BE865590A - ANTIBIOTIC COMPOSITIONS AND THEIR USE - Google Patents

Description

       

  "Antibiotic compositions and their use"

  
The present invention relates to a composition

  
pharmaceutical containing, as an active ingredient, the antibiotic

  
C-15003, as well as a method of treating human beings and

  
warm-blooded animals with one or more active tumors,

  
using this antibiotic C-15003.

  
For many years, research has been carried out

  
to find substances which, when administered to humans or warm-blooded animals with tumors, will prolong their lifespan.

  
It has now been found that the new antibiotic C-
15003 could achieve this goal.

  
Antibiotic C-15003 is a new compound with the following chemical structure:

  

 <EMI ID = 1.1>


  
In the context of the present invention, the expression “antibiotic C-15003” generally encompasses the three compounds corresponding to the preceding general formula (I), or else it relates to a mixture of two or three of these compounds. or

  
 <EMI ID = 2.1>

  
in general formula (I), the compound in which R represents

  
group

  

 <EMI ID = 3.1>


  
is called "antibiotic C-15003 P-3", or a

  
more abbreviated "C-15003 P-3"; the compound in which R represents the radical -CO-CH2-CH2-CH3 is called "antibiotic C-
15003 P-3 '", or, more abbreviated," C-15003 P-3' ";

  
compound in which R represents the radical

  

 <EMI ID = 4.1>


  
is ap-

  
peeled "antibiotic C-15003 P-4" or, more abbreviated, "C-15003 P-4".

  
Antibiotic C-15003 can be obtained, for example, by cultivating a microorganism belonging to the genus Nocardia and which is capable of producing antibiotic C-15003, in a culture medium containing sources of assimilable carbon. and digestible nitrogen sources until the antibiotic has practically accumulated there, and the antibiotic is recovered.

  
As an example of a strain of antibiotic-producing microorganism C-15003, there may be mentioned the strain of Actinomycete n [deg.] C-15003, which has been isolated from soil and others. samples when examining antibiotic-producing microorganisms.

  
The microbiological characteristics of the strain

  
n [deg.] C-15003 are studied by methods analogous to those pro-

  
 <EMI ID = 5.1>

  
tic Bacteriology 16, 313-340 (1966)]. The results of these observations at 28 [deg.] C for a period of 21 days are as follows:

  
1) Morphological characteristics:

  
The vegetative mycelium spreads well and grows in the form of branches, both on agar and in liquid medium. Many of the hyphae are between 0.8 and 1.2

  
 <EMI ID = 6.1>

  
bacteria-like fragments in the form of rods or short lengths of branching hyphae. The strain gives good growth on various taxonomic media, the aerated mycelium being superimposed on the vegetative mycelium, although it forms fre-

  
 <EMI ID = 7.1>

  
or spiral-shaped, loose being encountered on a few occasions. Microscopic examination of aged cultures reveals that only in a few cases do cells of the conidial type appear as chains, while cell suspensions obtained from the surface of these cultures, as has been possible

  
 <EMI ID = 8.1> <EMI ID = 9.1>

  
Electron microscopic examinations have shown that these bodies have smooth surfaces.

  
2) Cell constituents:

  
The strain is cultured, while being subjected to shaking, in ISP medium n [deg.] 1 to 28 [deg.] C for 66 to 90 hours, after which time the cells are collected and rinsed. It is examined by applying the method of B. Becker and Co. [Applied Microbiology 12, 421 (1964)] and the method of M. P. Lechevalier (Journal of Laboratory and Clinical Medicine 71, 134)
(1968)], the whole cells referred to above with regard to the composition of their diaminopimelic acid and their sugar. It is found that the first of these constituents is in the meso form, while spots which correspond to galactose and arabinose are detected.

  
3) Characteristics on taxonomic environments:

  
The strain shows good growth in various media, with the vegetative mycelium being colorless to pale yellow in the initial stages of culture and light yellowish brown to yellowish brown in later stages. The strain produces soluble pigments, ranging in color from yellow to yellowish brown, in various taxonomic media. The aerial mycelium is powdery and usually gives rise to moderate growth, ranging from white to yellow or light yellowish brown. The characteristics of the strain in various taxonomic media are given in Table 1.

Table 1: characteristics of the strain n [deg.] C-15003 in culture on various taxonomic media

  
(A) Sucrose Nitrate Agar:

  
 <EMI ID = 10.1>

  
amber (3 le), coremium-like body formation

  
Aerial mycelium (AD): poorly developed, white Soluble pigment (PS): none or pale yellowish brown
(B) Glycerol Nitrate Agar:

  
 <EMI ID = 11.1>

  
coremium

  
MA: moderate, white

  
PS: none
(C) Glucose and Asparagine Agar:

  
C: moderate, Marigold Brite (3 pa) * to Yellow Brite (2 pa) *.

  
MA: poorly developed, white

  
PS: Brite yellow (2 pa) *
(D) Glycerol and Asparagine Agar:

  
 <EMI ID = 12.1>

  
coremium

  
MA: poorly developed, white

  
PS: none
(E) Starch Agar:

  
 <EMI ID = 13.1>

  
PS: none
(F) Nutrient Agar

  
 <EMI ID = 14.1>

  
tion of coremium-type bodies

  
MA: poorly developed, white

  
PS: none (G) Calcium malate agar:

  
 <EMI ID = 15.1>

  
coremium-like body

  
MA: moderate, white to ivory Lt (2 ca) *

  
PS: none

  
(H) Yeast extract agar - malt extract

  
C: moderate, amber (3 le) * to yellow Brite (3 la) *, formation of

  
coremium-like body

  
 <EMI ID = 16.1>

  
PS: none
(I) Oatmeal Agar:

  
 <EMI ID = 17.1>

  
tion of coremium-type bodies

  
MA: poorly developed, white to light yellow

  
PS: none
(J) Peptone-yeast extract agar - iron

  
C: moderate, colonial yellow (2 ga)

  
MA: none

  
PS: colonial yellow (2 ga)

  
(K) Tyrosine Agar

  
 <EMI ID = 18.1>

  
tion of coremium-type bodies

  
 <EMI ID = 19.1>

  
PS: Camel (3 ie) *

  
Color codes according to the Color H &#65533; rmony Manual, 4th ed.

  
(Container Corporation of America, 1958).

  
4) Physiological characteristics

  
The physiological characteristics of the strain are shown in Table 2. Temperature range for growth:

  
 <EMI ID = 20.1>

  
good aerial growth on agar (ISP n [deg.] 2) is 20 to 35 [deg.] C.

  
Table 2: physiological characteristics of the strain n [deg.]

  
C-15003

  
Temperature range for growth: 12 to 38 [deg.] C.

  
Temperature range for aerial growth: 20 to 35 [deg.] C Gelatin liquefaction: positive

  
Starch hydrolysis: positive

  
Reduction of nitrates: positive

  
Peptonization of milk: positive

  
Milk coagulation: negative

  
Decomposition of casein: positive

  
Production of melanotic pigments:

  
 <EMI ID = 21.1>

  
positive (tyrosine agar)

  
Decomposition of tyrosine: positive

  
Decomposition of xanthine: negative

  
Decomposition of hypoxanthine: negative

  
Lysozyme tolerance: positive

  
Sodium chloride tolerance: 2%

  
5) Use of various carbon sources The use of various carbon sources is studied using a medium described by Pridham & Gottlieb [journal of Bacteriology 56, 107 (1948)] and a base medium corresponding to the same composition to which one added 0.1% yeast extract. The spectrum obtained is shown in Table 3.

  
Table 3: use of carbon sources by the strain

  
n [deg.] C-15003
 <EMI ID = 22.1>
 
 <EMI ID = 23.1>
 base medium with 0.1% yeast extract

  
Note: +++: abundant growth

  
++: good growth

  
+: growth

  
+: low growth
-: no growth 6) Other characteristics:

  
The cells were collected by the method described above in 2) and DNA was prepared by a method analogous to that of J. Marmur et Col. (Journal of Molecular Biology 3, 208, 1961). The G-C (Guanine-Cytosine) content of the DNA was of the order of 71 mol 96.

  
The gram stain of vegetative mycelium of this strain was positive.

  
The previous characteristics of strain n [deg.] C-15003 were then compared to the publications of SA Waksman "The Actinomycetes vol. 2" (Williams and Wilkins Co., 1961), and of RE Buchanan and NE Gibbons, "Bergey" s Manual of Determinative Bacteriology, 8th edition, 1974 ", as well as other publications.

  
Although this strain is believed to belong to group III of the genus Nocardia, the possibility of finding any species exhibiting the characteristics described so far among the known strains leads to the conclusion that this strain represents a new species of microorganism.

  
The strain n [deg.] C-15003 of the present invention has been deposited at the Institute for Research on Fermentations, Agency of Industrial Science and Technology (FERM) under the collection number 3992, at the Institute of Fermentation, Osaka (IFO) under

  
collection number IFO 13726 and to the American Type Culture Collection (ATCC), Maryland, U.S.A., under collection no. 31281.

  
Although strain n [deg.] C-15003 is a new species of the genus Nocardia, as has just been mentioned, it is susceptible, as microorganisms generally are, to undergo modifications. and mutations, either spontaneously or under the influence of a mutagen.

   As an example, the many variants of the strain which can be obtained by irradiation with X-rays, gamma rays, ultraviolet light, etc., by single-cell isolation, by culture on media containing various chemical substances, or by any other mutagenic treatment, as well as mutants arising spontaneously from the strain, should not be considered to represent any other distinct species and, therefore, any one may invariably be used for the purposes of the present invention. of those mutants and variants capable of producing C-15003 P-3, P-3 'and / or P-4.

   For example, subjecting strain n * C-15003 to various mutagenic treatments results in mutants practically devoid of the property of producing soluble pigments, mutants with support mycelia which are colorless, yellowish green, brown. reddish or orange-red, mutants whose hyphae are ready to fragment as bacillary elements or short branched hyphal fragments, as well as mutants with abundant white aerial mycelium or practically no aerial mycelium.

  
The medium used to cultivate such an antibiotic-producing strain C-15003 may be a liquid medium.

  
or any solid, as long as it contains nutrients which the strain can use, although it is preferable to use a liquid medium for heavy production tests. The medium can include sources of carbon and nitrogen which strain n [deg.] C-15003 can assimilate and digest, inorganic materials,

  
trace nutrients, etc. As an example of such carbon sources, there may be mentioned glucose,

  
lactose, sucrose, maltose, dextrin, starch, glycerol

  
 <EMI ID = 24.1>

  
(eg soybean oil, bacon oil, chicken oil, etc.). The sources of nitrogen can, for example, be meat extract, yeast extract, dried yeast, soybean meal, corn maceration liquor, peptone, seed meal. cotton wool, used molasses, urea, ammonium salts (e.g. ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.),

  
The medium may further contain sodium, potassium, calcium, magnesium, etc. salts, iron, manganese,. zinc, cobalt, nickel, etc., salts of phosphoric acid, boric acid, etc., as well as organic acid salts such as acetates and propionates. In addition, the medium may contain, in the form of supplements, various amino acids (eg glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (eg dipeptides,

  
 <EMI ID = 25.1>

  
pyrimidine and their derivatives), etc. With regard to the adjustment of the pH of the medium, an inorganic or organic acid, an alkali, a buffer substance, etc. can be added. Appropriate amounts of oils, fats, surfactants, etc., can also be added as defoamers.

  
The culture can be carried out under stationary conditions, shaking or under submerged aerobic conditions, or alternatively under any of the other culture conditions. For large scale production trials, it is best to use submerged aerobic culture. Although the cultivation conditions obviously depend on the state and composition of the medium, as well as the strain, method of cultivation and other factors, it is normally preferable to carry out the incubation at 20-35 [ deg.] C with an initial pH of about 7.0, or

  
 <EMI ID = 26.1>

  
re particularly suitable for an intermediate stage of culture, with an initial pH of 6.5 to 7.5. Although the incubation period may also vary depending on the same factors as those mentioned above, it is desirable to continue the incubation until the titer of the desired antibiotic substance is at the maximum. In the case of shake culture or aerobic submerged culture in liquid medium, the period required is normally in the range of 48 to 144 hours.

  
The potency of the antibiotic is examined with Tetrahymena pyriformis W, as the test microorganism. This is how we grew the microorganism in question above

  
 <EMI ID = 27.1>

  
1 g of yeast extract (Difco), 2 g of glucose, 1000 ml of distilled water and 10 ml of 1M phosphate buffer (pH 7.0) J at 28 [deg.] C for
44 to 48 hours, and that the potency of the antibiotic was determined by the serial dilution method with monitoring of turbidity of growth and effect on ascites tumor cells, and by means of thin layer chromatography, which will be described below.

  
The new antibiotics C-15003 P-3, P-3 'and / or P-4 are obtained and accumulate in the resulting fermentation broth, extracellularly and intracellularly.

  
These substances have also been detected by thin layer chromatography. This is how we separate the

  
of

  
fermentation broth in the form of cells and / a filtrate by filtration or centrifugation and the filtrate is extracted with the same volume of ethyl acetate. The same quart of acetone (70%) and water are added to the cells to the filtrate and, after stirring of

  
1 hour at 20 [deg.] C, the suspension is filtered. The acetone is separated from the filtrate and the resulting aqueous filtrate is extracted with ethyl acetate. Each of the extracts is concentrated to 1/100 of its volume and is subjected to thin layer chromatography on a glass plate with silica gel (Merck, Federal Republic of Germany ,,

  
 <EMI ID = 28.1>

  
methanol: 9/1). The power is determined as a function of the intensity of the spots detected by irradiation with ultraviolet light at 2537 Angstroms.

  
Since the C-15003 P-3, P-3 'and / or P-4, that

  
thus obtained in the fermentation broth, are lipophilic neutral substances, they can be recovered in a manner

  
 <EMI ID = 29.1>

  
which is normally used for the collection of microbial metabolites of this type. For example, one can use a method which uses the difference in solubility between the antibiotic and the impurities, means which make use of the adsorbent affinity of various adsorbents such as activated carbon, macroporous nonionic resins, silica gel, alumina, etc., a method for separating impurities by means of exchange resins; ions, etc., these different methods being applied singly, in combination and in an appropriate manner, or on several occasions.

  
Since, as already mentioned, the C-15003

  
 <EMI ID = 30.1>

  
cells, the antibiotics are separated and purified by means of such an adsorbent, if used, either directly or after solvent extraction in the case of the filtrate, or after solvent extraction in the case of microbial cells. The solvent extraction can be carried out by either of the following methods, or by other methods, such as, for example,

  
(1) solvent extraction from the culture broth prior to cell separation and (2) solvent extraction of the cells and filtrate obtained by a filtration, centrifugation or any other method. To extract the filtrate and the cells independently, the following method can advantageously be used.

  
Solvents suitable for the extraction of the filtrate are organic solvents immiscible in water, such as esters of fatty acids, for example ethyl acetate and amyl acetate, alcohols, for example butanol, halogenated hydrocarbons, for example chloroform, and ketones, for example methyl isobutyl ketone. The extraction is carried out at a pH close to neutrality and, preferably, the culture fluid previously adjusted to pH 7 is extracted with ethyl acetate.

  
The extract is washed with water and concentrated under reduced pressure. Then, a nonpolar solvent such as petroleum ether or hexane is added to the concentrate and the residue is recovered.

  
 <EMI ID = 31.1>

  
Thin layer chromatography, a number of spots are detected in addition to the antibiotic C-15003, the product 1 is then subjected to the following purification procedures. Thus, as a classical purification process, adsorption chromatography turns out to be interesting, and in this connection, one of the ordinary adsorbents such as silica gel, alumina, non-adsorbent resin can be used. macroporous ionic, etc. With regard to the purification of the crude product I, silica gel proves to be the most interesting. The development can be carried out, for example by starting with petroleum ether and hexane and the elution of the antibiotic C-15003 is carried out by the addition of a polar solvent, such as acetate. ethyl, acetone, ethanol or methanol.

   A typical process is column chromatography in which silica gel (Merck, Federal Republic of Germany, 0.05-0.2 mm) is used as the support and in which the hexane / acetate ratio is gradually increased. ethyl. The eluate is sampled and examined by means of thin layer chromatography and the fractions containing C-15003 are pooled and concentrated under reduced pressure. Then petroleum ether or hexane is added to the concentrate, so that the product is obtained.

  
II gross. Since this product still contains impurities, it is again purified as follows. For example, we can pu-

  
 <EMI ID = 32.1> <EMI ID = 33.1> using a different solvent system. The development system can, for this purpose, consist of a halogenated hydrocarbon such as dichloromethane or chloroform, with the addition of a polar solvent such as an alcohol, for example methanol or ethanol, a ketone, for example acetone or methyl ethyl ketone, etc. In this way, the antibiotic C-15003 is isolated.

  
The order of the solvent systems for the first and second silica salt columns can be reversed and, in addition, ordinary organic solvents can be used in conjunction with the foregoing systems, if necessary.

  
If a macroporous adsorbent resin is used as a purification medium for crude product II, the elution of the antibiotic C-15003 is carried out with a mixture of water and a lower alcohol, a ketone. lower or ester. The lower alcohol can, for example, be methanol, ethanol, propanol or butanol, and the lower ketone can, for example, be acetone or methyl ethyl ketone. The ester can, for example, be ethyl acetate. Following a characteristic process, the crude product II is dissolved in a mixture of methanol
(60%) and water and adsorbed on a column of Diaion HP-10 (Mitsubishi Kasei K.K.). The column is washed with a mixture of methanol (70%) and water and, then, the elution is carried out with a mixture of methanol (90%) and water. In this way, the antibiotic C-15003 is eluted from the column.

  
In each of the methods described above, the fractions containing the antibiotic C-15003 are combined and concentrated.

  
 <EMI ID = 34.1>

  
mes of ethyl acetate and the mixture is allowed to stand, so that crystals of antibiotic C-15003 separate. These crystals

  
 <EMI ID = 35.1>

  
te separated from each other by means of an adsorbent, such as one of those mentioned above. Thus, by using silica gel or a macroporous nonionic adsorbent resin and the foregoing solvents, the desired compounds can be eluted in fractions. If, for example, silica gel is used, development is carried out with hexane, ethyl acetate or a mixture of chloroform and methanol, so that C-15003 P-4, P -3 'and P-3 emerge in that order. After detection by means of thin-layer chromatography, the cor-

  
 <EMI ID = 36.1>

  
under reduced pressure and ethyl acetate is added to the concentrates. In this way, the different compounds can be obtained in the form of crystals. If a macroporous nonionic adsorbent resin is used, an elution can be used.

  
in gradient with a variable ratio of alcohol, ketone or ester / water. For example, by means of the gradient elution method involving the use of a mixture of methanol (60%) and water and a mixture of methanol (95%) and water, to which we have added 5% sodium chloride, the C-15003 P-3, P-3 'and P-4 emerge in the order mentioned. After sampling and detection by thin layer chromatography. each group of active fractions is concentrated under reduced pressure and crystallized from ethyl acetate.

   The isolated crystals include * ethyl acetate as a crystallization solvent and, after drying over phosphorus pentoxide at 70 [deg.] C for 8 hours, these crystals show the following physical and chemical properties (Table IV)
 <EMI ID = 37.1>
 <EMI ID = 38.1>
 
 <EMI ID = 39.1>
 Based on the foregoing molecular formula given above and the results of antimicrobial and antitumor activity given below, the antibiotic of the invention is compared to known groups of antibiotics. A search of the literature could not locate a distinct group similar to the antibiotic C-15003.

   However, a search for substances which could give ultraviolet absorptions similar to those of the antibiotic of the present invention among the constituents of plants and among other organic compounds of natural origin led to the groups of maytanacin and, based on more particularly on the molecular formulas involved, it was found that the antibiotic of the invention belonged to the group of compounds of the maytanacin type containing two nitrogen atoms. Maytanacin was obtained as a constituent of a plant and has been reported in the "Journal of American Chemical Society" 97, 5294 (1975). The mass spectrum of maytanacin is as follows:

  

 <EMI ID = 40.1>


  
The presence of m / e = 485,470 and 450 for the C-15003 P-3, P-3 'and P-4 is proof that these compounds have a backbone identical to that of maytanacin, these compounds not differentiating from the maytanacin than in the type of the acyl group in position 3. It is therefore clear that the antibiotic C-15003 is a new compound. When each of C-15003 P-3, P-3 'and P-4 is degraded with alkali and analyzed by gas chromatography to determine the carboxylic acids released therefrom , it was found that isobutyric acid, butyric acid and isovaleric acid could be obtained respectively from C-15003 P-3, C-15003 P-3 'and C -15003 P-4.

  
The structures based on the previous results are given below of the antibiotics C-15003 P-3, P-3 'and P-4.

  

 <EMI ID = 41.1>


  
The antibiotic C-15003 is endowed with activity prolonging the survival time of humans and warm-blooded animals with one or more tumors (eg, leukemia, sarcoma, mastocytoma, melanoma or cancer).

  
The antibiotic C-15003 also has a regressive effect on the growth of solid tumors subcutaneously, intramuscularly, as well as in other organs.

  
As an example of warm-blooded animals, there may be mentioned mammals.

  
In an acute toxicity test using mice as test animals, which involved intraperitoneal injections of antibiotic C-15003 P-3, P-3 'and P-4, all of these antibiotics were shown to be mon-

  
 <EMI ID = 42.1>

  
As to the method of administration to humans and warm-blooded animals with one or more tumors, antibiotic C-15003 is generally used in the form of injection. The injection can be performed intramuscularly, intrathoracic, intra-abdominal, intrauterine, intra-dura &#65533; the, intra-arterial, subcutaneous or intravenous.

  
Such an injection can be prepared in a manner known per se, i.e. the antibiotic C-15003 is dissolved or suspended in an aqueous liquid medium containing an ordinary so-lubricant, such as a alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol 200-300), a nonionic surfactant, or physiological saline and isotonic solution.

  
Typical examples of liquid medium consist of 1% ethanol and 0.85% physiological saline solution, 2.5% methanol and 0.85% physiological saline solution, as well as 1% Tween 80 and 0 , 85% physiological saline solution.

  
The concentration of the antibiotic C-15003 in the liquid is between 0.5 mcg / ml and 500 mcg / ml.

  
The injections can be sterilized and / or contain auxiliary substances, such as preservatives, stabilizers, wetting agents and emulsifying agents.

  
Antibiotic C-15003 is administered to a human or warm-blooded animal with tumors at a rate of approximately 0.8 to 50 mcg / kg / day.

  
Antibiotic C-15003 includes, as previously mentioned, antibiotics C-15003 P-3, P-3 'and P-4,

  
and in the pharmaceutical compositions of the present invention, only the antibiotic C-15003 P-3, C-15003 P-3 'or C-15003 P-4 can be used, or a mixture of one or more of these antibiotics (i.e. a mixture of antibiotics C-15003 P-3 and P-4, a mixture of antibiotics C-15003 P-3 'and P-4, a mixture of antibiotics C-15003 P-3 and P-3 ', or a mixture of the antibiotics C-15003 P-3, P-3' and P-4). In practice, it is preferable to use a mixture of the C- antibiotics
15003 P-3, P-3 'and P-4.

  
Each component or the mixture of components is not necessarily purified.

  
The following examples illustrate in more detail the preparation and use of the antibiotic C-15003, but do not in any way constitute a limitation to the invention; in these examples, parts are by weight unless otherwise indicated and the relationship between parts and parts by volume is a relationship between grams and milliliters, and percentages are by weight / volume, unless otherwise indicated.

  
It will be noted that in the context of the present invention,

  
 <EMI ID = 43.1>

  
read interchangeably.

  
All of the following experiments relating to the antitumor activity of the antibiotic C-15003 were tried according to the examination protocol and the methods for estimating antitumor agents described by the National Cancer Institute, National Institute of Health, USA from America (NCI protocols) [see Cancer Chemotherapy Reports (part 3), vol. 3, n [deg.] 2, 1972, pages

  
1 to 103].

  
B 16 melanoma and L 1210 leukemia obtained from the National Cancer Institute, U.S.A., in 1971 were maintained in C57BL / 6 and DBA / 2 mice, respectively [Cancer Chemotherapy Reports (part 1) vol. 50, n [deg.] 5, 1975; pages 919-928]. A leuce

  
 <EMI ID = 44.1>

  
in 1975 was maintained in DBA / 2 mice. P815 mastocytoma was also used in DBA / 2 mice, Ehrlich carcinoma and 180 sarcoma in JCR-JCL mice. Appropriate mice, male and female, 5 to 7 weeks old were used in each of the examples for each tumor.

  
Reference example 1 <EMI ID = 45.1>

  
Nocardia n [deg.] C-15003 (IFO 13726; FERM 3992 ATCC 31281), which was grown on a yeast extract-malt extract agar-based medium, was used to inoculate a fermenter with a capacity of 200 parts by volume. containing 40 parts by volume of a seed culture medium (2% glucose, 3% soluble starch, 1% crude soybean meal, 1% liquor maceration of mats, 0.5% polypeptone, 0.3% NaCl, 0.5%

  
 <EMI ID = 46.1>

  
rature of 28 [deg.] C for 48 hours so as to obtain an inoculum. A portion of 0.5 part by volume of the inoculum thus obtained is transferred into a fermenter with a capacity of 200 parts by volume containing 40 parts by volume of a fermentation medium composed of 5% dextrin, 3% mat maceration liquor,

  
 <EMI ID = 47.1>

  
nish an inoculum (seed culture).

  
By means of the serial dilution method using Tetrahymena pyriformis W as the test microorganism and the antibiotic C-15003 P-3 as the standard sample, the preceding culture was found to have a titre of 25 &#65533; g / ml.

Reference example 2

  
A portion of 10 parts by volume of the inoculum (in-

  
reference

  
seed) obtained in Example / 1, is transferred to a fermenter with a capacity of 2000 parts by volume containing 500 parts by volume of a seed culture medium (the same as above) and incubated at 28 [deg.] C for 48 hours. A 500 parts by volume portion of the resulting culture is transferred to a stainless steel tank with a capacity of 50,000 parts by volume containing 30,000 parts by volume of the seed culture medium and cultured at 28 [deg.] C, under aeration of 30,000 parts by volume / minute, stirring at 280 revolutions per minute (1 / 2DT) and an internal pressure of 1 kg / cm, so as to obtain a seed culture. This culture is used to inoculate a stainless steel tank with a capacity of

  
 <EMI ID = 48.1>

  
fermentation medium similar to that used in Example 1, at an inoculation rate of 10%. The inoculated medium is incubated at
28 [deg.] C, under an aeration of 100,000 parts by volume / minute, stirring at 200 revolutions per minute (1/2 DT) and an internal pressure of 1 kg / cm <2> for 90 hours. Using the same procedure as described in Example 1, the resulting culture was found to have a titer of 25 g / ml.

Example '' reference 3

  
To 95,000 parts by volume of the obtained culture is added.

  
reference

  
held in Example / 2, 2000 parts of Hyflo-supercel (Johnes and Manville Products, United States of America) and, after intense mixing, the mixture is filtered through a pressure filter to obtain 85000 parts by volume of filtrate and 32,000 parts of wet cells. The 85,000 parts by volume of the filtrate is stirred and extracted with 30,000 parts by volume of ethyl acetate. This process is repeated once again. The ethyl acetate layers are combined, washed twice with 30,000 parts by volume of water, dried by adding 500 parts of anhydrous sodium sulfate and concentrated under reduced pressure to 200 parts by volume. Petroleum ether is added to the concentrate and the resulting precipitate is collected by filtration (52 parts).

   This crude product I was stirred with 100 parts by volume of ethyl acetate and the insolubles were filtered off. The filtrate is stirred with 10 parts of silica gel (Merck, Federal Republic of Germany, 0.05-0.2 mm) and the ethyl acetate is separated off under reduced pressure. The residue is introduced at the top of a column of silica gel (400 parts by volume). Elution is carried out with 500 parts by volume of hexane, 500 parts by volume of a mixture of hexane and ethyl acetate (3/1), 500 parts by volume of a mixture of hexane and ethyl acetate (1/1),
500 parts by volume of a mixture of hexane and ethyl acetate
(1/3), 500 parts by volume of ethyl acetate and 1000 parts by volume of a mixture of ethyl acetate and methanol (50/1),

  
 <EMI ID = 49.1>

  
in volume.

  
A portion of one part by volume of each of the fractions is concentrated to dryness, and 0.1 part by volume of ethyl acetate is added to the concentrate so as to obtain a mixture. The mixture is distributed in the form of drops 2.5 cm from the lower edge of a glass plate based on silica gel (Merck, Federal Republic of Germany, 60 F 254 '0.25 mm, 20 x 20 ) and developed over a distance of about 17 cm with a solvent system formed from ethyl acetate and methanol (19/1). After development, detection is performed using ultraviolet light (2537A).

  
The active fractions n [deg.] 23 to 28 with an Rf of 0.6 to 0.65 are collected and concentrated under reduced pressure to about 20 parts by volume. We add to this concentrate

  
150 parts by volume of petroleum ether so as to obtain 15 parts of a crude product II.

Reference example 4

  
32,000 parts of the cells are extracted while shaking

  
reference

  
wet obtained in example / 3, with 50,000 parts by volume

  
 <EMI ID = 50.1>

  
filtered through a pressure filter. The extraction is repeated again with 50,000 parts by volume of a mixture of acetone (70%) and water and the subsequent filtration. The filtrates are combined and the acetone is separated by concentration under reduced pressure. The resulting aqueous system is passed through a column formed of 5,000 parts by volume of Diaion HP-10 (Mitsubishi

  
 <EMI ID = 51.1>

  
water and 50% aqueous methanol, this operation being followed by elution with 15,000 parts by volume of a mixture of methanol (90%) and water. The eluate is concentrated under reduced pressure to 3,000 parts by volume and shaken with 3,000 parts by volume of water and 3,000 parts by volume of ethyl acetate. The above process is repeated. The ethyl acetate layers are combined, washed with water, dried by the addition of anhydrous sodium sulfate and concentrated under reduced pressure to
200 parts by volume. After the addition of petroleum ether, the precipitate is collected by filtration (28 parts). We purify

  
the above product by means of a silica gel column and 8.0 parts of crude product II are recovered.

Reference example 5

  
1.5 parts of the crude product II obtained are dissolved in reference

  
Example / 3 in 10 parts by volume of ethyl acetate and the solution is stirred thoroughly with 4 parts of silica gel (Merck, Federal Republic of Germany, 0.05-0.2 mm). The ethyl acetate is separated off under reduced pressure. The residue is applied to the top of a column formed of 300 parts by volume of silica gel and the column is first washed with 500 parts by volume of chloroform and then eluted with 500 parts by volume of chloroform-methanol. (50/1), 500 parts by volume of chloroform-methanol (20/1) and 500 parts by volume of chloroform-methanol
(10/1). The eluate is collected in the form of fractions of 25 parts by volume.

  
A portion of 0.5 part by volume of each of the fractions is concentrated under reduced pressure. 0.05 part by volume of ethyl acetate was added to the concentrate, and the mixture was subjected as a sample to thin-layer chromatography with silica gel (developing system: chloroform-methanol-9 / 1).

  
o

  
Fractions Nos. 39 and 40 absorbing at 2537A in the Rf zone of 0.50-0.60 are collected and concentrated to dryness

  
under reduced pressure. To the residue is added 2 parts by volume of ethyl acetate and the mixture is allowed to stand, so that 0.150 crystals of antibiotic C-15003 are obtained.

  
The crystals of antibiotic C-15003 mentioned above (0.150 part) are dissolved in 15 parts by volume of methanol, this operation being followed by the addition of 0.300 part of sodium chloride and 15 parts by volume of water. A column is packed with 200 parts by volume of Diaion HP-10 (Mitsubishi Kasei K.K.) and is calibrated with 600 parts by volume of a mixture of methanol (50%) and water containing 5% NaCl. The sample solution prepared above is then passed through the column, and a gradient elution is carried out using 1,500 parts by volume of a mixture of methanol (60%) and water containing 5% NaCl and 1,500 parts by volume of a mixture of methanol (95%) and water. The eluate is collected in the form

  
of fractions of 15 parts by volume and each of the fractions is examined by means of thin-layer chromatography with

  
silica gel. Fractions 145 to 153 contain C-15003 P-3, fractions 167 to 180 contain C-15003 P-3 'and C-15003 P-4, and fractions 185 to 190 contain C-15003 P- 4.

  
Each group of fractions is concentrated and dissolved

  
by adding 50 parts by volume of water and 100 parts

  
by volume of ethyl acetate. The solution is shaken in a separating funnel, the aqueous layer is separated, and after washing with 2 portions of water of 50 parts by volume, the ethyl acetate layer is dried over anhydrous sodium sulfate, concentrated. and left to stand. In this way crystals are obtained from each group of fractions. The crystals are collected by filtration and dried.

  

 <EMI ID = 52.1>


  
of

  
 <EMI ID = 53.1>

  
(0.018 part) are dissolved in 0.3 part by volume of ethyl acetate and distributed in the form of drops in a line at a distance of 2.5 cm from the lower edge of a gel-based glass plate silica (Merck, Federal Republic of Germany, Keisel-

  
 <EMI ID = 54.1>

  
development using a mixture of ethyl acetate and methanol (19/1). After development to about a distance of 18 cm, the absorption bands at Rf 0.68 (P-4) and Rf 0.65
(P-3 ') are scraped off and each of them is independently extracted twice with ethyl acetate containing a small amount of water. The resulting ethyl acetate extract is washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure and allowed to stand.

  
0.010 part of C-15003 P-4 crystals and 0.003 part of C-15003 P-3 'crystals are obtained respectively from the fractions whose Rf is 0.68 and the Rf of 0.65.

  
Example of. reference 6

  
reference
1,000 parts by volume of the culture of Example / 2 are inoculated into a stainless steel tank with a capacity of 200,000 parts by volume containing 100,000 parts by volume of a seed culture medium and the medium is incubated. ino-

  
 <EMI ID = 55.1>

  
by volume / minute) and stirring (200 revolutions per minute) for 48 hours so as to obtain a seed culture. This seed culture is transferred to a stainless steel tank with a capacity of 2,000,000 parts by volume containing 1,000,000 parts by volume of a fermentation medium similar to that used in Example 1, at a transfer rate

  
 <EMI ID = 56.1>

  
for 90 hours. We see, using the process described

  
reference

  
 <EMI ID = 57.1>

  
ml.

  
900,000 parts by volume of the previous culture was added 900,000 parts by volume of acetone and, after stirring for 1 hour, 20,000 parts of Hyflo-Supercel (Johnes and Manville, USA). The mixture is stirred again and filtered through a pressure filter.

  
500,000 parts by volume of water are added to 1,700,000 parts by volume of the resulting filtrate and the mixture is extracted by means of a Podbielniak apparatus (Podbielniak, Inc.) with
1,000,000 parts by volume of ethyl acetate. The ethyl acetate layer is washed with water, dried by the addition of anhydrous sodium sulfate and concentrated under reduced pressure.

  
Petroleum ether is added to the concentrate, the resulting precipitate is collected by filtration and dried. 68 parts of crude product I. Then, as in Examples 3, 4 and 5, the crude product was purified to obtain 9.5 parts of C-15003 P-3, 0.300 part of C-15003 P-3 'and 2.5 parts of C-15003 P-4.

Example 1

  
Effect of C-15003 (a mixture of C-15003 P-3, C-15003 P-3 'and C-15003 P-4) on the survival time of mice carrying P388 leukemia

  
Mice (C57BL / 6 x DBA / 2) F1 (BDF1) (group of

  
3 to 5 mice), weighing 18 to 22 g, are inoculated intraperitoneally with 1 x 106 P388 leukemia cells on day 0.

  
Antibiotic C-15003 [a mixture of C-15003 P-3
(60 to 65% w / w), C-15003 P-4 (30 to 35% w / w) and C-15003 P-3 '] is suspended in a mixture of Tween 80 (1 %) and physiological saline solution (0.85%), and

  
The suspension is administered intraperitoneally to the animals inoculated with the leukemia cells (0.2 ml per mouse) once daily for 9 days 24 hours after tumor inoculation.

  
To estimate the antitumor activity of the antibiotic

  
the

  
than C-15003, we calculate / T / C in percent, which is the ratio x

  
100 of the mean survival time of mice carrying P388 leukemia cells treated (T) with the antibiotic C-15003 at

  
that of untreated control mice (C). It is considered, according to NCI standards, that a T / C ratio of more than 125% represents a positive antitumor activity.

  
TABLE 5

  

 <EMI ID = 58.1>


  
As the results show in Table 5,

  
1 administration of intraperitoneal daily doses of 1.6 to
25 mcg / kg of the antibiotic for 9 days prolonged the survival time of the treated mice significantly.

  
In particular, the most significant prolongation of the survival time of the treated mice is observed at a dose of

  
25 mcg / kg (optimal dose) (percentage T / C of 227).

Example 2

  
Effects of C-15003 P-3 and C-15003 P-4 on time

  
survival rate of mice carrying P388 leukemia cells

  
BDF1 mice (5 mice per test group), weighing 18-22 g, are inoculated intraperitoneally with

  
1 x 106 P388 leukemia cells on day 0. Each antibiotic is dissolved in methanol (2.5%) and physiological saline (0.85%) and the solution is administered intraperitoneally to mice (0.2 ml for each mouse) once daily for 9 days starting 24 hours after tumor inoculation.

  
As described in Example 1, it is considered, according to NCI standards, that a T / C ratio of more than 125% represents a positive antitumor activity. As summarized in Table 6, the ad-

  
 <EMI ID = 59.1>

  
nals of 1.6 to 25 mcg / kg or that: of C-15003 P-4 with doses

  
Daily intraperitoneal times of 0.8 to 50 mcg / kg prolong the survival / survival of treated mice significantly. A maximum effect is seen at a daily dose of 25 mcg / kg of each of the agents tested.

  
TABLE 6

  

 <EMI ID = 60.1>
 

Example 3

  
Effect of antibiotic C-15003 (a mixture of C-
15003 P-3, C-15003 P-3 'and C-15003 P-4) on survival time of mice with melanoma

  
B 16

  
One gram of the tumor is homogenized with 10 ml of a cold 0.85% physiological saline solution and 0.5 ml of this tumor homogenization product is inoculated intrape-

  
 <EMI ID = 61.1>

  
described in NCI protocols.

  
 <EMI ID = 62.1>

  
P-3 (60-65% w / w), C-15003 P-4 (30-35% w / w) and C-15003 P-3 '] is suspended in a mixture of Tween (1%) and physiological saline solution (0.85%) and the

  
 <EMI ID = 63.1>

  
once daily for 9 days, starting 24 hours after tumor inoculation (5 mice in each group and 15 mice per control group).

  
The% T / C ratio based on the mean survival time is calculated as described in Example 1, and a T / C ratio of more than 125% is considered to correspond to positive antitumor activity.

  
As shown in Table 7, the survival time of mice treated with the antibiotic C-15003 is significantly greater than that of untreated mice.

  
TABLE 7

  

 <EMI ID = 64.1>

Example 4

  
Effects of C-15003 P-3 and C-15003 P-4 on the survival time of mice carrying melanoma B 16 BDFl mice (5 animals per test group) are inoculated intraperitoneally with a suspension of melanoma B 16 following the same method as that described in Example 3.

  
Each antibiotic is dissolved -3 in a mixture of methanol (2.5%) and physiological saline solution (0.85%) and the solution is administered intraperitoneally to mice

  
(0.2 ml per mouse) once daily for 9 days, starting
24 hours after inoculation of the tumor.

  
As shown in Table 8, administration of C-
15003 P-3 or C-15003 P-4 with daily doses of 1.6 to

  
50 mcg / kg significantly prolongs the survival time of treated mice.

  
TABLE 8

  

 <EMI ID = 65.1>

Example 5

  
Effects of antibiotics C-15003 (a mixture of C-
15003 P-3, C-15003 P-3 'and C-15003 P-4), and C-
15003 P-3 on the survival time of mice carrying L1210 leukemia cells

  
Mice are inoculated intraperitoneally

  
 <EMI ID = 66.1>

  
ques L1210 on day 0.

  
Antibiotics C-15003 [a mixture of C-15003 P-3
(60-65% w / w), C-15003 P-4 (30-35% w / w) and C-15003 P-3 '], and C-15003 P-3 are dissolved respectively

  
in a mixture of methanol (2.5%) and physiological saline (0.85%) and each of the solutions is administered intraperitoneally to the mice inoculated with a tumor (0.2 ml per mouse) once per day for 9 days, starting 24 hours

  
after inoculation of the tumor.

  
To assess the antitumor activity of the test agents, the% T / C is calculated, which is the ratio x 100 of the mean survival time of mice carrying L1210 leukemia cells treated with the agents to that of untreated mice ( a

  
group of 25 mice), as described in the NCI protocols. It is considered that a T / C ratio of 120% corresponds to a minimum antitumor activity of the agents.

  
As shown in Table 9, the mixture of C-15003

  
of

  
P-3, C-15003 P-3 'and / C-15003 P-4, as well as C-15003 P-3 are found to be slightly effective in extending the

  
survival time of mice carrying L1210 leukemia cells.

  
TABLE 9

  

 <EMI ID = 67.1>

Example 6

  
Effects of C-15003 P-3 and C-15003 P-4 on survival time in mice with sarcoma 180

  
ICR-JCL mice (supplied by Japan Clea, Tokyo, 5 animals per test group) are inoculated intraperitoneally with 4 x 106 sarcoma 180 cells on day 0.

  
Each antibiotic is dissolved in a mixture of methanol (2.5%) and physiological saline solution (0.85%) and the solution is administered intraperitoneally. to tumor inoculated mice (0.2 ml per mouse) every day for 7 days, starting 24 hours after tumor inoculation.

  
The percentage of the T / C ratio, in mean survival time, is calculated as described in Example 1.

  
As shown in Table 10, the two agents are found to be particularly effective in prolonging the survival time of animals with sarcoma 180.

  
TABLE 10

  

 <EMI ID = 68.1>

Example 7

  
Effects of C-15003 P-3 and C-15003 P-4 on the survival time of mice carrying Ehrlich carcinoma ICR-JCL mice (5 animals per test group) are inoculated intraperitoneally with 4 x 106 Ehrlich carcinoma cells on day 0.

  
Each antibiotic is dissolved in a mixture of methanol (2.5%) and physiological saline (0.85%) and the solution is administered intraperitoneally to mice (0.2 ml per mouse) once a day for 7 days, starting 24 hours after tumor inoculation.

  
The percentage of the T / C ratio, in mean survival time, is calculated as described in Example 1.

  
As shown in Table 11, the two agents are shown to be particularly effective in prolonging the survival time of mice bearing Ehrlich carcinoma.


    

Claims (1)

TABLE 11 <EMI ID = 69.1> Example 8 Effects of C-15003 P-3 and C-15003 P-4 on the survival time of mice carrying mast cell P815 <EMI ID = 70.1> P815 tocytoma on day 0. Each antibiotic is dissolved in a mixture of methanol (2.5%) and physiological saline (0.85%) and the solution is administered intraperitoneally to tumor-inoculated mice (0.2 ml per animal) one after the other. times a day for 7 days, starting 24 hours after tumor inoculation. The percentage of the T / C ratio in mean survival time is calculated, as described in Example 1. As indicated in Table 12, the two agents are found to be particularly effective in prolonging the survival time of mice carrying P815 mast cell tumors at the doses used for this experiment. TABLE 12 <EMI ID = 71.1> 1. Pharmaceutical composition, characterized in that it comprises, as active ingredient, an effective amount of antibiotic C-15003. 2. Composition according to claim 1, characterized in that the active ingredient is the antibiotic C-15003-P-3. 3. Composition according to claim 1, characterized in that the active ingredient is the antibiotic C-15003 P-3 '. 4. Composition according to claim 1, characterized in that the active ingredient is the antibiotic C-15003 P-4. 5. Composition according to claim 1, characterized in that the active ingredient is a mixture of antibiotic C- 15003 P-3, antibiotic C-15003 P-3 'and antibiotic C-15003 P- 4. 6. Composition according to any one of claims 1 to 5, characterized in that the composition is in the form of an injection. 7. Composition according to claim 6, characterized in that the composition comprises antibiotic C-15003 and a liquid medium. 8. A method of treating human beings and warm-blooded animals presenting one or more tumors, characterized in that it consists in administering to them the antibiotic C-15003. 9. The method of claim 8, characterized in that the antibiotic C-15003 is the antibiotic C-15003 P-3. 10. The method of claim 8, characterized in that the antibiotic C-15003 is the antibiotic C-15003 P-3 '. 11. The method of claim 8, characterized in that the antibiotic C-15003 is the antibiotic C-15003 P-4. 12. The method of claim 8, characterized in that the antibiotic C-15003 is a mixture of antibiotic C- 15003 P-3, d. Antibiotic C-15003 P-3 'and antibiotic C-15003 P- 4. 13. Antibiotic compositions and their use, as described above, in particular in the Examples given. <EMI ID = 72.1>