RU2272643C2 - High-active crystalline preparation of cyclosporine a and method for its preparing - Google Patents

Abstract

FIELD: antibiotics, pharmacology of peptides.

SUBSTANCE: invention relates to high-active crystalline preparation of cyclosporine A and a method for its preparing. Invention relates to high-active crystalline preparation of cyclosporine A representing cyclic undecapeptide prepared from fungus of genus Tolypocladium inflatum subsporum blastosporum grown in cultural medium containing additionally rubomycin in the concentration 2 mcg/ml and zinc sulfate in the concentration 5 mg/ml. The fermentation medium comprises maltex and D,L-valine as main components wherein the final concentration of preparation in medium is 2.0-2.5 mg/ml. Product is isolated from fungus dried mycelium by extraction with petroleum ether before and after purification by chromatography method on silica gel and crystallized in system hexane - acetone (4:1). Invention provides the development of effective cyclosporine preparation wherein the content of cyclosporine A is 80-85%.

EFFECT: improved preparing method.

2 cl, 5 ex

Description

The invention relates to the field of pharmacology of peptides, more specifically to obtaining a highly active crystalline preparation of cyclosporin A and a method for its preparation.

Cyclosporins are a class of structurally similar cyclic poly-N-methylated undecapeptides. They include the natural cyclosporins A, B, C, D and G, as well as their derivatives dihydrocyclosporins, obtained by chemical modifications. The most famous among these antibiotics was cyclosporin A (CA), which has found application in surgical practice for organ and tissue transplants. Cyclosporin A is a neutral cyclopeptide containing 11 amino acids. This drug is a narrow-spectrum antifungal antibiotic, as it selectively inhibits the growth of Aspergillus niger and Curvularia lunata, while being inactive against yeast, gram-positive and gram-negative bacteria.

Cyclosporins represent a fundamentally new class of important immunosuppressive drugs. At the cellular level, their effect is extremely specific and is aimed at a subpopulation of T-lymphocytes. In clinical practice, cyclosporin A plays an important role in organ and tissue transplantation. The effectiveness of cyclosporin is currently being studied in a wide range of autoimmune and chronic inflammatory diseases, including idiopathic nephrotic syndrome, psoriasis, uveitis, insulin-dependent diabetes mellitus, ulcerative colitis, Crohn's disease, primary biliary cirrhosis, etc.

Cyclosporin as an antifungal and immunosuppressive antibiotic produced by fungal cultures of Cylindrocarpon lucidum (deposited in the United States in the Joint Department of Agriculture under the number NRRL 5760) and Tolypocladium inflatum (deposited in the same collection under the number NRRL 8044) was described in Switzerland patent No. 4, No. 284, 1974, No. 284. US analogue No. 41174527/76, A 61 K 37/00, C 07 C 103/52, 1976. In the scientific literature, cyclosporin, its producers, the method of biosynthesis and isolation were published by Dreyfuss M. Et al. J. Appl. Microb. 1976. - No. 3, - p. 125-133.

Further, other fungal cultures producing cyclosporin were patented and described: Tolypocladim grodes, T. Nubicola, T. Terricola, T. cyclindrosporum, T. tundrense, Chhaunopycnis alba, Aphanocladium sp., Beauveria bassiana, B. Brongniarti, Acremonium spp. Paecilomyces spp., Verticillium spp., Isaria felina, Fusarium spp., Trichoderma viride, Neocosmospora vasinfecta (Dreyfuss MM., Chapela IH. In The discovery of natural products with therapeutic Potential. Ed. Gullo VP., Butterworth-Heinemann, 1994, p. 49-80), Sesquicillopsis rosariensis (4895207/13, publ. 09/20/96, bull. 26b Dresden GmbH (DE), Tolypocladium inflatum subsp. blastosporum (ac USSR 1830947, 10.27.1988, A.S. USSR 1836425, 08/23/1993). The activity of the culture fluid is 600-1300 μg / ml during cultivation from 6 to 14 days 3150 mcg / mL after 14 days of culture strain Sesquicillopsis rosariensis F605. The patents described a process for preparing a complex of cyclosporins, wherein the cyclosporin A is 55-65%.

The aim of the present invention is to provide an effective preparation of cyclosporin and a method for its production, in which biosynthesis is carried out for 6-7 days, and the content of cyclosporin A at the end of fermentation is 2000-2500 μg / ml. A complex of cyclosporins is formed, in which cyclosporin A is 80-85%.

The predominant formation of cyclosporin A facilitates its purification and increases the yield of the target product.

The preparation of cyclosporin A was improved and modified, and crystalline forms of cyclosporin appeared (see patent RU 2002754, 1993, patent RU 2066687, 1996). For protection, mainly various modifications of the fungus of the genus Tolypocladium are grown, grown on various media (patent RU 2112807, 1998) and purified under various conditions (patent US 582655, 1995). The closest to the problem to be solved can be considered patent RU 2170741, 2001 (a method for producing cyclosporine) and patent RU 2182577, 2002 (a method for producing cyclosporin A of high purity).

An object of the present invention is to provide a purified crystalline preparation and method for producing cyclosporin A, with high specific activity per unit volume of culture medium (up to 2500 μg / ml).

The problem is solved in that a highly active crystalline preparation of cyclosporin A (HCFC) was created, which is a crystalline form of a cyclic undecapeptide obtained from a fungus of the genus Tolypocladium inflatum subsp. blastosporum, this genus of micro fungus is registered in the collection of the State Scientific Center for Antibiotics No. 330A, when cultured on standard agarized medium followed by fermentation, isolation, purification and crystallization, the target product is obtained, while the culture medium additionally contains 2 μg / ml rubomycin and 5 mg / ml zinc sulfate, and the fermentation medium contains maltex and D, L-valine as the main components, at a final concentration in the medium of HCFC 2.0-2.5 mg / ml, and the product is isolated from high celiac fungal mycelia by extraction with petroleum ether before and after purification by silica gel chromatography and crystallized in the hexane - acetone system (4: 1).

The HCVC product is a crystalline form of a cyclic undecapeptide, which is obtained from a fungus of the genus Tolypocladium inflatum subsporum blastosporum, registered in the collection of the State Scientific Center for Antibiotics No. 330A when cultured on standard media, characterized in that rubomycin 2 is additionally added to the agarized culture medium of Raistrik 0 μg / ml and zinc sulfate 5 mg / ml. Spore material with mycelium culture grown on agar medium at 24 ° C, seeded inoculum medium of the following composition, wt.%:

Soya flour 3.0-3.5 Glycerol 2.5-3.5 Potassium phosphate od unsubstituted 0.3-0.6 Sodium Chloride 0.5-0.8 Molasses 2.5-3.0 Tap water

The medium has a pH of 6.5.

The volume of medium in Erlenmeyer flasks with a capacity of 750 ml - 50 ml. Cultivation is carried out on a rocking chair at 200-220 rpm for 48 hours. The culture fluid from the seed flasks in the amount of 10% seeded fermentation medium of the following composition, wt.%:

Maltex 4.0-8.0 Soya flour 1.0-2.0 Yeast extract 2.0-6.0 Glycerol 1.0-1.5 Disubstituted Ammonium Phosphate 1.0-1.2 Sodium nitrate 0.2-0.5 Potassium phosphate monosubstituted 0.1-0.3 Tap water D, L-Valine 0.4-0.6

The medium has a pH of 6.5.

Fermentation is carried out in Erlenmeyer flasks with a capacity of 750 ml, the volume of the nutrient medium in the flask is 50 ml. Cultivation is carried out at 24 ° C for 6-7 days. The content of cyclosporin A in the culture fluid is 2000-2500 μg / ml.

A fermentation liquid with a final concentration of CA of 2.0-2.5 mg / ml is obtained, and the product is isolated from dried mycelium of the fungus by extraction with petroleum ether, followed by purification by chromatography on silica gel and crystallized in hexane - acetone system (4: 1).

Another object of the present invention is a method for producing such a preparation.

This object is achieved in that a method has been developed for producing a highly active crystalline preparation of HCFC described above, including cultivating a fungus on an agar medium, fermentation, isolation and purification, characterized in that the medium additionally contains rubomycin 2 μg / ml and zinc sulfate 5 μ / ml, and the fermentation medium contains maltex and D, L-valine as the main components, then the fungal mycelium containing the complex of cyclosporins A, B, C, D is separated from the native solution, the mycelium and the extract are dried m petroleum ether the desired product before and after purification by chromatography on silica gel or alumina, fractions containing cyclosporin A are combined and crystallized from hexane - acetone (4: 1), the content of the desired product is not less than 97.5%.

Another object of the invention is a method for producing a preparation.

The drug described above is prepared as follows:

- Obtaining agarized seed.

- Obtaining vegetative seed.

- Obtaining highly active culture fluid.

- Isolation: separation of mycelium containing 90% of the complex of cyclosporins A, B, C, D from the native solution, drying it, extraction of the complex from dried mycelium with petroleum ether or petroleum ether-acetone system (10: 1), chromatographic separation of the cyclosporins complex into a column with silica gel (aqueous silicic acid, kiesel gel), a petroleum ether - acetone system (4: 1), or an aluminum oxide column with a petroleum ether - acetone system (7: 3), hexane precipitation or crystallization in a hexane - acetone system (4: one).

The mycelium is separated from the culture fluid by centrifugation or filtration in a vacuum drum filter or on a frame filter press with a precoated layer of filter aid — filter perlite (about 10 grams per liter of culture fluid). The moisture content in the resulting mycelium is 75%. The wet biomass content of mycelium in the culture fluid is 20%. The filtered wet mycelium is dried in a dryer SP-30 in a fluidized bed at a temperature of 45-50 ° C. The moisture content in dried mycelium is about 6%. The output of the cyclosporin complex at the drying stage is about 80%. The content of cyclosporin A in dried mycelium is determined by HPLC (high performance liquid chromatography). The cyclosporin complex A, B, C, D is extracted from dried mycelium with petroleum ether (the boiling point of the used petroleum ether is 70-100 ° C). The use of petroleum ether is a feature of the described method, because CA is not soluble in petroleum ether, and its extraction occurs due to fatty impurities formed by the producer and contained in the mycelium, in which the cyclosporin complex is dissolved. Unlike other extractants, petroleum ether does not form emulsions during extraction and does not extract colored impurities. The output of cyclosporins at the extraction stage is 50-60% of the content in dry mycelium. The extract contains a complex of cyclosporins, in which the content of cyclosporin A is about 80%, C, B, D about 20%. To increase the yield at the extraction stage, a petroleum ether - acetone (10: 1) system is used as an extractant, however, the extract will contain a small amount of pigments, which leads to the need to introduce an additional chromatographic purification step. When the extract is passed through a column of silica gel under columnar conditions, fatty impurities are displaced with the resulting solution, and the cyclosporin complex is sorbed in the upper layer of silica gel. After washing the column with petroleum ether, the cyclosporin complex is eluted with a petroleum ether - acetone (4: 1) system. The eluate is collected fractionally and analyzed by TLC on Silufol plates in a benzene-acetone (2: 1) solvent system with the manifestation in iodine vapor and fixing the color of 0.01% in an aqueous solution of potassium permanganate or by HPLC. The fractions containing cyclosporin A are combined, concentrated in vacuo on a rotary evaporator. CA is precipitated with hexane or crystallized in a hexane-acetone system (4: 1), and a crude preparation with a content of cyclosporin A of at least 98.5% and an impurity of about 1.5% is obtained. Fractions with an impurity content of more than 1.5% are combined and subjected to repeated chromatographic purification on silica gel (in the system of petroleum ether - acetone (4: 1)) or on aluminum oxide (in the system of petroleum ether - ethyl acetate (7: 3)).

Analysis of drugs by HPLC. HPLC conditions: Zorbax C 18, 250 X 4.6 mm, 5 μm column. Thermostating temperature is 70 ° C. Detector 214 nm. Eluent: acetonitrile - water - phosphoric acid 70: 30: 0.05 (v / v). The flow rate of 2.0 ml / min.

The results of this analysis are confirmed by the following examples.

EXAMPLE 1

The agarized culture was grown on Raistrik medium containing rubomycin 2 μg / ml and zinc sulfate 5 μ / ml at 24 ° C for 10 days. The spore material with a mycelium of culture 330A is seeded with the seed medium of the following composition, wt.%: Soy flour - 3.0; Glycerin - 2.5; Potassium phosphate monosubstituted - 0.3; Sodium chloride - 0.5; Molasses - 2.5; Tap water; pH 6.5. The volume of medium in Erlenmeyer flasks with a capacity of 750 ml - 50 ml. Cultivation is carried out on a rocking chair at 200-220 rpm for 48 hours. The culture fluid from the seed flasks in the amount of 10% is seeded with a fermentation medium of the following composition, wt.%: Maltex - 4.0, Soy flour - 1.0; Yeast extract - 2.0, Glycerin - 1.0; Disubstituted ammonium phosphate - 1.0; Sodium nitrate - 0.2; Monosubstituted potassium phosphate - 0.1, Tap water; D, L-Valine - 0.4, pH 6.5.

Fermentation is carried out in Erlenmeyer flasks with a capacity of 750 ml, the volume of the nutrient medium in the flask is 50 ml. Cultivation is carried out at 24 ° C for 6-7 days. The content of cyclosporin A in the culture fluid is 2000-2500 μg / ml.

From one liter of culture fluid containing 2500 μg / ml cyclosporin A, the mycelium is separated by centrifugation or filtration and dried in a vacuum oven at 50 ° C for three hours. 150 g of dry mycelium are obtained. The cyclosporin complex from dry mycelium is extracted with stirring for three hours, first with three times the volume of petroleum ether (boiling point 70-100 ° C), then, after separation of the mycelium by centrifugation, two more volumes of petroleum ether. The extracts are combined and passed through silica gel, loaded in petroleum ether into a column of size (height to diameter) 30 cm: 4 cm. After passing the extract, the column is washed with petroleum ether. Fatty impurities are washed out with the resulting solution, and the cyclosporin complex A, C, B, D is sorbed in the upper layer of silica gel. The antibiotic is eluted using a petroleum ether - acetone (4: 1) system at a rate of 10 ml / hour. The analysis of fractions for the content of cyclosporins is carried out by TLC on silofol plates (Czechoslovakia Kavalier) in a benzene-acetone solvent system (2: 1). Cyclosporins are shown in iodine vapor with fixing the color of the spots in a 0.01% aqueous solution of potassium permanganate. Fractions with cyclosporin A without visual detection on the plates of other cyclosporins (B and D) are combined, concentrated in vacuo and precipitated with a 20-fold volume of hexane. The precipitate formed is filtered off, washed with hexane and dried in a vacuum oven at 50 ° C for two hours. Fractions with impurities of other cyclosporins are combined, concentrated in vacuo and subjected to repeated chromatographic purification under the same conditions. A drug with a content of cyclosporin A of at least 98.5% is combined. Obtain 1.0 g of the substance. The output of cyclosporin A is 50% of the content in dry mycelium.

EXAMPLE 2

Obtaining spore and vegetative inoculum is carried out, as in example 1. Fermentation is carried out on a medium of the following composition, wt.%: Maltex - 8.0, Soy flour - 2.0; Yeast extract - 6.0, Glycerin - 1.5; Ammonium phosphate disubstituted - 1.2; Sodium nitrate - 0.5; Monosubstituted potassium phosphate - 0.3, Tap water; D, L-Valine - 0.6, pH 6.5. Fermentation is carried out as in example 1. The content of cyclosporin A in the culture fluid is 2300-2500 μg / ml.

Obtaining a CA preparation from dried mycelium from one liter of culture liquid with an activity of 2 mg / kg is carried out under similar conditions on silica gel 60 (Merk) 0.04-0.05 μm in 60% yield and 99.7% cyclosporin A content.

EXAMPLE 3

The cyclosporin biosynthesis and isolation are carried out, as in examples 1 and 2. Chromatographic separation of 300 g of a complex of cyclosporins A, B, D (with a cyclosporin A content of about 80%) is carried out on a column of size (diameter to height) 1:20 with aluminum oxide (14 kg) using a solvent system of petroleum ether - ethyl acetate (7: 3). Pure fractions (after analysis by HPLC) are combined, concentrated, dried. 190 grams of cyclosporin A are obtained with an impurity content of less than 2.5%. The substance is purified by crystallization in hexane with acetone (4: 1). Get 150 grams of a substance with a content of cyclosporin A 98.6%. The output of cyclosporin A is 50% of the original substance.

EXAMPLE 4

Cyclosporin biosynthesis is carried out, as in examples 1 and 2. Mycelium from 140 liters of culture fluid containing 2 g / l of cyclosporin A is separated on a frame filter press or drum type filter with a precoated layer of filter aid — filter perlite (1%). The obtained wet mycelium in an amount of about 30 kg with a moisture content of 75% is dried in a dryer SP-30 with a fluidized bed at 50 ° C. Get 6 kg of dry mycelium. The content of cyclosporin A in terms of grams of dry mycelium is determined by HPLC. The loss of cyclosporin A during drying is 20%, the extraction of cyclosporin A from dry mycelium is carried out in a petroleum ether - acetone (10: 1) system in a reactor with stirring, first with three volumes of extractant (relative to the weight of the mycelium) for three hours. After separation of the first extract, the mycelium is re-extracted with two volumes of the same system.

A chromatographic column (diameter to height equal to 1:20) is loaded with a suspension of 14 kg of aqueous silicic acid in petroleum ether. After passing through the column of extracts (first and second), the chromatographic separation of the cyclosporin complex is carried out using a petroleum ether - acetone (4: 1) system, as described in example 1.

Analyze fractions for the content of cyclosporin A by HPLC. Pure fractions are combined, concentrated, and mixed fractions are subjected to repeated chromatographic purification under the same conditions. Concentrates containing cyclosporin A from two chromatographic columns are combined and crystallized in a hexane-acetone system (4: 1).

EXAMPLE 5

They carry out the biosynthesis, isolation and purification of cyclosporin, as in examples 1 and 2. Crystallization of CA.

The concentration of cyclosporin A in an amount of 130 g (content of cyclosporin A 98%) is dissolved in 0.3 l of acetone. The solution is filtered and slowly poured into a reactor containing 1.2 L of hexane while stirring. The reactor is cooled to 5 ° C. The crystalline precipitate of cyclosporin A is separated by filtration, washed with 0.2 l of chilled hexane and dried in a vacuum oven at 50-60 ° C until the moisture content in the substance is not more than 1%. A substance is obtained in an amount of 110 g with a cyclosporin A content of 99.1%. Yield 84% of the starting preparation.

Thus, the present invention is an improved method for producing a cyclosporin preparation, allowing to obtain a highly active cyclosporin A preparation in crystalline form.

Claims (2)

1. Highly active crystalline preparation of cyclosporin A (HCFC), which is a cyclic undecapeptide obtained from a fungus of the genus Tolypocladium inflatum subsporum blastosporum, registered in the collection of the State Scientific Center for Antibiotics No. 330A, when cultured on a standard agar medium followed by fermentation, isolation purification and crystallization, characterized in that the culture medium additionally contains rubomycin at a concentration of 2 μg / ml and zinc sulfate at a concentration of 5 mg / ml, and fermentation the medium contains maltex and D, L-valine as the main components, the final concentration in the medium being 2.0-2.5 mg / ml, and the product isolated from dried mycelium of the fungus by extraction with petroleum ether before and after purification by silica gel chromatography and crystallized in the hexane-acetone system (4: 1). 2. A method of obtaining a highly active crystalline preparation of HCFC described in claim 1, comprising cultivating the fungus on an agar medium, fermentation, isolation and purification, characterized in that the medium additionally contains 2 μg / ml rubomycin and 5 μ / ml zinc sulfate, and fermentation the medium contains maltex and D, L-valine as the main components, then the fungal mycelium containing the complex of cyclosporins A, B, C, D is separated from the native solution, the mycelium is dried and the target product is extracted with petroleum ether before and after purification by chromatography on silica gel or alumina, fractions containing cyclosporin A are combined and crystallized in a hexane-acetone system (4: 1) with a target product content of at least 97.5%.