DE1089928B - Production and extraction of the antibiotic BA-163 - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

German mj§m < patent office

kl. 30 h 6

INTERNAT. KL. C 12 d

EXPLAINING PUBLICATION 1089 928

P 23339 IVa / 30h

REGISTRATION DATE: AUGUST 8, 1959

NOTICE THE REGISTRATION AND ISSUE OF THE EXPLAINED: SEPTEMBER 29, 1960

The invention relates to the production, concentration and recovery of the antibiotic BA-163. That Antibiotic BA-163 is a valuable product for combating malignant tumors in humans and animals. It also has a destructive effect on gram-positive and gram-negative Bacteria that have uses as a therapeutic agent in veterinary medicine, industry and make agriculture suitable. It is also used as a disinfectant and for separating mixtures of microorganisms valuable for medical diagnostic and scientific purposes.

The newly discovered strains of the microorganism that are the starting microorganisms for manufacture are made from soil samples on an agar testicle won. The original isolates were selected on the basis of their antagonism to E. coli, where the cultures have been found to be different from the species which commonly produce antibiotics. Since they had tight threads and chains of spores that are typical of the genus Streptomyces, they were found on media as they are normally used to identify members of this genus. at BA-163 was the final evaluation of the media after a 2 week incubation period in the usual manner performed. For BA-4721 and X-13, the cultures were compared to the BA-163 culture in such a way that that they were planted side by side with BA-163 on the identification media.

Production and extraction of the antibiotic BA-163

Applicant:

Chas. Pfizer & Co., Inc., Brooklyn, N.Y. (V. St. A.)

Representative: Dr. W. Beil

and A. Hoeppener, lawyers,

Frankfurt / M.-Höehst, Antoniterstr.

Claimed priority: V. St. v. America August 8, 1958 and June 29, 1959

William Stephen Marsh, Ringwood, N.J., Aline Lucile Garretson, River Edge, N.J.,

and Koppaka Visveswara Rao, Pine Brook, N. J.

(V. St. Α.), Have been named as inventors

Table I.
Characteristics of a culture of Streptomyces flocculus, isolate BA-163

medium Growth
Degree Aerial mycelium Spurs
education Soluble
pigment Remarks yeast
extract
after
Pridham
Skinny
milk good up
out
draws
Well out
draws
Well weak
is missing none
pink Spore generation in moderately long straight or
wavy chains; Formation of sporophores individually
or in pairs on the threads; cylindrical spurs,
0.65 · 1.00 to 1.3Q μ, which were created by division;
Growth of the individual colonies in three conc
tric circles; the outer flat and the two
the inner one is raised and the middle one has a narrow collar
and the inside floury and velvety; few small ones
Spirals could be seen on the yeast agai;
strong unpleasant odor.
The vegetative mycelium was cream to pink in color; no
Curdling, peptonizing or hydrolyzing the milk;
no change in the p _H -value.

009 609/381

Table I (continued)

medium Growth
Degree Aerial mycelium Spurs
education Soluble
pigment Remarks Glucose Well Well; is missing none The vegetative mycelium is not visible; the backside Agar know up is whitish; the colonies correspond to those on the gray-white Yeast extract; other colonies have only one ring around the central raised part, or this is entirely absent; the colonies are floury and velvety. Nutrient agar moderate Well; is missing none The vegetative mycelium is not visible; the backside see you well White is white. Syn nearly thetical none Agai Calcium moderate Well; is missing none The vegetative mycelium is not visible; the backside Malate agar see you well White is white; the malate was not digested. Strength- weak weak; is missing none The vegetative mycelium is colorless; no hydrolysis. plates White Gelatin- moderate Well; is missing none The vegetative mycelium is brown; Back brown; plates see you well White Diameter of the liquefaction zone = 1.2 cm. Cellulose very scattered; white growth Potato moderate Well; is missing none The vegetative mycelium is colorless where it is visible; bits see you well White the reverse is very pale yellow. Dextrose weak scattered is missing none The vegetative mycelium was colorless, nitrates too Nitrate- Reduced nitrites. broth Emerson Well Well; is missing none Agar White to yellow-white Glucose moderate Well; is missing none The vegetative mycelium was brown; the back was Asparagine White yellowish brown; the colonies were pretty flat, with plates Except for the central, somewhat irregular pillars like growth.

The classification of cultures was carried out by Dr. John B. made routines, including the following Description comes from.

The newly discovered microorganisms were identified as new strains of Streptomyces flocculus (Duche). They are in the Chas culture collection. Pfizer & Co., Inc. designated Isolates BA-163, BA-4721, and X-13. Cultures of these organisms were deposited in the American Type Culture Collection and received the Numbers ATCC-13527, ATCC-13536 and ATCC-13535, respectively. The appearance of the BA-163 culture described in Table I after incubation on the listed media largely agrees with the description in Bergey's Manual of Determ native Bacteriology, 7th edition, agree, however, certain differences between the new and that of J. Duche in Encyclopedie Mycologique, 6: 1-372 (1934).

The Duche culture grew slowly while isolate BA-163 grew well in bulk, but where solitary Colonies were observed, they were small. Other differences are the production of a pink one Pigments in milk, while Duche describes only pink growth and no pigment. The isolate BA-163 did not peptonize within 2 weeks of milk. Slow peptonization was observed by Duche. The BA-4721 culture saw almost exactly like culture BA-163, so it can be assumed that they are the same Genus acts. It differed from the BA-163 culture only in that it was numerous, short, dense Had spirals.

Culture X-13 first exhibited a number of differences to culture BA-163, so that it was assumed to be either a variety of S. flocculus or maybe it was a new genus. Table II provides a comparison of culture X-13 as it first appeared of culture BA-163.

Cultures X-13 and BA-163 became multiple Months with special consideration of the shape of the spore chains in both. After the The various transfers made during this period were similar to cultures X-13 and BA-163 more and more until they finally appeared almost identical.

The changes were as follows: The biochemical and physiological properties of X-13 changed so that they resembled those of BA-163. The BA-163 and X-13 cultures both changed regarding the shape of the spore chains. The straight or wavy chains of BA-163 almost disappeared and became short chains in the form of curves or loops or very small dense spirals with an or two turns. X-13 had loops or incomplete spirals in some areas and in others Areas of dense spirals with one or two turns.

Table II
Characteristics of the culture of Streptomyces flocculus isolate X-13

medium Growth
Degree Aerial mycelium Spore color Soluble
pigment Remarks yeast
extract
after
Pridham Similar to BA-163, but less aerial mycelium;
abundant spores, long loose spirals, single,
in pairs or small vertebrae; oval spores 0.6 x 1.3 μ,
Education through division. Skinny
milk Similar to BA-163, but with lower growth and
darker color; Aerial mycelium pink-white. Glucose
Agar moderate moderate
White White is missing Vegetative mycol colorless to gray, reverse side cream
to "brownish, growth rather rough. Nutrient agar Similar to BA-163, but spores white, weak
Aerial mycelium. Syn
more thetic
Agar Similar to BA-163. Calcium
Malate agar weak weak,
White White brownish Vegetative mycelium cream-colored to brownish, reverse
side white to brownish; Malate digests a little. Cellulose no
growth Dextrose
Nitrate-
broth Similar to BA-163. Potato-
Bit Similar to BA-163, but weak aerial mycelium. Emerson
Agar moderate
see you well Well,
know up
light gray cream
Colours,
Gray Brown Vegetative mycelium colorless as visible; back
Brown. Glucose
Asparagine
plates Similar to BA-163, but dark pink, areas of the
vegetative mycelium. strength
plates Similar to BA-163. gelatin
plates Similar to BA-163.

It is obvious that a restriction to the above-mentioned organisms for the production of the antibiotic according to the invention BA-163 is not given. In particular, this includes mutants of these organism strains, by various methods, such as. B. exposure to X-rays or ultraviolet rays, Treatment with nitrogen mustard, single cell culture method, and the like.

The antibiotic BA-163 is effective as an antibiotic and antiprotozoal agent in vitro. The exact antibacterial picture of antibiotic BA-163 is given in Table III. It became an inhibition one Number of pathogens of clinical importance found in very low concentrations. Some of these organisms are e.g. B. Corynebacterium diphtheriae, Clostridium perfringens, Sahnonella typhosa, a number of strains of Proteus vulgaris and Micrococcus pyogenes, Neisseiia gonnorroene and Haemophilus innuenzae. Organisms that have some importance in agriculture, veterinary medicine and industry have and are sensitive to the antibiotic BA-163, are z. B. Listeria monocytogenes, Aerobacter aerogenes, Erwinia amylovora, Salmonella pullorum and Salmonella gallinarium.

Table III Sensitivity in vitro to the antibiotic BA-163

Microorganisms

Micrococcus pyogenes var. Aureus

Streptococcus pyogenes

Erysipelothrix rhusiopehthiae ... Corynebacterium diphtheriae ....

Listeria monocytogenes

Bacillus subtilis

Clostridium perfringens

Bacterium ammoniaegenes

Aerobacter aerogenes

Escherichia coli.

Escherichia coli 21

Proteus vulgaris

Proteus vulgaris 4

Proteus vulgaris 1

Proteus vulgaris 59

Sahnonella pullorum

Minimal

inhibiting

concentration

mcg / ml

0.39 1.56

12.5 0.09

50 0.09 0.09

50 3.12 0.78 6.25 6.25 3.12 6.25 6.25 0.78

Table III (continued)

Microorganisms

Salmonella gallinarium

Klebsiella pneumoniae

Neisseria gonnorrhoeae

Hemophilus influenzae

Shigella sonnei

Erwinia amylovara

Brucella bronchiseptica ...,.,.,

Malleomyces mallei

Desulfovibro desulfuricans

Vibro comma,

Pasteurella multocida

Candida albicans

Saccharomyces cerevisiae

Micrococcus pyogenes var. Aureus 376 Micrococcus pyogenes var. Aureus 400 Micrococcus pyogenes var. Aureus K 3 Micrococcus pyogenes var. Aureus K 4

Mycobaeterium 607

Mycobacterium berolinense

Aerobacter aerogenes

Pseudomonas aeruginosa

Salmonella typhosa

Minimal

inhibiting

concentration

mcg / ml

12.5 1.56

25 0.19 1.56

12.5 1.56

12.5 3.12 6.25 0.19

25th

50 0.78 0.39 1.56 0.78 0.19 0.19 1.56 3.12 0.78

The antibiotic BA-163 is remarkably effective in treating a number of different types of infection and malignancy, and shows great specific activity against tumors. Either the pure crystalline product or one of the unpurified forms of the product can be used for this purpose. Either a filtered fermentation liquid prepared from Streptomyces flocculus isolates BA-163, BA-4721, X-13 or a solid or a liquid concentrate obtained therefrom can be used for this purpose. Such preparations should have sufficient potency to deliver a daily dose of at least about 50 to 500 meg of the pure antibiotic BA-163 per kg of body weight to the disease carrier. To achieve this, a concentration of the active ingredient in the carrier material of at least about 0.0001 ° / ₀ is required. A non-toxic carrier material is of course used for administration to humans and animals. Taking non-toxic becomes a

ίο Understood carrier material that is non-toxic if it is is administered in an amount sufficient to deliver the above dose of antibiotic B-163. It can either be a pharmaceutical carrier material, be it a liquid or a solid,

such as B. water, aqueous ethanol, syrup, an isotonic salt solution or glucose, starch, lactose, calcium phosphate, etc., an animal feed or a mixture of different materials, for example, are present in a filtered fermentation liquid. Both oral and parenteral administration give good results. Parenteral administration is preferred until a satisfactory diet suitable for the patient is developed.
In order to demonstrate the anti-tumor activity of the new substance, the percentage inhibition of a number of tumors in mice and rats by various forms of the antibiotic BA-163 is given in Table IV. The value given in the column with the heading "Dose, mg / kg" relates to the total weight of the composition administered. It does not refer to the concentration of the active ingredient in it. Because of the relatively low concentration of the antibiotic BA-163 contained in it, the actual dose of a fermentation liquor dried by freeze is considerably greater than that of the pure crystalline material. The antibiotic BA-163 is therefore a valuable tool for research purposes, since more mice and rats are used in cancer research.

TabeUeIV

Anti-tumor effect of dried fermentation liquids and purified fractions

of the antibiotic BA-163

Sample and description dose
mg / kg Number of survivors inhibition
^q / o Tumor type *) Liquid (by freezing
dried up) 500 5/6 51 S-180 the same 100 6/8 81 HS.l the same 100 8/8 97 HS-I the same 300 6/8 99 HS-I the same 200 7/8 99 HS-I the same 100 8/8 91 HS-I the same 250 10/10 90 CA 755 the same 100 the animals survive the con
troll animals l, 7 times L 1210 Solvent extract in amor
pher raw form (about 30 Ge
weight percent pure) 2.0 5/6 59 P 180 the same 1.0 6/6 49 P 180 the same 0.5 7/8 98 HS-I the same 0.75 the animals survive the Kan
troll animals l, 7 times L 1210

Table IV (continued)

10

Sample and description dose
mg / kg Number of survivors inhibition
% Tumor type *) Pure crystalline active
material 0.5 4/6 57
(Average of
four attempts) P 180 Pure crystalline material 0.3 6/6 29 P 180 the same 0.4 6/6 46 P 180 the same 0.2 6/8 94 HS-I the same 0.1 8/8 96 HS-I the same 0.05 8/8 94 HS-I the same 0.01 1/8 98 HS-I the same 0.10 10/10 98 CA-755 the same 0.15 9/10 100 CA-755 the same 0.20 6/10 100 CA-755 the same 0.06 the animals survive the
troll animals l, 7 times L-1210 the same 0.125 the animals survive the
trudge l, 7 times L-1210 the same 0.25 the animals survive the con
troll animals l, 7 times L-1210 the same 0.2 6/8 84 HEP-3 the same 0.1 8/8 56 HEP-3

*) The following methods, listed in the literature, were used to assess the tumor: S-180-Sarcoma 180 (Reilly et al., Cancer Research, Vol. 13, p. 684 [1953]). HS-1 Human Sarcoma No. 1 (Toolan, supra, vol. 13, p. 389 [1953]). CA-755-Mammary adenocarcinoma 755 (Gellhorn et al. Cancer Research Supplement 3, p. 38 [1955]). L-1210 Lymphoid Leukemia 1210 (L. W. Law, Journal Nat. Cancer Inst., Vol. 10, pp. 179 [1949]). HEP-3-Human Epidermoid Carcinoma, No. 3, Toolan, op. a. O.

HS-I appears to be very sensitive to this antibiotic. At concentrations as low as 0.01 mg / kg per day were achieved inhibitions of 95 to 99 ° / _0th A similar effect was obtained when subcutaneous or intraperitoneal administrations were made. The same inhibition was achieved by oral administration of about 5 to 10 fold doses.

When HS-I tumors were found / days after implantation, large doses of about 0.2 to 0.3 mg / kg were required to achieve strong tumor growth inhibitions of 80 to 90 ° / ₀ . In the meantime administered doses of the antibiotic BA-163, beginning on or before the fourth day of implantation, showed very pronounced anti-tumor effects with hardly any tissue residues.

BA-163 shows none remarkable compared to S-180 Effect. Compared to L-1210, the remarkable ratio of survivors to control animals was of 1.7 received. Almost complete inhibition of CA-755 was seen in two experiments with doses of 0.1 and 0.15 mg / kg per day was found.

The antibiotic BA-163 with advantage; in combination with one or more other carcinostatic agents can be used. For this purpose are compositions with one Content from 10 to 90 percent by weight of the active Part of the antibiotic BA-163 valuable- acquaintances carcinostatic agents used with the antibiotic BA-163 in such compositions can be; are the means · of the type of nitrogen mustard, e.g. B. 6-mercaptopurine, 8-azaguanine, urethane, o-diazo-5-oxo-l-norleuzin (DON), azaserine, triethylene melamine, Mitomycin C, triethylenephosphoramide, 1,4-dimethylsulfonyloxybutane, the carcinostatic folic acid analogs, ethyl carbamate and similar substances.

The invention relates to the cultivation of Streptomyces nocculus isolate BA-163, Streptomyces flocculus BA-4721 and Streptomyces flocculus X-13 under certain conditions for the production of the antibiotic BÄ-163. Useful biologically active filtrates are prepared, for example, using an aqueous nutrient medium containing 10 g / l glucose, 15 g / l soybean meal, 5.0 g / l corn meal, 2.5 g / l soluble products from the distillation of spirits and 2.0 g / l contains calcium carbonate. This nutrient medium is adjusted of 7.0 before the treatment in the autoclave to a p _H value. The inoculation material can either consist of spore suspensions or a previously established growth of the organism. Fermentation is carried out at 28 ° C either by shaking culture or in a small vessel with mechanical ventilation for about 60 to 80 hours. The progress of fermentation is monitored by standard sampling procedures using B. subtilis, Staph. aüreus, No. 376, or E. coli, No. 21, followed as test organisms.

A large number of fermentation media have been tested with useful results. It's a medium required, its basic components from a nitrogen source, a carbohydrate source and from minerals

009 609/381

11 12

consists. Useful sources of nitrogen supply are measurements on a KBr bead containing 1% of the pro numberless proteinaceous substances, such as hydrolyzed casein: 706, 750, 784, 810, 823, 867, 915, 1000, various types, soybean meal, casein, soluble 1035 , 1072, 1081, 1095, 1176, 1203, 1227, 1289, 1342, products from brandy distillation or corn flour. 1377, 1406, 1445, 1464, 1504, 1585, 1603, 1675, 1715, Suitable carbohydrates provide e.g. B. Dextrose, Glycerin, 5 1730, 2793, 2890, 3175 and 3311 cm- ¹ . The curve is made of dextrin or starch. Less satisfactory er- shown in Fig. 1 in detail. The pure crystalline results will be obtained with mixtures of lactose and corn. Substance dissolved in methanol at an amount of 1%, obtained spring water. The above-mentioned substances are then the absorption maxima in the ultraviolet range often contain sufficient amounts of minerals to range of the spectrum at 245 ΐημ E = } £ = 760, the requirements of the organism for minerals to io 390 πιμ, E = ^ ₀ * = 344. satisfy at using a buffer without substantial additional mineral with a _pH value of 6, the absorption material appear to be added. maxima in the ultraviolet range at 245 ΐημ, Ε = } *,

After a fermentation of 60 to = 750 and 365 πιμ, E = Z ₀ * = 280.

80 hours a satisfactory efficiency of the antibiotic BA-163 is in water at neutral

Antibiotic BA-163 has been achieved, the 15 _pH value and acid p _H values only slightly soluble.

Fermentation liquid using 3 to Its acidic character is expressed by its proportion-

5 percent by weight of a diatomaceous earth filter aid film-wise greater solubility in aqueous sodium bicarbonate

trotted. The content of the filtrate of antibiotic can carbonate or buffers with a _pH value of 7.0

with an organic or more immiscible with water. In addition, the substance appears to be alkaline

Solvents, e.g. B. preferably butanol, methyl 20 Ph values to be unstable. She's in ether, benzene

isobutyl ketone or ethyl acetate or a little less and methylene chloride very weak and in ethyl acetate,

cheap with ether, benzene, toluene, methylene chloride, the lower alkanols from methanol to butanol and

Chloroform or carbon tetrachloride. Acetone sparingly soluble. It dissolves in more focused

About 30 ° / ₀ of the overall effectiveness of the antibiotic sulfuric acid and is a light yellow solution. One

remain in the mycelium cake when the fermentation liquid 25 alcoholic ferric chloride test solution takes a dark-

is filtered at a _pH value of 4.0. This part turns greenish brown in color when the antibiotic

is easily dissolved in it by extracting the cake with a BA-163. A reddish-brown watery one

lower alkanol, e.g. B. methanol, ethanol or iso solution turns light yellow when treated with sodium disulfite,

propanol, obtained. If this yellow disuL & t solution with with water not

During the solvent extraction to obtain the miscible solvents, in which antibiotic BA-163 from fermentation liquids, it is useful, the antibiotic BA-163 is soluble, such as. B. Äthylmäßig, this first acetate to a neutral or slightly acidic or chloroform, the extract was reddish brown to bring _pH value. It is a p _H range of 3.5 to and including a antibiotically effective solute. As the 7.2 preferred. The extraction proceeds relatively antibiotic BA-163 is an acidic substance, it forms more quickly and completely at a _pH value of 3.5 35 with basic substances such. B. Amines, Metallvonstätten than at 7.2. hydroxide and basic metal salts, salts. These

Obviously there is more than one antibiotic effective salt, which the antibiotic BA-163 with alkaline and substance in the fermentation of the Streptomyces flocculus- alkaline earth metals, z. B. Natiium, Potassium, Lithium, Isolate BA-163, BA-4721 and X-13 are produced. When calcium, magnesium, etc., are formed, ethyl acetate is used as an extractant because of their solubility, so becomes valuable as dosage forms. The properties and reactions of the BA-163 concentrated in the concentrate with five times its volume under reduced pressure and carried out in the other extract suggest a structure of the oxyquinone type. Isopropyl ether treated. Most of the active material for the technical production of the antibiotic is made from the organic solvent BA-163, a submerged culture is used in a usual mixture by shaking with successive 45 units. Appropriate containers have extracts a quantity of a phosphate buffer (p _H -Wert7,5), capacity 7560-756001 or more to a fresh amount of buffer with a an extract and are obtained with agitators and apparatus for asepnur light brown color. A substantial part of the table ventilation of the contents up to two or the antibacterial effectiveness, however, remains provided in the multiple volume of air per minute. An organic layer. The substance responsible for this effectiveness, which can be used for industrial large-scale production, appears to be a different one, but it is narrowly given above.

to be related antibiotic that has a somewhat The growth of the microorganism and the pro less acidic character than the antibiotic induction of the antibiotic usually achieve it BA-163. Maximum after about 60 to 80 hours at 28 ° C, as at

The antibiotic BA-163 is an acidic substance which has been found in one of the above test procedures. behaves like a weak monovalent acid. However, the influence changes in the used potentiometric titration in dioxane shows a measuring system, the aeration rate, stirring cash _pH value from 6.3 to 6.5. The time to reach the maximum composition often found by microanalysis, etc., is as follows: C = 58.3%, potency. In each case, a time of at least H = 4.5%, N = 10.9%, methoxyl = 18.0%, oxygen 60 about 24 hours is required. Aeration of the medium = 26.3%. The pure crystalline antibiotic BA-163 at submerged growth is kept at about ¹ Z ₂ to 2 volumes, has a dark maroon to black color air per volume of liquid per minute, and crystallizes from a mixture of acetone and aseptic conditions must of course during the ethyl acetate ( 1: 5) as a thin, elongated rhombohedral transfer of the inoculum and the growth of the plates, whose melting point to 27O ⁰ C 65 microorganism can be maintained at the 268th The lies. Individual crystals of the substance appear orange- removal of the mycelium and extraction of the antibiotic colors when they are carried out through a microscope according to the description above,
turning viewed from translucent light. It is similar to many carcinostatic agents that has the following absorption maxima with the following antibiotic BA-163 as well as its methyl ether a somewhat wavelength in the infrared region of the spectrum at 70 toxic material. However, therapeutic doses can be used

can be administered without any substantial adverse effect. This is illustrated by the data in Table V below. White Swiss mice received five injections of 1 cm ³ aqueous solution or suspension, one after the other, each day, each containing the same dose of the antibiotic BA-163 or its methyl ether. The following surviving animals were counted:

Table V

Toxicity of the antibiotic BA-163 and its methyl ether

BA-163 number of Methyl ether number of Above Above dosage living dosage living 3/6 0/3 0.75 mg / kg 5/6 25 mg / kg 2/3 0.5 mg / kg 6/6 15 mg / kg 2/3 0.375 mg / kg 10 mg / kg Untreated 6/6 Control attempt

example 1

A nutrient medium of the following composition was prepared, adjusted to a _pH value of 7.0 and sterilized:

Glucose 10 g / l

Soybean flour 15 g / l

Soluble products from the distillation of spirits 2.5 g / l

Dipotassium phosphate 5.0 g / l

Sodium chloride 2.0 g / l

Calcium carbonate 2.0 g / l

Tap water to replenish the volume

The inoculum is BA-163 manufactured by transfer of the growth of a strong versporten slant culture medium of Streptomyces flocculus isolate a part of this medium and incubating for about 36 to 40 hours at 28 ⁰ C on a rotary shaker. The majority of the medium is then inoculated by mixing it with 5 percent by volume of the inoculum so produced. The ventilation is carried out with about 2 volumes of air per minute, and it is stirred vigorously ^{at 26 to 30 ° C. during the incubation.} The progress of the fermentation is determined by testing the filtered liquid at certain intervals using Staph. aureus No. 376 and Ecoli No. 21 as the test organism. Typical zones of inhibition of the useful broth are 27.0 and 21.0 mm, respectively.

When the fluid thus prepared and nitrated is injected into mice which have sarcoma 180 tumors as determined by the method described by Reilly (op. Cit.), The tumor growth is inhibited to a considerable extent according to the Reilly standards. Similar results are obtained with one to two fold dilutions of the filtrate. The concentration of the antibiotic in such a filtrate is approximately 0.002 ° / _0th

Example 2

A fermentation liquid is prepared according to the method described in Example 1 and carried out Filtration purified using about 3 to 5 weight percent diatomaceous earth filter aid. That

Filtrate is then adjusted to a _pH value of 4.0 and extracted with one third of its volume, ethyl acetate: The ethyl acetate extract is then nitrided to free it of insoluble foreign matter, and then at 25 to 3O ⁰ C under reduced pressure to constricted a small volume. About 30 ° / ₀ of the produced antibiotic effectiveness remains in the mycelial cake. This is done by stirring the wet cake with methanol, filtering and concentrating the aqueous

ίο methanol extract up to. Evaporation of most of the methanol recovered. This concentrate is then adjusted to a _pH value of 4.0 and extracted with about an equal volume of ethyl acetate. The two ethyl acetate concentrates are then combined and treated with five times their volume of isopropyl ether. This solution is then shaken several times with a phosphate buffer having a _pH value of 7.5, until the final extract shows only a slight brown color. The combined aqueous

aa buffer extract is then adjusted .on a _pH value of 4.0 and extracted several times with methylene chloride. The concentration of the methylene chloride extract and careful treatment of the concentrate with ether lead to a "precipitation of the antibiotic BA-163 as a dark brown crystalline material in the form of thin elongated rhombohedral plates that have a dark maroon to brown color. Their melting point is 268 to 270 ° C.

_{3 <)} Analysis:

Found

C = 58.28%, H = 4.50 ° / ₀ ,
N = 10.93 o / _o .

Administering this material as in Example 1 at a dose of 0.5 mg / kg body weight to mice with the tumor S-180, one achieves a 57 ° / ₀ strength reduction of the average tumor weight of the treated mouse.

An O.OOOl% solution of the pure crystalline antibiotic BA-163 produced in this way in an isotonic Saline solution as a carrier is suitable for the parenteral treatment of sensitive infections and Tumors.

Example 3

The procedure of Example 2 is repeated with the following modification. The filtered liquid is adjusted to a _pH value of 7.0 and extracted repeatedly with 0.5 times its volume of butanol After a substantial amount of the product by extraction of the mycelial cake is recovered. An amorphous crude product by the procedure described in Table III type is obtained by treatment of the concentrated solvent extract with isopropyl ether and extraction with a phosphate buffer having a _pH value of 7.5. The acidification of the buffer extract and extraction of the same with methylene chloride gives a solution from which an amorphous product with a purity of about 30 percent by weight is obtained by removing the solvent.

Example 4

It becomes a fermentation liquid according to the example!

and aseptically filtered through a bacteriological filter. It gives a sterile filtrate, which frozen and converted from the frozen to a dried state. The dried residue consists of a product containing about 0.1 percent by weight of the antibiotic BA-163.

Claims (1)

15 16 Example 5 In a similar way, using Calcium hydroxide, magnesium hydroxide, sodium hy- The antibiotic BA-163 is based on the process of hydroxide and lithium hydroxide, the calcium, magnesium, of Example 2, but using the following sodium and lithium salts. Culture medium and the S. flocculus isolate X-13. 5 _,. . , " Example 8 Dextrose 10.0 g / l equivalent amounts of sodium acetate and anti- Diammomum phosphate 2.0 g biotikum BA-163 were dissolved in glacial acetic acid. The mixture "W ^el _,; _ 'ί ,, was frozen and dried in the frozen state, MaisqueUwasser 25 0 g / l _{Iff is the sodium salt of ^d it} antibiotic BA-163 to Give distilled water to make up the volume Example 6 If the S. flocculus isolate BA-163 is replaced by the claim:
S. ffocculus isolate BA-4721, you also get the 15th
Antibiotic BA463. . “The use of Streptomyces flocculus ATCC - ⁰⁶¹⁵ P ¹⁶¹ ' 13257, ATCC13535 and ATCC13536 or by their Equivalent amounts of pure crystalline anti-mutants or variants for the production of the antibiotic BA-163 (Example 2) and potassium hydroxide ao biotikums BA-163 by the usual fermentative route were dissolved in water. Fibers and insolubles under submerged aerobic conditions filtered off, the clear solution was frozen and, if the antibiotic was obtained by solubilization dried frozen state. The powder obtained means extraction and, if necessary, transferring the is the potassium salt of the antibiotic BA-163. Antibiotic in its salts or esters. 1 sheet of drawings © 009 609/381 9.60