GB1592264A - Treatment of tumor-carrying animal with antibiotic c-15003 - Google Patents

Description

(54) TREATMENT OF TUMOR-CARRYING ANIMAL WITH ANTIBIOTIC C-15003 (71) We, TAKEDA YAKUHIN KOGYO KABUSHIKI KAISHA also known as TAKEDA CHEMICAL INDUSTRIES LTD., a joint-stock company organised under the laws of Japan, of 27 Doshomachi 2-chome, Higashi-ku, Osaka, Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement :- This invention relates to the treatment of tumor-carrying animals with Antibiotic C-15003.

For years there has been a search for substances which, upon administration to warm-blooded animals carrying tumors, will prolong the survival time of the animal.

We have now found that novel Antibiotic C-15003 can fulfil this object.

The present invention provides a treatment for warm-blooded animals other than a human that carry one or more active tumors which involves the use of Antibiotic C 15003, and also provides a pharmaceutical composition having Antibiotic C-15003 as an active ingredient, together with a pharmaceutically acceptable carrier or diluent therefor.

Antibiotic C15003 is a novel compound and has the following chemical structure:

[wherein R represents

In the context of this invention, the term"Antibiotic C-15003"means, generic- ally, one or more of the three compounds having the above general fcrmula [I]. They may be utilized singly or as a mixture of any two or all three of said compounds. In general formula [I], the compound in which R is

is referred to herein as"Antibiotic C-15003 P-3"or more briefly as"C-15003 P-3" ; the compound in which R is-CO-CHz-CH2-CH3 is referred to herein as"Antibiotic C15003 P-3'"or, more briefly, as"C-15003 P-3"' ; the compound in which R is

is referred to herein as"Antibiotic C-15003 P-4"or, more briefly, as"C-15003 P-4".

The Antibiotic C-15003 can bc obtained, for example, by cultivating a microorganism belonging to the genus Nocardia and capable of producing Antibiotic C-15003 in a culture medium containing assimilable carbon sources and digestible nitrogen sources until the antibiotic is substantially accumulated therein, and recovering the antibiotic.

As an example of the Antibiotic C-15003-producing strain of microorganism, there may be mentioned an Actinoinycetes strain No. C15003 which was isolated from soil and other samples in screening for antibiotic-producing microorganisms.

The microbiological characteristics of Strain No. C-15003 were investigated by procedures analogues to those propose by Scherring & Gotdieb [International Journal of Systematic Bacteriology 16, 313-340 (1966)]. The results of observations ar 28 C over 21 days are as follows.

1) Morphological charactersstics : The vegetative mycelium extends well and develops into branches, both on agar and in liquid media. Many of the hyphae measure 0.8 to 1.2,, tm in diameter and, in certain instances, may divide into fragments resembling rod bacteria or branched short lengths of hyphae. The strain gives good growth on various taxonomical media, with the aerial mycelium being superimposed on the vegetative mycelium, although it fre quently forms coremia-like bodies (50-200 x 200-1000, tm) on which further aerial growth takes place. Many of the aerial mycelia are Hexuous or straight, or a lossely spiral-like configuration is encountered on a few occasions. Microscopic examination of aged cultures reveals that, only in a few cases, do the conidia-like cells occur in chains, while the cell suspensions obtained from the surfaces of such cultures, as microscopically examined, contain many elongated ellipsoidal (0. 8-1. 2 am x 4.8-6.8 um) and ellipsoidal (0.8-1.2 x 1.0-2.0 um) bodies resembling arthrospores.

Electron-microscopic examinations showed that these bodies had a smooth surface.

2) The constituents of cells : The strain was shake-cultured in a modified ISP No. 1 medium at 28 C for 66 to 90 hours, at the end of which time the cells were collected and rinsed. By the method of B. Becker et al. [Applied Microbiology 12, 421 (1964)] and the method of M. P. Lechevalier [Journal of Laboratory and Clinical Medicine 71, 934 (1968)], the above whole cells were examined for diaminopimelic acid and sugar composition.

The former was found to be of the meso-form, while spots were detected which corresponded to galactose and arabinose.

3) Characteristics on taxonomical media : The strain showed comparatively good growth on various media, with the vegetative mycelium being colourles to pale yellow in the initial phases of culture and light yellowish tan to yellowish tan in later phases. The strain produced soluble pigments, yellow to yellowish tan, in various taxonomical media. The aerial mycelium was powdery and generally gave moderate growth, being white to yellow or light yellowish tan.

The characteristics of the strain in various taxonomical media are set forth in Table 1 as follows : Table 1, Cultural characteristics o/Strain No. C-15003 on taxonotnical media.

(A) Sucrose nitrate agar: Growth (G); Moderate, Brite Melon yellow (3 ia) * to Amber tan (3 lc) *, coremia-like bodies.

Aerial mycelium (AM): Scant, white Soluble pigment (SP): None or pale yellowish tan (B) Glycerol nitrate agar: G: Moderate, Lt. Ivory (2 ca) *, coremia-like bodies AM: Moderate, white SP: None (C) Glucose asparagine agar: G: Moderate, Brite Marigola (3 pa) * to Brite yellow (2 pa) *.

AM: Scant, white SP: Brite Yellow (2 pa) * (D) Glycerol asparagine agar: G: Moderate, Lt. Ivory (2 ca) !, coremia-likc bodies AM: Scant, white SP: None (E) Starch agar: G: Moderate, Lt. Ivory (2 ca) * to Lt. Wheat (2 ea) *, coremia-like bodies formed.

AM: Abundant, Lt. Ivory (2 ca) * SP: None (F) Nutrient agar: G: Moderate, Lt. Ivory (2 ca) * to colonial Yellow (2 ga) *, coremia-like bodies formed AM: Scant, white SP: None (G) Calcium malate agar: G: Moderate Lt. Ivory (2 ca) * to Lt. Wheat (3 la) t, coremia-like bodies formed AM: Moderate, white to Lt. Ivory (2 ca) * SP: None (H) Yeast extract-malt extract agar: G: Moderate, Amber (3 lc) * to Brite Yellow (3 la) *, coremia-like bodies formed AM: Moderate, white to Lt. Ivory (2 ca) * SP : None (I) Oatmeal agar : G: Moderate, Lt. Ivory (2 ca) * to colonial Yellow (2 ga) *, coremia-like bodies formed AM: Scant, white to light yellow SP: None (J) Peptone yeast extract iron agar : G: Moderate, colonial yellow (2 ga) * AM: None SP: Colonial Yellow (2 ga) * (K) Tyrosine agar : G: Moderate, Lt. Ivory (2 ca) * to Lt. Melon Yellow (3 ea) *, coremia-like bodies formed AM: Moderate, white to Lt. Ivory (2 ca) * SP: Camel (3 ie) * The colour codes were according to the Color Harmony Manual, 4th ed. (Container Corporation of America, 1958).

4) Physiological characteristics : The physiological characteristics of the strain are shown in Table 2 below. The temperature range for growth was 12 C to 38 C. The temperature range in which good aerial growth occurred on agar (ISP No. 2) was 20 to 35 C.

Table 2, The physiological characteristics of Strain No. C-15003.

Temperature range for growth: 12 to 38 C Temperature range for aerial growth: 20 to 35 C Liquefaction of gelatin: Positive Hydrolysis of starch: Positive Reduction of nitrates: Positive Peptonization of milk: Positive Coagulation of milk: Negative Decompcsition of casein: Positive Production of Melanoid pigments: Negative (peptone yeast extract iron agar), posi tive (tyrosine agar) Deccmposition of tyrosine : Positive Deccmposition of xanthine: Negative Decompcsition of hypoxanthine: Negative Tolerance to lysozyme : Positive Tolerance to sodium chloride: 2% 5) Utilization of various carbon sources : The utilization of various carbon sources was investigated using a medium described in Pridham and Gottlieb [Journal of Bacteriology 56, 107 (1948)] and a basal medium of the same composition plus 0.1 % of yeast extract. The resulting spectrum is shown in Table 3 as follows: Table 3, The utilization of carbon sources by Strain C-I5003.

Sources of carbon GrowthSources of carbon Growth D-Xylose + + +* Raffinose + +* L-Arabinose + +Melibiose++ D-Glucose ++ ++ i-Inositol - D-Galactose + + D-Sorbitol D-Fructose +++ ++ D-Mannitol ++ ++ L-Rhamnose + + Glycerol- D-Mannose + + + + + Soluble starch + + Sucrose + + + + Control Lactose Maltose + Trehalose + + + * Basal medium with 0.1/yeast extract added Note : + + +: Luxuriant growth + + : Good growth +. Growth Poor growth - : No growth 6) Other Characteristics : The cells were harvested by the procedure previously described at (2) above and DNA was prepared by a procedure analogous to that of J. Marmur et al [Journal of Molecular Biology 3, 208,1961]. The G-C (Guanine-Cytosine) content of the DNA was found to be about 71 mole %.

Gram-staining of the vegetative mycelium of this strain was positive.

The above characteristics of Strain No. S-15003 were compared with the descriptions in S. A. Wakman's"The Actinomycetes Vol. 2" [The Williams and Wilkins Co., 1961]; R. E. Buchanan and N. E. Gibbons,"Bergey's Manual of Determinative Bacteriology, 8th ed. 1974" ; and other similar literature references.

Whilst this strain was thought to belong to Group III of the genus Nocardia, the failure to find any species having the characteristics so far described among the known strains led to the conclusion that this strain represented a novel species of microorganism.

The present strain No. C-15003 has been depesited at the Fermentation Research Institute, Agency of Industrial Science and Technology (FERM) under the receipt number of 3992; at the Institute for Fermentaticn, Osaka (IFO) under the accession number of IFO 13726 and at The American type culture Collection (ATCC) Maryland, U. S. A. under the accession number of 31281.

While Strain No. C-15003 is a ncvel species of the genus Nocatdia as just mentioned, it is liable, as are microorganisms generally, to undergo variations and mutations, either spontaneously or under the influence of a mutagen. For example, the many variants of the strain which are obtainable by irradiation with X-rays, gamma rays or ultraviolet light, by monocell isolation, by culture on media containing various chemicals, or by various other mutagenic treatments, as well as the mutants spontanecusly derived from the strain, should nct be ccnsidered to represent any cther distinct species but, rather, any of such variants and mutants capable of elaborating C-15003 P-3' and/or P-4 may be utilized for the purposes of this invention. Thus, for example, the subjection of Strain No. C-15003 to various mutagenic treatments yield mutants which substantially lack the ability to prcduce soluble pigments, mutants with substrate mycelia which are colourless, yellowish green, reddish tan or orange red, mutants whose hyphae are ready to fragment into bacillary elements or branched short hyphal frag ments, and mutants with abundant white aerial mycelia or substantially without aerial mycelia.

The medium employed for the cultivation of such an Antibiotic C-15003-pro ducing strain may be either a liquid er a solid medium only if it contains nutrients which the strain may utilize, although a liquid medium is preferred for high-production runs. The medium may comprise carbon and nitrogen sources which Strain No. C-15003 may assimiliate and digest, inorganic matter or trace nutrients. As examples of said carbon sources there may be mentioned glucose, lactose, sucrose, mal tose, dextrin, starch, glycerol, mannitol or scrbitol, fats and oils (e. g. soybean oil, lard oil or chicken oil). The nitrogen sources may be for example meat extract, yeast extract, dried yeast, soybean meal, corn steep liquor, peptone, cottonseed flour, spent molasses, urea or ammonium salts (e. g. ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium acetate). The medium may further contain salts of sodium, potassium, calcium or magnesium, salts of iron, manganese, zinc, cobalt or nickel, salts of phosphoric acid or boric acid, and organic acid salts such as acetates and propionates. Further, the medium may ccntain, as added, various aminoacids (e. g. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine or proline), peptides (e. g. dipeptides or tripeptides), vitamins (e. g. Bt, B2, nicotinic acid, B12, C or E) or nucleic acids (e. g. purine, pyrimidine and derivatives thereof). For the purpose of adjusting the pH of the medium, there may be added, for example, an inorganic or organic acid, or an alkali buffer. Suitable amounts of oils, fats or surfactants may also be added as antifoaming agents.

The cultivation may be conducted for example under srationary, shaking or sub merged aerobic cultural conditions. For high production runs, submerged aerobic culture is preferred. While the conditions of culture depend upon the condition and composition of medium, the strain, the cultural method other such factors, it is normally preferred to carry out incubation at 20 to 35 C with an initial pH of substantially 7.0.

A temperature of from 23 to 30 C is particu ! arly desirable in an intermediate stage of cultivation, with an initial pH of 6.5 to 7.5. While the incubation time is also variable acccrding to the same factors as were mentioned above, it is advisable to continue the incubation until the titer of the desired antibiotic product becomes a maximum. In the case of shaking culture or aerobic submerged culture in a liquid medium, the time required normally ranges from about 48 to 144 hours.

The potency of the antibiotic was assayed with Tetrcwhy1nens pyriformis W as an assay organism. Thus, the above microorganism was grown on a test medium [2.0 g of tryptosepeptone (Difco), 1 g of yeast extract (Difco), 2 g of glucose, 1000 ml of distilled water and 10 ml of 1M-phcsphate buffer (pH 7.0)] at 28 C for from 44 to 48 hours and the potency of the antibiotic was determined by the serial dilution method with monitoring of the turbidity of growth, the effect on ascites tumor cells and a thinlayer chromatographic, briefly TLC, assay to be described hereinafter.

The novel Antibiotic C-15003 P-3, P-3'and/or P-4 is produced and accu mulated in the resulting fermentation broth, both extracellularly and intracellularly.

These substances have also been detected by TLC. Thus, the fermentation broth is separated into cells and filtered by filtration or centrifuging and the filtrate is extracted with the same volume of ethyl acetate. The same amount cl 70^xi acetone-water as the nitrate is added to the cells and, after an hour's stirring at 20 C, the suspension is filtered. The acetone is removed from the filtrate and the resulting aqueous filtrate is extracted with ethyl acetate. Each of the extracts is concentrated by 1/100 by volume and subjected to thin-layer chromatography on a silica gel-glass plate (Merck, West Germany, Kieselgel 60 F,, J) 0. 25 mm, 20 x 20) (solvent system: chloroformmethanol=9 : 1). The potency was determined on the basis of the intensity of spits detected by irradiation with ultraviolet light at 2537 A.

Because C15003 P-3, P-3'and/or P-4, which are thus produced in the fermentation broth are lipophyl neutral substances, they can be conveniently recovered by the separation and purification procedures which are normally employed for the harvesting cf such microbial metabolites. For example, there may be employed a procedure which utilizes the difference in sclubility between the antibiotic and any impurity, means which utilize the adsorptive affinity of various adsorbents such as activated carbon, macrcporous non-ionic resins, silica gel or alumina, or a procedure for removing impurities by means of icn-exchange resins, alone or in a suitable combination or repetition of steps.

Since, as aforesaid, C-15003 P-3, P-3'and P-4 occur in both the filtrate and cells, the antibiotics are separated and purified by mcans wf such an adsorbent, if one is employed, either directly or after a solvent extraction in the case of the filtrate, or after a solvent extraction in the case of microbial cells. The solvent extraction may be performed by any of the following and other methods e. g. (1) solvent extraction from the culture broth prior to the separation of cells and (2) solvent extraction of the cells and the filtra. e obtained by filtration or centrifuging. To extra t the filtrate and cells independently, the following procedure may be advantageously used.

The solvents suitable for extraction of the nitrate are water-immiscible organic solvents such as fatty acid esters, e. g. ethyl acetate or amyl acetate ; alcohols, e. g. butanol ; halogenated hydrocarbons, e. g. chloroform ; and k : tones, e. g. methyl isobutyl ketone. The extraction is carried out at a pH near neutral and, preferably, the cul ure fluid previously adjusted to pH 7 is emru, : ted with ethyl acetate. The extract is washed with water and concentrated under reduced. esslire. Then a non-polar solvent such as petroleum ether or ;-hexane is added to the concentrate and the @rude product (1) containing the active ccmpcur. d is recovered. Because, on TLC, a number of spots are detected in addition to Antibiotic C-15003, the product (I) is sequentially subjected to the following purification prcceres. Thus, as a routine purification procedure, adsorption chromatography is useful and, for this purpose, one of those adsorbents such as silica gel, alumina or a macroporous non-. onc adsorbent resin may be employed. For purification from the crude product (I), silica gel is most useful. Development may be carried out, for example starti ;. g with petroleum ether and hexane, and elution of Antibiotic C-15003 is performed by the addition of a polar solvent such as ethyl acetate, acetcne, ethanol cr merhanol. In a iypical process, using silica gel (Merck, West Germany, 0. US-0. 2 mm) as a carrier, column chrcmatography is carried cut with a serial increase in the hexane to ethyl acetate ratio. The eluate is sampled and investigated by TLC C and the fractions containing C-15003 are pooled and concentrated under reduced pressure. Then, petroleum ether or hexane is added to the concentrate, whereby the crude product (II) is obtained. Since th : s product still contains impurities, it is further purified as follows. For example, the product (II) may be purified by means of a second silica gel column using a different solvent system. The developing system for this purpose may consist for example in a halcgenated hydrocarbon such as jjichlc. rcmethane or chloroform, with the addition of a polar solvent such as an alcool, e. g. methanol or ethanol or a ketone, e. g. acetone or methyl ethyl ketone. In this way, Antibiotic C-150G3 is isolated. The order of solvent systems for the first and second silica gel cclumns may be reversed and, in addition, ordinary organic solvents may be used in conjunction with the above systems if necessary.

Where a macroporous adsorbent resin is used as a purification means for the crude product (II), eluation of Antibiotic C-15003 is acccmplished with a mixture of water with a lower alcool, a lower ketone or an ester. The lower alcchul may be for example methancl, ethancl, propanol or butanol, and the lower ketone may be for example acetone or methyl ethyl ketone. The ester may be for example ethyl acetate. In a typical procedure, the crude product (II) is dissolved in 60,'mcthancl-water and adsorbed on a column of Diaion (Trade Mark) HP-10 (Mitsubish : Kasei K. K.). The column is washed with 70 /o methancl-water and elution is carried out with 90% meihanci- water. In this way, Antibiotic C-15003 is eluted frcm the column.

In either of the processes described above, the fractions containing Antibiotic C-15003 are pooled and concentrated under reduced pressure. To the dry product are added 5 to 8 volumes of ethyl acetate and the mixture is allowed to stand, whereupon crystals of Antibiotic C-15003 separate out. These crystals contain C-15003 P3, P3'and P-4. These compounds are then separated from each other by means of an adsorbent such as those mentioned hereinbefore. Thus, using silica gel or a macroporous non-ionic adsorbent resin and the above solvents, the desired compounds may be fractionally eluted. When, for example, silica gel is employed, development is carried out with hexane, ethyl acetate, or chloroform-methanol, whereby C-15003 P-4, P-3'and P-3 emerge in that crder.. After detecticn by TLC, the fractions corresponding to S15003 P-4, P-3'and P-3 are respectively concentrated under reduced pressure and ethyl acetate is added to the cencentrates. In this manner, the respective compounds can be obtained as crystals. When a macroporous non-ionic adsorbent resin is employed, gradient elution with a varying ratio of alcool, ketone or ester to water may be utilized. For example, by the gradient eluticn method involving the use cf 60% methanol-water and 955/O methanol-water, with 5% sodium chloride added, C-15003 P-3, P-3'and P-4 emerge in the order mentioned. After sampling and detection by TLC, each group of active fractions is concentrated under reduced pressure and crystallized from ethyl acetate. The isolated crystals include ethyl acetate as a solvent of crystallization and, after drying over phosphorus pentoxide at 70 C for 8 hours, show the following physical and chemical properties (Table 4).

Table 4

Antibiotic C-15003 P - 3 P - 3' P - 4 C32H43ClN2O9 = 635.169 C32J43ClN2O9 = 635.169 C33H45ClN2O9 = 649.196 m.p.( C) 190-192 182-185 177-180 Specific rotation -136 + 10 -134 + 10 -142 + 10 [α]D22 (C = 0.375 CHCl3) (C = 0.11 CHCl3 (C = 0.522 CHCl3) Elemental analysis C 60.06 60.09 60.65 Found (%) H 7.04 7.04 7.05 N 4.33 4.34 4.25 Cl 5.37 5.99 5.23 Elemental analysis C 60.51 60.51 61.05 Calcd. (%) H 6.82 6.82 6.99 N 4.41 4.41 4.32 Cl 5.58 5.58 5.46 Ultraviolet abstorpiton 233 (3025) 240(sh 28450) 233 (3055) 240(sh 28250) 233 (29900) 240(sh 28240) spectra nm (c) 252 (27640) 280 (5750) 252 (27600) 280 (5750) 252 (27590) 280 (5712) (in methanol) 288 (5700) 288 (5700) 288 (5680) Infrared absorption 1740, 1730, 1670, 1580, 1740, 1730, 1670, 1580, 1740, 1730, 1670, 1580, spectra 1445, 1385, 1340, 1255, 1445, 1385, 1340, 1255, 1445, 1385, 1340, 1255, (cm-1)KBr 1180, 1150, 1100, 1080 1180, 1150, 1100, 1080 1180, 1150, 1100, 1080 1038 1038 1038 NMR (ppm) 1.27 (d) (3H) 1.06(t) (3H) 1.03(d) (6H) 100MHz in CDCl3 1.28(d) (3H) Table 4 (Continued)

Antibiotic C-15003 P - 3 P - 3' P - 4 C32H43ClN2O9 = 635.169 C32J43ClN2O9 = 635.169 C33H45ClN2O9 = 649.196 Mass spectra (m/e) 573, 485, 470, 450 573, 485, 470, 450 587, 485, 470, 450 Solubility Insoluble in petr. ether, Insoluble in petr. ether, Insoluble in petr. ether, hexane & water, hexane & water, hexane & water, Sparingly soluble in Sparingly soluble in Sparingly soluble in benzene & ether. benzene & ether. benzene & ether.

Solbule in chloroform, Solbule in chloroform, Solbule in chloroform, ethyl acetate, acetone, ethyl acetate, acetone, ethyl acetate, acetone, ethanol, ethhanol, ethanol, ethhanol, ethanol, ethhanol, pyridine, tetrahydrofuran pyridine, tetrahydrofuran pyridine, tetrahydrofuran & dimethyl lsulfoxide. & dimethyl lsulfoxide. & dimethyl lsulfoxide.

Color reactions Dragendorff: Positive Dragendorff: Positive Dragendorff: Positive Beilstein: Positive Beilstein: Positive Beilstein: Positive Based on the molecular formula shown aboe and the antimicrobila and antitumor activity data given hereinaftert, the present antibiotic was compared with the known groups of antibiotics. The literature search fialed to locate a distict group similar to Antibiotic G-15003. Hweever, a search for substances that might give ultraiolet abroptions similiar to those of the present antibiotic among components of plant and other natually-occurring organic compounds led to the maytanacine groups and, based on the moleuclr formulae involved, in aprticular, it was assumed that the antibiotic belongs to the maytanacine group of compounds containing two nitrogen atoms.

Maytanacine was otained as a plant component and was reported in Journal of the American Cemical Scoiety 97, 5294 (1975). The mass spectrum of amytanacine is as follows.

The presence of m/e 485, 470 and 450 for C-15003 P-3, P-3'and P-4 convinces us at once that these compounds have a skeletal structure identical with that of may- tanacine, differentiating them from maytanacine in the kind of acyl group in the 3 pcsition. It is thus clear that Antibiotic C-15003 is a novel compound. When C-15003 P-3, P-3'and P-4 were each degraded with alkali and analysed by gas chromatography for the literated carboxylic acids, it was found that isobutyric acid, butyric acid and isovaleric acid were obtainable frcm C-15003 P-3, C15003 P-3' and C15003 P 1, respectively. The following formulae show the structures, based. on the above data, of S15003 P3, P3'and P4.

u,-, R M'Cf'C r'l * t CH 0 R AntibioticR o 30 N-C 1 CH CH3 P-3-CO-CH\ cx3 \ 3 CHI ouf P-3'-CO-CHz-CH2-CH3 N H CH, 33 3 3 CH, Antibiotic C-15003 is active to prolong the survival time of warm-blooded animals carrying one or more tumors (e. g. leukemia, sarcoma, mastocytoma, melanoma or carcinoma).

Antibiotic c-15003 also has a regressive effect on solid tumors growing sub cutaneously or intramuscularly, or in various organs.

As examples of said 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 showed an LD ; o value of more than 313 mcg/kg.

In a procedure for the administration to the warm-blooded animals carrying one or more tumors, Antibiotic 51003 is generally used in the form of an injection. The injection may be intromuscular, intrathoracic, intra-abdominal, intra-uterine, intra thecal, intra-artery, subcutaneous or intravenous.

Such an injection can be prepared in a per se conventional manner, that is to say Antibiotic C-15003 is dissolved or suspended in an aqueous liquid medium containing a conventional solubilizer such as an alcohol (e. g. thane !), a polyalcohol (e. g. propylene glycol cr polyethylene glycol 200-300), a non-ionic surface-active agent, physiological saline or an isotonic solution.

Typical examples of the liquid medium are 1 Xo ethanol-0. 85% physiological saline, 2.5% methanol-0.85 /O physiological saline, and 1 % Tween (Trade Mark) 10-0. 85% physiological saline.

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

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

Antibiotic C-13003 is administered to a warm-blooded animal carrying tumors at a dose of between 0.8 and 50 mcg/kg/day.

Antibiotic C-15003 includes, as mentioned above, Antibiotic C-130C3 P-3, P-3'and P-4, and, in the present pharmaceutical compositions, single component Antibiotic C-15003 P-3, P-3'or P-4 may be used, or mixtures of cnc or merle thereof (i. e., a mixture of Antibiotic C-10053 P-3 and P-4, a mixture of Antibiotic C-15003 P-3'and P-4, a mixture of Antibiotic C-15003 P-3, P3'and a mixture of Antibiotic C-15003 P-3, P-3'and P-4) may be used. In practice, a mixture of Antibiotic C-15003 P-3, P-3'and P-4 is preferred.

Each component or mixture thereof is not necessarily purified.

The following examples are further illustrative of the preparation and use of the Antibiotic C-15003 but are by no means limitative of the invention, wherein"part (s)" is based on weight unless otherwise noted and the relationship between"part (s)" and"part (s) by volume"corresponds to that between"gram (s)"and"milliliter (s)", and"%"is based on"weight/volume"unless otherwise noted.

For the purpose of this description, the terms tumor, neoplasia, leukemia, sarcoma or carcinoma are used interchangeably.

All of the following experiments on the anti-tumor activity of Antibiotic C-15003 were tested in accordance with the protocols for screening and evaluation methods of antitumor agents described by the National Cancer Institute, National Institute of Health, U. S. A. (NCI protocols). [See Cancer Chemotherapy Reports (Part 3) (Vol.

3, No. 2,1972, pages 1-103].

Melanoma B 16 and leukemia L 1210 obtained from the National Cancer Institute, U. S. A., in 1971 were maintained in C57BL/6 and DBA/2 m : ce [Cancer Chemo- therapy Reports (Part 1), Vol. 50, No. 5, 1975, pages 919-928], respectively.

Leukemia P388 provided by the Japan Cancer Chemotherapy Center in 1975 was maintained in DBA/2 mice. P815 mastocytoma in DBA/2 mice, Ehrlich carcinoma and sarcoma 180 in JCR-JCL mice were also used. Suitable strains of mice, female and male, 5 to 7 weeks old, to each tumor were used in each of the examples.

Reference Example 1.

Nocardia No. C-15003 (IFO 13726 ; FERM 3992 ; ATCC 31281) as grown on a medium (yeast extract-malt extract agar) was used to inoculate a 200 parts by volume fermenter containing 40 parts by volume of a seed culture medium (2% glucose, 3% soluble starch, 1% raw soybean meal, 1% corn steep liquor, 0.5% Polypepton, 0.3% NaCI and 05 /O CaCO3, pH 7. 0). The inoculated medium was incubated at 28 C for 48 hours to obtain an inoculum. A C. 5 part by volume portion of the inoculum thus obtained was transferred to a 200 parts by volume fermenter containing 40 parts by volume of a fermentation medium composed of 5% dextrin, 3% corn step liquor, 0.1% Polypepton and 0.5 % CaCO3 (pH 7.0), and cultivated at 28 C for 90 hours to give an inoculum (seed culture).

As determined by the serial dilution method using Tetrahymena pyriformis W as an assay organism and Antibiotic C-15003 P-3 as the standard sample, the above culture was found to have a titer of ZS s g/ml.

Reference Example 2.

A 10 parts by volume portion of the inoculum (seed) obtained in Reference Example 1 was transferred to a 2,000 parts by volume fermenter containing 500 parts by volume of a seed culture medium (the same as the above) and incubated at 28 C for 48 hours. A 500 parts by volume portion of the resulting culture was transferred to a 50, 000 parts by volume tank of stainless steel containing 30,000 parts by volume of a seed culture medium and cultivated at 28 C under aeration (30,000 parts by volume/min.), agitation [280 r. p. m. (1/2 DT)] and internal pressure (1 kg/cm2) te obtain a seed culture. This culture was used to seed a 200, 00 () parts by volume tank of stainless steel containing 100, 000 parts by volume of a fermentation medium similar to that used in Reference Example 1 at an inoculation rate of 10%. The inoculated medium was incubated at 28 C unde aertian (100,00 parts by volume/ min.), agitation [200 r. p. m. (1/2 DT)} and interral pressure (1 kg/cm2) for 90 hours. As determined by the same procedure as that described in Reference Example 1, the culture obtained above was found to have a titer of 25 . g/ml.

Reference Example 3.

To 95, 000 parts by volume of the culture obtained in Reference Example 2 were added 2, 000 parts of Hyflo-Supercel (Trade Mark, Johnes and Manville Products, U. S. A.) and, after thorough mixing, the mixture was filtered on a pressure filter to yield 85, 000 parts by volume of filtrate and 32,000 parts of moist cells. The filtrate (85, 000 parts by volume) was stirred and extracted with 30,000 parts by volume of ethyl acetic. This procedure was repeated once again. The ethyl acetate layers were pcoled, washed twice with 30. 000 parts by volume portions of water, dried by the addition of OO parts of anhydrous sodium sulphate and concentrated under reduced pressure to 200 parts by volume. Petroleum ether was added to the concentrate and the resulting precipitate was recovered by filtration (53 parts). This crude product (I) was stirred with 100 parts by volume of ethyl acetate and the insolubles were filtered off. The filtrate was stirred with 10 parts of silica gel (Merck, West Germany, 0. 05-0. 2 mm) and the ethyl acerate was removed under reduced pressure. The residue was applied to the top of a silica gel column (400 parts by volume). Elution was carried out with 500 parts by vo! ume of hexane, 500 parts by volume of hexaneethyl acetate (3: 1), 500 parts by volume of hexane-ethyl acetate (1: 1), 500 parts by volume of hexane-ethyl acetate (1 : 3), 500 parts by volume of ethyl acetate and 1,000 parts by volume of ethyl acetate-methanol (50: 1), with the eluate being collected in 100 parts by volume fractions. A one-part by volume portion of each fraction was concentrated to dryness, and 0.1 part by volume of ethyl acetate was added to the concentrate to give a mixture. The mixture was spotted at 2.5 cm from the bottom edge of a silica gel-gless plate (Merck, West Germany, 60 F234, 0. 25 mm, 20 x 20), and developed for about 17 cm with a solvent system of ethyl acetate-methanol (19: 1).

After development, detection was carried out with ultraviolet light (2537 i).

The active fractions No. 23-No. 28 of Rf 0.6-065 were collected and concentrated under reduced pressure to 20 parts by volume. To this concentrate were added 150 parts by volume of petroleum ether to yield 15 parts of a crude product (II).

Reference Example 4.

With stirring, 32,000 parts of the moist cells obtained in Reference Example 3 were extracted with 50,000 parts li volume of 70% acetone-water for 3 hours and then filtered on a pressure filter. The extraction with 50,000 parts bv volume of 70% acetone-water and subsequent filtration was repeated once again. The filtrates were pooled and the acetone was removed by concentration under reduced pressure. The resulting aqueous system was passed through a column of 5,000 parts by volume Diaion (Trade Mark) HP-10 (Mitsubishi Kaisei K. K.). The column was washed with 20,000 parts by volume of water and 50% aqueous methanol, followed by elution with 15,000 parts by volume of 90% methanol-water. The eluate was 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 procedure was repeated once again. The ethyl acetate layers were combine, washed with water, dried by the addition of anhydrous sodium sulphate and concentrated under reduced pressure to 200 parts by volume. Following the addition of petroleum ether, the precipitate was recovered by filtration (28 parts). The above product was purified by means of a column of silica gel to recover 8.0 parts of crude product (II).

Reference Example 5.

In 10 parts by volume of ethyl acetate were dissolved 1.5 parts of the crude product (II) obtained in Reference Example 3 and the solution was stirred well with 4 parts of silica gel (Merck, West Germany, 0.05-0.2 mm). The ethyl acetate was removed under reduced pressure. The residue was applied to the top of a column of 300 parts by volume silica gel and the column was first washed with 500 parts by volume of chloroform and then eluted with 500 parts by volume of chloroformmethanol (50: 1). 500 parts bv volume of chloroform-methanol (20: 1) and 500 parts by volume of chloroform-methanol (10: 1). The eluate was collected in 25 parts by volume fractions.

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

Fractions Nos. 39 and 40 abscrbing at 2537. i in the zone of Rf 0.50-0.60 were collected and concentrated to dryness under reduced pressure. To the residue there were added 2 parts by volume of ethyl acetate and the mixture was allowed to stand, whereupon 0.150 part of crystals of Antibiotic C-15003 was obtained.

The above crystals of Anitbiotic C-15003 (0.150 part) were dissolved in 15 parts by volume of methanGI, followed by the addition of 0.300 part of sodium chloride and 15 parts by volume of water. A column was packed with 200 parts by volume of Diaion (Trade Mark, HP-10, Mitsubishi Kasei K. K.), and calibrated with 600 parts by volume of 50% methanol-water containing S% of NaCI. The sample solution prepared above was passed through the column, and gradient elution was carried out using 1,500 parts by volume of 60% methanol-water containing 5% NaCI and 1,500 parts by volume of 95% methanol-water. The eluate was collected in 15 parts by volume fractions and each fraction was investigated by silica gel thin layer chromatography. Fractions 145 to 153 contained C-15003 P-3, fractions 167180 contained C-15003 P-3'and P-4 and fractions 185-190 contained C-15003 P-4.

Each group of fractions was concentrated and dissolved by the addition of 50 parts by volume of water and 100 parts by volume of ethyl acetate. The solution was shaken in a separatory funnel, the water layer was separated out and, after washing with two 50 parts by volume portions of water, the ethyl acetate layer was dried over anhydrous sodium sulphate, concentrated and allowed to stand. In the above manner, crystals were obtained from each group of fractions. The crystals were collected by filtration and dried.

C-15003 P-3 0. 070 part C-15003 P-3', P-4 0.018 part C-15003 P-4 0.015 part The mixed crystals of C-15003 P-3'and P-4 (0.018 part) were dissolved in 0.3 part by vclume of ethyl acetate and spotted in a line at a distance cf 2.5 cm from the bottom edge of a silica gel glass plate (Merck, West Germany, Kieselgel 60 F2 4 0. 25 mn, 20 x 20), fellowed by development with ethyl acetate-methancl 1).

After development to about 18 cm, the absorption bands at Rf 0.60 (P-4) and Rf 0.65 (P-3') were scraped off and each was independently extracted twice with ethyl acetate containing a small amount of water. The resulting ethyl acetate extract was washed with water, dried over anhydrous sodium sulphate, concentrated under reduced pressure and allowed to stand.

0.010 Part crystals of C-1S03 P-4 and 0.003 part crystals of C-15003 P-3'were obtained from the fractions of Rf 0.68 and Rf 0.65, respectively.

Reference Example 6.

1,000 Parts by volume of the culture of Reference Example 2 were inoculated into a 200,000 parts by volume tank of stainless steel containing 100,000 parts by volume of a seed culture medium. The inoculated medium was incubated at 28 C under aeration (100,000 parts by volume/min.) and agitation (200 r. p. m.) for 48 hours to prepare a seed culture. This seed culture was transferred to a 2,000,000 parts by volume tank of stainless steel containing 1,000, 000 parts by volume of a fermentation medium similar to that used in Reference Example 1 at a transplantation rate of 10%. Cultivation was carried out at 28 C under aeration (1,000,000 parts by volume/min.), agitation [120 r. p. m. (1/3 DT)] and internal pressure (1 kg/cm2) for 90 hours. The resulting culture was found to have a titer of 20 ug/ml as assayed by the assay procedure described in Reference Example 1.

To 900,000 parts by volume of the above culture were added 900, 000 parts by volume of acetone and, after an hour's stirring, 20,000 parts of Hyflo-Supercel (Trade Mark. Johnes & Manille, U. S. A.) were added. The mixture was further stirred and filtered on a pressure filter machine. To 1, 700, 000 parts by volume of the resulting filtrate were added 500, 000 parts by volume of water and, in a Podbielniak (Trade Mark) extractor (Podbielniak, Inc.) the mixture was extracted with 1,000,000 parts by volume of ethyl acetate. The ethyl acetate layer was washed with water, dried by the addition of anhydrous sodium sulphate and concentrated under reduced pressure. Petroleum ether was added to the concentrate and the resulting precipitate was recovered by filtration and dried. 68 parts of crude product (1) were obtained by the above procedure. Thereafter, as in Reference Examples 3,4 and 5, this crude produce was purified to yield 9.5 parts of C-15003 P-3, 0.300 part of C15003 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 leukemia P388-bearing mice.

(C57BL/6 X DBA/2F, (BDF), mice (in a group of 3 or 5 mice), weighing 18-22 g, were inoculated intraperitoneally with 1 X 10'cells of leukemia P388 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 C15003 P-3'] was suspended in 1%- Tween (Trade Mark) 80-0. 85% physiological saline, and the suspension was administered intraperitoneally to tumor inoculated animals (0.2 ml/each mouse) once daily for 9 days starting from 24 hours after tumor inoculation.

To evaluate the anti-tumor activity of Anitbiotic C-15003, the T/C% which is the ratio X 100 of the median survival time of P388-bearing mice treated (T) with Antibiotic C-15003 to that of untreated control (C) mice was calculated. More than 125 in T/C% was regarded as positive anti-tumor activity in accordance with NCI protocols.

Table 5 Median Dose No. of survival time mcg/kg/day mice (da) s) T/C oKo 25 3 24.5 227 12. 5 3 20.0 185 6.25 3 21. 5 l99 (Control) 11 10.8 50 5 9.5 82 25 5 22.5 194 12.5 5 22.5 194 6.25 5 22.5 194 3.13 5 19. 5 168 1. 6 5 15. 0 129 0.8 5 12.5 108 0. 4 5 12. 5 108 (Control) 20 11. 6 As shown by the results summarized in Table 5, the administration of daily intraperitoneal doses of 1.56-25 mcg/kg of the antibiotic for 9 days prolonged the survival time of treated mice significantly.

Especially, the mostt significant prolongation of survival time of treated mice was observe at a dose of 25 mcg/kg (optimal dose) (T/C % of 227).

Example 2.

Effects of C-15003 P-3 and C-15003 P-4 on the survival time of leukemia P388-bearing mice.

BDF, mice (5 mice per test group), weighing 18-22 g, were inoculated intra peritoneally with 1 X 106 cells of leukemia P388 on day 0. Each antibiotic was dissolved in 2.5% methanol-0. 83% physiogical saline and the solution was administered intraperitoneally to mice (0.2 ml/each mouse) once daily for 9 days starting from 24 hours after tumor inoculation.

As described in Example 1, more than 125 in T/C % was regarded as positive anti-tumor activity in accordance with NCI protocols. As summarized in Table 6, the administration of C-15003 P-3 with daily intraperitoneal doses of 1.6-25 mcg/kg or that of C-15003 P-4 with daily intraperitoneal doses of 0.8-50 mcg/kg prolonged the survival time of treated mice significantly. Maximum effect was observed at a daily dose of 25 mcg/kg of either test agent.

Table 6 Dose T/C % mcg/kg/day C-15003 P-3 C-15003 P-4 50 118 125 25 223 195 12.5 177 177 6.25 168 180 3.13 159 145 1.6 144 141 0.8 123 132 0.4 105 105 Example 3.

Ef3ect of Antihiotic C-15003 (a mixture of C-15003 P-3, C-15003 P-3'and C-15003 P-4) on the survival time of melanoma B 16-bearing mice.

One gram of the tumor was homogenized with 10 ml of cold 0.85% physiological saline and 0.5 ml of thois tumor homogenate was inoculated intraperitoneally into BDF, mice (5 animals per test group) as described in NCI protocols.

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'] was suspended in 1% Tween (Trade Mark) 80-0. 85% physiological saline, and the suspension was administered intraperitoneally to mice once daily for 9 days starting 24 hours after tumor inoculation (5 mice in each group and 15 mice in control group).

T/C % in median survival time was calculated as described in Example 1, and T/C % over 125 was regarded as positive anti-tumor activity.

As shown in Table 7, the survival time of treated mice with Antibiotic C-15003 was significantly longer than that of untreated mice.

Table 7 Median Dose No. of survival time mcg/kg/day mice (days) T/C % 24 5 29.5 159 12 5 31. 5 170 6 5 35.5 192 3 5 34.5 186 1. 5 5 29.0 157 Control 15 18.5 24 5 31. 5 213 12 5 28.5 193 6 5 22.5 152 3 5 16. 2 109 Contrat) 5 14.8 Example 4.

Effects of C-15003 P-3 and C-15003 P-4 on the survival time of melanoma B 16-bearing mice.

BDFI mice (5 animals per test group) were inoculated intraperitoneally with a suspension of B 16 melanoma by the same method as described in Example 3.

Each antibiotic was dissolved in 2. 5% methanol-0. 85% physiological saline and the solution was administered intraperitoneally to mice (0.2 ml/each mouse) once daily for 9 days starting 24 hours after tumor inoculation.

As shown in Table 8 below, the administration of C-15003 P-3 or C-15003 P-4 with daily doses of 1.6-50 mcg/kg prolonged the survival time of treated mice significantly.' Table 8 Dose T/C % mcg/kg/day C-15003 P-3 C-15003 P-4 50 > 219 > 219 25 > 219 > 219 12. 5 207 199 6.25 189 182 3.13 156 159 1. 6 128 122 0.8 116 103 0.4 105 104 Example 5.

Effects of Antibiotic 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 leukemia L1210-bearing mice.

BDFI mice (5 animals per test group) were inoculated intraperitoneally with I X 105 cells of leukemia L1210 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'], and C-15003 P-3 were respectively dissolved in 2. 5% methanol-0. 85% physiological saline, and each solution was administered intraperitoneally to tumor-inoculated mice (0.2 ml/mouse) once daily for 9 days starting from 24 hours after tumor inoculation.

To evaluate the anti-tumor activity of the test agents, the T/C % which is the ratio X 100 of the mean survival time of L1210-bearing mice treated with the agents to that of untreated mice (a group of 25 mice) was calculated as described in NCI protocols. A T/C % value of 120 was considered to be a minimum value in anti-tumor activity of the agents.

As shown in Table 9, the mixture of C-15003 P-3, C-15003 P-3'and C-15003 P-4 and C-15003 P-3 was effective in prolonging the survival time of L1210-bearing mice, slightly.

Table 9 Mean Dose No. of survival time Agent mcg/kg/day mice (days) T/C % the mixture of 40 5 12. 0 128 C-15003 P-3, C-15003 P-3'30 5 12. 2 130 and C-15003 P-4 20 5 11.0 117 10 5 10. 2 109 C-15003 P-3 40 5 11.0 117 30 5 12. 2 130 20 5 11. 2 119 10 5 10.4 111 Control 25 9.4 Example 6.

Effects of C-15003 P-3 and C-15003 P-4 on the sunva ! time of sarcoma 180-bearing mice.

ICR-TCL mice (purchased from Japan Clea, Tokyo, 5 animals per test group) were inoculated intraperitoneally with 4 x 10'cells of sarcoma 180 on day 0.

Each antibiotic was dissolved in 2.5% methanol-0. 85% physiological saline and the solution was administered intraperitoneally to tumor inoculated mice (0. 2 ml/each mouse) once daily for 7 days starting 24 hours after tumor inoculation.

T/C% in median survival time as described in Example 1 was calculated.

As shown in Table 10, both of the agents were significantly effective in the prolongation of survival time of sarcoma 180-bearing animals.

Table 10 Median Dose No. of survival time Agent mcg/kg/day mice (days) T/C % C-15003 P-3 25 5 24. 5 188 C-15003 P-4 25 5 33.5 258 Control 10 13. 0 C-15003 P-3 50 5 33.5 284 25 5 30.5 258 12.5 5 15. 5 131 6.25 5 17. 8 150 Control 10 11. 8 Example 7.

Effects of C-15003 P-3 and C--15003 P-4 on the survival time of Ehrlich carcinoma-bearing mice.

ICR-JCL mice (5 animals per test group) were inoculated intraperitoneally with 4 X 106 cells of Ehrlich carcinoma on day 0.

Each antibiotic was dissolved in 2.5% methanol < . 85% physiological saline, and the solution was administered intraperitoneally to mice (0.2 ml/each mouse) once daily for 7 days starting 24 hours after tumor inoculation.

The T/C% value in median survival time as described in Example 1 was calculated.

As shown in Table 11, both of the agents were significantly effective in the prolongation of the survival time of Ehrlich carcinoma-bearing mice.

Table l l Median Dose No. of survival time Agent mcg/kg/day mice (days) T/C % C-15003 P0-3 50 5 > 21. 5 > 671 25 5 > 21.5 > 171 12.5 5 > 21. 5 > 171 6.25 5 > 21. 5 > 171 C-15003 P04 25 5 > 21. 5 > 171 Centre) 10 12. 6 Example 8.

Effects of C-15003 P-3 and C-15003 P-4 on the survival time of P815 mastocytoma-bearing mice.

BDF1 mice (5 animals per test group) were inoculated intraperitoneally with 1 X 10'cells of P815 mastocytoma on day 0.

Each antibiotic was dissolved in 2.5% methanol-0. 85% physiological saline and the solution was administered intraperitoneally to tumor inoculated mice (0.2 m1/each animal) once daily for 7 days starting 24 hours after tumor inoculation.

The TC % value in median survival time as described in Example 1 was calculated.

As shown in Table 12, both of the agents were significantly effective in the pro longation of the survival time of P815 mastocytoma-bearing mice at the doses used in this experiment.

Table 12 Median Dose No. of survival time Agent mcg/kg/day mice (days) % C-15003 P-3 50 5 10.3 61 25 5 > 28.5 > 170 12. 5 5 > 28. 5 > 170 6.25 5 > 28.5 > 170 C-15003 P-4 25 5 > 28. 5 > 170 Control to 16. 8 Reference is directed to our co-pending Application No. 42822/1977 (Specification No. 1586688) which discloses a method of treating with Antibiotic C-15003 to effect tumour regression.

Claims (1)

1. WHAT WE CLAIM IS :

   1. A method of treating a warm-blooded animal other than a human carrying one or more tumors in which method Antibiotic C-15003 (as hereinbefore defined) is administered to the animal.

   2. A method as claimed in claim 1, wherein the Antibictic C-15003 is in the form of Antibiotic C-15003 (as hereinbefore defined) only.

   3. A method as claimed in claim 1, wherein the Antibiotic C-15003 is in the form of Anitbiotic C-15003 P-3' (as hereinbefore defined) only.

   4. A method as claimed in claim 1, wherein the Antibiotic C-15003 is in the form of Antibiotic C-15003 P-4 (as hereinbefore defined) only.

   5. A method as claimed in claim 1, wherein the Antibiotic C-15003 is a mixture of Antibiotic C15003 P-3 Antibiotic C-15003 P-3'and Antibiotic C15003 P,

   6. A pharmaceutical composition, which comprises as an active ingredient an effective amount of Antibiotic C-15003 (as hereinbefore defined), together with a pharmaceutically acceptable carrier or diluent therefor.

   7. A pharmaceutical composition as claimed in claim 6, wherein the active ingredient is Antibiotic C15003 P-3' (as hereinbefore dafined).

   8. A pharmaceutical composition as claimed in claim 6, wherein the active ingredient is Antibiotic C-15003 P-3' (as hereinbefore defined).

   9. A pharmaceutical composition as claimed in claim 6, wherein the active ingredient is Antibiotic C-15003 (P-4 (as hereinbefor edefined).

   10. A pharmaceutical composition as claimed in claim 6, wherein the active ingredient is a mixture of Antibiotic ; 15003 P-3, Antibiotic C-15003 P-3' and Antibiotic C-15003 P-4.

   11. A pharmaceutical composition as claimed in claim 6, wherein the composition is in the form of an injectable composition,

   12. A pharmaceutical composition as claimed in claim 11, wherein the composition comprises Antibiotic C-15003 and a liquid medium.

   13. A method according to claim 1, substantially as herein described with reference to any of the specific examples.

   15. A pharmaceutical composition as claimed in claim 6, substantially as herein described with reference to any of the specific examples.