Process for producing antibiotic complex having antitumoral effect

专利摘要:
An antitumor antibacterial complex having a quinoline nucleas referred to as BBM-928 is described which is produced by microbiological fermentation under controlled conditions using a species of actinomycetes. The bioactive components of the complex have pronounced antitumor activity and are moderately to weakly active against gram-positive and acid fast bacteria but practically inactive against gram- negative bacteria and fungi. Based on median survival time, the component designated BBM-928A is substantially more active than mitomycin C by a factor of 10 to 300 fold against intraperitoneally implanted mouse tumors.
公开号:SU999981A3
申请号:SU802906901
申请日:1980-04-01
公开日:1983-02-23
发明作者:Косияма Хидео；Сакаи Фумихиде；Окума Хироаки
申请人:Бристоль Мейерз Компани (Фирма)；
IPC主号:

专利说明:

slot but does not contain glycine. The hydrolysis of the whole cell revealed the presence of glucose, mannose and Mada .jpO3H (3-O-methyl-B-galactose).
Cultural and physiological properties.
Strain G-455-101 grows abundantly, forms a pink or grayish-pink aerial mycelium and gives a reddish water-insoluble pigment on nutrient-rich medium, such as agar with (Yeast extract and malt extract and agar with oatmeal. However, on mediums such as inorganic salts - starch agar, glycerin-ysparagin agar and tyrosine agar - the strain gives poor growth, forms white or beige. Rudimentary air glycelium and gives a small amount of reddish pigment. Melanoid pigment is not about It is produced on peptondrogeal iron agar and tyroain agar. It grows abundantly at 27 s and does not grow at or 50 ° C
Agar čapek. Growth is limited or absent. Substrate mycelium (reverse side is dark pink. Aerial mycelium is white to pale pink.
There is no soluble pigment.
Tryponton-yeast extract broth Growth moderate, cottony-lowered, sedimented and non-pigmented;
Agar with yeast and malt extract. Growth is plentiful. Substrate mycelium (reverse side) dark red to reddish-brown Aerial mycelium abundant / greyish-pink to purple-pink, no soluble pigment.
Oat agar. Growth is plentiful. The reverse side of the substrate mycelium is intensely yellowish-red. Air g "1 goal moderate, pink, soluble pigment grayish yellow
Inorganic salts are starchy agar. Growth is bad. The reverse side of the substrate mycelium is slightly yellowish-brown to dark red. Aerial mycelium is limited, white, to beige, there is no soluble pigment.
Glycerin-aspartic agar. Poe bad. The reverse side of the substrate mycelium is yellowish-pink to reddish-I. Aerial mycelium is limited, white, the pigment is absent.
Peptone yeast extract is a yellow agar. Growth is bad, folded. The reverse side of the mycelium 1 is intense reddish-orange. Aerial mycelium is absent, soluble pigment is slightly yellowish-orange
Tyrosine Agar. Growth is bad. The reverse side of the substrate mycelium is dark red. Aerial mycelium, limited white, no soluble pigment.
Glucose-ammonium salt agar. Growth is bad. The reverse side of the substrate mycelium is reddish brown. Aerial mycelium limited, light gray, no soluble pigment.
Agar Bennett. Growth is moderate. The reverse side of the substrate mycelium is reddish-brown. Aerial mycelium is a limited serovatorosis, there is no soluble pigment.
(Cultural signs are observed after incubation at 37 ° C for 3 weeks).
Biochemical properties.
Nitrate and nitrite restore casein in an agar medium and hydrolyzes weakly. Unbound milk coagulates. Gelatin does not dilute. Hydrogen sulfide from
Z-cysteine forms. Melanoids do not form. The reaction to catalase is positive, the reaction to oxidase is positive.
Well utilizes glycerin, D-xylose, D-ribose, Z-rhamnose,
D-galactose, D-fructose, D-mannose, D-mannitol, absorbs B (-) - arabinose, Z (+) - arabinose, D-glucose, cellobiose, trehalose, soluble starch, inositol, cellulose, chitin, keratin,
very poorly utilizes lactose, melibiosis (on Pridham-Gottlieb medium with the addition of 0.1% yeast extract, does not absorb Z (-) - cop6o3y, sucrose, raffinose, 0 (-) - melecitose,
Dulcite, D-sorbitol, salicin.
The method is carried out as follows.
 For the preparation of the anti-tumor antibiotic complex BBM-928
The strain of actinomycete G-455-101 is cultivated in an aqueous medium containing assimilable carbon and nitrogen sources under aerobic immersion conditions until accumulation of maximum antibiotic activity.
Starch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose, molasses, glycerin and others can be used as a carbon source in the medium. As a nitrogen source, you can use protein, protein hydrolyzate, polypeptides, amino acids, liquid from Grain 3 points, casein, urea and others, and as
mineral salts in the nutrient medium may include anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, nitrate and others. Cooling can be carried out at 20-45 ° C, the optimum cultivation temperature is 28-34 seconds, more preferably 30/32 seconds. The maximum accumulation of antibiotic is usually reached in about 4-6 days; cultivation, Cultivation is carried out under aeration and mixing conditions. If necessary, defoamers such as silicone oil, soybean oil, and lard can be added. Antibiotic activity was determined by agarodiffusion analysis on a paper disk using Sarcina lutea pH 9.0 as a test culture. The BBM-928 complex was isolated from the fermentation broth using conventional means, for example, by solvent extraction, purification was carried out by preparative countercurrent distribution and chromatographically. Example 1. Getting complex BBM-928. Agarova fermentation. The cultivation of agaric braid against the strain Aiiiflo na6ora species G-455-10I is used to inoculate a vegetative medium containing 2% soluble starch, 1% glucose, 0.5% pharmamedia (pharmaceutical medium, 0.5% yeast extract, 0.5% NZ-amine (type A) and 0.1% calcium carbonate, the pH being adjusted to 7.2 before sterilization. The seed culture is incubated for 72 hours on a rotating stirrer (250 rpm), and 5 ml of the growth culture is transferred to a 500 m Erlenmeyer flask containing 100 m of fermentation medium consisting of 2% soluble starch, 1% pharmaceutical, 0, 003% hpZOD 7H, and 0.4% calcium carbonate.The maximum accumulation of the VBM-928 complex is usually reached after about 5 days of shaking the culture. Fermentation in the tank. The seedling is shaken for 4 days in Erlenmeyer flasks and inoculated per 100 l of germination medium, consisting of 2.0% oat flour (produced by Quaker Products. Austria, 0.5% glucose, 0.2% dry yeast, 0.0008% MnCI / i 4H2.0, 0 , 0007% SyzOd-, 0.0002% ZnSO47HaO, and 0.0001% ReZod 7H.2p in a 200-liter tank fermenter for seeding, in which stirring is carried out at 200 revolutions per minute at 54 hours 15 A portion of the seed culture is then inoculated into 170 liters of fermentation medium containing 2.0% soluble starch, 1.0% pharmaceutical environment, 0.003% heptahydrate & zinc sulfate and 0.4% calcium carbonate in a 400-liter tank fermentor, which operates at 200 revolutions per minute at an aeration rate of 150 l / min. The pH value of the broth gradually increases as the process of fermentation proceeds and reaches 8.4-8.5 after 100-120 h, and during this period of time an antibiotic peak of 30 µg / mp is reached. Example 2. The selection of the complex BBM-928 by solvent extraction. The collected broth (170 L, pH 8.5) of Example 1 is filtered with a clarifying agent. Activity is found in both the mycelial sediment on the filter and in the filtrate. The mycelial sediment is extracted twice with a solvent representing a mixture of acetone and methanol (1: 1, 30 L X 2). The extracts are combined and evaporated under reduced pressure to obtain an aqueous concentrate, which is extracted with n-butanol. The broth filtrate is extracted twice with n-butanol (40 L x 2). Concentration of the combined n-butanol extracts under reduced pressure and freeze-drying the residue gave a pure purified solid (21.4 g). According to thin layer chromatography analysis data, this material is a complex consisting of three main components. A, B, and C, and the three minor components D, E, and F, which have differences ((s Rf, shown in Table 1). Table 1
BBM-928A BBM-928B BBM-928C BBM-928D
0.48 0.26 0.07 0.53
9L9981
BBM-928E BBM-928F
Detection using a UV scanner (Csimadzu C-910) at 345 cm,
n-Butanol-methanol-water (63:27:10).
XXX Xylene-methyl ethyl ketone-methanol (5: 5: 1). Example 3, Cleaning complex BBM-928. The crude complex prepared in Example 2 is purified using a preparative counter-dispersed distribution device (Mitamura, 100 ml / tube) using a solvent system: carbon-chloroform-methanol-water tetrachloride (5: 2: 5-1). After 50 transfers, the contents of tubes 5-20 are combined to form a pale yellow powder (4.4 g) containing components A, B, D, and Her F. This mixture is dissolved in a small amount of chloroform and passed through a silica gel column. -200 (500 n), which is pre-impregnated with ethyl acetate. The column was developed with ethyl acetate with an increasing amount of methanol (2-5%, v / v) and the fractions were metered in optical density at 345 nm. The ethyl acetate secondary component D is eluted first, and then component A Components E, B and F are eluted as follows in the indicated order at a methanol concentration of 3%. Each fraction containing the corresponding component is evaporated under reduced pressure, and the residue is crystallized with a mixture of chloroform and methanol. Similarly, the raw component preparation is obtained from tubes 21-35 with the above counter-flow distribution. Purification of component C was carried out by chromatography on silica gel and to 5 removal from: loroform-methanol. The outputs of components A, B, C, D, E and F are respectively 988 m, 420 mg, 848 mg, 130 mg, 119 mg and 114 mg. Example 4. Additional purification of the BBM-928A component of Example 3. The analysis of the thin-layer chromatography of the BBM-928A component of Example 3 (using the system and consisting of 10% methanol in toluene) showed.
Next is this bl.1
0.56
0.34 0.39 0.17 that the sample was not completely homogeneous due to the fact that it contains additional material immediately before the BBM-928A component. To achieve purification, the following steps are carried out. Step 1. The sample is chromatographed on silica gel using a linear gradient of chloroform to 6% methanol-chloroform. Fractions eluting between 2.4 and 3.3% methanol-chloroform (containing the BBM-928 A component plus some impurity) are sent to the next stage. Step 2. A concave gradient is created using three vessels, of which the first two contain 2% methanol-toluene, and the third 6% contain methanol-toluene. The composition of step 1, chromagrafted (silica gel column) on this gradient, produces a minor component that is eluted first, followed by elution almost immediately after the purified BBM-928 A component (referred to here as BBM-9-28 Ar) / Molecular AES BBM-928 r, defined by the Polissa Desorption Mass Spectrometry, is 1427, which corresponds to the empirical formula Ochi 70 NJ J O,. (MB 1427, 417). Elemental analysis (samples dried for 18 hours): Found (average of three determinations),%: C 53.85, H 5.51; N13.74, O (by difference) 26.90. Sat 14-18 over. Calculated,%: C 52.47; H 5.48, N 13.81 / O (by difference) 28.24. Physico-chemical properties of the components BBM-928 A, B, C and -D. 11 individual BBM-928 components show solubility and staining reactions similar to each other. For example, they are easily soluble in chloroform and methylene chloride, slightly.
soluble. benzene, ethanol, methanol, and n-butanol are insoluble in water and n-hexane. Positive reactions are obtained with ferric chloride and Ehrlich reagents, and negative analysis is found: C
53.19
n
5.40
N
12.92
on the difference (E 1% Lmax -
28.49 235 (586) in ethyl alcohol 264 (415) 345 (165)
234 (610)
in ethyl alcohol1 264 (410) 345 (165) 230 (564)
in ethyl alcohol hydroxide sodium 256 (763) 330 (180) 383 (170)
Molecular weight
(Eosmmeter, in a dreamIj) 1, 450
The NMR spectra of BBM-928 A, BBM-928 B and COMPU-928 C are very similar to each other, the only difference being the presence of acetyl groups in the A components (f (: 2.03 ppm, 2 mol, equivalent) and In Xtr: 2.05 ppm, 1 mol. Equivalent), but not in C. Using acetylation with acetic anhydride in pyridine, components BBM-928 A, B and C are introduced, respectively, 2.3 and
teline reactions with Tollens, Sakaguchi and nihydrin reagents.
The characteristic physico-chemical properties of the BBM-928 components are shown in Table. 2
table 2
51.77
50.75
5.29
5.25
13.55
12.5 &
29.39
31.42 235 (638) 235 (550) 264 (380) 264 (442) 345 (173) 345 (155)
234 (650)
234 (565) 264 (405) 264 (442) 345 (173) 345 (165) 230 (650) 230 (580)
J 256 (704)
256 (930) 330 (117) 330 (140) 383 (145) 383 (122)
1,470
55 4 molar equivalents of acetyl group. The three acetylation products thus obtained showed identical properties from those of the UV, IR and NMR spectra;
60 indicates that BBM-928 A is a mono acetyl derivative of the BBM-928 B component and diacetyl derivative of the BBM-928 e.
Acid hydrolysis of BBM-928 A yes (i5 shaft five UV-absorbing fragments soluble in n-butanol (I, fi, l lyf and U) and five water-soluble ninhydrin-positive substances NPV-1, 2, 3, 4 , and 5. Subsequently, substances are separated using a Dauex 50x4 screen and are not recognized as the following amino acids: (S-128) Compounds Rf NIP-1 NIP-2 NIP-3 NIP-4 NIP-5 Identification p) - Oxy-L-methylvaline (H - VdP) Glycine (Gly) gby Series (ser) Sarkozin (sar) (not identified) TLC, silica gel plate. 5-123: 10% ammonium acetate-methano 10% ammonia solution (9: 10: 1). It was shown that five UV absorbing fragments mentioned above have the following structures: Fragment 1., 4. 4. MS: m / s ZOSN) LYAoks 227, 233, 251, 345 them, CO-NH-CH-CO H CO
During acid hydrolysis (6H HC1), fragments 1, P, W, and Y gave the following 1U, Ser.
1U, Ser. NM-Val
1U
Series
-ser HM-val
p -Oxy-S-methylva1lin
Sarkozin
sar
decomposition products listed in Table 3 of Tables
1, HM-val
1, HM-val.Sar Fragment P CioH25N, OeMS: m / e 377 (M% 58), 234, 261, 345 named after Fragment Shake 230, 235, 262, 345 to them. CQ- () Fragment 1U N04 MCJ m / e 205 (M), 260, 354 nm. y Meon 230, 235, 262, 345 nm. Mattfc CH, / 5 C-OH CH-f -CO-CH, NH yH
Hydrolysis of BBM-928 A or BBM-928 C with 0.1 nOhm, sodium hydroxide at 25 ° C for 3 hours gives a fragment of U1 f ser, HM-val
and sar are as defined above, and gly represents glycine, and X represents an unidentified portion.
fragment 1U.
Processing fragment U1 0,1 n. HC1 when for 1 h gives a fragment of UE plus HM-val
WITH
about.
 w
Based on the results of the decomposition experiments described above, spectral data, microanalysis, and molecular weight determinations, it is considered that the following structures best represent BBM-92.8 A, B and C, R () Ri serine. .. siy glycine BBM-928. Ac AC sar sarcosine BBM-928B Ac H HMr-val-oxy-N-methyl BBM-928C H H Antimicrobial activity. The antimicrobial activity of the BBM-928 components is determined in relation to a variety of bacteria and fungi by the method of serial dilution of agar on nutrient agar at pH 7 using a Stier multi-inoculating device. The inoculum size is standardized to use 0.0025 ml of test organisms containing approximately 10 ml cells for all bacteria and fungi, excluding acid-resistant bacteria for which 10 cells / ml are used. The minimum inhibitory concentration of HMIK) was determined after incubation for overnight at 31 ° C. The BBM-92 component is moderately to weakly active against gram-positive and acid-starving bacteria, but the anti-negative bacteria and fungus are practically inactive. The activity of induction of bacterium phage is determined for the components of BBM-928 on lysogenic bacteria (1LB) up to a concentration of 100 mg / ml for the BBM-928 A, B and C components, no significant 1LB activity was demonstrated. 1. Antitumor activity. Comparative testing of BBM-928 components A, B, C and D with mitomycin C for anti-tumor activ. This is accomplished with intraperitoneally implanted tumors: P368 leukemia, L 1210 leukemia, B1b chalk cell, pulmonary carcinoma (LL) and sarcomatous ascites 180 (S180). Test solutions of the BBM-928 components in 0.9% saline solution containing 10% dimethyl sulfoxide and mitomycin C in a 0.9% physiological solution were administered once a day according to a dosage schedule varying from a single dose treatment. per day before multiple daily treatments. By varying the dosages, the minimum effective dose (MED) is determined, which gives an average survival time of at least 1.25 times greater than the control group.
15,99998116

权利要求:
Claims (1)
[1]
This degree of active formula is considered to be
This is a measure of significant antitumor activity. BBM-928A. Method for producing antibiotic
is markedly more active, the complex with the anti-tumor minomitsin C, with this factorial activity, is 10-300 times more pronounced, and due to the fact that the Actinoma strain depends on the tumor strain and dosage of Dura sp. G-445-101 is expressed in a heat-plot graph. Intraperitoneally nutrient medium containing
LOUKOVDA. carbon and nitrogen sources in aerobVBM-928 A, B, C, and D, and mitomycin C under inorganic conditions, followed by
defined by. the method of van der Werden and the 10th division of the antibiotic complex-iArch .Expt.path .pharmak 195, 389ca and / or its division into a compo-
(1940). A, B, C, D, E, and F.
The proposed method allows semi-information sources,
A new antibiotic complex taken into account in the examination of the high antitumor 5 Shoji, et al. G.Antibioties
activity. 14A, 335, 1961.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US4530835A|1985-07-23|CL-1577 Antibiotic compounds and their production

---

SU999981A3|1983-02-23|Process for producing antibiotic complex having antitumoral effect

---

EP0095154B1|1987-01-14|Biologically active ws 6049 substances, a process for the production thereof and their pharmaceutical compositions

---

US4524145A|1985-06-18|4'-Deschlororebeccamycin pharmaceutical composition and method of use

---

CA1291054C|1991-10-22|Antitumor antibiotics |

---

US4360458A|1982-11-23|Antitumor antibacterial agents

---

US4764602A|1988-08-16|Antibiotics, and their production

---

EP0110155A1|1984-06-13|Novel anthracycline derivatives, a process for preparing the same by a microorganism strain, the novel strain streptomyces cyaneus, and use of the anthracycline derivatives as medicaments

---

US3987029A|1976-10-19|Antibacterial antibiotics AM31α, AM31β and AM31γ

---

US4495286A|1985-01-22|Antibiotic complex producing bacterial culture

---

US4626503A|1986-12-02|Antitumor agents LL-D49194α1, LL-D49194β1, LL-D49194β2, LL-D49194β3, LL-D49194γ, LL-D49194δ, LL-D49194ε, LL-D49194ξ, LL-D49194η, LL-D49194ω1, LL-D49194ω2, and LL-D49194ω3

---

US4451456A|1984-05-29|Antitumor antibacterial agents

---

KR920001366B1|1992-02-11|Bbm-1675 new antitumor antibiotic complex

---

EP0206138B1|1989-03-08|Anthracycline compounds, a process for their preparation and their use as medicaments

---

US4552867A|1985-11-12|Antibiotic LL-BO1208α and LL-BO1208β, process of preparation, pharmaceutical composition and method of use

---

US4686308A|1987-08-11|Novel physiologically active substance MH435

---

US5849540A|1998-12-15|Thiodepsipeptide isolated from a marine actinomycete

---

EP0187049A2|1986-07-09|Micromonospora microorganisms and macrolide antibiotic production therewith

---

US4780416A|1988-10-25|Microorganism for BBM 928 production

---

US4631256A|1986-12-23|Fermentation process for BBM-928

---

Mizutani et al.1980|STUDIES ON THE IONOPHOROUS ANTIBIOTICS. XXIV LEUSERAMYCIN, A NEW POLYETHER ANTIBIOTIC PRODUCED BY STREPTOMYCES HYGROSCOPICUS

---

US4162305A|1979-07-24|Antibiotic XK-99 and process for production thereof

---

CA1235081A|1988-04-12|ANTITUMOR AGENTS LL-D49194 .alpha..SUB.1, LL-D49194 .beta.,LL-D49194 .beta..SUB.2, LL-D49194 UPSILON, LL-D49194DELTA, LL-D49194 EPSILON, LL-D49194 ZETA, ANDLL-D49194 ETA

---

US3577530A|1971-05-04|Janiemycin

---

US3647776A|1972-03-07|1-hydroxy- and acetyloxy-3-|-2-butanone

同族专利:

---

公开号 | 公开日

---

GR66663B|1981-04-07|

---

DE3012565A1|1980-10-23|

---

NO800967L|1980-10-03|

---

GB2050384B|1983-04-07|

---

DK153501B|1988-07-18|

---

ES8104411A1|1981-04-16|

---

IL59746D0|1980-06-30|

---

HU184256B|1984-07-30|

---

FI800973A|1980-10-03|

---

LU82318A1|1980-12-16|

---

AU533672B2|1983-12-08|

---

ATA180380A|1982-10-15|

---

BE882574A|1980-10-01|

---

JPH0341157B2|1991-06-21|

---

FR2452930B1|1983-12-30|

---

IE49191B1|1985-08-21|

---

AR223372A1|1981-08-14|

---

IL59746A|1983-02-23|

---

JPS63139192A|1988-06-10|

---

YU81280A|1983-09-30|

---

JPS63139191A|1988-06-10|

---

PH16970A|1984-04-27|

---

NO155780B|1987-02-16|

---

JPS6365679B2|1988-12-16|

---

FR2452930A1|1980-10-31|

---

SE8002521L|1980-10-03|

---

FI67403C|1985-03-11|

---

JPS55162752A|1980-12-18|

---

SE441930B|1985-11-18|

---

IE800598L|1980-10-02|

---

JPH0341475B2|1991-06-24|

---

YU41700B|1987-12-31|

---

CH647247A5|1985-01-15|

---

AT371144B|1983-06-10|

---

GB2050384A|1981-01-07|

---

AU5700280A|1980-10-09|

---

DK153501C|1988-11-28|

---

NO155780C|1987-05-27|

---

FI67403B|1984-11-30|

---

NL8001868A|1980-10-06|

---

DE3012565C2|1990-11-08|

---

DK138780A|1980-10-03|

---

ES490209A0|1981-04-16|

---

ZA801856B|1981-04-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US4416874A|1982-03-05|1983-11-22|Bristol-Myers Company|Injectable compositions of BBM-928A|

---

AU566569B2|1983-01-31|1987-10-22|Bristol-Myers Company|Luzopeptin e2|

---

DE3375076D1|1983-09-14|1988-02-11|Bristol Myers Co|Injectable compositions of bbm-928a|

---

US6749993B2|2002-02-06|2004-06-15|Konica Corporation|Planographic printing precursor and printing method employing the same|

---

JP2003300382A|2002-04-08|2003-10-21|Konica Minolta Holdings Inc|Imaging method using heat-transfer intermediate transfer medium|

---

JP2004188848A|2002-12-12|2004-07-08|Konica Minolta Holdings Inc|Print plate material|

---

JP2004322511A|2003-04-25|2004-11-18|Konica Minolta Medical & Graphic Inc|Printing method|

---

JP2006056184A|2004-08-23|2006-03-02|Konica Minolta Medical & Graphic Inc|Printing plate material and printing plate|

---

WO2007052470A1|2005-11-01|2007-05-10|Konica Minolta Medical & Graphic, Inc.|Lithographic printing plate material, lithographic printing plate, method for preparing lithographic printing plate, and method for printing by lithographic printing plate|

---

JP4878612B2|2008-07-16|2012-02-15|スタンレー電気株式会社|Vehicle signal lights|

---

US9052217B2|2012-11-09|2015-06-09|Honeywell International Inc.|Variable scale sensor|

法律状态:

优先权:

---

申请号 | 申请日 | 专利标题

---

US2648879A| true| 1979-04-02|1979-04-02|

---