temperature between 20 ⁰ C and 35 ⁰ C and recovery of the resulting product from the culture filtrate by means of extractive and absorptive steps, characterized in that - as Bestatin-formers the strain Streptomyces spec. AM 33378 used, - during a period of Cultivated 2 days to 6 days and - After the extraction step carried out with Wassernichtmischbaren alcohols extraction of the crude extract by washing with alkali or with water of dark pigments