EP1713455A2 - Procede pour produire du sorbicillactone a - Google Patents

Procede pour produire du sorbicillactone a

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Publication number
EP1713455A2
EP1713455A2 EP05701272A EP05701272A EP1713455A2 EP 1713455 A2 EP1713455 A2 EP 1713455A2 EP 05701272 A EP05701272 A EP 05701272A EP 05701272 A EP05701272 A EP 05701272A EP 1713455 A2 EP1713455 A2 EP 1713455A2
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EP
European Patent Office
Prior art keywords
sorbicillactone
derivatives
cells
ethyl acetate
mycelium
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05701272A
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German (de)
English (en)
Inventor
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
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johannes Gutenberg Universitaet Mainz
Julius Maximilians Universitaet Wuerzburg
Original Assignee
Johannes Gutenberg Universitaet Mainz
Julius Maximilians Universitaet Wuerzburg
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Publication of EP1713455A2 publication Critical patent/EP1713455A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/82—Benzo [b] furans; Hydrogenated benzo [b] furans with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/83—Oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C

Definitions

  • 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.
  • 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.
  • 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.
  • this object is first achieved by providing a method which comprises the following steps:
  • sorbicillactone A and derivatives thereof from the culture medium and / or the mycelia.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FCPC Fast Centrifugal Partitioning Chromatography
  • 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.
  • sorbicillactone A 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.
  • sorbicilllactone A 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 ⁇ g / 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 ⁇ g / 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 ⁇ g / 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 ⁇ g / 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.
  • 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).
  • 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 '
  • FIG. 1B shows the treatment of neurons with 200 ⁇ M serotonin (5-HT) and 2.5 mM
  • 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 ⁇ g / ml sorbicillactone A.
  • FIG. 2A shows the results after an incubation period of 4 hours.
  • 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 ⁇ g / ml sorbicillactone A.
  • FIG. 3A shows the results after an incubation period of 4 hours.
  • Example 1 Cultivation of the fungus Penicillium chrysogenum in saline media for the optimized production of sorbicillactone A
  • 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.
  • 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.
  • 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.
  • an exchange resin for example XAD-16
  • 100 g of an exchange resin for example XAD-16
  • 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.
  • 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 extracted extract already contains a mass content of up to 50% sorbicillactone A after concentration in a vacuum.
  • the extract is further purified with an additional liquid-liquid-chromatographic step.
  • FCPC Fast Centrifugal Partitioning Chromatography
  • HSCCC High Speed Countercurrent Chromatography
  • FCPC a two-phase solvent mixture is used.
  • the upper or lower phase can be used as a stationary phase.
  • HSCCC High Speed Countercurrent Chromatography
  • 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).
  • 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.
  • 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
  • Fig. 1 Structures of sorbicillactone A (2) and dihydrosorbicillactone A (3)
  • 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
  • a sample of the extract is first diluted with a methanol / water mixture.
  • 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
  • sorbicillactone A (2) 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 ⁇ L 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.
  • CD (c 0.2 in methanol): ⁇ 208 +11.1, ⁇ 23 ⁇ -12.6, ⁇ 278 +12.5, ⁇ 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 .
  • Fura-2-acetoxymethyl ester (Fura-2-AM) was co-labeled "Dulbecco's modified Eagle's Medium” by Molecular Probes (Leiden, The Netherlands)
  • 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].
  • HBSS Hank's Balanced Salt Solution
  • FCS fetal calf serum
  • the cells were plated in a chamber coated with poly-L-lysine (5 ⁇ g / ml, 300 ⁇ l / cm 2 ) with a cell density of 2.0 ⁇ 10 5 cells / cm 2 .
  • the DMEM / HG / 10% FCS medium was removed and replaced with DMEM / HG / serum-free medium.
  • 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 ⁇ M 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+ ] ⁇ .
  • the cells were cultivated on poly-L-lysine-coated borosilicate cover glasses in a 4-chamber system (Lab- Tek® Chamber Slide TM 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.
  • 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.
  • sorbicilllactone A 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 ⁇ g / mL sorbicillactone A for 4 and 24 hours. After the incubation, the cells were examined using the "comet assay". '
  • 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.
  • 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 ⁇ l ethidium bromide solution (20 ⁇ g / mL distilled water), photographed and evaluated.
  • 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-Plate ⁇ ) from Nunc (Wiesbaden, Germany). Sorbicillactone A of batch: 2208 / 2a was used.
  • sorbicilllactone A 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 ⁇ g / mL sorbicillactone A (1) for 4 and 24 hours. After the incubation, the cells were examined using the "Fast Microassay".
  • FCS fetal calf serum
  • PicoGreen pro 1 ml of lysis solution
  • the cells were then 40 lysed in the dark at room temperature for minutes.
  • Example 14 Detection of apoptosis triggered by sorbicillactone A (Cell Death Detection ELISA plus)
  • 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.
  • 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 ⁇ g / mL.
  • 100 ⁇ l of the various dihydrosorbicillactone A and sorbicillactone A solutions were pipetted into each 100 ⁇ l cell suspension ( ⁇ 10 3 cells) in a 96-well culture plate.
  • RPMI1640 medium was without Dihydrosorbicillacton and Sorbicillacton A used. The final concentrations were 30, 10 and 3 ⁇ g / 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 ⁇ l 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 ⁇ l of the supernatant from a cell lysis (see above) were added to each “well”. The samples were applied with 30 or 10 ⁇ g / 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 ⁇ l 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 ⁇ l incubation buffer each. After washing, 100 ⁇ l 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.
  • Sorbicillactone A induces apoptosis in L5178Y cells after 4 hours incubation at a concentration of 10 and 30 ⁇ g / 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.

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Abstract

L'invention concerne un procédé pour améliorer la culture de champignons Penicillium chrysogenum, en particulier la souche KIP 3201, ce qui permet une production optimisée du produit naturel antitumoral, le sorbicillactone A (2), et ses dérivés en grande quantité. L'invention concerne également des procédés améliorés pour extraire et pour purifier lesdits champignons hors de la biomasse des champignons et de leur milieu de culture. L'invention concerne, en outre, l'activité biologique du sorbicillactone A (2) et l'analyse de la génotoxicité du composé.
EP05701272A 2004-01-30 2005-01-31 Procede pour produire du sorbicillactone a Withdrawn EP1713455A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102004004901A DE102004004901B4 (de) 2004-01-30 2004-01-30 Verfahren zur Herstellung von Sorbicillacton A
PCT/EP2005/000923 WO2005072711A2 (fr) 2004-01-30 2005-01-31 Procede pour produire du sorbicillactone a

Publications (1)

Publication Number Publication Date
EP1713455A2 true EP1713455A2 (fr) 2006-10-25

Family

ID=34801370

Family Applications (1)

Application Number Title Priority Date Filing Date
EP05701272A Withdrawn EP1713455A2 (fr) 2004-01-30 2005-01-31 Procede pour produire du sorbicillactone a

Country Status (6)

Country Link
US (1) US20070135515A1 (fr)
EP (1) EP1713455A2 (fr)
JP (1) JP2007519414A (fr)
CA (1) CA2554733A1 (fr)
DE (1) DE102004004901B4 (fr)
WO (1) WO2005072711A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2014101490A (ru) 2011-06-19 2015-07-27 Эйбоджен, Инк. Устройства, растворы и способы для сбора образцов
PL3114225T3 (pl) * 2014-03-07 2021-11-08 Dna Genotek Inc. Sposób i kompozycja do stabilizacji kwasów nukleinowych w próbkach biologicznych

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4338356B2 (ja) * 2001-05-14 2009-10-07 シージェイ チェイルジェダン コーポレイション フラクトシルトランスフェラーゼを生成する微生物、及びそれを使用するフラクトオリゴ糖およびネオフラクトオリゴ糖の製造方法
DE10238257B4 (de) * 2002-08-21 2007-04-19 Johannes-Gutenberg-Universität Mainz Sorbicillacton A und Sorbicillacton-A-Derivate, Verfahren zu ihrer Herstellung und sie enthaltende Arzneimittel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005072711A2 *

Also Published As

Publication number Publication date
CA2554733A1 (fr) 2005-08-11
JP2007519414A (ja) 2007-07-19
US20070135515A1 (en) 2007-06-14
WO2005072711A3 (fr) 2005-09-15
DE102004004901B4 (de) 2006-01-05
DE102004004901A1 (de) 2005-08-25
WO2005072711A2 (fr) 2005-08-11

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