DE2746252A1 - ANTIBIOTIC C-15003 MEDICINAL PRODUCT FOR THE TREATMENT OF TUMOROUS WARM BLUETERS - Google Patents

Description

Dr.-Ing. by Kreisler f 1973

Dr.-Ing. K. Schönwald, Cologne Dr.-Ing. Th. Meyer, Cologne Dr.-Ing. KW Eishold, Bad Soden Dr. JF Fues, Cologne
Dipl.-Chem. Alek von Kreisler, Cologne Dipl.-Chem. Carola Keller, Cologne Dipl.-Ing. G. Selling, Cologne

5 COLOGNE 1 ^{13 oct} 1977 / Fu

DEICHMANNHAUS AT THE HAUPTDAHNHOF

Takeda Chemical Industries, Ltd.,

27, Doshomachi 2-chome, Higashi-ku, Osaka, Japan

Medicinal products for the treatment of tumorous warm-blooded animals containing antibiotic C-15003.

HU9840 / 0608

Telephone (02 21) 23 45 41 - 4 Telex: 888 2307 dopo d Telegram: Dompotenl Cologne

For years there has been research into substances which, after administration to V / poor bloods, are included People who are infected by tumors ensure that they have a longer lifespan.

It has now been determined that the new antibiotic C-15003 can achieve this goal.

The main purpose of the present invention is therefore the possibility of treating one or more active ones Warm-blooded animals with tumors with the antibiotic C-15003. Another object of the invention is to provide of pharmaceutical preparations which contain the antibiotic C-15003 as an active ingredient. ·

Antibiotic C-15003 is a new compound and has the following structural formula:

wherein R -CO-CH, -CO-CHp-CHp-CH or -CO-CH ₂ -CH ^N CH,

means.

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In the present specification the term "antibiotic C-15003" generally means the three compounds of the above-mentioned general formula I as a group or a mixture of two or three of said compounds or ultimately any of these compounds. With reference to the general formula I , the compound in which R is the radical

-CO-CH

CH,

represents, in the present specification referred to as "Antibiotic C-15003 P-3" or just "C-15003 P-3". The compound in which R represents the radical "-CO-CHp-CHp-CH," is referred to as "antibiotic C-15003 P-3" ¹ or just "C-15003 P-3" ^1. The compound in which R the rest

-CO-CH ₂ -CH

CH ₃

CH ₃

■ * represents is referred to in the present specification as "antibiotic C-15003 PV or just" C-15003 PV.

For example, antibiotic C-15003 can be obtained by using one of the genus Nocardia belonging microorganism which is able to produce antibiotic C-15003 in an assimilable carbon source and culture medium containing digestible nitrogen sources

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until the antibiotic is highly concentrated in it, after which it is isolated.

As an example of one that produces antibiotic C-15003 A strain of a microorganism can be called an actinomycete strain No. C-15003 denote which of the soil or other specimens when an antibiotic is sighted producing microorganisms has been isolated.

The microbiological properties of strain No. C-15003 were researched in a manner analogous to that proposed by Schirling & Gottlieb [International Journal of Systematic Bacteriology 16, 313-3 * 10 (1966)]. The results of these observations at 28 ^° C. over a period of 21 days can be found below.

1) Morphological properties

The vegetative mycelium expands and develops well to branch and this both on agar and in liquid medium, some of the hyphae have one Diameter from 0.8 to 1.2 ^ m and can vary in certain Cases divide to form fragments that resemble rod-shaped bacteria or branched short hyphae. Of the Stem shows good growth on various taxonomic media, with an air on the vegetative mycelium mycelium. Often times similar to coremia are formed

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Body (50-200 χ 200-1000 ^ m) on which more aerial niycel grows. Some of the aerial mycelia are convoluted, with straight or wide spiral-like configurations occasionally occurring. The microscopic examination of older cultures shows that the conidia-like cells are only present in chains in a few cases, while the cell suspensions obtained from the surfaces of such cultures lengthened variously when viewed under the microscope, ellipsoidal (0.8-1.2 ym χ ' 1.8-6.8 pm) and ellipsoidal (0.8-1.2 ^ m χ 1.0-2.0 / im) bodies that resemble arthrospores.

Observations under the electron microscope have shown that these bodies have smooth surfaces.

25 2) Cell Composition

The strain was in a modified ISP No. 1 medium grown for 66 to 90 hours at 28 ⁰ C with shaking, whereupon the cells were collected and rinsed. According to the method of B. Becker et al. [Applied Microbiology 1_2, '121 (1964)] and the method according to MP Lechevalier [Journal of Laboratory and Clinical Medicine 71, 93 ^ (1968)], the whole cells thus obtained were examined for their content of diaminopimelic acid and sugar composition.

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The acid precedes in the meso form while spots are observed which indicated galactose and arabinose.

3) Properties on taxonomic media

The strain showed relatively good growth on various media, the vegetative Mycelium is colorless to pale yellow in the initial stages of cultivation and pale yellowish-tan in the latter stages until it was yellowish tan. The strain produced soluble pigments that were found in various taxonomic media yellow to yellowish tan v / aren. The aerial mycelium was powdery and showed im. generally a massive growth white to yellow or light yellowish tan color. The characteristics of the strain in different taxonomic

! 5 media can be found in the following table I.

Table I.

Breeding characteristics of strain No. C-15003 on taxonomic media
(A) Sucrose Nitrate Agar:
Growth (G): lush, bright melon yellow (3 ia)

to amber (3 Ic), education of coremia-like structures

Aerial mycelium (AM): sparse, white

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Soluble pigment (SP): none or pale yellowish

tan

(B) glycerol nitrate agar:

G: massive, light ivory colored (2 ca), formation of 5 corenia-like structures

AM: massive, white SP: none

(C) Glucose Asparagine Agar:

G: massive, light colored marigolds (3 Pa) to 10 tendgelb luminaire (2 pa).

AM: sparse, white SP: bright yellow (2 pa)

(D) Glycerine Asparagine Agar:

G: massive, light ivory colored (2 ca), formation of 15 coremia-like bodies

AM: sparse, white SP: none

(E) Starch Agar:

G: massive, light ivory (2 ca) to light white (2 ea), formation of coremia-like

Bodies

AM: plentiful, ivory-colored (2 ca) SP: none

(F) nutrient agar:

² 5 G: massive, light ivory colored (2 approx) * to khaki yellow

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(2 ga), formation of coremia-like bodies AM: sparse, white SP: no

(G) Calcium Maleate Agar:

G: massive, light ivory colored (2 ca) to light white

zen-colored (2 ea), formation of coremia-like bodies

AM: massive, white to light ivory colored (2 ca) SP: none (H) Yeast Extract-Malt Extract Agar:

G: massive, amber (3 Ic) to bright yellow

(3 la), formation of coremia-like bodies AM: massive, white to light ivory colored (2 ca) SP: no

(I) Oatmeal Agar:

■ Y- G: massive, light ivory colored (2 ca) to khaki yellow

(2 ga), formation of coremia-like bodies AM: sparse, v / ice to light yellow SP: koin

(J) Peptone Yeast Extract Iron Agar: G: massive, khaki yellow (2 ga) AM: none

SP: khaki yellow (2 ga) (K) tyrosine agar:

G: massive, light ivory colored (2 ca) to light melo-

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AT THE:
SP:

neon yellow (3 ea), formation of coremia-like bodies

it massive, white to light ivory colored (2 ca)

γ
camel colored (3 ie)

Color code according to "Color Harmony Manual", Ί. Edition (container Corporation of America, 1958)

Ό Physiological properties

The physiological properties of the strain are shown in Table II below. The temperature range for the V / GROWTH was 12 to 38 ⁰ C. The temperature range in which a good V / rowth of aerial mycelium on agar (ISP Mo. 2) entered, was at 20 to 35 ⁰ C.

Table II

The physiological properties of strain No. C-I5OO3 were the following:

Temperature range for the growth: 12 to 38 ⁰ C temperature range for the growth of the air

myeels: 20 to 35 ⁰ C

Liquefaction of gelatin: positive

Starch hydrolysis: positive

Reduction of nitrates: positive

Peptonization of milk: positive

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Coagulation of milk: negative

Decomposition of casein: positive

Production of melanoid pigments: negative (peptone

Yeast extract- ^ iron agar),

positive (tyrosine agar)

Decomposition of tyrosine: positive

Decomposition of xanthine: negative

Decomposition of hypoxanthine: negative

Tolerance to lysozyme: positive

Tolerance on sodium chloride: 2 %

5) Use of different carbon sources

The use of different carbon sources was investigated using a medium as described in Pridham and Gottlieb [Journal of Bacteriology 5 ^ 5, 107 (1948)] and a base medium of the same composition plus 0.1 % yeast extract. The fourths shown in Table III below could be determined.

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- 12 -

Table III

Use of carbon sources by strain No. C-15003

Carbon source Growth Sources of carbon growth D-xylose + * + * Raffinose + + * L-arabinose 4- + Melibiosis' 4- + D-glucose i-inositol - - D-galactose + + D-sorbitol - - D-fructose ₊₊₊ 4-4- D-mannitol ++ ++ L-rhamnose 4- -I- Glycerin - + D-mannose 4: 4: 4: 4: 4 " soluble starch + + Sucrose 4r4: + + Blind test - - Lactose Maltose ± ⁺ Trehalose b ++ Basic medium at Addition of C ), 1 % yeast extract Note: 4 = 4: 4 :: lush growth 4-.b; good growth +: Growth +: 4th bad growth no growth

6) Other properties

The cells were collected according to the method given above under 2) and DNA according to an analogous method Method that of J. Marmur et al. [Journal of Molecular

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Biology, 3, 208, 1961]. The G-C (guanine cytosine) content the DNA was approximately 71 mol-Z.

The grief staining of the vegetative mycelium of this

Tribal was positive.
The above properties of strain No. C-15003 were identified in SA Waksman's "The Actinomycetes" Vol. 2 [The Williams and Wilkins Co., 1961]; RE Buchanan and NE Gibbons, "Bergey's Manual of Determinative Bacteriology, 8th Edition, 1974"; and other references

10 compared.

It was believed that this strain would belong to Group III of the genus Nocardia. But no species could be found among the known strains exhibited the properties described. This is to

! 5 conclude that this strain is a new type of microorganism represents.

Said strain No. C-15003 is with the Fermentation Research Institute, Agency of Industrial Science and Technology (FERM) "under number 3992, at the" Institute for Fermentation, Osaka, (IFO) "under the number IFO 13726 and" The American Type Culture Collection (ATCC), Maryland, U.S.A. "under number 31281

been.

While the No. C-15003, as just

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0_

believes that it is a new species of the genus Nocardia, it can, as microorganisms in general do, enter into variations and mutations, either spontaneously or under the influence of a mutagen. For example, the different variants of the strain obtainable by exposure to X-rays, γ-rays, ultraviolet light, etc., by monocell isolation, by culturing on media containing various chemicals, or by any other mutagenic treatment, as well as the Mutants which are spontaneously derived from the strain are essentially not considered to be representatives of any other particular species. Any variants and mutants capable of producing antibiotics C-15003 P-3, P-3 ¹ and / or PH can be used for the purposes of the present invention.

For example, strain No, C-15003 delivers by various mutagenic treatments mutants that do not produce any soluble pigments, mutants with substrate mycelia, v / which are colorless, yellowish green, reddish tan or orange-red, mutants whose hyphae can easily be divide into bacilli-like elements or branched, short hyphae, and mutants with abundant, white air mycelia or essentially without aerial mycelia. The medium used for breeding

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of a strain producing an antibiotic C-15003 can be any liquid or solid medium provided it contains nutrients usable by the strain. Preferably, however, a liquid medium will be used to achieve high production. The medium can contain carbon and nitrogen sources produced by strain no. C-15003 are assimilated and digested, contain inorganic substances, micro-nutrients, etc. As examples of possible carbon sources, there can be mentioned glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, etc., fats and oils such as soybean oil, bacon oil, chicken oil, etc., and the like. As nitrogen sources, for example, meat extract, yeast extract, dried yeast, soybean meal, corn steep liquor, peptone, cottonseed milk, treated molasses, urea, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc., and the like can be used. The medium can also contain sodium, potassium, calcium, magnesium salts etc., iron · *, magnesium · ", zinc", cobalt, nickel salts etc., salts of phosphoric acid, boric acid etc. and organic salts, for example acetates and propionates , contain. Furthermore, the medium can additionally contain various amino acids such as glutamic acid, aspartic acid, alanine, glycine, lysine, meth.ionine, proline cA ^ Ö-jq ffPPh ^ n Ϊ · ^Β · dipeptides, tripeptides

D _-

etc., vitamins such as vitamins B ₁ , B ₃ , nicotinic acid, BC, E etc., nucleic acids such as purine, pyrimidine and derivatives thereof. An inorganic or organic acid, an alkali, a buffering agent or the like can also be used to adjust the pH of the medium. Appropriate amounts of oils, fats, surfactants, etc. can also be added as anti-foaming agents.

The cultivation can take place under any stationary conditions, with shaking, under submerged aerobic conditions or under other cultivation conditions. In order to obtain a high yield, a submerged, aerobic culture will preferably be carried out. The cultivation conditions depend of course on the condition and composition of the medium, the strain, the cultivation method and other factors. It normally takes place at 20 to 35 ^° C. at an initial pH of approximately 7.0. The cultivation temperature used is preferably from 23 to 30 ^° C. with an initial pH of 6.5 to 7.5. The incubation time also varies depending on the factors mentioned above, with the incubation being continued until the titer of the desired antibiotic product has reached a maximum value. In the case of shaking cultures or aerobically submerged cultures

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_ ₁₇ . 27A6252

in a liquid medium the time normally required is about 8 to 1 1/2 hours.

The effectiveness of the antibiotic was tested with Tetrahymena pyriformis W as the test organism. The above-mentioned microorganism was placed on a test medium [2.0 g tryptose peptone (Difco), 1 g yeast extract (Difco), 2 g glucose, 1000 ml distilled water and 10 ml 1 molar phosphate buffer (pH 7 , 0)] at 28 ⁰ C during M to Ί8 hours allowed to grow, whereupon under the Wirkungsverinögen of the antibiotic by the series dilution method

Observation of the growth opacity caused by ascites tumor cells and determined by thin-layer chromatography, abbreviated TLC, according to the information described below.

The new antibiotics C-15003 P-3, P ~ 3 'and / or P- were preserved and enriched both extracellularly and intracellularly in the culture broth obtained.

These substances have also been detected by TLC. The fermentation broth was thus divided into cells and filtrate by filtration or centrifugation and the filtrate with the same volume of ethyl acetate extracted. Then, the same amount of 70% aqueous acetone as the amount of the filtrate was added to the cells

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given, whereupon, after stirring for 1 hour at 20 ^° C., the suspension was filtered. The acetone was removed from the filtrate and the aqueous filtrate obtained was extracted with ethyl acetate. Each of these extracts was concentrated in a volume ratio of 1: 100 and placed over a silica gel glass plate (Merck, German Federal Republic, Kieselgel 60 P ₃₅₁₁ , 0.25 mm, "20 20) (solvent system: chloroform and methanol in a mixing ratio of 9: 1) was subjected to thin-layer chromatography.

The C-15003 P-3, P-3 'and / or P- ¹ J produced in this way in the culture broth are lipophilic and neutral substances which can be isolated by the usual separation and purification methods when obtaining such microbial metabolites. For example, one can work in such a way that one takes advantage of the differences between the antibiotic and the impurities in terms of solubility. However , the fractional adsorption affinity of various adsorbents, such as activated carbon, macroporous, nonionic resins, silica gel, aluminum oxide, etc., can also be used. Furthermore, one can also remove the impurities with the help of ion exchange resin ^! remove.

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But you can finally also use other methods or the aforementioned methods in a suitable combination or but apply repeatedly.

Since, as mentioned above, C-15OO3 P-3, 5 P-3 ¹ and P- ¹ I in both the filtrate and in

If the cells are present, antibiotics will be removed with the help of such an adsorbent, if one is used, either directly or after extraction with a solvent in the case of a filtrate or after extraction with a solvent, in the case of microbial cells, separate and clean. The extraction with a solvent can be done by any of the following methods, or by any other method, for example (1) by solvent extraction from the culture broth prior to separating the cells and (2) by solvent extraction the cells and the filtrate obtained by filtration, centrifugation or in another way, be performed. In order to extract the filtrate and the cells independently of one another, it is advantageous to use the

Use the following method.

Solvents suitable for extracting the filtrate are organic, which are immiscible with water / water Solvents such as e.g. Fatty acid esters such as ethyl acetate or amyl acetate, alcohols such as butanol, halogenated Hydrocarbons, such as chloroform, and ketones,

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e.g., methyl isobutyl ketone. The extraction takes place at one the pH value close to the neutral pH value and preferably the culture liquid previously adjusted to a pH value of 7 extracted using ethyl acetate. The extract is made with water washed and concentrated under reduced pressure. The concentrate is then mixed with a non-polar solvent, e.g. petroleum ether or hexane, and the crude product I, which contains the active compound, is obtained Since, in addition to the antibiotic C-15003, a certain number of spots were found in thin-layer chromatography is, the product I is then subjected to the following purification: process. It shows that a routine ge purification method, especially adsorption chromatography, is valuable, whereby for this purpose one of the usual adsorbents, such as silica gel, aluminum oxide, a macroporous, nonionic adsorption resin, etc., can be used. To purify the crude product I silica gel is particularly valuable. The development can take place, for example, starting from petroleum ether and hexane, while the elution of the antibiotic C-15003 by adding a polar solvent, such as ethyl acetate, acetone, ethanol or methanol, takes place. Used according to a typical procedure one silica gel (Merck, German Federal Republic, 0.05 to 0.2 mm) -as a carrier and leads the column Chrornato-

graphie with a mixture of hexane and ethyl acetate, the hexane proportion increasing successively. The eluate is collected and checked by TLC, showing the antibiotic C-15003-containing fractions are collected and concentrated under reduced pressure. The concentrate is then added with petroleum ether or hexane to give the crude product II. Because this product still contains impurities contains, it is further purified in the manner described below. So you can use the product II for example with the aid of a second silica gel column using a different type of solvent system clean. The developing system used for this purpose can be made of a halogenated hydrocarbon, e.g. Dichloromethane or chloroform, if a polar solvent is added, such as an alcohol, e.g. ethanol or methanol, a ketone, e.g. acetone or methyl ethyl ketone, or the like. exist. In this way the antibiotic C-15003 can be isolated. The journey 1 The order of the solution systems for these first and second silica gel columns can also be reversed, whereby one moreover, if necessary, common organic solvents can also be used in conjunction with the above systems.

If a macroporous adsorption resin is used for cleaning the raw product II, this takes place

25 the elution of antibiotic C-15003

with a mixture of water with a lower Al- _; alcohol, a lower ketone, or an ester. The lower alcohol can be, for example, methanol, ethanol, propanol or butanol, and the lower ketone, for example, acetone or methyl ethyl ketone. The ester can be, for example, ethyl acetate. According to a typical procedure, the crude product II is dissolved in 60 % aqueous methanol and adsorbed on a column of Diaion HP-IO (Mitsubishi Kasei KK). This column is then washed with 70 % aqueous methanol and eluted with 90% aqueous methanol. This is how antibiotic C-15003 gets out of the column

eluted.

In each of the above methods, the fractions containing the antibiotic C-15003 are collected and concentrated under reduced pressure. The dried product is then mixed with 5 "to 8 times the volume of ethyl acetate and the mixture is left to stand, whereupon crystals of the antibiotic C-15003 separate. These crystals contain C-15003 P-3, P-3 ¹ and PI. These compounds are then separated from one another with the aid of an adsorbent, for example of the type mentioned above

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be eluted. For example, using silica gel, so the development is carried out using hexane, ethyl acetate or a mixture of chloroform and methanol to give 0-15003-P ¹ I, P-3 ¹ and P-3 are obtained in the order mentioned. After thin-layer chromatography has taken place, the fractions corresponding to 0-15003-P- ^, P-3 'and P-3 are concentrated under reduced pressure and ethyl acetate is added to the concentrates. In this way, the corresponding compounds are obtained in the form of crystals. If a macroporous, non-ionic adsorption resin is used, then a gradual elution with fluctuating proportions of alcohol, ketone or ester in relation to the water can be used. When applying the step iron elution method using 60 # aqueous methanol and 95 # aqueous methanol with the addition of 5 % sodium chloride, C-15003 P-3, P-3 ¹ and pi are obtained in the order mentioned above . After treating by thin layer chromatography, each group of the active fractions is concentrated under reduced pressure and made to crystallize from ethyl acetate. The isolated crystals containing ethyl acetate as a crystallization and facing the Troc ¹ NEN over phosphorus pentoxide for 8 hours at 70 ⁰ C the following physical and chemical properties.

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Table K

α co co

ο cn ο co

C ₃₂ H P -
,., ClN ₀ O
Ό 2 3
₉ = 635, 169 Antibiotic C-1 ^ 003 3 '
₉ = 635, 169 CHCl ₃ ) P -
C ₃₃ H ₄₅ ClN ₂ O 4th 65 190 - 1 92 ⁰ C P - 182-185 ⁰ C. 09 177 to 180 ⁰ C 05 Melting point ( ⁰ C) -136 ° + 10 °
(c = 0.37 5 CHCl ₃ ) -134 ° + 10 °
(c = 0, ll 02 -142 ° + 10 °
(c = 0.522 CHCl ₃ ) 25th Specific
22 °
Rotation [q] _d C. 60, 06 60, 34 60, 23 Elemental analysis H 7, 04 7, 99 7, 05 Found: (?) N 4, 33 4, 51 4, 99 Cl 5, 37 5, 82 5, 32 C. 60, 51 6O ₃ 41 61 ·, 46 Elemental analysis H 6, 82 53 6, Ber .: {%) N 4, 41 4, 4, Cl 5, 58 5 5 ₃

Ultraviolet absorber
tior.sspektrum nn (£)
(in methanol) Antibiotic C-15003 P - 3
C ₃₂ K, ₃ ClN ₂ O _g = 635.169 P - 3 '
C ₃₂ H ₄₃ ClN ₂ O _g = 635.169 P - 4
C ₃₃ H ₄₅ ClN ₂ O _g = 649.196 Infrared absorber
range of functions
(cm " ¹ ) KBr 233 (30250) 2iO'sh 28 ^ 50)
252 (27640) 280 (5750)
288 (5700) 233 (30155) 24o (sh 23250)
252 (27600) 280 (5750)
288 (5700) 233 (29900) 24O (sh 28240)
252 (27590) 280 (5712)
288 (5680) magnetic nuclear
sonan spectra
(ppm)
ICOMHz in CDCl, 1740, 1730, 1670, 1560,
1445, 1385, 1340, 1255,
1180, 1150, 1100, 1080,
1038 1740, 1730, 1670, 1580,
1445, 1335, 1340, 1255,
1180, 1150, '1100, 1080,
1038 1740, 1730, 1670, 1580,
1445, 1385, 1340, 1255,
1180, 1150, 1100, 1080,
1038 OO
C. Mass spectra (m / e) 1.27 (d) (3H)
1.28 (d) (3H) 1.06 (t) (3H) 1.03 (d) (6H) 9840/0 solubility 573, 485, 470, 450 573, 485, 470, 450 587, 485, 470, 450 cn
ο
W. Color reactions In petroleum ether, hexane
and water insoluble,
in benzene and ether
sparingly soluble, in
Chloroform, ethyl acetate
did, acetone, Aethar.ol,
Methanol, pyridine, Te
trahydrofuran and prostitute
Thylsulfoxide soluble insoluble in petroleum ether,
Hexane and water, spar
Lich soluble in benzene
and ether, soluble in.
Chloroform, ethyl acetate,
Acetone, ethanol, metha
nol, pyridine, tetrahydro
furan and dimethyl sulf
oxide insoluble in petroleum ether,
Hexane and water. Spar
Lich soluble in benzene
and ether. Soluble in
Chloroform, ethyl acetate
did, acetone, ethanol,
Methanol, pyridine, Te
trahydrofuran and dimen-
methyl sulfoxide Dragendorff: positive
Beilstein: positive Dragendorff: positive
Beilstein: positive Dragendorff: positive
Beilstein: positive

On the basis of the above molecular formula and the antimicrobial and tumor-preventing efficacy values given below, the new antibiotic was compared with the known groups of antibiotics. No group similar to the antibiotic C-15003 could be identified in the literature. Research into substances which could provide ultraviolet absorption values similar to the present antibiotic and which would be of vegetable or natural, organic origin led to the maytanacic groups. In particular, on the basis of the molecular formulas in question, it was assumed that the antibiotic belongs to the maytanacic group of compounds which contain 2 nitrogen atoms. Maytanacin was obtained as a herbal component. About this is in Journal of American Chemical Society 9_7_, 5294

(1975) reported. The mass spectrum for maytanacin is the following:

⁺ * 485-C1

55 5 450

545 + 485 ( b ) = 470 Ca) = H ₂ O UNCO O
(j R-C-OH

The values tn / e 485, 470 and 450 for C-15003 P-3, P-3 ¹ and P-4 lead to the conviction that these compounds have an identical skeletal structure to that of maytanacin with the difference in that in the 3-position existing

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Acyl group. It is clear from this that the antibiotic C-15003 is a new compound. If C-15003 P-3, P-3 ¹ and Ρ-Ί are treated with an alkali and analyzed by gas chromatography with respect to the released carboxylic acids, it is found that isobutyric acid, butyric acid and isovaleric acid from C-15003 P-3 , C-15003 P-3 'and C-15003 PI are available. Fig. 1 shows the structures based on the above data

for C-15003 P-3, P-3 'and P-I. Fig. 1

CH _x O

P-3 -CO-CH

CH-

P-3 ¹ -CO-CH ₂ -CH ₂ -CH ₃ PH -CO-CH ₂ -CH

CH-

CH-

The antibiotic C-15003 is able to extend the survival time of warm-blooded animals, i.e. of animals and People who have one or more tumors, for example Leukemia, sarcomas, mastocytomas, melanomas or carcinomas suffer

The antibiotic C-15003 also has a regressive effect on solid tumors, which are subcutaneous, intramuscular and grow in other organs.

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Examples of warm-blooded animals are mammals

and called people.

In an acute toxicity test using mice as test animals, intraperitoneal Identify injections of antibiotics C-15003 P-3, P-3 ', and P-H that consistently an LD ^ value of more than 313 mcg / kg could be achieved.

For the administration of the antibiotic C-15003 to warm-blooded animals, who suffer from one or more tumors are used in the general of the injection form.The injection can be intramuscular, intrathoracic, intraabdominal, intrauterine intrathecally, intraarterially, subcutaneously or intravenously

Such an injection means can be produced in a manner known per se. For example the antibiotic C-15003 in an aqueous liquid medium containing a conventional solubilizing agent, e.g. an alcohol such as ethanol, a polyalcohol such as propylene glycol, polyethylene glycol 200 to 300, a nonionic surfactant, a physiological saline solution and / or contains, dissolve or suspend an isotonic solution.

Typical examples of a liquid medium are a 0.85% physiological saline solution which contains 1 % ethanol or 2.5 % methanol, or a 0.85% physiological saline solution which contains 1 % Tween 80.

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The concentration of antibiotic C-15003 in the liquid is between 0.5 mcg / ml and 500 mcg / ml. The injection solutions or suspensions can be sterilized and / or auxiliary substances, such as e.g. Contain preservatives, stabilizers, wetting agents and emulsifiers.

The antibiotic C-15003 is used in tumor-containing warm-blooded animals in doses between 0.8 and 50 mcg / kg / Administered on the day.

As already mentioned, antibiotic C-15003 contains antibiotics C-15003 P-3, P-3 'and PI. In the pharmaceutical preparations according to the invention, a single component of said antibiotic, namely antibiotic C-15003 P-3, P-3 ^? or P-4. However, mixtures of one or more of these components can also be used, ie a mixture of antibiotics C-15003 P-3 and P-4, a mixture of antibiotics C-15003 P-3 'and P- ¹ I, a mixture of antibiotics C. -15003 P-3 and P-3 ¹ and a mixture of the antibiotics C-15003 P-3, P-3 ¹ and P- ¹ I. In practice, a mixture of the antibiotics C-15003 P-3, P-3 ¹ and PU.

The individual components or a mixture thereof do not necessarily have to be cleaned.

The following examples explain the production and use of the antibiotic C-15003, without

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old, used for each tumor.

Reference example 1

Nocardia No. C-15003 (IFO 13726; FEHH 3992; ATCC 31281), which is based on a

was dium (yeast extract-malt extract agar) pjezüchtet been used, barrel end at a 200 parts by volume fermenter vessel containing ¹ IO parts by volume of a seed culture medium (2% glucose, 3% soluble starch, 1% of raw soybean flour, 1 % Corn swelling liquid, 0.5 % polypeptone, 0.3 % NaCl, 0.5 % CaCO, pH 7.0). The inoculated medium was ^{incubated for 48 hours at 28 °} C. in order to obtain an inoculum. 0.5 parts by volume of the inoculum thus obtained were in a 200 parts by volume fermentation vessel which contained 40 parts by volume of a fermentation medium which was composed of 5 % dextrin, 3 % corn steep liquor, 0.1 % polypeptone and 0, 5% Calciurncarbonat (pH 7.0) was contained, is introduced, and cultured for 90 hours at 28 ⁰ C in order to obtain in this way an inoculum (seed culture).

As with the serial dilution method using Tetrahymena pyriformis V / as the test organism

and C-15003 P-3 was found to be the standard sample

the above culture had a titer of

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- 30 -

restrict. The parts are in each case parts by weight, unless otherwise stated. The ratio between parts and moles corresponds to those between g and ml. The term "%" refers to "w / v" unless otherwise stated.

In the present description, the terms tumor, neoplasm, leukemia, sarcoma or carcinoma to equate each other more or less.

All subsequent experiments for the cytostatic effects of antibiotic C-15003 were carried out according to the information in the National Cancer Institute, National Institute of Health, U.S.A. (NCI protocols), (cf. Cancer Chemotherapy Reports (Part 3) Vol. 3, No. 2, 1972, pp. 1-103) regarding the breeding and evaluation of the Cytostatics tested.

Melanomas B 16 and leukemias L 1,210 obtained from the National Cancer Institute, U.S.A. in 1971 were reported in C57BL / 6 and DBA / 2 mice [Cancer Chemotherapy Reports (Part 1) Vol. 50, No. 5, 1975, pp. 919-9281.

in 1975

Leukemia Received through the Japan Cancer Chemotherapy Center / P388 were maintained in DBA / 2 mice. P8l5-

and
Mastocytoma in DBA / 2 mice / Ehrlich's carcinoma and sarcoma 180 in JCR-JCL mice were also used. In each of these examples, a suitable strain of mice of male or female origin, lasting 5 to 7 weeks

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25 pt; / ml .

Reference example 2

Inoculum (inoculum) obtained 10 parts by volume of according to Reference Example 1 were added to a 2000 parts by volume comprehensive fermenter vessel, a Sanienkulturmediums same composition as which contained 500 parts VoI. above, followed by poring during Ί8 hours at 28 ⁰ C. 500 parts by volume of the culture thus obtained were placed in a 50,000 parts by volume stainless steel tank containing 30,000 parts by volume of seed culture medium, followed by cultivation with aeration (30,000 parts by volume / minute) at 28 ⁰ C and stirred at an internal pressure of 1 kg / cm ² at 280 revolutions per minute (1/2 DT) in order to obtain a seed culture in this way.

This culture was used to inoculate a stainless steel tank with a capacity of 200,000 parts by volume containing 100,000 parts by volume of a fermentation medium of similar composition as in Reference Example 1 above, with an inoculation rate of 10 % . The inoculated medium was incubated under aeration (100,000 'Vol. Parts by / minute) at 28 ⁰ C and gen at an internal pressure of 1 kg / cm ² for 90 hours at 200 Umdrehu stirred per minute (1/2 DT). In determining

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of the titer in the same way as in Example 1, it was found that the culture obtained in this way had a titer of 25 ^ ig / rnl.

Reference example 3

95,000 parts by volume of the according to the information in reference example 2

culture obtained were mixed with 2000 parts of Hyflo ~ superce.] M (Johnes and Manville Products, U.S.A.) and the mixture after thorough mixing over a pressure filter filtered, 85,000 parts by volume of filtrate and 32,000 parts of moist cells were obtained. The filtrate (85,000 parts by volume) was stirred and extracted with 30,000 parts by volume of ethyl acetate. This measure was taken one more time repeated. The ethyl acetate layers were collected, washed twice with 30,000 Vol.rTcilen V / water each time, dried by adding 500 parts of anhydrous sodium sulfate and reduced to 200 parts by volume under reduced pressure constricted. Then petroleum ether was added to the concentrate and the resulting sediment was filtered won (53 parts). This crude product I was stirred with 100 parts by volume of ethyl acetate, whereupon the insoluble Components were filtered off. The filtrate was coated with 10 parts of silica gel (Merck, German Federal Republic, 0.05 up to 0.2 mm) and the ethyl acetate under reduced

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tem pressure removed. The residue was on top of a silica gel column ('100 parts by volume) added. Then with 500 VoI, parts Hexane, 500 parts by volume of a mixture of hexane and ethyl acetate (mixing ratio 3: 1), 500 parts by volume a mixture of hexane and ethyl acetate (mixing ratio 1: 1), 500 parts by volume of a mixture of hexane and Ethylacetaf (mixing ratio 1: 3), 500 parts by volume of ethyl acetate and 1000 parts by volume of a mixture of ethyl acetate and methanol (mixing ratio 50: 1) eluted, the eluate being collected in fractions of 100 parts by volume became.

1 part by volume of each fraction was concentrated to dryness, whereupon 0.1 part by volume of ethyl acetate was added to the concentrate added. The mixture was then spread on a 2.5 cm above the lower edge of a silica gel glass plate (Merck, Dundes Republik Germany, 60 F ^, 0.25 mm, 20 χ 20) lying Place applied and in a length of about 17 cm with a solvent system of ethyl acetate and Methanol (mixing ratio 19: 1) developed. After development, ultraviolet light (2537a) determined the desired product.

The active factions No. 23-28 with Rf 0.6-0.65 were collected and reduced under reduced pressure to approx 20 vol. Parts concentrated. These concentrates were each

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because 150 parts by volume of petroleum ether are added, with 15 parts « of a crude product II.

Reference example 4

32,000 parts of the moist obtained according to Reference Example 3 Cells were extracted with stirring with 50,000 parts by volume of 70% aqueous acetone for 3 hours and then extracted filtered on a pressure filter. The extraction using 50,000 parts by volume of 70% aqueous acetone and the subsequent Piltrations were repeated one more time.

The filtrates were collected and the acetone was removed by concentration under reduced pressure. The resulting, watery System was passed through a column of 5,000 parts by volume of Diaion HP-IO (Mitsubishi Kasei K.K.). the Column was washed with 20,000 parts by volume V / water and 50 # aqueous methanol and then with 15,000 Parts by volume of 90 # aqueous methanol eluted. The eluate was concentrated under reduced pressure to 3,000 parts by volume and mixed with 3,000 parts by volume of water and 3,000 parts by volume Shaken ethyl acetate. This way of working became once more repeated. The ethyl acetate layers were combined, washed with water by adding anhydrous sodium sulfate dried and concentrated under reduced pressure to a volume of 200 parts. Then became petroleum ether

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added and the resulting precipitate

collected by filtration (28 parts).

This product was purified using a silica gel column to obtain 8.0 parts of crude product II.

5 Reference example 5

In 10 parts by volume of ethyl acetate, 1.5 parts of the crude product II obtained according to Reference Example 3 above dissolved and the solution was stirred thoroughly with Ί parts of silica gel (Merck, Federal Republic of Germany, 0.05 to 0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was on top of a column of 300 parts by volume Introduced silica gel and washed the column first with 500 parts by volume of chloroform and then with 500 parts by volume of a mixture of chloroform and methanol (mixing ratio 50: 1), 500 parts by volume of a mixture of chloroform and methanol (mixing ratio 20: 1) and 500 parts by volume of a mixture of chloroform and methanol (10: 1) eluted. These eluates were collected in fractions of 25 parts by volume.

Each 0.5 part by volume of each fraction was concentrated under reduced pressure. The concentrate was mixed with 0.05 parts by volume of ethyl acetate and the mixture of thin layer chromatography (development system:

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. - 37 -

27A6252

Mixture of chloroform and methanol in a mixing ratio of 9: 1).

The 2537 Å absorbing fractions Nos. 39 and 40 in the zone of Rf 0.50-0.60 became collected and concentrated to dryness under reduced pressure. The residue was then mixed with 2 parts by volume Ethyl acetate is added and the mixture is left to stand, whereupon 0.150 parts of antibiotic C-15003 in the form of Crystals received.

The above crystals of antibiotic C-15003 (0.150 parts) were dissolved in 15 parts by volume of methanol, and then 0.300 parts of sodium chloride and 15 parts by volume of water were added. A column filled with 200 parts by volume of Diaion HP-IO (Mitsubishi Kasei KK) I was charged with 600 parts by volume of 50 # aqueous methanol containing 5 % sodium chloride. The solution thus prepared was passed through a column and gradient elution was carried out using 1500 parts by volume of 60 T aqueous methanol containing 5 % sodium chloride and 1500 parts by volume of 95 S aqueous methanol. The eluate was collected in various fractions of 15 parts by volume each and each fraction was checked by thin-layer chromatography using silica gel. Fractions 1'15 to 153 contained C-15003 P-3, fractions I67 to 18O contained C-15003 P-3 'and PI and the fraction

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Functions 185 to 190 contained C-1503 P-I.

Each group of the fractions was concentrated and by adding 50 parts by volume of water and 100 parts by volume Dissolved ethyl acetate. The solution was shaken in a separatory funnel and the aqueous layer separated. After two Washing with 50 parts by volume of water each time, the ethyl acetate layer was dried over anhydrous sodium sulfate, narrowed and left to stand. In this way, crystals were obtained in each group of the fractions. These Crystals were collected by filtration and dried.

C-15003 P-3 0.070 parts

C-15003 P-3 ¹ , P- ¹ »0.018 parts C-15003 P- ¹ I 0.015 parts

The mixed crystals of C-15003 P-3 ¹ and P-4 (0.018 parts) were dissolved in 0.3 parts by volume of ethyl acetate and placed on a line at a distance of 2.5 cm above the lower edge of a silica gel glass plate (Merck , German Federal Republic, silica gel 60 F _{2rJ (} 0.25 mm, 20 χ 20) applied, which was followed by development with a mixture of ethyl acetate and flethanol (mixing ratio 19: 1). After this development to about 18 cm, the absorption band at Rf 0 , 68 (P-4) and Rf 0.65 (P-3 ') scraped off and each band independently extracted twice with ethyl acetate containing a little water each time.

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dried anhydrous sodium sulfate, concentrated under reduced pressure and left to stand.

0.010 part of the crystals of C-15003 P-Ί and 0.003 part of the crystals of C-15003 P-3 ¹ were obtained from the fractions having Rf 0.68 and Rf 0.65, respectively.

Reference example 6

1000 parts by volume of the culture according to reference example 2 were inoculated with 1000 parts by volume of the culture medium according to reference example 2 in a stainless steel tank which had a capacity of 200,000 parts by volume and contained 100,000 parts by volume of a seed culture medium to obtain and angciiiipfte medium under aeration (100,000 Vol. parts by / minute), and incubated under stirring at 200 rpm for 48 hours at 28 X is a seed culture. This seed culture was placed in a stainless steel tank having a capacity of 2,000,000 parts by volume containing 1,000,000 parts by volume of a fermentation medium having the same composition as in Example 1 at a transplantation rate of 10 % . The culture was performed under aeration (1,000,000 VoI. parts / min) at 28 ⁰ C. vfährend 90 hours with stirring at 120 revolutions per minute (1/3 DT) and at an internal pressure of 1 kg / cm ^2. The resulting culture showed when tested

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according to the method according to reference example 1 a titer of

20 µg / ml.

900,000 parts by volume of the culture liquid thus obtained

were mixed with 900,000 parts by volume of acetone and after Stirring for 1 hour with 20,000 parts of Hyflo-Supercel (Johnos & Manville, U.S.A.). This mixture was further stirred and filtered in a pressure filter machine.

1,700,000 parts by volume of the filtrate obtained were mixed with 500,000 parts by volume V / water and the mixture was extracted in a Podbielniak device (Podbielniak, Inc., USA) with 1,000,000 parts by volume of ethyl acetate. The ethyl acetate layer was washed with water, dried by adding anhydrous sodium sulfate and concentrated under reduced pressure. Petroleum ether was added to the concentrate and the resulting precipitate was collected by filtration and dried. In this way, 68 parts of the crude product I were obtained. This crude product was then purified according to the information given in Examples 3, 1 and 5. In this way, 9.5 parts of C-15003 P-3, 0.300 parts of C-15003 P-3 ¹ and 2.5 parts of C-15003 P- ¹ * were obtained.

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example 1

Effect of C-15003 (a mixture of C-15003 P-3 , C-15003 P-3 ^f and C-15003 P- ¹ O on survival in mice suffering from leukemia P388

(C57BL / 6 χ DBAZS) F ₁ (BDF ₁ ) mice (in a group

of 3 or 5 mice) with a weight of 18 to 22 g were intraperitoneally with 1 χ 10 cells leukemia P388 am Day 0 inoculated.

The antibiotic C-15003 [a mixture of C-15003 P-3 (60 to 65% by weight), C-15003 P- ² I (30 to 35% by weight) and C-15003 P-3 '] was suspended in a 1 ^ -Tween 80 containing 0.85 2-physiological saline and the suspension was vaccinated intraperitoneally tumor-containing animals (0.2 ml / mouse) once a day for 9 days, the treatment was started 2M hours after the tumor vaccination .

In order to evaluate the cytostatic effect of the antibiotic C-15003, the T / C %, ie the ratio χ 100 of the average survival time of the P388-containing mice which had been treated with the antibiotic C-15003 (T), was that of the untreated mice (C) calculated. A value of more than 125 for T / C % was considered positive in terms of the cytostatic effect according to the NCI standard.

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"I ₄ 2 -

Table 5 average T / C % good survival
duration in days 227 dose number 24 _? 5 185 incg / kg / day Mice 20 _; 0 199 25th 3 21.5. 12.5 3 10 8 82 6.25 3 194 (Blind test) 11 22 _; 5 194 50 5 22.5 194 25th 5 22.5 168 12.5 5 19.5 129 6.25 5 15.0 108 3.13 5 12.5 108 1.6 5 12.5 0.8 5 11.6 0.4 5 (Blind test) 20th

As can be seen from the results in Table V, intraperitoneal administration of daily dosages is possible from 1.56 to 25 mcg / kg of the antibiotic for 9 days an increased survival time in the treated mice to effect.

The most significant lifespan extension of the treated mice was found at a dose of 25 mcg / kg (optimal dose) (T / C % of 227).

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Example 2

Effects of C-15003 P-3 and C-15003 P- ^ on the survival time in mice suffering from leukemia P388

BDF ₁ mice (5 mice per test group) weighing 18 to 22 g were administered intraperitoneally with 1 χ 10

from
Cells / leukemia P388 inoculated on day 0. Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5 % methanol, and the solution was intraperitoneally administered to mice (0.2 ml / mouse) once a day for 9 days, the administration being 2½ hours took place after tumor inoculation.

As has been described in Example 1, more than 125 in T / C % according to the NCI method was considered to be positive with regard to the cytostatic effect. As can be seen from Table 6, intraperitoneal administration of C-15003 P-3 prolonged at daily dose levels of 1.6 to 25 mcg / kg or of C-15003 P- ¹ I at daily dose levels of 0.8 to 50 mcg / kg the survival time of the treated mice in a significant way. The maximum effect was observed at a daily dose level of 25 mcg / kg for each of the aforementioned test agents.

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Table 6

αν
C-15003 P-3 c-15003 p-4 dose 118 125 50 223 .195 25th 177 177 12.5 168 . 180 6.25 159 145 3.13 144 141 123 I32 0.8 105 IO5 0.4 Example 3

Effect of the antibiotic C-15003 (a mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P- ¹ I) on the survival of mice infected with Mclanoma B16

1 g of the tumor was homogenized with 10 ml of a cold, 0.85% physiological saline solution, whereupon 0.5 ml of this homogenized material containing the tumor was given intraperitoneally in BDF mice (5 animals per test group) according to the information in the NCI directives.

The antibiotic C-15003 [a mixture of

C-15003 P-3 (6O - 65% by weight?), C-15003 P-1 (30 - 35% by weight *)

0.85 % - and C-15003 P-3 '] was suspended in a physiological saline solution containing 1% Tween 80 and the suspension was administered intraperitoneally to mice once a day for 9 days, the treatment after 2k hours

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tumor inoculation (5 mice in each group and 15 mice in the blind experiment) was started.

T / C % for the mean survival time was calculated according to the information in Example 1. T / C % greater than 125 was considered a positive cytostatic effect

As can be seen from Table 7, the

Survival of those treated with antibiotic C-15003 Mice elongated significantly, in contrast to the untreated mice.

Table 7

dose number 5 mcg / kg / lap; Mice 5 24 5 5 12th 5 5 6th 5 3 5 1.5 5 (Blind test) 15 24 12th 6th 3

average survival time in days

T / C

29.5 31.5 35.5 34.5 29 _t O

18.5

159 170 192 186 157

(Blind test) 15

31.5 28.5 22.5 16.2 14.8

213 193 152 109

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example

Effects of C-15003 P-3 and C-15003 P-H on the survival time of mice suffering from melanoma B 16

BDF ₁ mice (5 animals per test group) were inoculated intraperitoneally with a suspension of B 16 melanoma in the same manner as in Example 3.

Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5 % methanol, and the solution was intraperitoneally administered to the mice (0.2 ml / mouse) once a day for 9 days, the administration after the lapse of 24 hours after tumor inoculation was started.

As can be seen from Table 8, the administration of C-15003 P-3 or C-15003 PH at daily dose levels of 1.6 to 50 meg / kg caused a significantly increased survival time in the treated mice.

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_ 1.7 -

Table 8

¹ J? * ^1. * C-15003 P-3

mcG / kß / day ^x

T / C

C-15003 P-4

50
25th

12.5

6.25
3.13

0.8

> 219

> 219

207

189

128 116 IO5

> 219> 219 199 182 159 122 103 104

Example 5

Effects of antibiotic C-15003 (a mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P- ¹ Q and C-15003 P-3 on survival in mice suffering from leukemia L1210

BDF, mice (5 animals per test group) were intraperitoneally treated with 1 × 10 cells of leukemia L1210 during the day vaccinated.

The antibiotic C-15003 [a mixture of

C-15003 P-3 (60-65 wt.?), C-15003 PI (30-35 wt.?) And C-15003 P-3 ¹ I and C-15003 P-3 were each in a 0 , 85? -Igen physiological saline solution, polygamy 2.5? Methanol contained, dissolved and each solution once a day for 9 days on mice vaccinated with the tumor (0.2 ml / mouse)

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administered intraperitoneally, the administration beginning 2'I hours after tumor inoculation.

In order to evaluate the cytostatic effect of the test agents, the T / C % was recorded according to the information in NCI methodology 5. As already mentioned, this is the ratio χ 100 of the mean survival time of mice suffering from L1210 which had been treated with the agents and this in comparison to untreated mice (a group of 25 mice). T / C % of

10 120 was considered to be the minimum value for the cytostatic effect of the agents.

As can be seen from Table 9, the mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P- ² J and C-15003 P-3 was in terms of the prolongation of the survival time

15 of mice infected with L1210 were effective.

Table 9

middle

Number of doses ^you rchschnittlimcg / kg / Τθί- _{M 'use} ^che survival T / C%

duration in days

The mixture of
C-15003 P-3,
C-ISC03 p-3
and C-15003
T " ¹ » ft 40
30th
20th ■ 5
5
5 12 0
12.2
11.0 128
I30
117 P-4 10 5 10.2 109 C-15003 P-3 40 5 11.0 117 30th 5 12 _; 2 I30 20th 5 11.2 119 10 5 10.4 111 (Blind test) - 4UL 25th
a.ft δfj / ng 9.4
well >

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Example 6

Effects of C-15003 P-3 and C-15003 P - * < on the survival time of mice suffering from sarcoma 180

ICR-JCL mice (supplied by the Japan Clea,

g Tokyo, 5 animals per test group) were with 4 × 10 cells

Sarcoma 180 vaccinated intraperitoneally on day 0.

Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5 % methanol, and the solution was administered intraperitoneally to the tumor-vaccinated mice (0.2 ml / mouse) once a day for 7 days, v / if the administration was started 2H hours after tumor inoculation.

The T / C Ϊ value for the mean survival time was calculated according to the information in Example 1.

As can be seen from Table 10, both were agents for increasing the survival time of significantly effective in animals suffering from sarcoma 180.

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Table 10

middle

DoFis number of average ■ p.cg / kg / fög Mice before survival T / C

duration in days

C-15003 P-3
C-15005 P-4-
(Blind test) 25th
25th 5
5
10 24.5
33.5
13.0. 188
258 C-15003 p-3
(Blind test) 50
25th
12th
6th 5
5
5 5
25 5
10 33.5
30.5
15.5
17.8
5.1.8 284
258
I3I
I50 Example 7 Effects from C- 15003 P-3 and C-15003 P- ¹ I. on the Ueber lifetime of Mice suffering from Ehrlich's carcinoma

ICR-JCL mice (5 animals per test group) were ^{inoculated intraperitoneally with 1} I χ 10 cells of Ehrlich's carcinoma on day 0,

Each antibiotic was dissolved in a 0.85 ίί strength physiological saline solution containing 2.5 % methanol, and the solution was administered intraperitoneally to mice (0.2 ml / mouse) once a day for 7 days, with the administration 2 * Started 4 hours after tumor inoculation.

The T / C £ value for the mean survival time

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was calculated as in Example 1.

As can be seen from Table 11, both agents were effective in terms of increasing the survival time was significantly effective in mice bearing Ehrlich's carcinoma.

Table 1 at number 1 8th T / C % Mice average dose good survival middle mc ^ / kg / fög 5 duration in days VJl > 21.5 > 171 C-15003 P ~ 3 50 VJI > 21.5 25th 5 > 21.5 > 171 12.5 5 > 21.5 6.25 10 > 21.5 c-15003 p-4 25th game 12.6 (Blind test)

Effects of C-15003 P-3 and C-15003 P- ¹ ^ on the survival of mice with P815 mastocytoma

BDF, mice (5 animals per test group) were with 1 × 10 cells of P815 mastocytoma inoculated intraperitoneally on day 0.

Each antibiotic was dissolved in a 0.85% physiological saline solution containing 2.5 % methanol.

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held, dissolved and the solution was administered intraperitoneally to tumor-vaccinated mice (0.2 ml / animal) once a day for 7 days , the administration being started after 24 hours after tumor inoculation had taken place.

The value T / C % for the mean survival time was calculated as in Example 1 according to the information.

As can be seen from Table 12, both were agents in terms of increasing the survival time of P815 mastocytoma diseased mice at the dosage levels used in this experiment Way effective.

dose Table 12 average T / C % ; / kg / day good survival Attz ahl duration in days 61 Medium _mC o 50 Mice 10, -3 > 170 25th > 28 _; 5 > l? 0 C-15003 P-3 } 2 _; 5 VJ! > 28 _? 5 > 170 6.25 5 > 28 _? 5 > 170 25th 5 > 28.5 VJI 16.8 C-15003 P-4 VJI (Blind test) 10

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Claims (7)

Claims 1. Pharmaceutical preparation, characterized in that it is an effective amount of antibiotic C-15OO3 preferably in addition to customary auxiliaries and / or carriers contains. 2. Pharmaceutical preparation according to claim 1, characterized in that that the active ingredient is the antibiotic C-15003 P-3. 3. Pharmaceutical preparation according to claim 1, characterized in that the active substance is the antibiotic C-15003 P-3 ¹ . 4. Pharmaceutical preparation according to claim 1, characterized in that that the active substance is the antibiotic C-15OO3-P-4. 5. A pharmaceutical preparation according to claim 1, characterized in that the active substance is a mixture of the antibiotic C-15003 P-3, antibiotic C-15003 P-3 ¹ and antibiotic C-15003 P-4. 6. Pharmaceutical preparation according to claim 1, characterized in that that it is in a form suitable for injection. / 7. Pharmaceutical preparation according to claim 1, characterized in that that it contains a liquid medium in addition to an antibiotic C-15003. 809840/0608