EP1713455A2

EP1713455A2 - Method for producing sorbicillactone a

Info

Publication number: EP1713455A2
Application number: EP05701272A
Authority: EP
Inventors: Gerhard Bringmann; Gerhard Lang; Tobias Gulder; Karsten Schaumann; Werner E. G. Mueller; Sanja Perovic-Ottstadt; Ruediger c/o Leibniz-Institut für Meeres- STOEHR; Jutta Institut Für Meereskunde WIESE; Rolf Institut für SCHMALJOHANN; Johann Institut für Meereswissenschaft IMHOFF
Original assignee: Johannes Gutenberg Universitaet Mainz; Julius Maximilians Universitaet Wuerzburg
Current assignee: Johannes Gutenberg Universitaet Mainz; Julius Maximilians Universitaet Wuerzburg
Priority date: 2004-01-30
Filing date: 2005-01-31
Publication date: 2006-10-25
Also published as: CA2554733A1; DE102004004901A1; US20070135515A1; WO2005072711A3; JP2007519414A; DE102004004901B4; WO2005072711A2

Abstract

The invention relates to an improved method for cultivating the fungus Penicillium chrysogenum, especially strain KIP 3201, for producing the antitumoral natural substance Sorbicillactone A of formula (I) and its derivatives in an optimized manner and in large amounts. The invention also relates to an optimized method for obtaining and purifying said substance from the fungal biomass and from the culture medium. The invention also describes the biological activity of Sorbicillactone A of formula (I) and investigations relating to the genotoxicity of the compound.

Description

Process for the preparation of sorbicillactone A

The present invention relates to a process for the optimized production of the biologically active compound sorbicillactone A and derivatives thereof by growing Penicillium chrysogenum, in particular strain KIP 3201. The invention further relates to a process for purifying large amounts of sorbicillactone A and derivatives thereof from the culture medium and the fungal biomass and the use of sorbicillactone A for the treatment of diseases and infections.

Background of the Invention

There are two main ways to search for new, biologically active compounds. The isolation of active natural products from biological systems on the one hand, as well as the synthesis of novel compounds, partly inspired by secondary metabolites from nature, on the other. Of particular interest is the study of the metabolites of macro and microorganisms in difficult to access and. extreme habitats. For example, marine eukaryotic organisms, especially sponges, are a rich source of bioactive substances (Sarma AS, Daum T, Müller WEG (1993) Secondary metabolites from marine sponges. Academy of Non-Profit Sciences in Erfurt, Ullstein-Mosby Verlag, Berlin). The reason for this is the sessile way of life of these organisms. They rely on the transport of their food, floating particles (algae, bacteria or fungi) in the water. These particles are bubbled in through a constant stream of water. This leads to an increased risk of bacterial or fungal infections. For this reason, sponges rely on efficient defense mechanisms to protect against such infections from biologically active secondary metabolites. However, it often remains unclear whether these substances are formed by the sponge itself or by one of the numerous microorganisms associated with the sponge. It is therefore particularly interesting to isolate these microorganisms living in the sponge and to study their metabolite spectrum. Accordingly, it is a major goal of research to develop methods that enable these microorganisms to be grown in culture in order to make their biologically active secondary metabolites available in sufficient quantities.

Today it can be assumed that only about 5% of the microorganisms occurring in marine eukaryotic organisms can be cultivated. It can therefore be assumed that their potential is still largely untapped. For the successful and sustainable use of these microorganisms as sources of new pharmaceuticals, it is important to develop cultivation methods which are characterized in that the production of the desired metabolites is increased as much as possible, while the formation of the undesired by-products is largely prevented.

The range of applications for commercial use of the bioactive secondary metabolites is broad and ranges from the treatment of neurodegenerative diseases, bacterial, viral and fungal infections to the treatment of tumors.

There is also an intensive search for those bioactive substances which develop a high specificity against defined tumors and at the same time are opportunistic infections, e.g. B. by viruses.

The bioactive substance sorbicillactone A and derivatives thereof were recently isolated from a Penicillium strain which was isolated from a marine sponge of the genus Ircinia (DE 102 38 257.3). It was found that sorbicillactone A and derivatives thereof have unforeseeable pronounced antitumor and antiviral as well as anti-inflammatory properties. Up to now, it has not been possible to scale up the processes used to prepare this substance and its derivatives. However, the reliable production of large amounts of sorbicillactone A and derivatives thereof for promising use in the Treatment of diseases such as cancer and inflammation is urgently needed.

It is therefore the object of the present invention to provide a process for the optimized production of the natural product sorbicillactone A and derivatives thereof, and for the extraction of sorbicillactone A and derivatives thereof from the culture medium and the fungal biomass and for the purification of large amounts of sorbicillactone A and derivatives thereof to deliver.

According to the invention, this object is first achieved by providing a method which comprises the following steps:

a) cultivating a fungus of the genus Penicillium at 20-25 ° C. in a suitable growth medium at a salt concentration of 2-5% until a compact surface mycelium is formed,

b) raising the temperature to 28-35 ° C and further incubating for 5-10 days,

c) separating the culture fluid from the mycelium, and

d) extracting sorbicillactone A and derivatives thereof from the culture medium, and optionally,.

e) underlay the mycelium with fresh medium with a reduced salt concentration of 0.5-1.5% and incubate at 28-35 ° C for 3-8 days,

f) repeating steps c) and d), and if appropriate,

g) repeating steps e) to f), and.

h) extracting sorbicillactone A and derivatives thereof from the culture medium and / or the mycelia. According to the invention, it is therefore a method in which the mushroom is grown under special conditions which bring about an increased yield and at the same time increased production rates. So growth and production of sorbicillactone A and precursors or derivatives thereof are controlled by varying the culture conditions and z. B. initiated and stimulated by adding NaCI. In particular, the production is accelerated in that conditions for optimal growth and optimal production conditions are successively realized in step processes, for. B. by changing the incubation temperature from, for example, 20-25 ° C to 28-35 ° C and changing the salt concentration from 2-5% to 0.5-1.5%. At other incubation temperatures, little or no production of sorbicillactone A and / or derivatives thereof was found. Surprisingly, the method of the present invention thus reliably provides large amounts of sorbicillactone A and derivatives thereof.

A method according to the invention is preferred in which the fungus Penicillium chrysogenum, in particular the strain KIP 3201, is used for production.

In a further aspect of the present invention, it is a method which is characterized in that an increase in the yield of sorbicillactone A is achieved in that the growth media by varying the substrates and substrate concentrations, such as. B. by addition ^ of pyruvate, glutamate, proline and acetate, but also of sorbicillin and other biosynthetic precursors of sorbicillactone A, are changed.

In a preferred embodiment, it is a process in which sorbicillactone A and derivatives thereof are produced in a flat bed process. Production of sorbicillactone A and derivatives thereof can be further accelerated in that the liquid phase from the cells at suitable times is separated and the cells supplied with fresh medium are stimulated to production again. Suitable times are, for example, 3-10 days.

A type of inoculum is preferred in the form of a solid-bound form of the fungus, the inoculum of which is floating on solid bodies, for. B. cereal grains or polystyrene balls, is presented.

A method is further preferred in which a decrease in the surface mushroom mycelium is prevented. This can be done by integrating a carrier device for stabilizing the surface mycelium into the cultivation container. A suitable form of a carrier device is, for example, a network.

A preferred method is characterized in that the sorbicillactone A produced and derivatives thereof are bound directly from the culture media to a solid exchanger. This bound form is then used for purification in further steps. The elution from the exchanger can be carried out using organic solvents such as methanol, ethanol, ethyl acetate, heptane or acetonitrile. In this way, sorbicillactone A and derivatives thereof can be obtained from the exchanger in a highly enriched form.

Another preferred method is characterized in that the sorbicillactone A produced and derivatives thereof are extracted from the fungal mycelium which has been separated from the culture medium by adding organic solvents. Ethyl acetate is preferably used.

In a further aspect of the present invention, it is a method which is characterized in that a further extraction of the crude extracts is carried out. The raw extracts can be acidified and then sorbicillactone A and derivatives. of which are extracted using organic solvents. Ethyl acetate is also preferably used. It is also advantageous to perform an optimized purification of the extracts using Fast Centrifugal Partitioning Chromatography (FCPC). Furthermore, an optimized purification of the extracts by means of gel chromatography, e.g. B. on Sephadex LH-20, preferred. Various organic solvents can be used to elute sorbicillactone A and derivatives thereof.

A preferred aspect of the present invention is a process which is characterized in that it comprises derivatizing sorbicillactone A to sorbicillactone A methyl ester.

It was found that neuronal cells incubated with sorbicillactone A showed a sharp drop in the intracellular calcium level after the addition of L-glutamic acid and serotonin (5-HT). L-glutamic acid and serotonin, as neurotransmitters, are etiologically involved in a number of neurodegenerative diseases. Because of these properties, sorbicillactone A or derivatives thereof could be used in the treatment of neurodegenerative diseases and the associated symptom complexes.

The genotoxicity of sorbicilllactone A was also tested on L5178Y mouse lymphoma cells (ATCC CRL 1722). It was found that no DNA strand breaks were induced in cells incubated with 1, 3 and 10 μg / ml sorbicillactone A. In contrast, a significant increase in the proportion of DNA strand breaks was found after 24 hours of incubation at a concentration of 30 μg / ml of sorbicillactone A. Because of these properties, it is advantageous to use sorbicillactone A or derivatives thereof in the treatment of leukemia.

Sorbicillactone A also induces apoptosis in L5178Y cells after 4 hours of incubation at a concentration of 10 and 30 μg / mL. Because of these properties, the use of sorbicillactone A or derivatives thereof is preferred in the treatment of leukemia. Furthermore Sorbicillacton A or derivatives thereof in the treatment of viral infections ₇ are applied. Details of this are described in the examples of DE 102 38 257.3.

Another aspect of the present invention relates to methods for producing a pharmaceutical composition, wherein sorbicillactone A or derivatives thereof are formulated together with suitable pharmaceutically acceptable auxiliaries and additives. Pharmaceutical compositions in which sorbicillactone A or derivatives thereof are present in an amount such that there is a concentration range between 0.3 and 30 μg / ml during the treatment in vivo are preferred.

Another aspect of the present invention relates to the fungus strain of the genus Penicillium chrysogenum KIP 3201. This was deposited on January 14, 2004 with the German Collection of Microorganisms and Cell Cultures GmbH under the number DSM 16137.

In the context of the present inventions, a "derivative" is intended to be a compound derived from the general formula 1, which is substituted, for example, by various of the radical groups specified for R 1 to R and X or Y, and mixtures of various of these compounds, which, for example, belong to a "personalized" medicament that is tailored to the particular illness to be treated and / or the patient can be processed on the basis of diagnostic or data on the success or course of treatment. A derivative is also to be understood as a compound of the sorbicillactone A class, which is derived from other (eg) marine organisms. can be isolated as the one mentioned here (by way of example).

The present invention will now be further described by the following examples with reference to the accompanying figures, without, however, being restricted thereto. It shows

Figure 1 shows the influence of sorbicillactone A on the intracellular calcium level of neuronal cells.

Figure 1A shows the treatment of neurons with 200 uM L-glutamic acid (L-Glu) and ^'

2.5 mM Ca ²⁺ (•), the neurons treated with 10 μg / mL sorbicillactone A (o) 5 min before adding 200 μM L-Glu and 2.5 mM CaCl ₂ (at time 10 min) were preincubated. The change kinetics were measured continuously over approximately 20 minutes. Both the mean (n = 19) and standard deviation (± SE) were determined.

FIG. 1B shows the treatment of neurons with 200 μM serotonin (5-HT) and 2.5 mM

Ca ²⁺ (•), the neurons treated with 10 μg / mL sorbicillactone A (o) being preincubated 5 min before the addition of 200 μM 5-HT and 2.5 mM CaCl ₂ (at the time 10 min) , The change kinetics were measured continuously over approximately 20 minutes. Both the mean (n = 21) and standard deviation (± SE) were determined.

FIG. 2 shows the results of the "comet assay" after the incubation of L5178Y mouse lymphoma cells (ATCC CRL 1722) with 1, 3 and 10 μg / ml sorbicillactone A. FIG. 2A shows the results after an incubation period of 4 hours. Figure 2B shows the results after 24 hours of incubation. The mean value (A: n = 25, B: n = 29) and the standard deviation (± SD) were determined. FIG. 3 shows the results of the “Fast Microassay” after the incubation of L5178Y mouse lymphoma cells (ATCC CRL 1722) with 1, 3, 10 and 30 μg / ml sorbicillactone A. FIG. 3A shows the results after an incubation period of 4 hours. Figure 3B shows the results after 24 hours of incubation. The mean (A: n = 5, B: n = 7) and the standard deviation (± SD) were determined.

Example 1: Cultivation of the fungus Penicillium chrysogenum in saline media for the optimized production of sorbicillactone A

In order to optimize the production of sorbicillactone A, media and culture conditions have been established that differ from the usual ones, e.g. as a result of that. cultivated at elevated salt levels and temperatures that are unusual for this organism.

Spore suspensions prepared under standard conditions are used as inoculum for cultivation, which are first grown on agar plates with Wickerham medium with 1.5% Bactoagar at room temperature for 1.4 days. The spores are suspended in a solution of sea water (34%) and glycerol (2: 1), adjusted to a standard titer, portioned and frozen in suitable portions and stored at -20 ° ^C. The inoculum for large approaches to mass cultures is. prepared by sterilizing cereal grains by autoclaving, adding medium and inoculating with fungal spores. After an incubation period of 14 days at 20 ° C., the grains are dried and stored at room temperature until use. The culture media are inoculated with these spore preparations.

A modified version of Wickerham's medium with the following composition is used for production: 3 g yeast extract, 6 g malt extract, 5 g peptone, 10 g glucose, 25 g NaCI in 1000 mL aqua dest. The pH is adjusted to 5.5.

The medium is placed in culture vessels with a filling height of 4 cm, autoclaved (20 min. At 121 ° C), inoculated after cooling with spore suspension and incubated for 7 days at 22 ° C until a compact surface mycelium is formed. Then the Temperature increased to 31 ° C and incubated for another 7 days. Then the culture fluid, which contains the majority of the sorbicillactone (1) produced, is separated from the mycelium and further processed for substance extraction. The mycelium is again underlaid with the same amount of fresh medium with a slightly different composition. The medium is used as listed above, but with 5 g NaCl, 15 g glucose per liter and without malt extract. The medium is warmed to incubation temperature before filling and the mycelia are incubated for 5 days. Then the same procedure is repeated. Sorbicillactone A (1) is extracted from the culture fluids and the mycelia by separate processes.

Example 2: Extraction of sorbicillactone A from the fungal biomass

The mycelium is separated from the culture medium by a fine-mesh network ^', added with 3 mL of ethyl acetate per gram of biomass and extracted. The extracts are filtered and concentrated on a rotary evaporator. The concentrate is stored at '5 ° C or -20 ° C until further purification.

Example 3: Extraction of sorbicillactone A from the culture medium

100 g of an exchange resin, for example XAD-16, are added to the culture medium separated from the mycelium with gentle stirring. After loading with the substances, the XAD-16 is filtered off from the medium and extracted successively with methanol / water (1: 1) and methanol. The extract is concentrated on a rotary evaporator and the methanol-free concentrate at 5 ° C or - ^C stored 20 C until further purification.

Example 4: Determination of the Sorbicillactone A Content by HPLC-UV

The content is determined on an analytical HPLC with a diode array detector (gradient: 80% A: 20% B to 20% A: 80% B in 20 min. With A = water + 0.05% TFA and B = acetonitrile + 0.05% TFA) on a Waters Symmetry C-18 reversed-phase column at a flow of 0.4 mL / min. The integration of the peak area is at a wavelength of 370 nm, since the absorption of dihydrosorbicillactone A (3) is practically zero at this wavelength, and thus the error in the content determination is minimized. The concentration in the crude extracts is determined by diluting the respective extract with a methanol / water mixture (1: 1) by a factor of 150, in the case of the extracts of the various purification stages by diluting by a factor of 10. The determination of the sorbicillactone content A (2) both in the crude extracts and in the individual extracts of the various purification stages is done with the aid of a calibration line from measurements with samples of differently concentrated solutions of sorbicillactone A (2) in methanol.

Example 5: Purification of the crude extract obtained from the culture medium

The aqueous crude extract of the fungal cultures of Penicillium chrysogenum is extracted with ethyl acetate to remove unwanted by-products such as meleagrin. The ethyl acetate phase obtained is discarded. The aqueous phase is acidified with phosphoric acid (pH = 2) and extracted exhaustively with ethyl acetate. Sorbicillactone A can be converted practically quantitatively into the ethyl acetate phase of the acidic extraction. The extracted extract already contains a mass content of up to 50% sorbicillactone A after concentration in a vacuum.

Example 6: Further Purification of the Extract by Fast Centrifugal Partitioning Chromatography (FCPC)

The extract is further purified with an additional liquid-liquid-chromatographic step. The new method of so-called Fast Centrifugal Partitioning Chromatography (FCPC) is used. With this method, as with common liquid-liquid chromatographic methods, such as High Speed Countercurrent Chromatography (HSCCC), a two-phase solvent mixture is used. The upper or lower phase can be used as a stationary phase. Unlike the HSCCC, the FCPC does not use a capillary coil, but one with several hundred Separation chambers provided rotor worked. The distribution of the substances contained in the extract between the mobile and the stationary phase takes place in these directly connected chambers. The system is rotated rapidly during the separation (1200-1400 rpm). On the one hand, depending on the flow direction, the desired phase is retained in the rotor of the FCPC and on the other hand the separation of the two phases is accelerated by the centrifugal force. This enables the use of high flow rates and thus the throughput of large amounts of substance in a short time. ' The distribution coefficient K of the desired substance between the two phases should be in the range between 0.7 and 4.5. If the K is smaller, the substance elutes too quickly, so that no separation takes place. At higher K, however, the retention time is too long for the rapid purification of large amounts of extract. In the case of sorbicillactone A, the use of a solvent mixture of heptane / ethyl acetate / methanol / water (42% / 58% / 42% / 58%) at a flow of 6 to 7 ml per min., A speed of 1200 rpm and the Use of the upper phase as a stationary phase is particularly advantageous. 1 mL of concentrated phosphoric acid is also added per liter of solvent mixture. When using a 200 mL FCPC rotor, a purification of up to 1.5 g extract per run is possible. With these settings, including preparation and rinsing times, only 90 minutes are required per separation. The fractions containing sorbicillactone A are freed from the organic solvents and the remaining aqueous acidic phase is exhaustively extracted with ethyl acetate. After concentration in vacuo, extracts with a mass content of sorbicillactone A of up to 70% are obtained.

Example 7: Obtaining pure sorbicillactone A using gel chromatography

The most difficult step in obtaining pure sorbicillactone A (2) is the separation of the structurally very similar but five times less active compound dihydrosorbicillactone A (3). 2 3

Fig. 1. Structures of sorbicillactone A (2) and dihydrosorbicillactone A (3)

Due to the fact that the two compounds differ only in the degree of saturation at C-2 'and C-3' in the sorbyl side chain, the physical properties of the molecules, such as mass or polarity, are used in the separation of substance mixtures , almost identical. However, the separation can be carried out by gel chromatography on Sephadex LH-20 material with methanol as the eluent. Because of the great similarity of the molecules, however, the use of a very long column system (greater than or equal to 6 m) is necessary. As a MPLC system, this column system is supplied with solvent via a pump. The flow rate of the eluent is approx. 10 mL per min. The two lactones 2 and 3 are sufficiently separated from each other, with 3 eluting faster. About 70% of the applied, contaminated sorbicillactone A (2) is obtained in clean fractions per separation. The mixed fractions contaminated with 3 can be easily cleaned by re-chromatography on Sephadex LH-20.

Example 8: Determination of the percentage ratio of sorbicillactone A (2) and dihydrosorbicillactone A (3) in the extracts by HPLC-UV

To determine the percentage ratio of sorbicillactone A (2) and dihydrosorbicillactone A (3) in the crude extracts, a sample of the extract is first diluted with a methanol / water mixture. The sample is on an analytical HPLC with a diode array detector under isocratic conditions (water / acetonitrile / TFA = 70/30 / 0.05%) on a Waters Symmetry-C-18 reversed-phase column at a flow of 0.4 mL / min examined. One achieves one for the integration of the peak areas the connections sufficient separation. The integration of the peak areas is carried out at a wavelength of 220 nm, since the absorption of the two lactones (2, 3) is approximately equally strong at this wavelength. This method can also be used to investigate the purity of the fractions from gel chromatography.

Example 9: Determination of the purity of the fractions from the gel chromatography by means of UV absorption experiments

In order to accelerate the analysis of the fractions from the last purification step, a method was sought that did not require chromatographic methods. SiGh found it practical to record the UV absorption of the corresponding fractions both at 300 nm and at 430 nm and to use the quotient from the two measured values to check the purity. The undesired impurity dihydrosorbicillactone A (3) elutes before sorbicillactone A (2) and, like sorbicillactone A (2), absorbs strongly at 300 nm, but practically not at a wavelength of 430 nm. When a constant quotient of the two measured values is reached, it can be assumed that the fractions only contain clean sorbicillactone A (2). These results could be verified by control measurements. be confirmed under the conditions described in Example 8. The measurement time for the analysis of all fractions in a separation can be reduced from several hours in HPLC analysis to just a few minutes.

Example 10: Derivatization of sorbicillactone A (2) to the methyl ester 4

The derivatization of sorbicillactone A (2) to the methyl ester 4 was interesting because the compound has become an internal standard for determining the concentration in e.g. B. blood serum is suitable, on the other hand to consider structure-activity relationships. 30 mg of sorbicillactone A (2) were dissolved in 5 ml of methanol and 200 μL of concentrated sulfuric acid were added. After stirring at room temperature for 6 hours, 100 ml of water were added and the mixture was extracted twice with 100 ml of ethyl acetate each time. After evaporating the organic phases in vacuo the residue was purified by preparative HPLC. This gave 18.6 mg of a yellow amorphous substance.

Physical and spectroscopic data of sorbicillactone A methyl ester (4): The compound is present as a yellow amorphous solid.

166-170 ° C (THF).

[a $ = -558 ° (c = 0.2 in methanol).

CD (c = 0.2 in methanol): Δε ₂₀₈ +11.1, Δε ₂₃ ι -12.6, Δε ₂₇₈ +12.5, Δε ₃₇₀ -13.0.

IR (KBr): v = 3333 (br.), 2931 (w), 1783 (m), 1730 (m), 1681 (s), 1612 (s), 1552 (s), 1442 (m), 1415 ( m), 1384 (m), 1350 (s), 1310 (s), 1198 (m), 1176 (m), 1065 (m) cm ^"1 .

MS (ESI, positive): m / z 432 [M + H] ⁺ .

Table 1. NMR data of sorbicillactone A methyl ester (1) in THF-d ₈ position ^a C [ppm] H [ppm] HMBC 1 99.5 2 192.0. 3 110.8 4 166.7 5 81.1 6 53.2 3.47 s 1,2,4,5,7,9, 11, 1 '7 -23.5 1.55 s 4,5,6 8 7.3 1.52 s 2,3,4 9 60.0 10 173.0 11 -26.0 1.42 s 6.9, 10 r 169.7 .2 '121.7 6.39 d 1', 3 ', 4' 3 '139.1 7.20 dd 1', 3 ', 5' 4 '131.9 6.28 ddd 2', 6 '. 5 '137.0 6.09 m 3', 6 '6' 18.6 1.82 dd 4 ', 5' 1 "162.3 2" 136.3 6.71 d 1 ", 3", 4 "3" 130.3 6.52 d 1 ", 4" 4 "165.9 COO e 51.8 3.69 s 4 "1'-OH 16.606s NH 7.66 s 9th, 10.11, 1"

Example 11: Detection of the bioactivity of sorbicillactone A

A) Measurements

Material: The same materials were used as in Perovic et al. (1998, Mech. Aging

Dev. 101: 1-19). Fura-2-acetoxymethyl ester (Fura-2-AM) was co-labeled "Dulbecco's modified Eagle's Medium" by Molecular Probes (Leiden, The Netherlands)

4.5 g / L glucose ^' (DMEM / HG), L-glutamic acid (L-Glu) and serotonin (5-HT) from Sigma- Aldrich (Taufkirchen, Germany) and the antibodies mice anti-neurofilament (68 kDa) and Mäu ^' se-Aπti-Giial-Fibrillary-Acidic-Protein (GFAP) from Röche Diagnostics (Mannheim, Germany). Sorbicillactone A of batch: 2208 / 2a was used.

Primary neurons: The cortical cell culture was created from the brains of 17-18 day old rat embryos using a modified procedure [Freshney (1987) Culture of specific cell types. In: Culture of Animal Cells. A Manual of Basic Technique, AR Liss, New York, S, 25.7-288; Perovic et al. (1994) Eur. J. Pharmacol. (Mol. Pharmacol. See.) 288: 27-33]. After isolation, the gerebral hemispheres were placed in "Hank's Balanced Salt Solution" (HBSS) without Ca ²⁺ and Mg ²⁺ and then the neuronal cells in HBSS with the aid of 0.025% (w / v) trypsin (10 min .; The proteolytic reaction was terminated with 10% (v / v) fetal calf serum (FCS). The single cell suspension was centrifuged and the resulting pellet in "Dulbecco's modified Eagles's Medium" with 4.5 g / L glucose (DMEM / HG) (contains 2 mM L-glutamine, 100 mU / L insulin and 10% (v / v) FCS). The cells were plated in a chamber coated with poly-L-lysine (5 μg / ml, 300 μl / cm ² ) with a cell density of 2.0 × 10 ⁵ cells / cm ² . After two days the DMEM / HG / 10% FCS medium was removed and replaced with DMEM / HG / serum-free medium. Two weeks after isolation, the immune staining was carried out using anti-neurofilament (68 kDa) as a marker for neurons and anti-GFAP as a marker for glial cells. The cultures contained> 80% neurons; the remaining 20% were GFAP-positive cells, mainly astrocytes (Ushijima et al. (1995) Eur. J. Neurosci. 7: 1353-9). The neurons were kept in an atmosphere of 95% air and 5% CO ₂ at 37 ° C.

Loading neurons with Fura-2-AM: The intracellular Ca ²⁺ concentration ([Ca ²⁺ ] j) was determined by fluorescence measurements. The decisive factor was the ratio of the absorption of the Ca ²⁺ indicator dye fura-2-AM at 340 and 380 nm (Grynkiewicz et al. (1985) J. Biol. Chem. 260: 3440-50). The neurons were at 6 μM Load Fura-2-AM in DMEM / HG / serum-free medium (mixed with 1% (w / v) bovine serum albumin) at 37 ° C for 60 min. After incubation, the cells were washed twice with medium and incubated for a further 45 min at 37 ° C. This incubation period is sufficient to load the neurons (inactive Fura-2-AM) and. to hydrolyze the acetoxymethyl ester (active Fura-2).

Calcium calibration curve: A calcium calibration curve was calculated using the methods of Grynkiewicz et al. (1985, J. Biol. Chem. 260: 3440-50). created. Fluorescence images were obtained for each buffer at 340 and 380 nm. The quotient from the two fluorescence spectra (340/380 nm) was calculated and displayed as a calibration curve. The quotient (340/380 nm [ratio value]) of 1.0 corresponds to 228 nM [Ca ²⁺ ] ι.

Changes in the calcium level in neurons: In all experiments, the cells were first loaded with Fura-2-AM and then with different substances (in Locke's solution; 154 mM NaCI; 5.6 mM KCI; 3.6 mM NaHCO ₃ ; 5.6 mM glucose and 10 mM Hepes; pH 7.4; without CaCl ₂ ) stimulated. Sorbicillactone A was dissolved in a concentration of 10 mg / mL in 100% (v / v) DMSO and stored at -20 ° C. In the first approach, neurons were stimulated with 0.1% (v / v) DMSO (control) for 5 min; after 10 min, 200 μM L-glutamic acid (L-Glu), 200 μM serotonin (5-HT) and 2.5 mM CaCl _{2 were added} to the cells. In the second approach, the effect of L-Glu, 5-HT and 2.5 mM CaCl ₂ on the calcium level of pre-incubated neurons (each 5 min with 10 μg / mL sorbicillactone A) was tested. The [Ca ²⁺ ] j level was measured in all experiments for at least 20-25 min.

To determine the [Ca ²⁺ ] j, the cells were cultivated on poly-L-lysine-coated borosilicate cover glasses in a 4-chamber system (Lab- ^Tek® Chamber Slide ™ system; Nunc, Wiesbaden, Germany). The fluorescence measurements were carried out using an “inverted-stage” microscope (Olympus IX70) with apochromatically reflected light and the fluorescence objective UApo40X / 340. The cells were alternately exposed to light of wavelengths 340 and 380 nm using a computer-controlled narrowband interference filter in front of a 100 -W xenon lamp lit. In addition, a 0.25 ND filter was used at 380 nm Fluorescence emissions at 510 nm were recorded with a CCD camera (model C2400-87; Hamamatsu, Herrsching, Germany). The images were computerized with the Argus 50 imaging system, Hamamatsu, digitized as 256 x 256 pixels with 8-bit arrays. The fluorescence quotient 340/380 nm was determined by dividing the image pairs.

Statistics: The results were evaluated using the paired "Student's t-test" (Sachs (1984) Applied Statistics. Springer, Berlin).

B) Results Influence of sorbicillactone A on the [Ca ²⁺ ] _r level in neuronal cells: The

Addition of 10 μg / mL sorbicillactone A showed almost no effect on [Ca ²⁺ ] j in neuronal cells.

Influence of L-glutamic acid on [Ca ²⁺ Ji levels in the presence or absence of sorbicillactone A in neuronal cells: Incubation of the cells with 200 μM. L-

Glutamic acid and 2.5 mM Ca ²⁺ (Figure 1A) gave a strong increase in [Ca ²⁺ ] j after 10 min. At the end of the measurement, the 340/380 nm values increased by 1.715 + 0.081 (307.1%). The control was assumed to be 100%. Preincubation of the neurons with 10 μg / mL sorbicillactone A caused a decrease in the [Ca ²⁺ ] j level after the addition of 200 μM L-Glu and 2.5 mM CaCl ₂ . The drop in the [Ca ²⁺ ] _r level was significant and was 46.7% at 10 μg / mL (p <0.001, Figure 1A).

Influence of serotonin on [Ca ²⁺ ] j levels in the presence or absence of sorbicillactone A in neuronal cells: Addition of 200 μM serotonin (5-HT) and 2.5 mM Ca ²⁺ to the cells (FIG. 1B) resulted a sharp increase in [Ca ²⁺ ] ι after 10 min. The 340/380 nm values. increased by 0.971 ± 0.112 (219.9%). The control. was assumed to be 100%. The decrease in the free calcium concentration in neurons after preincubation with 10 μg / mL sorbicillactone A and subsequent incubation with 200 μM of 5-HT was significant; in comparison to the control, the values had dropped by 77.6% (FIG. 1B). C) Conclusion It was found that cells incubated with sorbicillactone A showed a sharp drop in intracellular calcium levels after the addition of L-glutamic acid and serotonin (5-HT). L-glutamic acid and serotonin, as neurotransmitters, are etiologically involved in a number of neurodegenerative diseases. Because of these properties, sorbicillactone A can be used in the treatment of the diseases mentioned and the associated symptom complexes.

Example 12: Detection of the genotoxicity of sorbicillactone A: "Comet assay"

Material: "Normal Melting Agarose" (NMA, Cat. No. 840041) and "Low Melting Agarose" (LMA, Cat. No. 870081) were from Biozym (Hamburg, Germany), Triton x-100 from Fluka (Buchs SG , Switzerland), RPMI1640 medium, DMSO and EDTA from Sigma-Aldrich (Taufkirchen, Germany), NaCI and Tris from Roth (Karsruhe, Germany) and fetal calf serum (FKS) from Gibco (Karlsruhe, Germany). Sorbicillactone A of batch: 2208 / 2a was used.

Cells: The genotoxicity of sorbicilllactone A was tested on L5178Y mouse lymphoma cells (ATCC CRL 1722). As described (Müller et al., 1979, Cancer Res. 39: 1102-1107), the cells were cultured in RPMI1640 medium with 10 mM Hepes, to which 10% fetal calf serum (FCS) had been added. 0 ⁴ cells / mL was chosen as the inoculum concentration. The cells were incubated with 1, 3 and 10 μg / mL sorbicillactone A for 4 and 24 hours. After the incubation, the cells were examined using the "comet assay". '

Alkaline electrophoresis: The slides (Superfrost) were cleaned with acetone. 1.0% "normal melting agarose" (NMA) in 1 x PBS was boiled and then about 600 μl agarose was applied to a slide. The slides were dried briefly (~ 5 min) at 50 ° C. and overnight at room temperature The 0.7% "Low Melting Agarose" (LMA) would then be prepared in distilled water. The second layer on the slides consisted of 200 ul LMA. The slides were incubated at 4 ° C for 10 minutes. The 3rd layer consisted of 10 μl cell suspension (7 × 10 ⁴ cells / mL) plus 60 μl LMA. After incubation with sorbicillactone A, the cells were washed once with 1 × PBS and centrifuged at 800 × g for 5 min at 4 ° C. The cell number was diluted to 7 × 10 ⁴ cells / mL. After application, the slides were incubated at 4 ° C for 10 minutes. The last LMA layer (~ 100 μl) was applied before cell lysis. The cells were lysed in the dark by incubation in lysis solution (10 mM Tris, 100 mM EDTA, 2.5 M NaCl; pH = 10) with 10% (v / v) DMSO and 1% (v / v) Triton x- 100 for 1 hour at 4 ° C. The slides were transferred directly from the lysis into the electrophoresis chamber and incubated for 20 min in the electrophoresis buffer (30 mM NaOH, 1 mM EDTA; pH 13.8). The electrophoresis was constantly adjusted to 0.75 V / cm (~ 300 mA) and carried out under ice cooling for 30 min. For neutralization, the slides were washed for 5 minutes with neutralization buffer (400 mM Tris; pH 7.5) at room temperature and then for 5 minutes. dewatered with 95% ethanol and air dried in the dark. The slides were stained with 60 μl ethidium bromide solution (20 μg / mL distilled water), photographed and evaluated. The values are given as "Extent tail moment" (Bihari et al., 2002, Croatica Chemica Acta 75: 793-804; Müller et al., 1979, Cancer Res. 39: 1102-1 107). A large numerical value therefore indicates to a high level of disintegration of the DNA in the cells. In normal cells the values are around 0.

Results: After 4 hours (FIG. 2A), the incubation of the L5178Y cells with 1, 3 and 10 μg / mL sorbicillactone A showed no significant change in the “extent tail moment”. At these concentrations, sorbicillactone A did not induce any DNA strand breaks after 24 hours of incubation with sorbicillactone A, no significant changes in the "Extent tail moment" in L5178Y cells could be determined (FIG. 2B).

Conclusion: It was found that cells which were incubated with sorbicillactone A showed no significant increase in the "Extent tail moment" after the addition of 1, 3 and 10 μg / mL. Example 13: Detection of the genotoxicity of sorbicillactone A: "Fast Microassay"

Material: PicoGreen was from Molecular Probes (Leiden, the Netherlands), RPMI1640 medium from Sigma-Aldrich (Taufkirchen, Germany), fetal calf serum (FKS) from Gibco (Karlsruhe, Germany) and black microtiter plates (96-WeII-Plateπ) from Nunc (Wiesbaden, Germany). Sorbicillactone A of batch: 2208 / 2a was used.

Cells: The genotoxicity of sorbicilllactone A was tested on L5178Y mouse lymphoma cells (ATCC CRL 1722). As described (Müller et al., 1979, Cancer Res. 39: 1102-1107), the cells were cultivated in RPMI1640 medium to which 10% fetal calf serum (FCS) had been added. 10 ⁴ cells / mL was selected as the inoculum concentration. The cells were incubated with 1, 3, 10 and 30 μg / mL sorbicillactone A (1) for 4 and 24 hours. After the incubation, the cells were examined using the "Fast Microassay".

"Fast Microassay": The method was carried out according to a modified method (Batel et al., 1999, Anal. Biochem., 270: 195-200). The cells were washed twice with 1 × PBS. After centrifugation, the pellet was washed in 1 x PBS and diluted to 10 ⁴ cells / mL 25 μl of the cell suspension (approximately 2.5 × 10 ⁴ cells per well) were prepipetted into black microtiter plates (96-well plates, Nunc, Wiesbaden) 25 μl of 1 x PBS was present, then 25 μl of lysis solution (7 M urea, 0.1% (w / v) SDS, 0.2 M EDTA, pH = 10.0) were mixed with PicoGreen (20 μl conc. PicoGreen pro _. 1 ml of lysis solution) was pipetted into the microtiter plate. the cells were then 40 lysed in the dark at room temperature for minutes. After incubation, the denaturation is carried out (unwinding) of the DNA by addition of the alkaline solution freshly prepared. This is prepared by mixing 20 mL of 20 mM EDTA and 32 mL 20 mM EDTA / 100 mM NaOH to a pH of 12.3 Di e Measurement was carried out immediately in a fluoroscan at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, every 3 to 5 min, for a total of 20 min. long. Results were reported as Strand Scission Factors (SSF) indicated and calculated after a denaturation time (unwinding time) of 20 min according to the following equation: SSF = log (% dsDNA sample /% dsDNA control).

Results: Influence of sorbicillactone A on the SSF value in L5178Y cells: The addition of 1, 3, 10 and 30 μg / mL of sorbicillactone A showed almost no effect on the SSF values in L5178Y cells after 4 hours of incubation ( Figure 3A). A significant increase in SSF values (proportion of DNA strand breaks) was observed after 24 hours with the addition of 3 and 30 μg / ml (p <0.001) sorbicillactone A (FIG. 3B). Incubation of the L5178Y cells with 1 to 10 μg / mL sorbicillactone A showed a slight but no significant increase in the SSF values.

Conclusion: Sorbicillactone A induced in L5178Y cells after 24 hours incubation at a concentration of 30 μg / mL DNA strand breaks. Because of these properties, sorbicillactone A can be used in the treatment of leukemia.

Example 14: Detection of apoptosis triggered by sorbicillactone A (Cell Death Detection ELISA plus)

Material: RPMI1640 medium, Hepes buffered, was from Sigma-Aldrich (Taufkirchen, Germany), fetal calf serum (FKS) from Gibco (Karlsruhe, Germany) and Cell Death Detection ELISA plus from Röche (Mannheim, Germany, Cat.No. 1774425 ) based. Sorbicillactone A of batch: 2208 / 2a was used.

Incubation and processing of the cells: The L5178Y mouse lymphoma cells (ATCC CRL 1722) were counted and adjusted to a cell number of 10 ⁴ cells / ml. The sorbicillactone A and dihydrosorbicillactone A stock solution (10 mg / mL in DMSO) was diluted with medium (RPMI / Hepes with 10% FCS) to concentrations of 60, 20 and 6 μg / mL. 100 μl of the various dihydrosorbicillactone A and sorbicillactone A solutions were pipetted into each 100 μl cell suspension (~ 10 ³ cells) in a 96-well culture plate. As a negative control, RPMI1640 medium was without Dihydrosorbicillacton and Sorbicillacton A used. The final concentrations were 30, 10 and 3 μg / mL sorbicillactone A and dihydrosorbicillactone A. Four parallel batches were investigated. The cells were incubated at 37 ° C for 4 hours. After the incubation, the culture plate was centrifuged at 200 xg (~ 1200 rpm) for 10 min at room temperature. The supernatant was carefully pipetted off and the cells were covered with 200 μl lysis buffer (Roche kit). They were then lysed at room temperature for 30 minutes. After lysis, the culture plate was centrifuged again at 200 xg (~ 1200 rpm) for 10 min at room temperature.

ELISA: Two strips with eight “wells” each were removed from the streptavidin ELISA plate of the Roche kit. 20 μl of the supernatant from a cell lysis (see above) were added to each “well”. The samples were applied with 30 or 10 μg / mL sorbicillactone A and dihydrosorbicillactone A as 4-fold, all others as double determinations. In addition to these samples, a positive control (DNA-histone complex) was applied. 80 μl of immunoreagent were then added to each “well”. This solution was composed of incubation buffer, anti-histone biotin and anti-DNA peroxidase. The incubation buffer was used as blank. The ELISA plate was kept in a shaking incubator at room temperature for 2 hours The supernatants were then carefully removed and the “wells” were washed 3 times with 250 μl incubation buffer each. After washing, 100 μl of ABTS solution were added to each “well”. The cells were then incubated for 30 minutes in the dark at room temperature. The measurement was carried out in a multiscan at an emission wavelength of 405 nm.

Results: Addition of 3 μg / mL sorbicillactone A to the cells (Table 2) showed no increase in apoptosis cells after 4 hours of incubation. The values 91.2% are in the range of the negative control (= 100%) (Table 2).

Table 2.

After incubation (4 hours) of the L5178Y cells with 10 and 30 μg / mL sorbicillactone A, the values rose significantly to 283.8% and 322.1% (p <0.001, Table 2). The negative control was assumed to be 100%.

Table 3.

13,952.4% 2,930 ± 0.245 positive control

100.0% 0.021 ± 0.002 negative control

The addition of 3, 10 and 30 μg / mL dihydrosorbicillactone A showed no induction of apoptosis after 4 hours of incubation in L5178Y cells (Table 3). The values had dropped below the control range to 62 to 67%.

Conclusion: Sorbicillactone A induces apoptosis in L5178Y cells after 4 hours incubation at a concentration of 10 and 30 μg / mL. Based on these Properties Sorbicillacton A can be used in the treatment of leukemia. Dihydrosorbicillactone A does not induce apoptosis in L5178Y cells under the experimental conditions chosen.

Claims

claims

1. A process for the production of sorbicillactone A or derivatives thereof, comprising the steps of:

b) raising the temperature to 28-35 ° C and further incubating for 5-10 days,

c) separating the culture fluid from the mycelium, and

d) extracting sorbicillactone A and derivatives thereof from the culture medium, and, if appropriate,

f) repeating steps c) and d), and if appropriate,

g) repeating steps e) to f), and

h) extracting sorbicillactone A and derivatives thereof from the culture medium and / or the mycelia.

2. The method according to claim 1, wherein the fungus is Penicillium chrysogenum, in particular the strain KIP 3201.

3. The method according to any one of the preceding claims, wherein various additives can be added to the suitable growth media, such as pyruvate, glutamate, proline, acetate, sorbicillin or other biosynthetic precursors of sorbicillactone A.

4. The method according to any one of the preceding claims, wherein the production takes place in the flat bed process.

5. The method according to any one of the preceding claims, wherein the inoculum is a solid-bound form of the fungus.

A method according to claim 5, wherein the solids to which the fungus is attached are buoyant solids, e.g. Cereal grains or polystyrene balls.

7. The method according to any one of the preceding claims, wherein a carrier device for stabilizing the surface mycelium is introduced into the cultivation container.

8. The method according to claim 7, wherein the carrier device is a network.

9. The method according to any one of the preceding claims, wherein sorbicillactone A or derivatives thereof are extracted from the fungal mycelium separated from the culture medium by adding ethyl acetate.

10. The method according to any one of claims 1-8, wherein sorbicillactone A or derivatives thereof are bound directly from the culture medium to a solid exchanger and further purified from this bound form.

11. The method of claim 10, wherein the solid exchanger is the Amberlite XAQ-16 exchange resin.

12. The method according to claim 10 or 11, wherein the loaded solid exchanger is filtered off from the medium and sorbicillactone A or derivatives thereof are eluted with organic solvents.

13. The method according to claim 12, wherein the organic solvents are methanol, ethanol, ethyl acetate, heptane or acetonitrile.

14. The method according to any one of claims 10-13, wherein sorbicillactone A or derivatives thereof are extracted from the crude extract with organic solvents.

15. The method according to claim 14, wherein the crude extract is brought to pH 2 with phosphoric acid and then extracted with ethyl acetate.

16. The method according to any one of the preceding claims, wherein the extracts are purified by means of FCPC (Fast Centrifugal Partitioning Chromatography).

17. The method according to claim 16, wherein a solvent mixture of heptane, ethyl acetate, methanol and water with the addition of 1 ml / l of concentrated phosphoric acid at a flow of 6-7 ml / min and a speed of 1200 revolutions per minute, and wherein the upper is used as a stationary phase.

18. The method according to any one of the preceding claims, wherein the extracts are purified by gel chromatography on Sephadex LH-20 using an organic solvent.

19. Procedure. according to claim 18, wherein sorbicillactone A is eluted with methanol.

20. A method for producing sorbicillactone A methyl ester comprising the steps of:

a) preparing sorbicillactone A as described in claims 1-19, b) adding concentrated sulfuric acid to sorbicillactone A dissolved in methanol, c) stirring at room temperature for 6 h, d) adding water, e) extracting with ethyl acetate, f) evaporating the organic phases in vacuo, and g) cleaning the residue by preparative HPLC.

21. A method of manufacturing a pharmaceutical comprising the steps of:

a) preparing sorbicillactone A or derivatives thereof as described in claims 1-19, and b) formulating a pharmaceutical composition using pharmaceutically acceptable auxiliaries and additives.

22. A method for producing a pharmaceutical according to claim 21, characterized in that sorbicillactone A or derivatives thereof are present in an amount such that a concentration range between 0.3 and 30 μg / ml is present during the treatment in vivo.

23. Use of sorbicillactone A or derivatives thereof as triggers of apoptosis in diseased cells, in particular tumor cells.

24. Use of sorbicillactone A or derivatives thereof in the treatment of leukemia.

25. Use of sorbicillactone A or derivatives thereof in the treatment of neurodegenerative diseases.

26. Use of sorbicillactone A or derivatives thereof in the treatment of bacterial and fungal infections.

27. Mushroom strain of the genus Penicillium. chrysogenum KIP 3201, with the deposit number DSM 16137.