DD295870A5 - METHOD FOR MICROBIAL RECOVERY OF CYCLOSPORINS - Google Patents

Abstract

The invention describes a technical production process for the microbial recovery of cyclosporins, which is made possible in an effective manner only by the use of a new, hitherto unknown fungal strain, which has been taxonomically determined as Sesquicilliopsis rosariensis G. ARNOLD. Due to the chemotherapeutic and pharmacological properties of cyclosporins, in particular cyclosporin A, the field of application of the invention lies primarily in the field of human medicine, where the class of drugs is increasingly of interest as a remedy. {Microbiology; Fermentation; Fungal strain; Sesquicilliopsis rosariensis G. ARNOLD; cyclic peptide; cyclosporine; Cyclosporin A}

Description

Field of application of the invention

The invention relates to a novel process for the preparation of cyclic peptides, one of these compounds, the cyclosporin A, by its chemotherapeutic and pharmacological properties, especially the pronounced immunomodulatory effect in general medicine preferred but increasingly in transplantation medicine as a pharmaceutically interesting substance and as a remedy gained worldwide importance.

Characteristic of the known state of the art

The cyclosporins belong to a novel group of peptides which are mentioned for the first time in the patents CH-PS 589716 and CH-PS 603790 and their chemical structure and biological activity, for example in the monograph of BOREL (Ciclosporin, Progress in Allergy 38, Karger- Verlag 1986).

It is known that in the industrial production process, the ability of microorganisms to synthesize naturally occurring cyclosporins is exploited by using these strains as product formers in a fermentation process while maintaining well known and usual culture conditions.

However, the extremely small number of available and hitherto technically usable producer strains must be assessed as a disadvantage.

While the microorganism Tolypocladium inflatum GAMS, NRRL 8044-the hitherto only known producer of industrial significance-and the strains Fusarium solani MARTIUS MCI-1549 and MCI-1550 (JP-OS 55-138523,

1980) are capable of product formation in submerged culture, the synthesis of cyclosporins in Cylindrocarpon lucidum BOOTH, NRRL 5760 only in Emers culture. However, the most economical production method is submerged fermentation.

According to DREYFUSS et al. (Europ. J. Appl. Microbiol., 3, 1976, 125-133) are intechn. Fermentors with strain Tolypocladium inflatum GAMS achieved biological yields of 200mg cyclosporin A per liter of culture on the 12th day of fermentation, while AGATHOS et al. (J. of Industrial Microbiology 1,1986, 39-48) with the same strain gives values of maximum 180 mg CY A / l.

By means of special investigations, however, KOBEL and TRABER (Europ. J. Appl. Microbiol. Biotechnol., 14,1982, 237-240) were able to achieve a further increase in cyclosporin Α synthesis by feeding special amino acids whose industrial use must be ruled out. When feeding 8g threonine or 8g valine, cyclosporin A received 396mg / l resp.

319mg / l received.

In the case of immobilizing the cells of Tolypocladium inflatum GAMS, FOSTER et al. (Biotechnology Letters 5, 10.10.1983, 693-696) max. Yields of 240 mg / l on the 18th culture day or 300 mg / l for untreated starting material.

With the application DD-285793 a method was proposed, according to which the Emersproduzent Cylindrocarpon lucidum BOOTH, NRRL 5760 in the form of its variants forms up to 260 mg / l cyclosporin A.

In terms of economical use of the producer strains, these space-time yields are extremely unsatisfactory for a technical production process which, moreover, has a disadvantageous effect with regard to the immediate subsequent isolation and purification of the various cyclosporins, in particular cyclosporin A, which inevitably includes active ingredient losses ,

Object of the invention

The object of the invention is to provide therapeutically valuable cyclic peptides of the substance group of cyclosporins with an economically favorable production process in high biological yield.

Explanation of the essence of the invention

The invention has for its object to expand the number of available and capable of Cyclosporinsynthese producer strains and to enable them in technically relevant order of magnitude to a high product synthesis. According to the invention the object is achieved by using for fermentation a new strain of the fungus species Sesquicilliopsis rosariensis G.ARNOLD or a strain variant obtained therefrom by mutagenesis and / or selection as a producer strain. Surprisingly, from a natural habitat of the subtropical climate zone, it has been possible to isolate a new, once unique microorganism that not only accumulates the desired substance group as a metabolite.

but this and in particular the cyclosporin A forms after appropriate strain conditioning as a result of mutagenesis and selection processes in submerged culture in a completely unexpectedly high order of magnitude in the context of a multi-stage technical fermentation process.

The novel microorganism used in the present process for the preparation of cyclosporins is deposited in the ZIMET Depository for Microorganisms, Jena-GDR, under number IMET-43888; as well its highly productive trunk line F 605, which received the deposit number IMET-43887.

Of course, the invention also includes all by mutation and / or selection of the wild strain or o.g. Mutants emerged strain variants.

The original microorganism is a member of fungi isolated from a soil sample of Cuba in the Sierra del Rosario and taxonomically assigned to a new genus and species called Sesquicilliopsis rosariensis G.ARNOLD.

The organism grows on a variety of nutrient media, which contain the usual nutrients for mushrooms.

When grown on malt agar in a natural day-and-night rhythm, there are approximately 8 cm large colonies within 10 days with weak, slightly fuzzy, white aerial mycelium, which extends to the colony edge. Ring formation is weak in the outer parts of the colony. A faint scent of binoculars is noticeable. The aerial mycelium consists of septate, branched hyphae, which appear white in mass. The ascending to upright conidiophores are usually strong and more or less whorled or irregularly branched, carry at their branches several pharyids, which are formed by Übergipfelung, which are colorless, pretty and slender designed.

The conidia are formed in succession at the top of the phialides; they are oblong, unicellular, hyaline, glued smoothly and thinly to a small head. Chlamydospores were not observed.

Of all similar genera with phialidic, conidiogenic cells (e.g., Fusarium with microconidia, Gliocladium, Verticillium, Sesquicillium, Sympodiophoya), the Sesquicilliopsis rosariensis G.ARNOLD strain differs due to the peculiarities noted, but especially due to the manner in which the conidiogenic cells are arranged. A detailed description of the strain and other morphological details of the taxonomic classification of the tribe are currently being included in a running monograph series for Cuban mushrooms.

The cultivation of the new strain Sesquicilliopsis rosariensis G.ARNOLD and its isolated lines takes place under aerobic conditions.

After culturing the strain on suitable agar media, the spores are transferred into a liquid, previously sterilized medium, the resulting oycell being isolated in a manner known per se at temperatures below CJ ° C., preferably 24 ° C., for a maximum of 18 days. preferably 12-14 days, shaken in glass flasks, glass fermentors and / or in fermenters made of highly refined steel alloys or stirred, cultured in a submerged manner.

The nutrient medium consists of carbon and nitrogen sources as well as the usual mineral salts and trace elements. As carbon source, especially maltose, glucose and sucrose can be used; As a nitrogen source, in particular complex, amino acid-rich substrates such. As peptones, dry yeast and corn steep liquor. The cyclosporines formed by the microorganism used according to the invention are not secreted after their synthesis into the liquid, aqueous environment surrounding the strain and therefore have to be extracted from the mycelium. Cyclosporin A and other cyclosporins are detected by adding 1: 5 methanol to the culture suspension, homogenizing the mycelium with the Ultra-Turrax and shaking out the methanol extracts concentrated to dryness several times with methylene chloride / water. The subsequently combined extracts, which are likewise concentrated to dryness, can then be taken up in a mixture of acetonitrile / water and, in a manner known per se, using HPLC against standard methods, based on the publication by KREUTZIG (J. of Chromatography 290, 1984, 181-186). Pure substance can be exactly determined qualitatively and quantitatively.

After extraction of larger amounts of the active substance-containing mycelion separated from the culture solution and multiple purification steps of the extraction residue, the various cyclosporins and preferably cyclosporin A can be obtained in high yield as a high-purity substance which can be uniquely identified by conventional analytical methods.

embodiments

1. 500 ml Erlenmeyer wide-mouth flasks contain per 100 ml of the nutrient medium of the following composition

75g / l maltose

25g / l casein peptone

1 g / l KH ₂ PO ₄

2.5 g / L KCl in deionized water, pH 5.5.

Each flask is inoculated with a spore suspension (1x10 ⁷ spores / ml) as an inoculum of the strain Sesquicilliopsis rosariensis G.ARNOLD IMET # 43888, which is made with sterile isotonic saline from a 10-14 day old culture on malt agar.

After 3 days incubation at 24 "C on a shaker at a frequency of 190U · min" ^1, the pre-culture is transferred to fresh medium of the same quality in a ratio of 1:10 and cultured again in the same conditions compliance. The cyclosporin A content determined on the 12th day was a maximum of 213 mg / l of culture suspension.

2. Cultivation and cultivation of the producer strain is carried out as described in the first embodiment, but in contrast, the high-performance strain F 605, IMET 43887 obtained from the wild-type strain IMET 43888 by mutagenesis and selection is used.

The maximum proven content of cyclosporin A on the 12th day of culture was 1890 mg / l.

3. Cultivation and cultivation of the strain F 605, IMET 43887 is carried out as described in the first embodiment, however, in contrast, the producer strain after the preculture in a 5-I or. Transferred 12 l laboratory fermenter and in the same medium or in another medium, which, for. B. contains glucose instead of maltose, cultured aerobically.

The fermentation of the strain carried out for up to 15 days resulted in cyclosporin Α values up to a maximum of 2460 mg / l.

4. According to a carried out according to Embodiments 1-3 two-stage Impfmaterialanzucht in shake flask or Ruhrfermentor the strain F 605, IMET 43887 was used as an inoculum for the production stage in a 450-l fermenter made of alloy steel and transferred in such a way that Start of fermentation 0.1 g / l mycelium dry mass in the 75 g / l glucose, 25 g / l casein peptone, 2.5 g / l KCl, 1 g / l KH ₂ PO ₄ and tap water, previously sterilized 60 'at 120 ⁰ C nutrient solution, were.

Already after 12 days of cultivation with purely batchwise process control, 860 mg of cyclosporin A per liter of culture were measured. The product formation rate reached against fermentation end was 0.006 g / Lh for cyclosporin A.

The cultivation took place at 24 ° C. Oxygen limitation was avoided. The constructional and operational parameters guaranteed oxygen transport coefficients (K _L a) of more than 40 h " ¹ .

Claims (3)

1. A process for the microbial production of cyclosporins, characterized in that one uses for fermentation a new strain of the fungal species Sesquicilliopsis rosariensis G. ARNOLD or a strain obtained therefrom by mutagenesis and / or selection as a producer strain. 2. The method according to claim 1, characterized in that one uses the new strain of the fungus species Sesquicilliopsis rosariensis G.ARNOLD IMET 43888. 3. The method according to claim 1, characterized in that one uses the new strain of the fungus species Sesquicilliopsis rosariensis G.ARNOLD F 605, IMET 43887.