NL8302610A - ANTITUMOR ANTIBIOTIC; METHOD FOR PREPARING THEREOF ACTINOMADURA MICROORGANISM; THERAPEUTIC TREATMENT; PHARMACEUTICAL PREPARATION. - Google Patents
ANTITUMOR ANTIBIOTIC; METHOD FOR PREPARING THEREOF ACTINOMADURA MICROORGANISM; THERAPEUTIC TREATMENT; PHARMACEUTICAL PREPARATION. Download PDFInfo
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- NL8302610A NL8302610A NL8302610A NL8302610A NL8302610A NL 8302610 A NL8302610 A NL 8302610A NL 8302610 A NL8302610 A NL 8302610A NL 8302610 A NL8302610 A NL 8302610A NL 8302610 A NL8302610 A NL 8302610A
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- bbm
- actinomadura
- antibiotic
- tumor
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Classifications
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- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/06—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using actinomycetales
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/03—Actinomadura
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/825—Actinomadura
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- Tropical Medicine & Parasitology (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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- Compounds Of Unknown Constitution (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
s * ) t VO 5005 is *) t VO 5005 i
Antitumor-antibioticum; werkwijze voor het bereiden daarvan; actino-madura microörganisme; therapeutische behandeling; farmaceutisch preparaat.Anti-tumor antibiotic; method for preparing it; actino-madura micro-organism; therapeutic treatment; pharmaceutical preparation.
De uitvinding heeft betrekking op een nieuw antitumor-antibioticum, alsmede op zijn produktie en winning.The invention relates to a new anti-tumor antibiotic, as well as to its production and recovery.
Het antitumor-antibioticum volgens de onderhavige uitvinding, BBM-1644, is een nieuw lid uit de groep eiwit antitumor-antibio-5 tica, waarvan neocarzinostatine, macromomycine en auromycine voorbeelden zijn.The anti-tumor antibiotic of the present invention, BBM-1644, is a new member of the protein anti-tumor antibiotic group, of which neocarzinostatin, macromomycin and auromycin are examples.
Neocarzinostatine (ookwel zinostatine genoemd) is een zuur eiwitmacromolecuul met een molecuulgewicht van 10.700, dat bestaat uit een enkele polypeptideketen van 109 aminozuren, die door twee disul-10 fidebruggen zijn verknoopt. De produktie van neocarzinostatine door fermentatie van stammen van Streptomyces carzinostaticus var. neocarzino-staticus wordt beschreven in het Amerikaanse octrooischrift 3.334.022 en in J. Antibiotics 18: 68 - 76 (1965). De aminozuurvolgorde van neocarzinostatine wordt beschreven in Cancer Treatment Reviews 6: 239 - 249 (1979).Neocarzinostatin (also called Zinostatin) is an acidic protein macromolecule of 10,700 molecular weight consisting of a single 109 amino acid polypeptide chain cross-linked by two disulfide bridges. The production of neocarzinostatin by fermentation of strains of Streptomyces carzinostaticus var. neocarzino statist is described in U.S. Pat. No. 3,334,022 and in J. Antibiotics 18: 68-76 (1965). The amino acid sequence of neocarzinostatin is described in Cancer Treatment Reviews 6: 239 - 249 (1979).
15 Macromomycine is een neutraal of zwakzuur polypeptide met een molecuulgewicht van ongeveer 15.000. De produktie van macromomycine door fermentatie van Streptomyces macromomyceticus (NIHJ MC-8-42) wordt beschreven in het Amerikaanse octrooischrift 3.595.954 en in J. Antibiotics 21: 44 - 49 (1968). De zuivering van macromomycine en ken-20 merkende gegevens voor de gezuiverde verbinding worden beschreven in J. Antibiotics 29; 415 - 423 (1976).Macromomycin is a neutral or weakly acidic polypeptide with a molecular weight of about 15,000. The production of macromomycin by fermentation of Streptomyces macromomyceticus (NIHJ MC-8-42) is described in U.S. Patent 3,595,954 and in J. Antibiotics 21: 44-49 (1968). The purification of macromomycin and characterizing data for the purified compound are described in J. Antibiotics 29; 415-423 (1976).
Auromomycine is een zwakzuur polypeptide met een molecuulgewicht van ongeveer 12.500 en een isoëlectrisch punt van pH 5,4.Auromomycin is a weakly acidic polypeptide with a molecular weight of about 12,500 and an isoelectric point of pH 5.4.
Het bestaat uit 16 verschillende aminozuren. De isolatie van auromomycine 25 uit de cultuurvloeistof van Streptomyces macromomyceticus en kenmerkende eigenschappen van het gezuiverde produkt worden beschreven in J. Antibiotics 32; 330 - 339 (1979).It consists of 16 different amino acids. The isolation of auromomycin 25 from the culture fluid of Streptomyces macromomyceticus and characteristics of the purified product are described in J. Antibiotics 32; 330-339 (1979).
BBM-1644 kan worden onderscheiden van bekende polypeptide antitumor-antibiotica zoals neocarzinostatine, macromomycine en 30 auromomycine door fysisch-chemische eigenschappen zoals molecuulgewicht, aminozuurgehalte en papier-electroforese.BBM-1644 can be distinguished from known polypeptide anti-tumor antibiotics such as neocarzinostatin, macromomycin and auromomycin by physicochemical properties such as molecular weight, amino acid content and paper electrophoresis.
Door de onderhavige uitvinding wordt een nieuw eiwit- 8302610 v » i 2 antitumor-antibioticum verschaft, dat hierin wordt aangeduid als BBM-1644, welk antibioticum bereid wordt door een nieuwe stam te kweken van Actinomadura, welke aangeduid wordt als Actinomadura sp. stam H710-49 (ATCC 39144) in een waterig voedingsmedium, dat assimileerbare bronnen 5 van koolstof en stikstof bevat onder ondergedompelde aerobe omstandigheden totdat een aanzienlijke hoeveelheid BBM-1644 door het organisme in het cultuurmedium is geproduceerd en het BBM-1644 desgewenst uit het cul-tuurmedium te winnen. De uitvinding omvat het BBM-1644 antibioticum in verdunde oplossing, als een ruw concentraat, als een ruwe vaste stof en 10 als een gezuiverde vaste stof.The present invention provides a novel protein 8302610 v212 anti-tumor antibiotic, herein referred to as BBM-1644, which antibiotic is prepared by cultivating a new strain of Actinomadura, which is referred to as Actinomadura sp. strain H710-49 (ATCC 39144) in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under submerged aerobic conditions until a significant amount of BBM-1644 has been produced by the organism in the culture medium and the BBM-1644 from the culture if desired. winning medium. The invention includes the BBM-1644 antibiotic in dilute solution, as a crude concentrate, as a crude solid, and as a purified solid.
Pig. 1 toont het infrarood absorptiespectrum van BBM- 1644 (KBr korrel).Pig. 1 shows the infrared absorption spectrum of BBM-1644 (KBr grain).
Fig. 2 toont de ultraviolet absorptiespectra van BBM-1644 in water, 0,01N HC1 en 0,01N NaOH.Fig. 2 shows the ultraviolet absorption spectra of BBM-1644 in water, 0.01N HCl and 0.01N NaOH.
15 De uitvinding heeft betrekking op een nieuw eiwit anti- tumor-antibioticum, dat hierin wordt aangeduid als BBM-1644 alsmede op zijn bereiding door fermentatie van een nieuwe stam van Actinomadura aangeduid als Actinomadura sp. stam H710-49. Het bovengenoemde organisme werd geïsoleerd uit een bodemmonster dat in West-Duitsland was verza-20 meld. Een biologisch zuivere vultuur van het organisme is gedeponeerd bij de American Type Culture Collection, Washington D.C., en aan de permanente verzameling van microörganismen toegevoegd als ATCC 39144.The invention relates to a novel protein anti-tumor antibiotic, herein referred to as BBM-1644, as well as to its preparation by fermentation of a new strain of Actinomadura designated Actinomadura sp. strain H710-49. The above organism was isolated from a soil sample collected in West Germany. A biologically pure culture of the organism has been deposited with the American Type Culture Collection, Washington D.C., and added to the permanent collection of microorganisms as ATCC 39144.
BBM-1644 belemmert de groei van verscheidene gramposi-tieve en zuurbestendige. bacteriën. Het antibioticum vertoont tevens 25 faag inducerende eigenschappen in lysogene bacteriën en belemmert de groei van lymfatische en vaste tumoren zoals P388 leucemie in muizen.BBM-1644 stunts the growth of several gram positive and acid resistant. bacteria. The antibiotic also exhibits phage-inducing properties in lysogenic bacteria and inhibits the growth of lymphatic and solid tumors such as P388 leukemia in mice.
Het nieuwe antibioticum kan derhalve worden gebruikt als een antibac-terieel middel of als een antitumor middel voor de inhibitie van tumoren bij zoogdieren.The new antibiotic can therefore be used as an antibacterial agent or as an anti-tumor agent for tumor inhibition in mammals.
30 De actinomyceten· stam nr. Η71Ό-49 werd geïsoleerd uit een bodemmonster en volgens conventionele procedures geprepareerd als een biologisch zuivere cultuur voor karakterisering. De stam H710-49 vormt zoals substraat als luchtmycelia. Het substraatmycelium is lang, vertakt en niet tot korte filamenten gebroken. Korte sporenketens wor-35 den gedragen op de top of monopodiale tak van het luchtmycelium. De sporenketens bevatten 2 tot 15 sporen in een keten (meestal 4 tot 8 sporen) 8302610 f # 3 en zijn recht, gehoekt of lusvormig van uiterlijk. De sporen hebben een wratachtig oppervlak en zijn ovaal tot elliptisch (0,5 0,6 x 0,7 (\j 1,2 ^um) van uiterlijk met een rond of gepunt uiteinde. Rijpe sporen worden vaak door lege hyfae afgescheiden. Terminale zwellingen van hyfae 5 worden zo nu en dan waargenomen op het substraatmycelium in Czapek's agar en Bennett's agar. Beweeglijke sporen, sporangia of sclerotische granules worden in geen enkel onderzocht medium waargenomen.The actinomycetes strain No. Η71Ό-49 was isolated from a soil sample and prepared according to conventional procedures as a biologically pure culture for characterization. The strain H710-49 forms like substrate as aerial mycelia. The substrate mycelium is long, branched and not broken into short filaments. Short spore chains are carried on top or monopodial branch of the aerial mycelium. The track chains contain 2 to 15 tracks in a chain (usually 4 to 8 tracks) 8302610 f # 3 and are straight, angled or looped in appearance. The spores have a wart-like surface and are oval to elliptical (0.5 0.6 x 0.7 (1.2 µm) in appearance with a rounded or pointed end. Mature spores are often separated by empty hyphae. swellings of hyphae 5 are occasionally observed on the substrate mycelium in Czapek's agar and Bennett's agar Movable spores, sporangia or sclerotic granules are not observed in any of the media tested.
Anders dan gewone soorten van het genus Streptomyces, groeit de stam H71Q-49 langzaam en vormt hij in chemisch gedefinieerde 10 media en natuurlijke organische media een armzalig luchtmycelium. De kleur van het luchtmycelium is wit en verandert na de vorming van sporen in havermeelagar, anorganische zouten-zetmeelagar en glycerol-asparagine-agar in een roze-kleurige tint. De massakleur van het substraatmycelium is kleurloos, geel, roodachtig bruin of donker grijsachtig bruin. Mela-15 noidepigment wordt niet geproduceerd, maar een citroengeel diffundeer-baar pigment wordt waargenomen in glycerol-asparagine-agar, tyrosine-agar en Pridham-Gottlieb's bas aal-agar, aangevuld met een van glycerol, L-arabinose, D-xylose, L-rhamnose, D-glucose, D-fructose, trehalose en D-mannitol. De stam H710-49 groeit bij 20°C, 28eC en 37°C, maar niet 20 bij 10°C of 41°C. Hij is gevoelig voor NaCl bij 10% maar niet bij 7%, en resistent tegen lysozyme bij 0,001%. D- Galactose, D-mannose, sucrose, raffinose en inositol worden door de stam niet gebruikt. Cultuureigen-schappen en fysiologische eigenschappen van de stam H710-49 worden respectievelijk in de tabellen A en B getoond. Het patroon van het gebruik door 25 de stam van de koolstofbron wordt in tabel C getoond.Unlike ordinary species of the genus Streptomyces, the strain H71Q-49 grows slowly and forms a poor aerial mycelium in chemically defined media and natural organic media. The color of the aerial mycelium is white and, after the formation of spores, changes to oatmeal agar, inorganic salt-starch agar and glycerol-asparagine agar to a pinkish hue. The mass color of the substrate mycelium is colorless, yellow, reddish brown or dark grayish brown. Mela-15 noide pigment is not produced, but a lemon-yellow diffusible pigment is observed in glycerol asparagine agar, tyrosine agar and Pridham-Gottlieb's base eel agar supplemented with one of glycerol, L-arabinose, D-xylose, L-rhamnose, D-glucose, D-fructose, trehalose and D-mannitol. The strain H710-49 grows at 20 ° C, 28eC and 37 ° C, but not 20 at 10 ° C or 41 ° C. It is sensitive to NaCl at 10% but not 7%, and resistant to lysozyme at 0.001%. D-Galactose, D-mannose, sucrose, raffinose and inositol are not used by the strain. Culture properties and physiological properties of the strain H710-49 are shown in Tables A and B, respectively. The pattern of use by the strain of the carbon source is shown in Table C.
TABEL ΛTABLE Λ
XX
Cultuureigenschappen van stam H710-49Culture properties of strain H710-49
XXXX
Trypton-gistextract G slecht tot matig; floccose, bouillon (ISP No. 1) gesedimenteerd en niet gepigmen- teerd.Trypton yeast extract G bad to moderate; floccose, broth (ISP No. 1) sedimented and unpigmented.
30 Sucrose-nitxaat agar G gering (Czapek's agar) R kleurloos tot lichtoranjegeel (73)xxx A gering? wit (263) D geen 35 . Glucose-asparagine agar G slecht R gelig wit (92) tot diep oranjegeel (69) ____é 8302610 4 . o TABEL A (Vervolg)30 Sucrose nitxate agar G low (Czapek's agar) R colorless to light orange yellow (73) xxx A low? white (263) D none 35. Glucose asparagine agar G poor R yellowish white (92) to deep orange yellow (69) ____ é 8302610 4. o TABLE A (Continued)
Cultuureigenschappen van stam H710-49 A zeer gering; wit (263) D geenCulture characteristics of strain H710-49 A very low; white (263) D none
Glycerol-asparagine agar G slecht tot matig 5 (ISP) No. 5) R lichtgeel (89) tot donker oranje geel (72) A slecht; wit (263) tot lichtgelig- roze (31) D heldergeel (83) 10 anorganische zouten-zetmeel G slecht tot matig agar (ISP No. 4) R kleurloos tot diepgeel (85) A slecht; roze-wit (9) tot lichtgelig- roze (31) D geen 15 Tyrosine agar (ISP No. 7) G matig R bruinig-oranje (54) tot matig rood achtig bruin (43) A slecht; wit (263) tot lichtgeel (89) D sterk geel (84) 20 Voedingsagar G slecht tot matig R gelig wit (92) tot matig gelig bruin (77) A slecht; wit (263) D geen 25 Gistextract-moutextract G matig agar (ISP No. 2) R donkergeel (88) tot donkerbruin (59) A gering; wit (263) D licht olijfbruin (94) havermeelagar (ISP No. 3) G slecht 30 R kleurloos A slecht; wit (263) tot roze-wit (9) D geenGlycerol asparagine agar G bad to moderate 5 (ISP) No. 5) R light yellow (89) to dark orange yellow (72) A poor; white (263) to pale yellowish-pink (31) D bright yellow (83) 10 inorganic salts starch G poor to moderate agar (ISP No. 4) R colorless to deep yellow (85) A poor; pinkish-white (9) to slightly yellowish-pink (31) D none 15 Tyrosine agar (ISP No. 7) G moderate R brownish-orange (54) to moderate reddish brown (43) A poor; white (263) to light yellow (89) D strong yellow (84) 20 Nutritional agar G poor to moderate R yellowish white (92) to moderate yellowish brown (77) A poor; white (263) D none 25 Yeast extract malt extract G moderate agar (ISP No. 2) R dark yellow (88) to dark brown (59) A low; white (263) D light olive brown (94) oatmeal agar (ISP No. 3) G poor 30 R colorless A poor; white (263) to pink-white (9) D none
Bennett's agar G matig R grijzig gelig bruin (80) tot donker- 35 grijzig bruin (62) A zeer gering; wit (263) D matig olijfbruin (95) 8302610 « · 5 TABEL· A (Vervolg)Bennett's agar G moderate R greyish yellowish brown (80) to dark greyish brown (62) A very small; white (263) D moderate olive brown (95) 8302610 «· 5 TABLE · A (Continued)
Cultuureigenschappen van stam H710-49 Pepton-gistextract- G slecht ijzeragar (ISP No. 6) R grijzig geel (90) tot donker grijzig bruin (62) 5 A slecht; wit (263) D geen tot matig gelig bruin (77) * waargenomen na incubatie bij 28eC gedurende 3 weken uu afkortingen: G - groei; R - kleur achterkant, A - luchtmycelium; D - diffundeerbaar pigment 10 ^^leur en getal tussen haakjes volgen de kleurstandaard in "Kelly, K. , L·. & D. B. Judd: XSCC-NBS color-name charts illustrated with Centroid Colors. U.S. Dept, of Comm. Circ. 553, Washington, D.C., Nov., 1975" 8302610 6 * 4 μ Φ c ο> φ φ Μ +) 4J c Μ Μ Φ M<l)n3cnOrOfOn3 Π ΙβΝΙβ,-ΡιΡβ'Ο'Β' •Η Ο'-ηρΟ'ΗΦΦΦ +J (0 -Ρ -Ρ I Ή (0 -Ρ | ί-| φ 3 -Ρ -Ρ -Ρ ,-) ΟΦ-ΡΠΜΟϋϋϋ g Ο) 4J ΦβΟΙΟΛΦΦΦ 5 en φ ρ -ρ φ φ υ mum -Η I φ Λ! ·ΡΜΡϋΐ3·Ρ·Ρ-Ρ-Ρ OH S HH ΧΟ-ΡΟΗΦ*** 0)(0 o aai φμχοΟ'ΦφφφCulture properties of strain H710-49 Pepton yeast extract G bad iron agar (ISP No. 6) R greyish yellow (90) to dark greyish brown (62) 5 A poor; white (263) D no to moderate yellowish brown (77) * observed after incubation at 28eC for 3 weeks hh abbreviations: G - growth; R - back color, A - air mycelium; D - diffusible pigment 10 ^^ leur and number in brackets follow the color standard in "Kelly, K., L ·. & DB Judd: XSCC-NBS color-name charts illustrated with Centroid Colors. US Dept, or Comm. Circ. 553 , Washington, DC, Nov., 1975 "8302610 6 * 4 μ Φ c ο> φ φ Μ +) 4J c Μ Μ Φ M <l) n3cnOrOfOn3 Π ΙβΝΙβ, -ΡιΡβ'Ο'Β '• Η Ο'-ηρΟ 'ΗΦΦΦ + J (0 -Ρ -Ρ I Ή (0 -Ρ | ί- | φ 3 -Ρ -Ρ -Ρ, -) ΟΦ-ΡΠΜΟϋϋϋ g Ο) 4J ΦβΟΙΟΛΦΦΦ 5 and φ ρ -ρ φ φ υ mum - Η I φ Λ! ΡΜΡϋΐ3 · Ρ · Ρ-Ρ-Ρ OH S HH ΧΟ-ΡΟΗΦ *** 0) (0 o pat φμχοΟ'Φφφφ
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TABEL CTABLE C
Gebruik van koolstofbronnen door stam H710-49 Glycerol + i D(-)-arabinose L(+)-arabinose + 5 D-xylose + D-ribose + L-rhamnose + D-glucose -+ D-galactose 10 D-fructose + D-mannose L(-)-sorbose Sucrose Lactose 15 Cellobiose +Use of carbon sources by strain H710-49 Glycerol + i D (-) - arabinose L (+) - arabinose + 5 D-xylose + D-ribose + L-rhamnose + D-glucose - + D-galactose 10 D-fructose + D-mannose L (-) - sorbose Sucrose Lactose 15 Cellobiose +
MelibioseMelibiosis
Trehalose +Trehalose +
Raffinose D (+)-melezitose 20 oplosbare zetmeel +Raffinose D (+) - melezitose 20 soluble starch +
Cellulose -Cellulose -
DulcitolDulcitol
Inositol D—mannitol + 25 D-sorbitolInositol D-mannitol + 25 D-sorbitol
Salicine waargenomen na incubatie bij 28eC gedurende 3 weken. ·Salicin observed after incubation at 28 ° C for 3 weeks. ·
Basaal medium: Pridham-Gottlieb anorganisch medium.Basal medium: Pridham-Gottlieb inorganic medium.
Gezuiverde celwand van stam H710-49 bevat mesodiami-30 nopimelinezuur maar mist glycine. Het hele cellen-hydrolysaat vertoont de aanwezigheid van madurose (3-O-methyl-D-galactose), glucose, ribose en een kleine hoeveelheid mannose. De celwand-samenstelling en hele cel-suikercomponenten van de stam H710-49 wijzen uit dat de stam behoort tot celwand-type ΙΙΙβ.Purified cell wall of strain H710-49 contains mesodiami-30 nopimelic acid but lacks glycine. The whole cell hydrolyzate shows the presence of madurose (3-O-methyl-D-galactose), glucose, ribose and a small amount of mannose. The cell wall composition and whole cell sugar components of the strain H710-49 indicate that the strain belongs to cell wall type ΙΙΙβ.
8302610 .........^ * - 88302610 ......... ^ * - 8
De bovenstaand beschreven eigenschappen van de stam H710-49 lijken op die van het genus Actinomadura. Volgens de numerieke taxonomie van Actinomadura en verwante actinomyceten door Goodfellow et al. in J. Gen. Microbiol. 112: 95 - 111 (1979), worden de meeste 5 Actinomadura species van bodemoorsprong ingedeeld in Cluster No. 7 onder de beschreven 14 clusters. Stam No. H710-49 is het meest verwant aan de species van Cluster 7. Nonomura en Ohara in jJ. Ferment. Technol.The properties of the strain H710-49 described above resemble those of the genus Actinomadura. According to the numerical taxonomy of Actinomadura and related actinomycetes by Goodfellow et al. In J. Gen. Microbiol. 112: 95-111 (1979), most 5 Actinomadura species of soil origin are classified in Cluster No. 7 among the 14 clusters described. Strain No. H710-49 is most closely related to the species of Cluster 7. Nonomura and Ohara in jJ. Ferment. Technol.
49: 904 - 912 (1971) hebben vijf saprofytische species beschreven van het genus Actinomadura en Nonomura [J. Ferment. Technol. 52: 71-77 10 (1974)3 en Preobrazhenskaya et al. [Actlnomycetes and Related Organisms 42: 30 - 38 (1977)] hebben de identificatie en indeling van Actinomadura species beschreven. Als resultaat van een vergelijking met bekende Actinomadura species die in de literatuur zijn beschreven, wordt stam H710-49 geacht te bhoren tot een nieuwe species van Actinomadura, die 15 lijkt op A. roseola, A. salmonea, A. vinacea of A. corallina beschreven in bovenstaande Preobrazhenskaya et al. referentie en in Japanse Kokai 55/94391.49: 904-912 (1971) have described five saprophytic species of the genus Actinomadura and Nonomura [J. Ferment. Technol. 52: 71-77 10 (1974) 3 and Preobrazhenskaya et al. [Actomomycetes and Related Organisms 42: 30-38 (1977)] have described the identification and classification of Actinomadura species. As a result of a comparison with known Actinomadura species described in the literature, strain H710-49 is believed to belong to a new species of Actinomadura, resembling A. roseola, A. salmonea, A. vinacea or A. corallina described in the Preobrazhenskaya et al. reference above and in Japanese Kokai 55/94391.
Aangenomen wordt dat voor de produktie van BBM-1644, de onderhavige uitvinding ondanks de beschrijving in detail onder verwij-20 zing naar de bepaalde stam Actinomadura sp. stam H710-49 (ATCC 39144), niet tot dit microörganisme of tot volledig door de hierin beschreven cultuureigenschappen beschreven microörganismen beperkt is. Het is in het bijzonder de bedoeling dat de uitvinding de stam H710-49 en alle natuurlijke en kunstmatige BBM-1644-producerende varianten en mutanten 25 daarvan omvat.·It is believed that for the production of BBM-1644, the present invention despite the description in detail with reference to the particular strain Actinomadura sp. strain H710-49 (ATCC 39144), is not limited to this microorganism or to microorganisms fully described by the culture properties described herein. In particular, the invention is intended to encompass the strain H710-49 and all natural and artificial BBM-1644 producing variants and mutants thereof.
Het BBM-1644 antibioticum volgens de onderhavige uitvinding kan worden bereid door een BBM-1644-producerende stam van het genus Actinomadura, bij voorkeur een stam van Actinomadura sp. met de identificerende eigenschappen van ATCC 39144 of een mutant daarvan, in .The BBM-1644 antibiotic of the present invention can be prepared by a BBM-1644 producing strain of the genus Actinomadura, preferably a strain of Actinomadura sp. with the identifying properties of ATCC 39144 or a mutant thereof, in.
30 een conventioneel waterig voedingsmedium te kweken. Het organisme wordt gegroeid in een voedingsmedium dat bekende voedingsbronnen voor actino-myceten bevat, d.w.z. assimileerbare bronnen van koolstof en stikstof plus optionele anorganische zouten en andere bekende groeifactoren. Ondergedompelde aerobe condities worden bij voorkeur gebruikt voor de produk-35 tie van grote hoeveelheden antibioticum, hoewel voor de produktie van beperkte hoeveelheden ook oppervlakteculturen en flessen kunnen worden 8302610 ο 9 gebruikt. De algemene procedures die voor het kweken van andere actino-myceten worden gebruikt, zijn op de onderhavige uitvinding van toepassing-Het voedingsmedium dient een gepaste assimileerbare kool-stofbron te bevatten zoals glycerol, L(+)-arabinose, D-xylose, D-ribose, 5 D-glucose, D-fructose, oplosbare zetmeel, D-mannitol of cellobiose. Als stikstofbronnen kunnen ammoniumchloride, ammoniumsulfaat, ureum, ammo-niumnitraat, natriumnitraat, enz. alleen of in combinatie met organische stikstofbronnen zoals pepton, vleesextract, gistextract, maisweekwater, sojabonenpoeder, katoenzaadmeel, enz. worden toegepast. Indien nodig 10 kunnen ook anorganische voedingszouten worden toegevoegd om bronnen te verschaffen voor natrium, kalium, calcium, ammonium, fosfaat, sulfaat, chloride, bromide, carbonaat, zink, magnesium, mangaan, kobalt, ijzer, en dergelijke.30 to cultivate a conventional aqueous nutrient medium. The organism is grown in a nutrient medium containing known dietary sources for actino mycetes, i.e. assimilable sources of carbon and nitrogen plus optional inorganic salts and other known growth factors. Submerged aerobic conditions are preferably used for the production of large amounts of antibiotics, although for the production of limited amounts, surface cultures and bottles can also be used. The general procedures used for culturing other actino mycetes apply to the present invention. The nutrient medium should contain an appropriate assimilable carbon source such as glycerol, L (+) - arabinose, D-xylose, D- ribose, D-glucose, D-fructose, soluble starch, D-mannitol or cellobiose. As nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. can be used alone or in combination with organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep water, soybean powder, cottonseed meal, etc. Inorganic food salts can also be added as needed to provide sources of sodium, potassium, calcium, ammonium, phosphate, sulfate, chloride, bromide, carbonate, zinc, magnesium, manganese, cobalt, iron, and the like.
De produktie van het BBM-1644 antibioticum kan bij elke 15 temperatuur worden uitgevoerd die tot een bevredigende groei van het producerende organisme leidt, bijvoorbeeld 20 - 37°C, en wordt geschikt uitgevoerd bij een temperatuur vein ongeveer 27 - 32 °C. Gewoonlijk wordt een optimale produktie verkregen in schudkolven na incubatieperioden van ongeveer 6-7 dagen. Wanneer een tankfermentatie moet 20 worden uitgevoerd, is het gewenst dat een vegetatief inoculum wordt geproduceerd in een voedingsbouillon door de bouilloncultuur te enten met een hellende of bodemcultuur of een gelyofiliseerde cultuur van het organisme. Nadat op deze wijze een actief inoculum is verkregen, wordt dit op aseptische wijze naar het fermentatietankmedium overgebracht. De anti-25 bioticumproduktie kan worden gevolgd door de papierschijf-agardiffusie-analyse waarbij Bacillus subtilis M45 [Ree mutant; Mutation Res. 16; 165 - 174 (1972)] als het testorganisme wordt gebruikt.The production of the BBM-1644 antibiotic can be carried out at any temperature that leads to a satisfactory growth of the producing organism, for example 20-37 ° C, and is conveniently carried out at a temperature of about 27-32 ° C. Usually optimal production is obtained in shake flasks after incubation periods of about 6-7 days. When a tank fermentation is to be performed, it is desirable that a vegetative inoculum be produced in a nutrient broth by inoculating the broth culture with an inclined or bottom culture or a lyophilized culture of the organism. After an active inoculum is obtained in this manner, it is aseptically transferred to the fermentation tank medium. The anti-biotic production can be followed by the paper disc agar diffusion analysis in which Bacillus subtilis M45 [Ree mutant; Mutation Res. 16; 165-174 (1972)] when the test organism is used.
Wanneer de fermentatie compleet is, bevindt het BBM-1644 zich voornamelijk in het vloeibare deel van de gefermenteerde bouillon 30 na afscheiding van het vaste deel door filtratie of centrifugeren. De geoogste bouillon kan derhalve door centrifugeren in myceliumkoek en bovenstaande bouillon worden gescheiden. Het filtraat wordt daarna geconcentreerd en aan dialyse onderworpen tegen kraanwater door een semi-permeabel membraan zoals een cellofaanbuis om permeabele verontreini-35 gingen te verwijderen. De binnengehouden oplossing (na verwijdering van onoplosbare materialen) welke het BBM-1644 bevat, kan daarna worden ver- 8302610 10 zadigd met een uitzout-reagens zoals ammoniumsulfaat om BBM-1644 neer te laten slaan als een ruwe vaste stof. Deze vaste stof kan in water worden opgelost en door dialyse tegen kraanwater worden ontzout.When the fermentation is complete, the BBM-1644 is mainly located in the liquid part of the fermented broth 30 after separation of the solid part by filtration or centrifugation. The harvested broth can therefore be separated by centrifugation in mycelium cake and the above broth. The filtrate is then concentrated and dialyzed against tap water through a semi-permeable membrane such as a cellophane tube to remove permeable contaminants. The contained solution (after removal of insoluble materials) containing the BBM-1644 can then be saturated with a salting reagent such as ammonium sulfate to precipitate BBM-1644 as a crude solid. This solid can be dissolved in water and desalinated by tap water dialysis.
Verdere zuivering van het ruwe BBM-1644 kan worden ge-5 realiseerd door conventionele procedures die bij andere zure polypepti-den worden toegepast. Bijvoorbeeld kan de waterige oplossing die BBM-1644 bevat, worden geadsorbeerd op een ionenuitwisselaar zoals DEAE-Sephadex, DEAE-cellulose, CM-Sephadex of CM-cellulose en worden geëlu-eerd met een neutrale zoutoplossing. Bij voorkeur worden opvolgende 10 chromatografische stappen toegepast met een gradiëntconcentratie van de als het eluens toegepaste zoutoplossing. Vervolgens worden waterige fracties die het gezuiverde BBM-1644 bevatten, tot een droge toestand geconcentreerd, bijvoorbeeld door lyofilisatie.Further purification of the crude BBM-1644 can be accomplished by conventional procedures used in other acidic polypeptides. For example, the aqueous solution containing BBM-1644 can be adsorbed on an ion exchanger such as DEAE-Sephadex, DEAE cellulose, CM-Sephadex or CM cellulose and eluted with neutral saline. Preferably, subsequent chromatographic steps are used with a gradient concentration of the saline used as the eluent. Aqueous fractions containing the purified BBM-1644 are then concentrated to a dry state, for example by lyophilization.
BBM-1644 wordt na lyofilisatie als een amorf wit poe-15 der geïsoleerd. Bij onderzoek met papier-electroforese onder hoge spanning (4500 V in 0,05M barbitalbuffer bij pH 8,6), migreert BBM-1644 als en zuur waarbij het. na 1 uur 8,7 cm naar de anode loopt. BBM-1644 vertoont niet een bepaald smeltpunt en ontleedt geleidelijk boven 240°C.BBM-1644 is isolated as an amorphous white powder after lyophilization. When tested with high voltage paper electrophoresis (4500 V in 0.05M barbital buffer at pH 8.6), BBM-1644 migrates as an acid to form the. after 1 hour runs 8.7 cm to the anode. BBM-1644 does not show a particular melting point and gradually decomposes above 240 ° C.
Het is oplosbaar in water, maar praktisch onoplosbaar in gewone organi- ; 20 sche oplosmiddelen zoals methanol, ethanol, aceton, ethylacetaat en n- • 26 hexaan. Het antibioticum vertoont een optische rotatie van [a]^ = -75,6° in 0,25%'s waterige oplossing. Zoals in fig. 2 getoond, vertoont 1% het UV-spectrum van BBM-1644 absorptiemaxima bij 275 nm (E j 8,2) en 310 nm (E*!* 4,6, schouder) in waterige oplossing. Het vertoont een lcm 25 nagenoeg identiek UV-spectrum in water en in 0,0IN HC1 oplossing, maar 1% slechts een enkel maximum bij 285 nm (E, 8,9) in 0,01N NaOH oplossing.It is soluble in water, but practically insoluble in ordinary organics; 20 solvents such as methanol, ethanol, acetone, ethyl acetate and n-26 hexane. The antibiotic shows an optical rotation of [α] 25 = -75.6 ° in 0.25% aqueous solution. As shown in Fig. 2, 1% shows the UV spectrum of BBM-1644 absorption maxima at 275 nm (Ej 8.2) and 310 nm (E *! * 4.6, shoulder) in aqueous solution. It shows a 1 cm 2 almost identical UV spectrum in water and in 0.0IN HCl solution, but 1% only a single maximum at 285 nm (E, 8.9) in 0.01N NaOH solution.
lcmlcm
Het IR-spectrum van BBM-1644, gemeten in KBr, wordt getoond in fig. 1.The IR spectrum of BBM-1644, measured in KBr, is shown in Figure 1.
Het spectrum laat de aanwezigheid zien van NH- en OH-groepen (3300 ^ -1 -1 2980 cm ) en amide-groepen (1650 en 1540 cm ). Het antibioticum geeft 30 positieve reacties ten opzichte van Folin-Lowry, xantoproteïne, biureet en ninhydrine reagentia en ontkleurt een kaliumpermanganaat-oplossing. Het is negatief ten opzichte van anthron-en Sakaguchi-reacties. Bij co-chromatografische behandeling over Sephadex G-75 met ovalbumine (mole-cuulgewicht 43.000), chymotrypsinogeen (25.000) en ribonuclease A 35 (13.700), wordt BBM-1644 net na chymotrypsinogeen geëlueerd en zijn molecuulgewicht wordt daarom geschat op ongeveer 22.000. Elementair- 4 8302610 11 analyse van BBM-1644 wijst een koolstofgehalte van 46,60%, een water-stofgehalte van 6,45%, een stikstofgehalte van 13,34% en een zwavel-gehalte van 0,20% uit. De aanwezigheid van 13 soorten aminozuren in het BBM-1644 molecuul werd door aminozuur-analyse geopenbaard zoals ge-5 toond in tabel D. Basische aminozuren zoals lysine, histidine en arginine zijn in BBM-1644 niet aanwezig.The spectrum shows the presence of NH and OH groups (3300 ^ -1 -1 2980 cm) and amide groups (1650 and 1540 cm). The antibiotic gives 30 positive reactions to Folin-Lowry, xantoprotein, biuret and ninhydrin reagents and decolorizes a potassium permanganate solution. It is negative towards anthron and Sakaguchi reactions. By co-chromatography on Sephadex G-75 with ovalbumin (molecular weight 43,000), chymotrypsinogen (25,000) and ribonuclease A 35 (13,700), BBM-1644 is eluted just after chymotrypsinogen and its molecular weight is therefore estimated to be about 22,000. Elemental analysis of BBM-1644 indicates a carbon content of 46.60%, a hydrogen content of 6.45%, a nitrogen content of 13.34% and a sulfur content of 0.20%. The presence of 13 kinds of amino acids in the BBM-1644 molecule was revealed by amino acid analysis as shown in Table D. Basic amino acids such as lysine, histidine and arginine are not present in BBM-1644.
TABEL DTABLE D
Aminozuursamenstelling van BBM-1644 χAmino acid composition of BBM-1644 χ
Aminozuur Relatieve samenstelling _ van aminozuren_ 10 Alanine 8,8Amino acid Relative composition of amino acids Alanine 8.8
Aspartinezuur 6,0 half-Cystine 1,0Aspartic Acid 6.0 Half-Cystine 1.0
Glutaminezuur 5,7Glutamic acid 5.7
Glycine 8,7 15 Isoleucine 2,3Glycine 8.7 15 Isoleucine 2.3
Leucine 1,0Leucine 1.0
Fenylalanine 1,4Phenylalanine 1.4
Proline 4,1Proline 4.1
Serine 1,6 20 Threonine 7,2Serine 1.6 20 Threonine 7.2
Tyrosine 0,6Tyrosine 0.6
Valine 11,1 het gehalte aan leucine werd arbitrair genomen als 1,0.Valine 11.1 the leucine content was taken arbitrarily as 1.0.
BBM-1644 is behoorlijk stabiel in het pH-gebied van 25 2aj9, maar de stabiliteit neemt voorbij dit.pH-gebied scherp af. De waterige oplossing van BBM-1644 is gedurende 2 uur bij 50°C bij neutrale pH stabiel. Bij blootstelling aan ultraviolet licht, gaat de antibiotische werkzaamheid van BBM-1644 binnen 20 minuten verloren.BBM-1644 is quite stable in the pH range of 2aj9, but stability decreases sharply beyond this.pH range. The aqueous solution of BBM-1644 is stable at 50 ° C for 2 hours at neutral pH. When exposed to ultraviolet light, the antibiotic efficacy of BBM-1644 is lost within 20 minutes.
De bovenstaand beschreven fysisch-chemische eigenschap-30 pen van BBM-1644 wijzen uit dat het een lid is van de eiwit antitumor-antibioticum-groep die neocarzinostatine, macromomycine en auromomycine omvat. BBM-1644 kan echter van de bekende eiwit antitumor-antibiotica worden onderscheiden door zijn molecuulgewicht, aminozuurgehalte en papiar-electroforese. De papier-electroforetische molibileiten van BBM-1644, _i 8302610 12 neocarzinostatine en macromomycine worden in tabel E getoond.The physicochemical properties of BBM-1644 described above indicate that it is a member of the protein anti-tumor antibiotic group comprising neocarzinostatin, macromomycin and auromomycin. However, BBM-1644 can be distinguished from the known protein anti-tumor antibiotics by its molecular weight, amino acid content and papiar electrophoresis. The paper electrophoretic properties of BBM-1644, 8302610 12 neocarzinostatin and macromomycin are shown in Table E.
TABEL E Papier-electroforese*TABLE E Paper Electrophoresis *
Mobiliteit (mm vanaf de opbrengplaats) BBM-1644 +87 5 Neocarzinostatine +31Mobility (mm from application site) BBM-1644 +87 5 Neocarzinostatin +31
Macromomycine -20 * 4.500 V, 1 uur; Barbital buffer pH 8,6 , De neocarzinostatine-groep van antibiotica vertoont gewoonlijk 2 üV-absorptie-maxima bij ongeveer 275 en 350 nm, terwijl 10 BBM-1644 de üV-maxima heeft bij ongeveer 275 en 310 nm. Onlangs is be schreven in Biochem. Res. Commun. 95:' 1351 - 1356 (1980) dat het maximum bij 350 nm van de neocarzinostatine antibiotica te wijten kan zijn aan de niet-eiwit chromoforen die voor hun biologische werkzaamheid essentieel zijn. Opgemerkt wordt dat BBM-1644 een ander chromofoor heeft 15 dan de bekende neocarzinostatine-groep antibiotica.Macromomycin -20 * 4,500 V, 1 hour; Barbital buffer pH 8.6. The neocarzinostatin group of antibiotics usually exhibits 2 µV absorption maxima at about 275 and 350 nm, while 10 BBM-1644 has the µV maxima at about 275 and 310 nm. Recently described in Biochem. Res. Commun. 95: 1351-1356 (1980) that the maximum at 350 nm of the neocarzinostatin antibiotics may be due to the non-protein chromophores essential for their biological activity. It is noted that BBM-1644 has a different chromophore than the known neocarzinostatin group of antibiotics.
De antibacteriële werkzaamheid van BBM-1644 werd bepaald door de in serie uitgevoerde tweevoudige agar-verdunningsmethode. Voedingsagarmedium werd gebruikt voor grampositieve en gramnegatieve bacteriën; voedingsagarmedium dat 4% glycerol bevatte voor zuurbesten-20 dige bacteriën en Sabouraud agarmedium voor schimmels. De activiteit werd uitgedrukt als minimum inhibitore concentratie (MIC) in het agarmedium en de resultaten worden in tabel F getoond samen met die vein neocarzinostatine. BBM-1644 vertoonde een sterke inhibitore werkzaamheid tegen grampositieve en zuurbestendige bacteriën, maar vertoonde 25 geen inhibitie van de groei van gramnegatieve bacteriën en schimmels.The antibacterial activity of BBM-1644 was determined by the serial two-way agar dilution method. Nutrient agar medium was used for gram-positive and gram-negative bacteria; nutrient agar medium containing 4% glycerol for acid-resistant bacteria and Sabouraud agar medium for fungi. The activity was expressed as minimum inhibitory concentration (MIC) in the agar medium and the results are shown in Table F together with that of neocarzinostatin. BBM-1644 showed strong inhibitory activity against gram-positive and acid-resistant bacteria, but did not inhibit the growth of gram-negative bacteria and fungi.
Het antibacteriële spectrum van BBM-1644 lijkt op dat van neocarzinostatine, terwijl de intrinsieke werkzaamheid van BBM-1644 in sommige van de testorganismen sterker is dan die van laatstgenoemde produkt.The antibacterial spectrum of BBM-1644 is similar to that of neocarzinostatin, while the intrinsic efficacy of BBM-1644 is stronger in some of the test organisms than that of the latter.
30 8302610 1330 8302610 13
TABEL· FTABLE · F
In vitro antimicrobiële activiteit _MIC in mcg/ml_In vitro antimicrobial activity _MIC in mcg / ml_
Test organisme BBM-1644 NeocarzinostatineTest organism BBM-1644 Neocarzinostatin
Staphylococcus aureus FDA 209P 0,4 1,6 5 Staphylococcus aureus Smith 0,2 0,8Staphylococcus aureus FDA 209P 0.4 1.6 5 Staphylococcus aureus Smith 0.2 0.8
Streptococcus pyogenes A202Q1 6,3 3,1Streptococcus pyogenes A202Q1 6.3 3.1
Micrococcus luteus PCI 1001 1,6 1,6Micrococcus luteus PCI 1001 1.6 1.6
Micrococcus flavus D12 1,6 1,6Micrococcus flavus D12 1.6 1.6
Bacillus subtilis PCI 219 0,8 3,1 10 Escherichia coli NIHJ > 100 > 100Bacillus subtilis PCI 219 0.8 3.1 10 Escherichia coli NIHJ> 100> 100
Klebsiella pneumoniae D-ll >100 .> 100Klebsiella pneumoniae D-ll> 100.> 100
Proteus vulgaris A9436 >100 > 100Proteus vulgaris A9436> 100> 100
Pseudomonas aeruginosa A9930 >100 >100Pseudomonas aeruginosa A9930> 100> 100
Mycobacterium smegmatis 607 D87 12,5 50 15 Mycobacterium phlei D88 3,1 12,5Mycobacterium smegmatis 607 D87 12.5 50 15 Mycobacterium phlei D88 3.1 12.5
Candida albicans IAM 4888 > 100 > 100Candida albicans IAM 4888> 100> 100
Het vermogen van BBM-1644 on profagen te induceren in lysogene bacteriën (ILB) werd bepaald door de methode van Lein et al. (Nature 196: 783 - 784, 1962) waarbij neocarzinostatine als referentie-20 verbinding werd gebruikt. De plague-telling werd uitgevoerd op agarpla-ten die testmateriaal (T) en controlemateriaal (C) bevatten. Een T/C-verhouding van de plaque-tellingen van meer dan 3 werd significant geacht en de ILB-activiteit werd uitgedrukt als de minimum inducerende concentratie van de testverbinding. Zoals getoond in tabel G, als de 25 ILB-activiteit van BBM-1644 vergelijkbaar met die waargenomen met neo-carzinostatine, waarbij de minimum inducerende concentratie 1 microgram/ ml was.The ability of BBM-1644 to induce prophages in lysogenic bacteria (ILB) was determined by the method of Lein et al. (Nature 196: 783 - 784, 1962) using neocarzinostatin as the reference compound. Plague counting was performed on agar plates containing test material (T) and control material (C). A T / C ratio of the plaque counts greater than 3 was considered significant and the ILB activity was expressed as the minimum inducing concentration of the test compound. As shown in Table G, the ILB activity of BBM-1644 was comparable to that observed with neo-carzinostatin, where the minimum inducing concentration was 1 microgram / ml.
TABEL· GTABLE · G
Inductie van lysogene bacteriën door BBM-1644 _ILB-activiteit (T/C)1 30 100 10 _1_ 0,1 mcg/ml BBM-1644 10,4 10,3 6,0 1,2Induction of lysogenic bacteria by BBM-1644 _ILB activity (T / C) 1 30 100 10 _1_ 0.1 mcg / ml BBM-1644 10.4 10.3 6.0 1.2
Neocarzinostatine 28,6 20,4 10,8 0,7 8302610 significante activiteit: T/C ^ 3,0 14Neocarzinostatin 28.6 20.4 10.8 0.7 8302610 significant activity: T / C ^ 3.0 14
De antitumor-werkzaamheid van BBM-1644 werd bepaald in muizen (BDF. stam) tegen lymfocytische leucemie P388. Elke nuis werd 1 5 intraperitoneaal geënt met drie keer 10 cellexj van de tumor. Getrapte » doses van het antibioticum werden 24 uur na het implanten van de tumor 5 intraperitoneel aan de muizen toegediend. De behandelingen werden eenmaal per dag gegeven op de dagen 1, 4 en 7 (q3d x 3 schema) of 9 opvolgende dagen (qd 1 —> 9 schema) . Neocarzinostatine werd ter vergelijking getest als referentie antitumor-middel en de resultaten zijn samengevat in tabel H. BBM-1644 was zeer werkzaam tegen de muisleucemie in een 10. dosisgebied van 0,03 fv 1,0 mg/kg/dag volgens beide behandelingen. De antitumor-werkzaamheid van BBM-1644 was ongeveer hetzelfde als (qd 1 —* 9 schema) of drie keer zo sterk als (q3d x 3 schema) dan die van neocarzinostatine in termen van minimum effectieve dosis.The anti-tumor activity of BBM-1644 was determined in mice (BDF strain) against lymphocytic leukemia P388. Each tube was inoculated intraperitoneally with three times 10 cells of the tumor. Staged doses of the antibiotic were administered intraperitoneally to the mice 24 hours after implantation of the tumor. Treatments were given once a day on days 1, 4 and 7 (q3d x 3 schedule) or 9 consecutive days (qd 1 -> 9 schedule). Neocarzinostatin was tested for reference as a reference anti-tumor agent and the results are summarized in Table H. BBM-1644 was highly effective against mouse leukemia in a dose range of 0.03 fv 1.0 mg / kg / day by both treatments. The anti-tumor efficacy of BBM-1644 was approximately the same as (qd 1 - * 9 schedule) or three times as strong (q3d x 3 schedule) than that of neocarzinostatin in terms of minimum effective dose.
TABEL HTABLE H
Antitumor-activiteit tegen leucemie P388 15 _T/C (%) in MST*_Anti-tumor activity against leukemia P388 15 _T / C (%) in MST * _
Dosis in mg/kg/dag, ip, q3d x 3 _1 0^2 £2 0,03 0,01 BBM-1644 188 188 138 125 113Dose in mg / kg / day, ip, q3d x 3 _1 0 ^ 2 £ 2 0.03 0.01 BBM-1644 188 188 138 125 113
Neocarzinostatine 163 150 125 113 20 _T/C (%) in MST_Neocarzinostatin 163 150 125 113 20 _T / C (%) in MST_
Dosis in mg/kg/dag, ip, qd 1—> 9 0,3 0,1 0,03 0,01 BBM-1644 125 175 138 113Dose in mg / kg / day, ip, qd 1 -> 9 0.3 0.1 0.03 0.01 BBM-1644 125 175 138 113
Neocarzinostatine 163 138 125 113Neocarzinostatin 163 138 125 113
HH
25 median survival time (middelbare overlevingstijd); significante activiteit: T/C £ 125%25 median survival time (middle survival time); significant activity: T / C £ 125%
De antitumor-werkzaamheid van BBM-1644 werd ook aangetoond door een tweede test tegen P388 leucemie in muizen, waarvan de resultaten onderstaand in tabel I zijn getoond. Details van de in deze 30 test toegepaste methoden zijn beschreven in Cancer Chemother. Rep.3: 1 - 87 (deel 3), 1972.The anti-tumor activity of BBM-1644 was also demonstrated by a second test against P388 leukemia in mice, the results of which are shown in Table I below. Details of the methods used in this assay are described in Cancer Chemother. Rep. 3: 1 - 87 (Part 3), 1972.
8302810 — fk 158302810 - fk 15
TABEL ITABLE I
Effect van BBM-1644 op P388 leucemieEffect of BBM-1644 on P388 leukemia
' Effect AWCEffect AWC
Materiaal Behandelings— Dosis, IP MST MST gm Overlevenden schema mg/kg/in 3 dagen %T/C d.6 dag 5(30) 5 Neocarzino- D.l,4&7 1,6 8,0 89 -4,8 6/6 statine o,8 10,5 117 -4,3 6/6 0,4 15,5 172 -3,2 6/6 0,2 15,0 167 -2,5 6/6 0,1 13,5 150 -1,2 6/6 10 ‘ 0,05 12,0 133 -1,7 6/6 BBM-1644 D.l 2,56 9,0 100 - 4/6 1,28 14,0 156 -4,1 6/6 0,64 14,5 161 -4,9 6/6 0,32 16,5 182 -4,8 6/6 15 0,16 14,0 156 -4,1 6/6 0,08 12,0 133 -2,8 6/6 0,04 10,5 117 -2,7 6/6 0,02 11,0 122 -2,5 6/6 D.l,4&7 1,28 23,5 261 -3,3 6/6 20 0,64 19,0 211 -3,5 6/6 0,32 18,5 206 -4,3 6/6 0,16 14,5 161 --3,8 6/6 .Material Treatment— Dose, IP MST MST gm Survivors schedule mg / kg / in 3 days% T / C d.6 day 5 (30) 5 Neocarzino- Dl, 4 & 7 1.6 8.0 89 -4.8 6/6 Statin 0.8 10.5 117 -4.3 6/6 0.4 15.5 172 -3.2 6/6 0.2 15.0 167 -2.5 6/6 0.1 13.5 150 -1.2 6/6 10 '0.05 12.0 133 -1.7 6/6 BBM-1644 Dl 2.56 9.0 100 - 4/6 1.28 14.0 156 -4.1 6 / 6 0.64 14.5 161 -4.9 6/6 0.32 16.5 182 -4.8 6/6 15 0.16 14.0 156 -4.1 6/6 0.08 12, 0 133 -2.8 6/6 0.04 10.5 117 -2.7 6/6 0.02 11.0 122 -2.5 6/6 Dl, 4 & 7 1.28 23.5 261 -3, 3 6/6 20 0.64 19.0 211 -3.5 6/6 0.32 18.5 206 -4.3 6/6 0.16 14.5 161 --3.8 6/6.
0,08 12,0 133 -3,3 6/6 0,04 12,0 133 -2,9 6/6 25 0,02 10,0 111 -2,5 6/6 0,01 ΓΟ,Ο 111 -1,9 6/6 QD 1—* 9 0,64 8,0 89 -5,2 6/6 0,32 10,0 111 -4,2 6/6 0,16 19,0 211 -4,3 6/6 30 0,08 18,0 200 -4,2 6/6 0,04 15,5 172 -3,5 6/6 0,02 13,0 144 -3,3 6/6 0,01 12,0 133 -2,2 6/6 0,005 11,0 122 -1,1 6/6 8302610 16 TABEL I (Vervolg)0.08 12.0 133 -3.3 6/6 0.04 12.0 133 -2.9 6/6 25 0.02 10.0 111 -2.5 6/6 0.01 ΓΟ, Ο 111 -1.9 6/6 QD 1— * 9 0.64 8.0 89 -5.2 6/6 0.32 10.0 111 -4.2 6/6 0.16 19.0 211 -4, 3 6/6 30 0.08 18.0 200 -4.2 6/6 0.04 15.5 172 -3.5 6/6 0.02 13.0 144 -3.3 6/6 0.01 12.0 133 -2.2 6/6 0.005 11.0 122 -1.1 6/6 8302 610 16 TABLE I (Continued)
Effect van BBM-1644 op P388 leucemie *Effect of BBM-1644 on P388 leukemia *
Effect AWCEffect AWC
Materiaal Behandelings- Dosis, IP MST MST gm Overlevenden _schema_mg/kg/inj dagen %T/C d.6 dag 5(30) 7 5 Controle 10 pekel 8,0 - - 10/10 106 pekel 9,0 - -0,3 20/20 105 pekel 11,0 - - 10/10 104 pekel 14,0 - - 10/10 6Material Treatment Dose, IP MST MST gm Survivors _scheme_mg / kg / inj days% T / C d.6 day 5 (30) 7 5 Control 10 brine 8.0 - - 10/10 106 brine 9.0 - -0, 3 20/20 105 brine 11.0 - - 10/10 104 brine 14.0 - - 10/10 6
Tumor inoculum: 10 ascites cellen, ip (plus titratie) o 10 gastheer . CDF^ + muizen.Tumor inoculum: 10 ascites cells, ip (plus titration) o 10 host. CDF ^ + mice.
Tox : -C 4/6 muizen levend op dag 5.Tox: -C 4/6 mice alive on day 5.
Evaluatie : MST = median survival time.Evaluation: MST = median survival time.
Effect : % T/C = (MST behandeld/MST controle) x 100.Effect:% T / C = (MST treated / MST control) x 100.
Criteria : % T/C ^ 125 beschouwd als significante antitumor acti- 15 viteit.Criteria:% T / C ^ 125 considered significant anti-tumor activity.
De acute toxiciteit van BBM-1644 werd bepaald in muizen (dd Y stam) door enkelvoudige intraperitoneale toediening, waarbij het LD^q berekend werd als 5,8 mg/kg.The acute toxicity of BBM-1644 was determined in mice (dd Y strain) by single intraperitoneal administration, calculating the LD ^ q as 5.8 mg / kg.
Zoals bovenstaand getoond bezit BBM-1644 sterke anti-20 bacteriële werkzaamheid tegen grampositieve en zuurbestendige bacteriën en is het derhalve bruikbaar in de therapeutische behandeling van zoogdieren en andere dieren tegen door dergelijke bacteriën veroorzaakte 'infe ctieziekten. Bovendien kan het worden gebruikt voor andere conventionele toepassingen van antibacteriële middelen zoals het desinfecteren 25 van medische en tandheelkundige apparatuur.As shown above, BBM-1644 has strong anti-bacterial activity against gram-positive and acid-resistant bacteria and is therefore useful in the therapeutic treatment of mammals and other animals against infectious diseases caused by such bacteria. In addition, it can be used for other conventional antibacterial applications such as disinfection of medical and dental equipment.
De inductie van profagen in lysogene bacteriën en de uitgesproken antitumor-werkzaamheid die tegen P388 leucemie in muizen zijn aangetoond, wijzen uit dat BBM-1644 ook therapeutisch bruikbaar is voor de inhibitie van de groei van tumoren bij zoogdieren.The induction of prophages in lysogenic bacteria and the pronounced anti-tumor activity demonstrated against P388 leukemia in mice indicate that BBM-1644 is also therapeutically useful for inhibiting tumor growth in mammals.
30 De onderhavige uitvinding verschaft derhalve een werk wijze voor het therapeutisch behandelen van een gastheerdier, dat door een bacteriële infectie of door kwaadaardige tumor is aangedaan, waarbij aan deze gastheren effectieve antibacteriële of tumor-inhiberende dosis 8302610 17 van BBM-1644 of een farmaceutisch preparaat daarvan wordt toegediend.The present invention therefore provides a method of therapeutically treating a host animal affected by a bacterial infection or a malignant tumor, wherein these hosts have effective antibacterial or tumor inhibitory dose 8302610 17 of BBM-1644 or a pharmaceutical composition of which is administered.
In een ander aspect verschaft de onderhavige uitvinding een farmaceutisch preparaat, dat een effectieve antibacteriële of turaor-inhibiërende hoeveelheid van BBM-1644 omvat in combinatie met een inerte 5 farmaceutisch aanvaardbare drager of verdunningsmiddel. Deze samenstellingen kunnen in elke farmaceutische vorm worden gebracht die geschikt is voor parenterale toediening.In another aspect, the present invention provides a pharmaceutical composition comprising an effective antibacterial or turaor inhibiting amount of BBM-1644 in combination with an inert pharmaceutically acceptable carrier or diluent. These compositions can be made into any pharmaceutical form suitable for parenteral administration.
Preparaten volgens de uitvinding voor parenterale toediening omvatten steriele waterige of niet-waterige oplossingen, sus-10 pensies of emulsies. Ze kunnen ook worden bereid in de vorm van steriele vaste samenstellingen die in steriel water, fysiologische zoutoplossing of enig ander steriel injecteerbaar medium onmiddellijk voor gebruik kunnen worden opgelost.Compositions of the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or any other sterile injectable medium immediately before use.
Eet zal duidelijk zijn dat de feitelijk geprefereerde 15 hoeveelheden van het toegepaste BBM-1644 antibioticum zullen variëren afhankelijk van de in het bijzonder gekozen samenstelling, de toedienings-wijze en de bepaalde plaats, de gastheer en de ziekte die behandeld worden. Vele factoren die de werking van het ·genesmiddel beïnvloeden, zullen door de deskundigen in aanmerking worden genomen zoals bijvoor-20 beeld de leeftijd, het lichaamsgewicht, het geslacht, het dieet, de toedieningstijd, de toedieningsroute, de uitscheidingssnelheid, de conditie van de gastheer, combinaties van geneesmiddelen, reactiegevoelig-heden en de ernst van de ziekte. De toediening kan continu of periodiek binnen de maximaal toegelaten dosis worden uitgevoerd. Optimale toedie-25 ningssnelheden voor een bepaalde set condities kunnen door de deskundigen worden vastgesteld met conventionele tests voor het bepalen van de dosering met het oog op de bovenstaand gegeven richtlijnen.It will be appreciated that the actually preferred amounts of the BBM-1644 antibiotic used will vary depending on the particular composition selected, the mode of administration and the particular site, host and disease being treated. Many factors affecting the action of the drug will be considered by those skilled in the art, such as age, body weight, sex, diet, time of administration, route of administration, rate of secretion, condition of the host , combinations of drugs, sensitivities to reaction and disease severity. Administration can be carried out continuously or periodically within the maximum allowable dose. Optimal delivery rates for a given set of conditions can be determined by those skilled in the art with conventional dosing assays in accordance with the guidelines given above.
De volgende voorbeelden worden ter toelichting gegeven en beogen niet de omvang van de uitvinding te beperken. DEAE cellulose 30 is een diëthylaminoethyl-ion -uitwisselingscellulose. Sephadex G-50 is een filtratiegel, die door Pharmacia Fine Chemicals, Ine. wordt gemaakt. DEAE Sephadex A-50 is een diëthylaminoethyl anione uitwisselings-The following examples are given for illustrative purposes and are not intended to limit the scope of the invention. DEAE cellulose 30 is a diethylaminoethyl ion exchange cellulose. Sephadex G-50 is a filtration gel produced by Pharmacia Fine Chemicals, Ine. is made. DEAE Sephadex A-50 is a diethylaminoethyl anione exchange
ÖÊDÖÊD
gel, die door Pharmacia Fine Chemicals, Ine. wordt gemaakt. Sephadex^ is een merk van Pharmacia Fine Chemicals, Ine.gel, supplied by Pharmacia Fine Chemicals, Ine. is made. Sephadex ^ is a brand of Pharmacia Fine Chemicals, Ine.
35 Voorbeeld IExample I
Fermentatie van BBM-1644Fermentation of BBM-1644
Een agar-helling met goed gerealiseerde groei van 3302610 --- 18An agar slope with well-realized growth of 3302610 --- 18
Actinomadura sp. H710-49 werd gebruikt voor het enten van een entmedium (100 ml in een 500 ml Erlenmeyer-kolf) dat 1% mannitol, 2% pepton en 1% gistextract bevatte, waarbij de pH vóór de sterilisatie werd inge- t steld op 7,2. De entcultuur werd gedurende 72 uur bij 32°C op een rote-5 rende schudmachine (250 omwentelingen per minuut) geincubeerd en 5 ml van de cultuur werd naar het tweede entmedium (100 ml) overgebracht, dat uit dezelfde samenstelling bestond als het eerste entmedium. Er werd onder dezelfde omstandigheden gekweekt als gebruikt werden voor de eerste entcultuur. Men gebruikte 5 ml van de aldus bereide inoculum-10 groei om fermentatie te starten in 500 ml Erlenmeyer-kolven die 100 ml fermentatiemedium bevatten met een samenstelling van 2,5% mannitol, 0,5% glucose, 1% sojabonenmeel, 0,5% pepton, 1% vleesextract, 0,3% CaCO^ en 0,2% NaCl. Fermentatie wordt uitgevoerd bij 28°C op een roterende schudmachine met 250 omwentelingen per minuut rotatie. De antibioticum-15 produktie werd gevolgd door de papierschijf-agardiffusie-analyse waarbij Bacillus subtilis M45 (Ree mutant) als het testorganisme werd gebruikt. De antibioticum-activiteit in de cultuurbouillon nam geleidelijk toe met het voortschrijden van de fermentatie en bereikte na ö'Vj 7 dagen ongeveer 300 microgram/ml.Actinomadura sp. H710-49 was used to inoculate a seeding medium (100 ml in a 500 ml Erlenmeyer flask) containing 1% mannitol, 2% peptone and 1% yeast extract, adjusting the pH to 7 before sterilization. 2. The seed culture was incubated on a rotary shaker (250 rpm) for 72 hours at 32 ° C and 5 ml of the culture was transferred to the second seed medium (100 ml), which consisted of the same composition as the first seed medium. . Cultivation was carried out under the same conditions as were used for the first seed culture. 5 ml of the inoculum-10 growth thus prepared was used to start fermentation in 500 ml Erlenmeyer flasks containing 100 ml fermentation medium with a composition of 2.5% mannitol, 0.5% glucose, 1% soybean meal, 0.5 % peptone, 1% meat extract, 0.3% CaCO2 and 0.2% NaCl. Fermentation is performed at 28 ° C on a rotary shaker with 250 revolutions per minute of rotation. Antibiotic production was followed by the paper disc agar diffusion analysis using Bacillus subtilis M45 (Ree mutant) as the test organism. The antibiotic activity in the culture broth gradually increased with the fermentation progressing, reaching about 300 micrograms / ml after 7 days.
20 Voorbeeld IIExample II
De geoogste bouillon (18 1), die met de fermentatie • volgens voorbeeld 1 was verkregen, werd onder toepassing van een scherpte-centrifuge-apparatuur (Kokusan No. 4A) in myceliumkoek en bovenstaande bouillon gescheiden. Het filtraat werd beneden 40°C geconcentreerd tot 25 1/10 van het oorspronkelijke volume 'en het concentraat werd aan dialyse onderworpen door een cellofaan-buis (Union Carbide) tegen kraanwater in een koude kamer. De binnengehouden oplossing werd geconcentreerd in ongeveer 1,5 1 waarna deze hoeveelheid gecentrifugeerd werd met 8.000 G om onoplosbare materialen te verwijderen. De heldere bovenstaande vloei-30 stof werd verzadigd met ammoniumsulfaat en liet men gedurende 5 uur bij 5°C staan. Het gevormde neerslag werd door centrifugeren verzameld, opgelost in 300 ml water en door dialyse tegen kraanwater ontzout.The harvested broth (18 L) obtained with the fermentation of Example 1 was separated into mycelium cake and the above broth using a sharp centrifuge equipment (Kokusan No. 4A). The filtrate was concentrated below 25 ° C to 25 1/10 of the original volume and the concentrate was dialyzed through a cellophane tube (Union Carbide) against tap water in a cold room. The contained solution was concentrated to about 1.5 L and this amount was centrifuged with 8,000 G to remove insolubles. The clear supernatant was saturated with ammonium sulfate and allowed to stand at 5 ° C for 5 hours. The precipitate formed was collected by centrifugation, dissolved in 300 ml of water and desalted by dialysis against tap water.
De aan dialyse onderworpen oplossing (700 ml) bevat 22 g ruwe vaste stof van BBM-1644 zoals door lyofilisatie van een deel van de oplossing 35 werd aangetoond. De rest van de oplossing werd gebruikt voor een daaropvolgende zuivering zonder concentratie om ontleding te vermijden.' De antibioticum-oplossing werd door een kolom van DEAE-cellulose (Cl , 400 8302610 19 ml) gevoerd en de kolom werd met 1 1 water gewassen en met 1/15M fosfaatbuffer (pH 7,0) die 0,3M natriumchloride bevatte, ontwikkeld. De actieve fracties werden gecombineerd (300 ml),«aan dialyse onderworpen gedurende 18 uur tegen kraanwater en chromatografisch behandeld op 5 een kolom van DEfiE-cellulose( 400 ml) die in evenwicht gebracht was met 1/15M fosfaatbuffer (pH 7,5). De kolom werd ontwikkeld met dezelfde bufferoplossing die een toenemende hoeveelheid natriumchloride bevatte (0 oj 0,2M). Het actieve eluaat werd door dialyse ontzout en op een kolom van DEAE-Sephadex A-50 (17 ml) geladen. De kolom werd ontwikkeld 10 met 1/15M fosfaatbuffer (pH 7,5) welke een gradiëntconcentratie van natriumchloride bezat (0^ 0,3M). De actieve fracties zoals bepaald door B. subtilis M45:analyse werden elk aangebracht en aan dialyse onderworpen tegen lopend water gedurende 18 uur. De ontzoute oplossing werd chromatografisch behandeld op een kolom van DEAE-Sephadex A-50 (18 ml) 15 gebruikmakend van een 1/15M-fosfaatbuffer (pH 7,0)-NaCl (0 ^ 0,3M) sys-teen als eluens. De gepaste fracties werden verzameld, geconcentreerd tot 10 ml en op een kolom van Sephadex G-50 gebracht om te worden ontzout. De kolom werd door gedeloniseerd water geëlueerd en het actieve eluaat gelyofiliseerd waarbij 120 mg van een wit poeder werd verkregen.The dialysed solution (700 ml) contains 22 g of BBM-1644 crude solid as demonstrated by lyophilization of part of the solution. The rest of the solution was used for subsequent purification without concentration to avoid decomposition. The antibiotic solution was passed through a column of DEAE cellulose (Cl, 400 8302610 19 ml) and the column was washed with 1 L of water and developed with 1 / 15M phosphate buffer (pH 7.0) containing 0.3M sodium chloride . The active fractions were combined (300ml), dialyzed for 18 hours against tap water and chromatographed on a column of DEfiE cellulose (400ml) equilibrated with 1 / 15M phosphate buffer (pH 7.5) . The column was developed with the same buffer solution containing an increasing amount of sodium chloride (0 oj 0.2M). The active eluate was desalted by dialysis and loaded onto a column of DEAE-Sephadex A-50 (17 ml). The column was developed with 1 / 15M phosphate buffer (pH 7.5) which had a gradient concentration of sodium chloride (0 ^ 0.3M). The active fractions as determined by B. subtilis M45: analysis were each applied and dialyzed against running water for 18 hours. The desalted solution was chromatographed on a column of DEAE-Sephadex A-50 (18 ml) using 1 / 15M phosphate buffer (pH 7.0) -NaCl (0 ^ 0.3M) system as eluent. The appropriate fractions were collected, concentrated to 10 ml and placed on a column of Sephadex G-50 to be desalted. The column was eluted by deionized water and the active eluate lyophilized to give 120 mg of a white powder.
20 Het aldus verkregen monster van BBM-1644 was homogeen zoals werd aangetoond door polyacrylamide-gel-electroforese.The sample of BBM-1644 thus obtained was homogeneous as demonstrated by polyacrylamide gel electrophoresis.
----- 8302610----- 8302610
Claims (9)
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| JPS606194A (en) * | 1983-06-23 | 1985-01-12 | Meiji Seika Kaisha Ltd | Novel antibiotic substance sf-2288 and its preparation |
| GB8425685D0 (en) * | 1984-10-11 | 1984-11-14 | Lepetit Spa | Antibiotic a 40926 complex |
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| JPS56113791A (en) * | 1980-02-15 | 1981-09-07 | Kaken Pharmaceut Co Ltd | Novel antibiotic and its preparation |
| CA1198386A (en) * | 1981-10-22 | 1985-12-24 | Donald B. Borders | ANTIBACTERIAL AGENTS LL-C23024.beta. AND IOTA AND MICROORGANISM ACTINOMADURA YUMAENSE NRRL 12515 |
| US4578271A (en) * | 1982-05-24 | 1986-03-25 | Fujisawa Pharmaceutical Co., Ltd. | Biologically active WS 6049 substances, a process for the production thereof and their pharmaceutical compositions |
| NZ208013A (en) * | 1983-05-16 | 1987-07-31 | Bristol Myers Co | Antitumour antibiotic bbm-1675 and production by cultivating actinomadura verrucosospora |
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