DD285793A5 - METHOD FOR MICROBIAL RECOVERY OF CICLOSPORINES - Google Patents

Abstract

The invention describes the microbiological recovery of the cyclic peptides cyclosporin A, B and C, which is selectively possible by using specific variants isolated from the fungus strain Cylindrocarpon lucidum. The field of application of the invention is primarily in the field of human medicine, since, as a result of the chemotherapeutic and pharmacological properties of cyclosporins, in particular cyclosporin A, this compound is increasingly of interest as a remedy. {Microbiology; Fungal strain; Strain variants; Cylidrocarpon lucidum; Drug production; cyclic peptide; Cyclosporin A; Cyclosporin B; Cyclosporine C; Remedies; Human Medicine}

Description

Field of application of the invention

The invention relates to a process for the preparation of cyclic peptides, wherein one of these compounds, the cyclosporin A, by its chemotherapeutic and pharmacological properties is increasingly of interest as a remedy.

The active ingredient cyclosporin A is especially useful in the therapy of such human diseases in which immune reactions play a role. Thus, the applicability relates to autoimmune diseases of various kinds (e.g.

Multiple Sclerosis, Uveitis, Type I Juvenile Diabetes, Anemia, Psoriasis, and Others. a.), on the anti-inflammatory effect (eg.

Arthritis and rheumatic diseases), antiparasitic action (e.g., malaria, schistosomiasis, filariasis, coccidiomycosis) and other uses.

However, the current main field of application is the use of the immunosuppressive substance in a variety of transplantations (e.g., kidney, heart, lung, bone marrow, etc.) (to avoid rejection of implanted tissues and organs.

Characteristic of the known state of the art

The cyclosporins belong to a novel group of cyclic peptides which are found as naturally occurring metabolic products of microorganisms and are mentioned for the first time in CH-PS 17111-73 and CH-PS 14043-74, respectively.

In addition to the Ciclosporins present in nature, there are also semisynthetic compounds, which, however, have so far played no role in terms of therapeutically relevant substances.

The most important compound of this substance group is cyclosporin A (WHO name ciclosporin). Other naturally occurring, formed by microorganisms Ciclosporine are z. The compounds cyclosporin B (Ala'-cyclosporin), cyclosporin C (Thr ² ciclosporin), cyclosporin D (Val'-cyclosporin) and others.

So far, the following microorganisms are known that are able to synthesize Ciclosporine:

Cylindrocarpon lucidum BOOTH, NRRL 5760

(DD-PS 115695, 1974) Tolypocladium inflatum GAMS, NRRL 8044

(DD-PS 115695, 1974) Fusarium solani MARTIUS (Satcardo emend., Synder et Hansen)

MCI 1549undMCI-1550

(JP-Os 55-138523, 1980)

In the mentioned DD-PS 115695 the microbial recovery of cyclosporins A and B using the strain Cylindrocarpon lucidum NRRL 5760 is described. The recovery and isolation of cyclosporin C from the same parent material under identical culture conditions is illustrated in CH-PS 141195.

In the characterization of the NRRL 5760 producer strain, it is assumed that this strain is a new, previously unknown representative within the taxonomic species description for Cylindrocarpon lucidum. Substantial justification for the delimitation of the strain to the well-known species conception (see: C. BOOTH, 1966, The Genus Cylindrocarpon, Mycological Papers No. 104,21) is the description of the size of the macroconidia and the determined growth rate of individual colonies on potato dextroso agar.

To obtain the individual Ciclosporine from the strain NRRL 5760, the growth of the microorganism takes place in emerscr production culture, ie on the surface of a liquid nutrient medium. The producer strain is incubated for 11 days at 27 ⁰ C, followed by the harvest of the culture and processing of the active ingredient-containing mycelium. Details of the proportionate content of the individual Ciclosporine after completion of the fermentation at the beginning of the chemical workup of the culture are not known.

Own investigations for the reproduction of the patent data with the help of the strain NRRL 5760 confirm in principle the known manufacturing process with regard to Bildupgsvermögen and proof of the different Ciclosporinverbindungen. As a disadvantage, however, in the field of microbiological culture control, conspicuous process fluctuations have been recorded in a number of experimental batches under constant cultivation conditions. This was evident, among other parameters, in particular with regard to the amount of cyclosporin A formed. The irregularities which occurred in the microbiological production culture also led to changed conditions for the isolation and purification of the active ingredient, which generally made standardization of the production process more difficult.

Zelf of the invention

The object of the invention is to develop a stable, reproducible microbiological production process which allows the cyclic peptide cyclosporin A to be obtained while simultaneously increasing the biological yield level of the fermentation.

Explanation of the essence of the invention

The invention has for its object to develop a technical method for the producer strain NRRL 5760, which eliminates the shortcomings of the aforementioned known solution.

According to the invention the object is achieved in that one uses and cultivates as vaccine obtained by selection variants of the strain NRRL 5760. In this case, the variant "f" is preferably used for the preparation of cyclosporin A and the variant "b" is used for the preparation of cyclosporin C.

Interestingly, in addition to a significantly improved performance base material - which concerns especially the synthesis of cyclosporins A and C- also negative for cyclosporin A formation variant was obtained, which forms the known drugs only in the slightest traces. It is striking that this variant occurs more or less frequently and the product yield of the starting strain NRRL 5760, which thus proves to be a heterogeneous strain, can influence in various unfavorable ways.

The preservation of these, extremely distinctive variants-also with regard to morphological criteria-was not to be expected insofar as the strain NRRL5760 was hitherto in no way known to be uneven in the art. In addition, the occurrence of a morphological and physiological variability was not described, and in the present method was no effect on the genetic starting material, eg. B. by mutagenic chemicals, irradiation o. Ä. Made

w. earthen.

The variants isolated and cultivated according to the invention from the strain NRRL 5760 essentially correspond to the characteristics of the parent description (growth rate, size of the macroconidia) known in the cited patents.

Deviant and new is the macroscopic assessment of the strain, in particular the characteristic of its isolated single colonies, which allow a distinctive distinction of the variants, which, however, on the quoted potato dextrose agar hardly, but iodine, for example, on a Nährmodium containing casein peptone and glucose, especially works well (see Appendix 1, description of the variants).

In addition, there are differences in sporulation behavior (see Appendix 2). However, the decisive criterion in law characterization of the isolated variants - in terms of the manufacturing process - the new educational asset for the individual cyclosporins.

This different synthetic ability of the individual variants recognized under uniform and comparable conditions of cultivation can thus be exploited, both with the aim of increasing the hitherto most important cyclosporin compound, cyclosporin A, and with regard to the deliberate selection of a particular one Variant from the viewpoint of the priority of obtaining a particularly desired Ciclosporinverbindung, z. As the cyclosporin C, which also shows immunosuppressive activity.

Independently of this, a change in the medium of the production culture led to a general increase in product outlays.

The isolation of the variants was carried out after cultivation of the parent strain under aerobic and sterile culture conditions on several media with different C and N sources. The isolates, as is common in microbiological processes, are subject to constant testing and, where appropriate, a subsequent selection procedure prior to their use in the production process of the production culture in order to assure the purity and stability of the biological material.

The aerobic, emerse production culture is done by cultivating a defined amount of spores obtained from a stem guide on solid medium and transferred to a liquid, sterile nutrient medium containing known assimilable sources of carbon and nitrogen as well as mineral salts and trace elements.

The propagation of the parent strain NRRL 5760 or its variants is carried out in Erlenmeyerkolban or penicillin bottles at a temperature between 2O ⁰ C and 3O ⁰ C over a period of 7 to 18 days.

After completion of the fermentation, the extraction of Ciclosporine from the culture is carried out by commonly used methods. The determination of ciclosporins is carried out by thin layer chromatography and HPLC analysis in comparison with standard Roinsiibstanz.

The following examples serve to illustrate the invention in comparison to the known prior art, without limiting it thereto.

1st embodiment

Spores of strain NRRL 5760, stored under liquid nitrogen, are suspended in sterile 0.9% IgM NaCl solution and transferred to a solid medium containing either the extract of 100g grated potatoes, 20g glucose and 30g agar or 20g malt extract, 4g yeast extract and 30g agar per liter of tap water.

After a period of incubation of 10 days at 27 ° C. and intermediate storage of the culture at 4 ^° C. for at least 8-10 days, the strain is retransmitted using the spores, which in turn are suspended in 0.9% NaCl solution, either by application of the Lindtnerschan droplet Verdiahrenü to monospores or isolated by Ausplatten appropriate dilutions isolated from each other on a medium for about 3-5 days at 27 ⁰ C containing per liter of demineralized water 10 g of casein peptone, 20g glucose, 1.8g NaCl and 30g agar.

From the set grown single colonies are then isolated from the parent strain deviating lines and

again passagated separately on the same medium.

Cultures kept in this way are stable over several passages and provide the starting material for the

respective production culture.

In the case of a phenotypic change in the isolates during the cultivation, which usually results in a shift

Ciclosporinspectrum goes along, these are again to win according to the above description.

2nd embodiment (reference example)

Penicillin bottles each containing 700 ml of a nutrient solution content, per liter of demineralized water 30 g of sucrose, 10g of corn steep liquor (corresp. About 0.6 g nitrogen), 3g of NaNO _3, 1 g K ₂ HPO _4, 0.5 g MgSO ₄ x 7 H ₂ 0, 0.5 g KCl and 0.01 g FeSO ₄ × 7 H, 0 are inoculated with 7 ml of a conidia suspension (about 5 × 10 ⁷ conidia per ml) of the strain NRRL 5760 and incubated at 27 ⁰ C for 14 days. The separated from the culture broth and homogenized with the Ultraturrax mycelium is separated after extraction with 90% methanol by filtration from the solvent and the filtrate concentrated to dryness at room temperature. After taking up the residue in a mixture of acetonitrile / water, the active ingredient is determined by HPLC analysis

 The proven level of cyclosporin A is on average at 125 mg per liter batch volume.

3.Ausführungsbeispiel

Penicillin bottles with each 900ml or 100ml Erlenmeyer wide-mouth flask, each containing 75ml content of a nutrient solution, which - in contrast to the 2nd embodiment - per liter of demineralized water 40g sucrose, 15g corn steep liquor (corresponding to about 0.9g nitrogen), 3g NaNO ₃ , 1g K ₂ HPO ₄ , 0.5g MgSO ₄ x 7H ₂ .0.5g KCl and 0.01g FeSO ₄ .7H ₂ O are mixed with 9ml and 0.75ml of a conidia suspension (about 1-5 x 10 ⁷ conidia per ml) of the strain NRRL 5760 and incubated for 14 days at 27 ⁰ C. The mycelium worked up according to the second exemplary embodiment gives a content of cyclosporin A of on average 190 mg per liter of batch volume.

4th embodiment

The conditions of the culture control are also selected as described in the third embodiment, but in contrast to that the variant "w" of the strain NRRL5760 isolated according to the 1st embodiment is used. The determined content of cyclosporin A is below 10 mg on the 14th day of culture per liter batch volume Ciclosporins B and C could not be detected during the cultivation.

5th embodiment

The conditions of the culture control are also selected as described in the third exemplary embodiment, but in contrast to this, the variant 1 h used is isolated variant "h" of strain NRRL 5760. The content of cyclosporin A is on average 110 mg per liter of batch volume

 The ratio of ciclosporins A. B, C among each other is 80:10:10.

6.Ausführungsbelsplel

The conditions of the culture run are also selected as described in the 3rd embodiment, but in contrast to that, the variant 1 according to Example 1 is used isolated strain "f" of the strain NRRL 5760. The content of cyclosporin A is on average 260 mg per liter of batch volume

 The ratio of Ciclosporine A, B, C with each other is about 85: 5: 10.

7.Ausführungsbelsplel

The conditions of the culture control are also selected as described in the third exemplary embodiment, but in contrast thereto the variant "b" of the strain NRRL 5760 isolated according to Example 1 is used. The content of cyclosporin A is on average 130 mg per liter of batch volume However, the cyclosporin C content averages at 300 mg per liter batch volume.

Cyclosporin B is only formed in trace amounts below 10 mg per liter of batch volume, i.e. the ratio of cyclosporins A and C with each other is about 30:70.

Attachment 1

Description of the phenotype of the variants isolated from strain NRRL 5760 (single colony) Variant "f" flat, punctiform in the center, mycelium thinly wooled, greyish-beige, Bottom orange with bright edge

"B" flat, finely textured surface, without woolly aerial mycelium,

Colored circularly from gray-beige to pink-orange, bottom pink-orange, light edge

"W" hat-like raised with flat rim, woolly aerial mycelium, reir white,

Underside light gray-brown

"H" flat, radial furrows, circularly folded in the center, fine woolly, white to light beige,

Bottom light brown beign Culture medium: (data per liter)

20g glucose, 10g caseinpaste, 1.8g NaCl and 30g thread agglomerator 2

Table: Conidia formation of isolated variants of the strain NRRL 5760 after 7 days of culture at 27 ° C.

parent material Konidienzahlpro Konidienzahlpro colony cm ¹ colony NRRL5760 3,3x10 ' 1,1x10 ' Variant "f" 3.5x10 " 1,1x10 ' "B" 2,3x10 ' 1.0x10 ' "W" 1.5x10 ' 5,7x10 ⁵ "H" 1.5x10 * 7.4 χ 10 ⁴

Growing medium: see Appendix 1

Claims (3)

1. A process for the microwave extraction of Ciclosporinen by fermentation of the strain Cylindrocarpon lucidum NRRL 5760 using conventional Nährlösungsbestandteile, characterized in that one uses as a seed material obtained by selection variants of the strain NRRL 5760 and cultured. 2. The method according to claim 1, characterized in that one uses the isolated variant "f" and cultured for the production of cyclosporin A as inoculum. 3. The method according to claim 1, characterized in that one uses the isolated variant "b" and cultured for the production of cyclic sporin C as inoculum.