CH632010A5 - Process for the preparation of a novel antibiotic - Google Patents
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- CH632010A5 CH632010A5 CH745777A CH745777A CH632010A5 CH 632010 A5 CH632010 A5 CH 632010A5 CH 745777 A CH745777 A CH 745777A CH 745777 A CH745777 A CH 745777A CH 632010 A5 CH632010 A5 CH 632010A5
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- cyclosporin
- preparation
- novel antibiotic
- new antibiotic
- nutrient medium
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- 230000003115 biocidal effect Effects 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- ZNVBEWJRWHNZMK-SYOLRUPNSA-N (3s,6s,9s,12r,15s,18s,21s,24s,30s,33s)-33-[(e,1r,2r)-1-hydroxy-2-methylhex-4-enyl]-1,4,7,10,12,15,19,25,28-nonamethyl-6,9,18,24-tetrakis(2-methylpropyl)-3,21,30-tri(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontane-2,5,8,11,14,17,20,2 Chemical compound C\C=C\C[C@@H](C)[C@@H](O)[C@@H]1N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C(=O)[C@H](C(C)C)NC1=O ZNVBEWJRWHNZMK-SYOLRUPNSA-N 0.000 claims abstract description 20
- 108010019594 cyclosporin D Proteins 0.000 claims abstract description 20
- 235000015097 nutrients Nutrition 0.000 claims abstract description 10
- 241001149960 Tolypocladium inflatum Species 0.000 claims abstract description 5
- 241000233866 Fungi Species 0.000 claims description 4
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 230000002538 fungal effect Effects 0.000 abstract 1
- 229960003444 immunosuppressant agent Drugs 0.000 abstract 1
- 230000001861 immunosuppressant effect Effects 0.000 abstract 1
- 239000003018 immunosuppressive agent Substances 0.000 abstract 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 206010003246 arthritis Diseases 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000001506 immunosuppresive effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229930105110 Cyclosporin A Natural products 0.000 description 2
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 2
- 108010036949 Cyclosporine Proteins 0.000 description 2
- 208000009386 Experimental Arthritis Diseases 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 229910000831 Steel Inorganic materials 0.000 description 2
- 230000000274 adsorptive effect Effects 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- DKPFZGUDAPQIHT-UHFFFAOYSA-N butyl acetate Chemical compound CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 2
- 229960001265 ciclosporin Drugs 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- KDCIHNCMPUBDKT-UHFFFAOYSA-N hexane;propan-2-one Chemical compound CC(C)=O.CCCCCC KDCIHNCMPUBDKT-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000010959 steel Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- JTOKYIBTLUQVQV-QRVTZXGZSA-N (3s,6s,9s,12r,15s,18s,21s,24s,30s,33s)-30-[(1r)-1-hydroxyethyl]-33-[(e,1r,2r)-1-hydroxy-2-methylhex-4-enyl]-1,4,7,10,12,15,19,25,28-nonamethyl-6,9,18,24-tetrakis(2-methylpropyl)-3,21-di(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontan Chemical compound C\C=C\C[C@@H](C)[C@@H](O)[C@@H]1N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C(=O)[C@H]([C@@H](C)O)NC1=O JTOKYIBTLUQVQV-QRVTZXGZSA-N 0.000 description 1
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000223211 Curvularia lunata Species 0.000 description 1
- JTOKYIBTLUQVQV-UHFFFAOYSA-N Cyclosporin C Natural products CC=CCC(C)C(O)C1N(C)C(=O)C(C(C)C)N(C)C(=O)C(CC(C)C)N(C)C(=O)C(CC(C)C)N(C)C(=O)C(C)NC(=O)C(C)NC(=O)C(CC(C)C)N(C)C(=O)C(C(C)C)NC(=O)C(CC(C)C)N(C)C(=O)CN(C)C(=O)C(C(C)O)NC1=O JTOKYIBTLUQVQV-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000035584 blastogenesis Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 150000008280 chlorinated hydrocarbons Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 108010019248 cyclosporin C Proteins 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 239000013003 healing agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
- C07K7/645—Cyclosporins; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The novel antibiotic cyclosporin D having immunosuppressant and anti-inflammatory action (Formula I) <IMAGE> and process for the preparation of the novel antibiotic by culturing a strain of the fungal species Tolypocladium inflatum Gams on or in a nutrient medium and then isolating the novel antibiotic cyclosporin D from the nutrient medium by methods known per se and purifying it.
Description
**WARNUNG** Anfang DESC Feld konnte Ende CLMS uberlappen **.
PATENTANSPRUCH
Verfahren zur Herstellung des neuen Antibiotikums Cyclosporin D (Formel 1),
EMI1.1
dadurch gekennzeichnet, dass man einen Stamm der Pilzspecies Tolypocladium inflatum Gams auf oder in einem Nährmedium züchtet und hierauf das neue Antibiotikum Cyclosporin D aus dem Nährmedium isoliert und reinigt.
Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung des neuen Antibiotikums Cyclosporin D (Formel I).
EMI1.2
Erfindungsgemäss gelangt man zum neuen Antibiotikum
Cyclosporin D, indem man einen Stamm der Pilzspecies Tolypocladium inflatum Gams auf oder in einem Nährmedium züch- tet, das neue Antibiotikum aus der Fermentationsbrühe auf an sich bekannte Weise durch extraktive und/oder adsorptive Arbeitsmethoden isoliert und hierauf chromatographisch oder mittels Gegenstromverteilung reinigt.
Ein Stamm der Pilzspecies Tolypocladium inflatum Gams wurde in der DOS 2455859 beschrieben. Er wurde aus einer in Norwegen gesammelten Erdprobe isoliert und eine Kultur davon beim United States Department of Agriculture (Northern Research and Development Division), Peoria, llI., USA am 12August1974 unter der Nummer NRRL 8044 deponiert.
Das neue Antibiotikum Cyclosporin D kann man beispielsweise in der Weise herstellen, dass ein flüssiges Medium mit einer Konidien- und Mycelsuspension des Stammes NRRL 8044 beimpft und die Kultur 11 bis 18 Tage, vorzugsweise 13 Tage, bei 25 bis 30 , vorzugsweise bei 27 , und bei einem pH-Wert von 4,3 bis 6,2, vorzugsweise 5,6, in einem Stahlfermenter unter Rühren (170 UPM) und Belüftung (1 Liter Lufti MinSLiter Nährlösung) inkubiert wird.
Sobald eine maximale Menge an Antibiotikum produziert ist, wird dieses aus der Kulturbrühe durch extraktive undloder adsorptive Methoden auf an sich bekannte Weise gewonnen.
Charakterisierung von Cyclosporin D: Farblose, prismatische Kristalle; Smp. 148-151 "C = = - 2450 (C = 0,52 in Chloroform) [a = - 2110 (c = 0,51 in Methanol) Löslichkeit:
Cyclosporin D ist leicht löslich in Alkoholen, Äther, Aceton und chlorierten Kohlenwasserstoffen; praktisch unlöslich in Wasser und gesättigten Kohlenwasserstoffen.
Das neue Antibiotikum Cyclosporin D zeichnet sich durch interessante chemotherapeutische und pharmakologische Eigenschaften aus und kann daher als Heilmittel verwendet werden. So hemmt es das Wachstum von Aspergillus niger und Curvularia lunata. Insbesondere zeichnet sich die Substanz Cyclosporin D durch eine immunosuppressive und entzündungshemmende Wirkung aus.
Die immunosuppressive Wirkung von Cyclosporin D kann wie folgt gezeigt werden: a) Im Lymphozytenstimulationstest nach Jänossy wird in vitro in Konzentrationen von 0,01 bis 10,0 ugiml eine starke Hemmung des H3-Thymidin-Einbaus, der Proliferationsrate und der Blastogenese von mit Concanavalin A stimulierten Lymphozyten aus Mäusemilz festgestellt.
b)Hämagglutinationstest an der Maus: Erfasst werden die gegen Schaferythrozyten gebildeten Antikörper. Die Hemmung wird im suppressiven Index (ski) ausgedrückt, wobei die unbehandelte Kontrolle mit 1,00 angegeben wird; Sl = 0,53 nach p. o.5x250 mg/kg.
Aufgrund ihrer immunosuppressiven Wirkung kann Cyclosporin D zur Prophylaxe und Behandlung von Krankheiten, die mit der Beeinflussung der Abwehrreaktion im negativen Sinn zusammenhängen, angewandt werden.
Das neue Antibiotikum besitzt ebenfalls eine Arthritis-hemmende Wirkung. So wirkt es z. B. im Freund-Adjuvans-Arthritis-Latenzzeitversuch an der Ratte in Dosen von etwa 50 mg/kg KörpergewichtlTag stark schwellungshemmend.
Eine ähnliche Wirkung wird im Freund-Adjuvans-Arthritis Therapieversuch an der Ratte in Dosen von etwa 50 mg/kglTag beobachtet.
Aufgrund seiner Arthritis-hemmenden Wirkung kann das Antibiotikum zur Prophylaxe und Behandlung von Arthritis und rheumatischen Krankheiten angewandt werden.
Die zu verwendenden Dosen variieren naturgemäss je nach Art des Antibiotikums, der Administration und des zu behandelnden Zustandes. Im allgemeinen werden jedoch bei Testtieren befriedigende Resultate mit einer Dosis von 10 bis 200 mglkg Körpergewicht erzielt. Diese Dosis kann nötigenfalls in 2 bis 3 Anteilen oder auch als Retardform verabreicht werden.
Für grössere Säugetiere liegt die Tagesdosis bei etwa 50 bis 900 mg. Für orale Applikationen können die Teildosen beispielsweise etwa 25 bis 300 mg des neuen Antibiotikums neben festen und flüssigen Trägersubstanzen enthalten.
Als Heilmitel kann das neue Antibiotikum allein oder in geeigneter Arzneiform mit pharmakologisch indifferenten Hilfsstoffen verabreicht werden.
In dem nachfolgenden Beispiel, das die Erfindung näher erläutern, ihren Umfang aber in keiner Weise einschränken soll, erfolgen alle Temperaturangaben in Celsiusgraden.
Beispiel:
500 Liter einer Nährlösung, die pro Liter 40 g Glucose, 5 g Caseinpepton. 5 g MgSO4- 7dz0, 2 g KH2PO4,3 g NaNO3,0,5 g KCI, 0,01 g FeSO4 und entmineralisiertes Wasser enthält, werden mit 50 Liter einer Vorkultur des Stammes NRRL 8044 angeimpft und in einem Stahlfermenter unter Rühren (170 UPM) und Belüftung (1 Liter LuftlMinJLiter Nährlösung) 13 Tage bei 27 inkubiert (siehe DOS 2 455 859).
Die Kulturbrühe wird mit der gleichen Menge n-Butylacetat ausgerührt, nach Abtrennung der organischen Phase wird diese im Vakuum konzentriert und der Rohextrakt durch 3stufige Verteilung zwischen Methanolwasser (9:1) und Petroläther entfettet. Die methanolische Phase wird abgetrennt, im Vakuum konzentriert und das Rohprodukt durch Zugabe von Wasser ausgefällt. Das nach der Filtration gewonnene Material wird an der 5- bis 7fachen Menge Sephadex LH-20 mit Methanol als Elutionsmittel chromatographiert. Die Spitzenfraktionen werden anschliessend an Kieselgel 60, Korngrösse 0,0630,20 mm (Merck) mit Hexanaceton (2:1) chromatographiert, wobei die zuerst eluierten Fraktionen vorwiegend Cyclosporin A und Cyclosporin D enthalten, die später eluierten Anteile vorwiegend Cyclosporin C.
Zur weiteren Reinigung werden die Cyclosporin A- und D-haltigen Fraktionen aus der 2- bis 2,fachen Menge Aceton bei - 15 kristallisiert und anschliessend durch zweimalige Chromatographie an Kieselgel 60, Korngrösse 0,063-0,20 mm (Merck) weiter aufgetrennt, wobei die mit Hexanaceton (2:1) zuerst eluierten Fraktionen Cyclosporin D in stark angereicherter Form enthalten. Diese werden in der doppelten Menge Aceton gelöst und bei - 15 kristallisieren lassen. Das dabei erhaltene Rohkristallisat von Cyclosporin D wird zur weiteren Reinigung in der 10fachen Menge Aceton gelöst, mit 2 Gewichtsprozent Aktivkohle versetzt und wäh- rend 5 Minuten auf 60 erwärmt.
Das nach Filtration über Talk erhaltene klare und beinahe farblose Filtrat wird auf ein Drittel des Volumens eingeengt und auf Raumtemperatur erkalten lassen, wobei Cyclosporin D spontan auskristallisiert. Durch Ste henlassen bei - 17 wird die Kristallisation vervollständigt. Die durch Abfiltrieren gewonnenen Kristalle werden mit wenig eiskaltem Aceton gewaschen und anschliessend im Hochvakuum bei 80" während 2 Stunden getrocknet.
** WARNING ** beginning of DESC field could overlap end of CLMS **.
PATENT CLAIM
Process for the preparation of the new antibiotic cyclosporin D (Formula 1),
EMI1.1
characterized in that a strain of the fungus species Tolypocladium inflatum Gams is grown on or in a nutrient medium and then the new antibiotic cyclosporin D is isolated from the nutrient medium and purified.
The present invention relates to a process for the preparation of the new antibiotic cyclosporin D (formula I).
EMI1.2
According to the invention, the new antibiotic is obtained
Cyclosporin D, by cultivating a strain of the fungus species Tolypocladium inflatum Gams on or in a nutrient medium, isolating the new antibiotic from the fermentation broth in a manner known per se by extractive and / or adsorptive working methods and then purifying it chromatographically or by means of countercurrent distribution.
A strain of the fungus species Tolypocladium inflatum Gams has been described in DOS 2455859. It was isolated from a soil sample collected in Norway and a culture of it was deposited with the United States Department of Agriculture (Northern Research and Development Division), Peoria, llI., USA on August 12, 1974 under number NRRL 8044.
The new antibiotic cyclosporin D can be prepared, for example, by inoculating a liquid medium with a conidia and mycelium suspension of the strain NRRL 8044 and the culture for 11 to 18 days, preferably 13 days, at 25 to 30, preferably at 27, and at a pH of 4.3 to 6.2, preferably 5.6, in a steel fermenter with stirring (170 rpm) and aeration (1 liter of Lufti MinSLiter nutrient solution).
As soon as a maximum amount of antibiotic is produced, this is obtained from the culture broth by extractive and / or adsorptive methods in a manner known per se.
Characterization of Cyclosporin D: Colorless, prismatic crystals; 148-151 "C = = - 2450 (C = 0.52 in chloroform) [a = - 2110 (c = 0.51 in methanol) Solubility:
Cyclosporin D is easily soluble in alcohols, ether, acetone and chlorinated hydrocarbons; practically insoluble in water and saturated hydrocarbons.
The new antibiotic Cyclosporin D is characterized by interesting chemotherapeutic and pharmacological properties and can therefore be used as a remedy. It inhibits the growth of Aspergillus niger and Curvularia lunata. In particular, the substance cyclosporin D is characterized by an immunosuppressive and anti-inflammatory effect.
The immunosuppressive effect of cyclosporin D can be shown as follows: a) In the lymphocyte stimulation test according to Janosy, a strong inhibition of H3-thymidine incorporation, the proliferation rate and the blastogenesis of with concanavalin is shown in vitro in concentrations of 0.01 to 10.0 µg A stimulated lymphocyte from mouse spleen was found.
b) Hemagglutination test on the mouse: The antibodies formed against sheep erythrocytes are recorded. The inhibition is expressed in the suppressive index (ski), the untreated control being given as 1.00; Sl = 0.53 according to p. o.5x250 mg / kg.
Due to its immunosuppressive effect, cyclosporin D can be used for the prophylaxis and treatment of diseases which are related to the negative effects of the defense reaction.
The new antibiotic also has an arthritis-inhibiting effect. So it works z. B. in Freund's adjuvant arthritis latency experiment on the rat in doses of about 50 mg / kg body weight per day, strongly inhibiting swelling.
A similar effect is observed in Freund's adjuvant arthritis therapy experiment in rats at doses of approximately 50 mg / kg / day.
Due to its arthritis-inhibiting effect, the antibiotic can be used for the prophylaxis and treatment of arthritis and rheumatic diseases.
The doses to be used naturally vary depending on the type of antibiotic, the administration and the condition to be treated. In general, however, satisfactory results are obtained with test animals with a dose of 10 to 200 mglkg body weight. If necessary, this dose can be administered in 2 to 3 portions or as a slow-release form.
For larger mammals, the daily dose is around 50 to 900 mg. For oral applications, the partial doses can contain, for example, about 25 to 300 mg of the new antibiotic in addition to solid and liquid carriers.
As a healing agent, the new antibiotic can be administered alone or in a suitable pharmaceutical form with pharmacologically indifferent auxiliaries.
In the following example, which explains the invention in more detail but is not intended to restrict its scope in any way, all the temperatures are given in degrees Celsius.
Example:
500 liters of a nutrient solution containing 40 g glucose, 5 g casein peptone per liter. Containing 5 g MgSO4-7dz0, 2 g KH2PO4.3 g NaNO3.0.5 g KCI, 0.01 g FeSO4 and demineralized water are inoculated with 50 liters of a pre-culture of strain NRRL 8044 and in a steel fermenter with stirring (170 rpm ) and aeration (1 liter air / min / liter of nutrient solution) for 13 days at 27 (see DOS 2 455 859).
The culture broth is stirred with the same amount of n-butyl acetate, after the organic phase has been separated off, it is concentrated in vacuo and the crude extract is degreased by 3-stage distribution between methanol water (9: 1) and petroleum ether. The methanolic phase is separated off, concentrated in vacuo and the crude product is precipitated by adding water. The material obtained after the filtration is chromatographed on the 5- to 7-fold amount of Sephadex LH-20 with methanol as the eluent. The top fractions are then chromatographed on silica gel 60, particle size 0.0630.20 mm (Merck) with hexane acetone (2: 1), the fractions which eluted first contain mainly cyclosporin A and cyclosporin D, the later eluted parts predominantly cyclosporin C.
For further purification, the cyclosporin A- and D-containing fractions are crystallized from 2 to 2 times the amount of acetone at -15 and then further separated by two chromatography on silica gel 60, particle size 0.063-0.20 mm (Merck), whereby the fractions first eluted with hexane acetone (2: 1) contain cyclosporin D in a highly enriched form. These are dissolved in twice the amount of acetone and left to crystallize at - 15. The crude crystallizate of cyclosporin D obtained in the process is dissolved in 10 times the amount of acetone for further purification, 2% by weight of activated carbon is added and the mixture is heated to 60 for 5 minutes.
The clear and almost colorless filtrate obtained after filtration over talc is concentrated to a third of the volume and allowed to cool to room temperature, cyclosporin D spontaneously crystallizing out. The crystallization is completed by leaving at - 17. The crystals obtained by filtering are washed with a little ice-cold acetone and then dried in a high vacuum at 80 "for 2 hours.
Claims (1)
Priority Applications (25)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH745777A CH632010A5 (en) | 1977-06-17 | 1977-06-17 | Process for the preparation of a novel antibiotic |
| DE19782819094 DE2819094A1 (en) | 1977-05-10 | 1978-04-29 | CYCLOSPORIN DERIVATIVES, THEIR USE AND MANUFACTURING |
| DK188778A DK148751C (en) | 1977-05-10 | 1978-05-01 | PROCEDURE FOR THE PREPARATION OF CYCLOSPORINE DERIVATIVES |
| SE7805055A SE438860B (en) | 1977-05-10 | 1978-05-02 | PROCEDURE FOR THE PREPARATION OF CYCLOSPORIN D |
| FI781348A FI59814C (en) | 1977-05-10 | 1978-05-02 | FRAMEWORK FOR CYCLOSPORINE DETERMINATION OF THERAPEUTIC THERAPY |
| US05/902,794 US4220641A (en) | 1977-05-10 | 1978-05-04 | Organic compounds |
| IT49208/78A IT1104195B (en) | 1977-05-10 | 1978-05-05 | CYCLOSPORIN DERIVATIVES THEIR PREPARATION AND THEIR APPLICATION AS MEDICATIONS |
| NL7804846A NL7804846A (en) | 1977-05-10 | 1978-05-05 | CYCLOSPORINE DERIVATIVES AND METHODS FOR PREPARING AND USING THESE DERIVATIVES. |
| IL54666A IL54666A (en) | 1977-05-10 | 1978-05-08 | Cyclosporin derivatives, their production and pharmaceutical compositions containing them |
| NZ193028A NZ193028A (en) | 1977-05-10 | 1978-05-08 | Cyclosporin d and pharmaceutical compositions |
| IE1924/81A IE46884B1 (en) | 1977-05-10 | 1978-05-08 | Cyclosporin derivatives |
| IE932/78A IE46883B1 (en) | 1977-05-10 | 1978-05-08 | Cyclosporin derivatives |
| ES469567A ES469567A1 (en) | 1977-05-10 | 1978-05-08 | Organic compounds |
| PT68013A PT68013B (en) | 1977-05-10 | 1978-05-08 | PROCESS FOR THE PREPARATION OF NOVEL ORGANIC COMPOUNDS WITH PHARMACEUTICAL EFFECT |
| PH21111A PH13973A (en) | 1977-05-10 | 1978-05-08 | Cyclosporin derivatives and pharmaceutical compositions-containing them |
| GB6316/80A GB1591934A (en) | 1977-05-10 | 1978-05-08 | Cyclosporin derivatives |
| JP5412578A JPS53139789A (en) | 1977-05-10 | 1978-05-09 | Improvement of organic compound |
| CA000302914A CA1117046A (en) | 1977-05-10 | 1978-05-09 | Organic compounds |
| FR7813625A FR2390420A1 (en) | 1977-05-10 | 1978-05-09 | NEW CYCLOPEPTIDIC DERIVATIVES, THEIR PREPARATION AND THEIR APPLICATION AS MEDICINAL PRODUCTS |
| AU35947/78A AU526582B2 (en) | 1977-05-10 | 1978-05-09 | Cyclosporin d |
| US06/126,215 US4289851A (en) | 1977-05-10 | 1980-03-03 | Process for producing cyclosporin derivatives |
| FI801682A FI63562C (en) | 1977-05-10 | 1980-05-26 | FRAMEWORK FOR THE PROCESSING OF THERAPEUTIC THERAPY OF ISOCYCLOSPORIN D |
| FI801681A FI63561C (en) | 1977-05-10 | 1980-05-26 | FRAMEWORK FOR THE PREPARATION OF THERAPEUTIC THERAPEUTIC DIHYDROCYCLOSPORIN D |
| SE8401134A SE8401134L (en) | 1977-05-10 | 1984-03-01 | PROCEDURE FOR THE PREPARATION OF DIHYDRO-CYCLOSPORIN D |
| SE8401135A SE8401135L (en) | 1977-05-10 | 1984-03-01 | PROCEDURE FOR PRODUCING ISO CYCLOSPORIN D |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH745777A CH632010A5 (en) | 1977-06-17 | 1977-06-17 | Process for the preparation of a novel antibiotic |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CH632010A5 true CH632010A5 (en) | 1982-09-15 |
Family
ID=4325421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CH745777A CH632010A5 (en) | 1977-05-10 | 1977-06-17 | Process for the preparation of a novel antibiotic |
Country Status (1)
| Country | Link |
|---|---|
| CH (1) | CH632010A5 (en) |
-
1977
- 1977-06-17 CH CH745777A patent/CH632010A5/en not_active IP Right Cessation
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PFA | Name/firm changed |
Owner name: SANDOZ AG TRANSFER- NOVARTIS AG |
|
| PL | Patent ceased |