DE1111341B - Production and production of actinomycin P - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY KL.30h 6

EMTERNAT.KL. C 12 d

GERMAN

PATENT OFFICE

EXPLAINING EDITORIAL 1111341

P24574IVa / 30h

REGISTRATION DATE: MARCH 10, 1960

NOTICE
THE REGISTRATION
AND ISSUE OF
EDITORIAL: JULY 20, 1961

The invention relates to the antibiotic actinomycinP ₂ in raw and in purified form and its production, concentration and isolation. Actinomycin P ₂ is suitable for combating malignant tumors in animals and also in humans. In addition, it has a growth-inhibiting effect on gram-positive bacteria, which makes it suitable for many types of application in human and veterinary medicine, industry and agriculture. It is also suitable as a disinfectant and for separating mixtures of microorganisms for medical diagnostic and research purposes.

The newly discovered microorganism that is used in the process according to the invention, differs from other common antibiotic producing species. As it is the weak hyphae and Exhibited chains of spores, typical of members of the genus Streptomyces, it was planted on media, which are generally used to identify organisms of this genus. Final exams of the Media were run after 2 weeks of appropriate incubation.

The newly discovered microorganism was identified as a new strain of Streptomyces aurefaciens Duggar. It's in the Chas culture collection. Pfizer & Co., Inc., specified as Culture A-500. A culture of this microorganism has been deposited in the American Type Culture Collection, Washington, D. C; there it was given the number 13,336. The appearance of the culture described in Table I agrees well with the description of this species, but has the following minor differences: it produced a soluble pink pigment in milk, while for S. aureofaciens no color is described; they manufacture and obtain actinomycin P ₂

Applicant:

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

Representative: Dr. W. Beil, A. Hoeppener and Dr. H. J. Wolff, lawyers,

Frankfurt / M.-Höchst, Antoniterstr. 36

Claimed priority: V. St. v. America March 10, 1959

Koppaka Visveswara Rao, Pinebrook, N.J.,

William Stephen Marsh, Ringwood, N. J., and Donald Walter Renn, New Milford, N. J.

(V. St. A.),
have been named as inventors

showed an orange-colored to orange-brown, soluble pigment on potato culture media, while Duggar stated: "Color of culture unchanged"; the pigment of the cultures changed in different Media from yellow-brown or yellow-green to yellow and orange-brown, whereas S. aureofaciens a "golden yellow" Has pigment as described.

Table I.
Characteristic properties of Streptomyces aureofaciens, ATCC 13336

medium growth Aerial mycelium Spurs
education Soluble pigment Remarks Skimmed
milk
Glucose agar small amount
Well moderately white-gray
Well; know up
yellow-gray, will
darker no
Gray pinkish yellow brown
deep yellow brown Vegetative mycelium cream-colored
to pale yellow to pin
head-sized dark gray
Points; Milk not curdled,
peptonized or hydrolyzed;
p _H value not changed.
Vegetative mycelium not recognizable
bar; Bright yellow reverse side.

109 648/356

Table I still

medium growth Aerial mycelium Spurs
education Soluble pigment Remarks Nutrient agar poor until poor, mostly on dark bright yellow Vegetative mycelium colorless where moderate Reason of Gray visible; Back yellow to Oblique olive gray. soil white to completely dark Gray More synthetic moderate moderate to good, dark yellow Vegetative mycelium colorless, see above Agar white to light Gray visible from afar; Underside yellow gray to darker to olive gray. Gray Calcium malate Well Well; know up Gray yellow Vegetative mycelium yellow, so far agar light gray to visible; Back yellow; Malate dark gray digested. Strength- poor is missing is missing no Vegetative mycelium yellow; Under Tile side yellow; Zone partial hy drolysis, 2.0 cm diameter. Gelatin- poor very poor; is missing no Vegetative mycelium light yellowish Tile know until completely Brown; Reverse light yellowish dark gray Brown; Liquefaction zone, Diameter 1.5 cm. Cellulose out Well; know up dark nothing, yes Vegetative mycelium not recognizable draws dark gray Gray Edge of ge bar. strained Strei fens yellow-orange potato out moderate to good; light gray yellow Vegetative mycelium golden yellow to bits draws white to yellow deep reddish orange; Back lich to light gray side reddish orange. Dextrose Well Well; know up dark yellow Vegetative mycelium colorless to Nitrate broth dark gray Gray yellow; Nitrates become Ni kick reduced. Emerson agar out Well; know up yellowish yellowish brown Vegetative mycelium not recognizable draws yellowish gray Gray bar; Reverse yellowish brown. Glucose- poor until very sparse; is missing nothing Vegetative mycelium colorless to Asparagine moderate yellowish white yellow; Yellow back. Tile Suitable for spore formation neten media; Become spurs in long intertwined ket th formed, oval to cylindrical drisch, 1.0 to 1.3 x 1.6 to 1.9 μ, formed by division.

It goes without saying that the production according to the invention of the antibiotic, actinomycin P ₂ , is not restricted to the organisms mentioned. Above all, it is intended to include mutants obtained by various means such as X-ray irradiation or ultraviolet treatment, mustard gas bodies, single cell culture methods, and the like.

Actinomycin P _{2 has} shown strong efficacy in the treatment of a wide variety of infections, including tumors and other malignancies. For these purposes, either the pure, crystalline substance or one of the raw forms described in more detail below can be used. The selected substance must be largely free of the known toxic actinomycin B. It must contain so much antibiotic that the patient is given a daily dose of at least 50 to about 500 γ pure actinomycin P ₂ per kilogram of body weight. Actinomycin P ₂ as a pure crystalline material or in one of its crude forms can be used in medicinal compositions in conjunction with a therapeutically acceptable carrier. In general, a composition of this type contains at least 0.0001 percent by weight antibiotic, but can also contain 90 percent by weight or even more. A composition containing less than 0.0001% may be effective, but is generally unsatisfactory because it requires the administration of an unusually large amount of vehicle. The term therapeutically acceptable carrier in this invention includes the usual carrier substances, such as humans and animals

can be administered safely. They can be liquid or solid. It can e.g. B. Carriers such as water, aqueous ethanol, simple syrup, isotonic saline or glucose, Starch, lactose, calcium phosphate, etc. can be used.

In animals it may be beneficial to mix the active substance in the feed. The active substance can administered orally or parenterally. In the initial stage of treatment, parenteral administration

move forward until a suitable treatment is found for the patient; after that you can continue to do so or choose a different route of administration.

In order to make the effectiveness of the antibiotic according to the invention against tumors clear, the percentage inhibitory effect of different types of the agent according to the invention with a number Tumors in mice and rats are given in Table II.

Table II Antitumor Activity

Sample and description dosage
(mg / kg) Mortality
rate Percentage comparison 72
(Average of five attempts) Art
of the tumor * Actinomycin P ₂ (raw) 0.25
0.3
0.40
0.60 0/10
0/10
0/6
0/6 27
40
54
55
(Average of two attempts) CA-755
CA-755
Sa-180
Sa-180 0.75 1/6 13th Sa-180 0.2 Animals lived 1.75 times longer than comparison animals
(Average of two attempts) L-1210 0.1 0/8 HS No. 1

Effective minimum concentration 0.03 y / cm ³ HeLa

Actinomycin P ₂ (pure
crystalline material) 0.375
0.3 0/10
0/10 12th
24
(Average of three attempts) 0/6
1/6 57
54 CA-755
CA-755 0.25 0/10 40
(Average of two attempts) Animals lived 0.7 times longer than comparison animals CA-755 0.5 0/6 58
(Average of two attempts) Sa-180 0.6
0.75 0/6
1/6 33
58 Sa-180
Sa-180 0.45 Animals lived 1.3 times longer than comparison animals L-1210 0.375 Animals lived 1.6 times longer than comparison animals
(Average of two attempts) L-1210 0.25 Animals lived 1.7 times longer than comparison animals L-1210 Minimum effective concentration 0.05 to 0.10 jVcm ³ HeLa Actinomycin P ₁ (purer
crystalline substance 0.1
0.05 Sa-180
Sa-180 0.25 L-1210

Effective minimum concentration 0.02 y / cm ^s HeLa

* Methods given in the literature for evaluating experiments on tumors are the following: S-180 (Sarcoma 180) - Reilly et al., Cancer Research, 13, p. 684 (1953). HS No. 1 (Human sarcoma No. 1) - To ο lan, a. a. O., 13, p. 389 (1953). CA-755 (Mammary adenocarcinoma 755) - Gi 11 Hör et et al., Cancer Research Supplement, IH, p. 38 (1955). L-1210 (Lymphoid leukemia 1210) - L. W. Law, Journal Nat. Cancer Inst., 10, p. 179 (1949).

In tumor therapy, actinomycin P _{2 can} advantageously be used together with one or more cancer-inhibiting agents. Compositions containing 10 to 90 percent by weight of active ingredients of this antibiotic are suitable for this purpose. Anti-cancer agents which can be used in such compositions with this antibiotic are the mustard gas type anti-cancer agents, 6-mercaptopurine, 8-azaguanine, urethane, 6-diazo-5-oxo-l-norleucine (DON), azaserine,

Triethylenmelamine, Mitomycin C, Triäthylenphosphoramid, 1,4-Dimethylsulfonyloxybutan and the anti-cancer folic acid analogs, ethyl caramate

and similar.

One uses to produce the antibiotic

an aqueous nutrient medium containing 10 g glucose, 15 g soybean oil, 50 g corn flour, 2.5 g soluble residues per liter from alcohol distillation (Distillers Solubles) and 2 g calcium carbonate. This nutrient medium is applied to the autoclave before treatment

p _H value placed 7.0. Either spore suspensions or previously grown cultures of the organism can be used as inoculation material. Fermentation is carried out at 28 ° C by shaking culture or fermentation in small, mechanically ventilated vessels for about 60 to 80 hours. The course of fermentation is observed by experimental methods with normal platelets using B. subtilis or Staph. aureus, no.376.

A variety of fermentation media have been studied and found to be satisfactory. It is a medium is required, which consists mainly of a nitrogen source, a carbohydrate and minerals consists. The nitrogen sources that meet the requirements include a large number of proteinaceous sources Substances like different types of hydrolyzed caseins, soybean meal, casein, soluble residues from alcohol distillation, corn flour, etc. Suitable sources of carbohydrates are dextrose, glycerin, Dextrin, starch, etc. Less satisfactory results will be obtained with mixtures of lactose and swollen Corn scored. The above substances often contain enough minerals to meet the mineral needs of the To cover the organism without adding large amounts of mineral components have to.

Once a satisfactory level of antibiotic activity is achieved, the fermentation liquor is filtered after fermentation for 60 to 80 hours using 3 to 5 percent by weight diatom filter aid. The antibiotic content of the filtrate can be removed from this with the aid of a water-immiscible, organic solvent, such as preferably butanol, methyl isobutyl ketone, ethyl acetate or, less satisfactorily, ether, benzene, toluene, methylene chloride, chloroform or carbon tetrachloride. A portion of the total antibiotic substance remains in the mycelial cake, if the culture medium is filtered at a _pH value of 4.0. This part can easily be obtained by extracting the cake with a lower alkanol such as methanol, ethanol or isopropanol.

It appears that during the fermentation process of Streptomyces aureofaciens ATCC 13,336 more than forms an antibiotic substance.

The desired substance can be isolated by a variety of means such as those already indicated. The use of n-butanol is recommended as a cheap method for isolating the active substances as an extractant. They can be obtained by simply reducing the solvent Pressure is evaporated; but in order to keep the decomposition within the smallest possible limits, it is if also not necessary, but preferable to azeotropically remove the butanol solution. This is achieved by continuously adding small amounts of water while the extractant is under reduced Pressure is distilled. At atmospheric pressure, the azeotropic mixture contains 38% water and 62% butanol. The mixing ratio can be slightly different at reduced pressure, but the effect is the same. When most of the butanol is removed, the remaining solution becomes with the mycelium extract, preferably a methanol extract, combined and the mixture under reduced Pressure concentrated almost to dryness. It is in a chromatographically suitable solution, e.g. B.

Ethyl acetate, added and the solution moistened with a with the chosen solvent Acid washed alumina column passed. Approximately one uses to make the column 5 to 15 grams of clay per gram of raw antibiotic. This column is with the solvent - preferably Ethyl acetate - washed until the dark red strip of actinomycin B is completely eluted. The elution is continued by gradually increasing the methanol content in a mixture increased from methanol and ethyl acetate. The fractions are examined by paper chromatography. Fractions containing 5 to 10% methanol - ethyl acetate usually also contain the most of the valuable antibiotic according to the invention. These factions are being united, almost concentrated to dryness and crystallized from a mixture of methylene chloride.

This product is crude actinomycin P ₂ , in effect

a mixture of actinomycin P ₁ , actinomycin P ₂ and actinomycin P ₃ . It can be used medicinally, that is, to treat humans and animals that have a tumor. That the various fractions are present in the raw mixture

as are detected by paper chromatography with a benzene-formamide mixture. The paper sheets are immersed in a mixture of methanol and formamide, ratio 1: 1, and soaked. Then the samples are applied and developed with benzene saturated with formamide. Under these circumstances, the following R _r values resulted for the actinomycin mixture: P ₁ = 0.8 to 1.0, P ₂ = 0.5 to 0.6, P ₃ = 0.0 to 0.2. In contrast to the known actinomycins such as actinomycin B, C and D, the types of actinomycins according to the invention do not follow the solvent front of the system.

A comparison with the known types of actinomycin has shown that actinomycin P _{2 has} the following differences compared to these:

_{1. The presence of the following amino acids in actinomycin P 2 was} determined by hydrolysis for 16 hours with 6N HCl at 100 ° C and comparison by paper chromatography:

«-Hydroxyproline,

Valine,

Sarcosine,

Threonine,

(Traces of proline).

2. The elemental analysis of three specific samples obtained from different fermentation processes resulted in the following average values in very good agreement: C = 56.19, H = 7.29, N = 12.37, 0 (difference) = 24.15. An N-methyl determination gave the average value 12.5.

3. Due to the presence of -hydroxyproline in actinomycin P ₂ , the possibility that this is described in German patents 944 395, 934715, 925 064 or 930 579 as actinomycin C ₁ , C ₂ or C ₃ is excluded. the

Separation methods and systems described therein are not the same as those used with actinomycin P ₂ .

"Α. With respect to German Patent 912,010 (Actinochrysin) is to say that the actinomycin P is not eluted from Merck-alumina in the conditions described therein _2. The melting point of actinomycin P ₂ is lower than Actinochrysin.

5. A comparison with actinomycin F ₁ and F ₃ (German Auslegeschrift 1021131) shows that the analysis of P ₂ agrees with F ₁ , but this does not contain hydroxyproline and therefore cannot be the same substance as P ₂ . The analysis of F ₃ and the amino acid content show that this is not identical to P ₂ .

For the industrial production of actinomycin P ₂ , immersion cultures are used in the customary devices known to the person skilled in the art. Suitable containers have a size between 7500 and 76001 or more and are equipped with suitable agitators and devices for aseptic ventilation of the contents with up to 2 volumes or more air per minute. A suitable medium for broad-based production is indicated above. The cultivation of the microorganism and the production of the antibiotic normally reach ^{their peak after about 60 to 80 hours at 28 °} C., as was determined by one of the above-mentioned test methods. However, differences in equipment used, levels of ventilation, agitation, etc. often affect the time it takes to reach maximum activity. A period of at least about 24 hours is required in any case. For aeration of the medium, about ¹ hour to 2 volumes of air per minute and per volume of fermentation liquid are carried out with submerged cultivation. Of course, aseptic conditions must be observed during the transfer of the inoculum and the cultivation of the microorganism. The mycelium is removed and the antibiotic is obtained as described.

Like many cancer-inhibiting agents, actinomycin P _{2 is} somewhat toxic. Therapeutic dosages, however, can be administered without substantial deleterious effect, as evidenced by the data in Table III. In Swiss white mice, five successive daily injections of 1 cm ^{3 of} aqueous solutions containing the specified antibiotic doses were made. Mortality rate and average weight loss are given.

f P ₂ (raw) r P., (purer
more crystalline Tabel III Weight
loss Actinomycin Material) Toxicity
Dose: mg / kg Mortal
rate 1.2 5/6 - 1.0 3/6 - 0.8
0.6 1/6
0/6 P ₁ (purer 0.4 0/6 - crystalline ■ 0.2 0/6 Material) 2.5 6/6 5 2.0 4/6 3.6
4.1
4.0 1.1
1.0
0.9 1/6
1/6
0/6 4.0 0.8 0/6 3.7 0.7 0/6 2.5 0.6 0/6 0.2 6/6 5.7 0.15 5/6 5.1 0.125 2/6 3.7 0.1 3/6 0.3 0.05 0/6

From these data it can be seen that the actinomycins of the invention are much less toxic than the known types of actinomycin B, C and D.

Example I.

A nutrient medium having the following composition is prepared brought to _pH value 7.0 and sterilized:

Cerelose 10 g / l

Corn starch 10 g / l

N-Z-amine B 5 g / l

Curbay 5 g / l

Sodium chloride 5 g / l

Soybean oil 15 g / l

Calcium carbonate lg / 1

Tap water to make up for the volume
to fill up.

The inoculum is produced by growing a culture medium from Streptomyces aureofaciens ATCC 13 336, which shows good sporulation, brought into contact with part of this medium will. It is incubated in a rotating shaker for about 36 to 40 hours at 28 ° C.

The main part of the medium is then inoculated by adding 5 percent by volume of the so produced Inoculums is mixed. About 2 volumes of air per minute per volume of the are used for ventilation Medium passed through, and during the incubation at 26 to 30 ° C is stirred vigorously. The progression The fermentation is carried out by using platelet testing of the filtered medium at intervals pursued by B. subtilis as a test organism. The fermentation product is obtained after 48 to 72 hours.

The fermentation liquor is then filtered using diatomaceous earth as a filter aid; the filtrate is extracted with half the volume of n-butanol. The mycelium cake is extracted without interruption with 500 cm ^{3 of} methanol. The n-butanol is evaporated under reduced pressure and the methanol extract is added. The combined extracts are evaporated almost to dryness and taken up with ethyl acetate. The ethyl acetate is passed over acid-washed clay which has been moistened with additional ethyl acetate. Approximately 5 to 15 grams of clay per gram of raw material is used. The column is washed by adding more ethyl acetate until the dark red band of actinomycin B is completely eluted. The column is then washed with various methanol-ethyl acetate mixtures, each mixture containing progressively more methanol. The fractions, which contain 5 to 10% methanol, are combined and evaporated almost to dryness under reduced pressure. Der.Rückstand is recrystallized from a mixture of methylene chloride and ether. The product is obtained by filtration. It contains actinomycin P ₂ , as well as a smaller amount of actinomycin P ₁ and actinomycin P ₃ . Not enough tetracycline or chlortetracycline is found in the fermentation media for it to be separated.

These three components are separated by column division chromatography. A mixture of benzene (41), formamide (50 cm ³ ) and Celite 545 (100 g) is stirred to a homogeneous slurry, poured into a suitable column and inflated by air pressure

109 648/356

pressed tight. Celite545 is the trade name for a form of diatomaceous earth. The actinomycin mixture (5 g) is dissolved in formamide (10 ml) and the solution is ^{shaken with benzene (200 cm 3} ) and Gelite 545 (20 g). This slurry is brought to the top of the packed column and compressed. Then the column is developed with benzene saturated with formamide. Three bands could be distinguished on the column, the migration of which was followed with the help of the optical density at 440 ταμ ίο and the activity against B. subtilis. Actinomycin P ₂ appeared as the second and largest fraction. The first fraction, actinomycin P ₁ , and the third fraction, actinomycin P ₃ , were enriched and the corresponding types of actinomycin were recrystallized from a mixture of methylene chloride and ether.

After concentration and recrystallization, the largest fraction, actinomycin P ₂ , was purified by countercurrent distribution using one or a successive combination of the following systems: (1: 1) benzene-formamide; (4: 2: 1.1: 2) isopropyl ether-ethyl acetate-pyridine-water and (2: 2: 1.2: 2) pyridine-water-ligroin-benzene. The fractions containing actinomycin P ₂ are concentrated and the residue is recrystallized from acetone and ether (1: 9). The antibiotic is deposited in bright red, rectangular platelets that melt at 242 to 245 ° C (decomposition). The elemental analysis shows the following results: C = 56.2%, H = 7.3%, N = 12.4%. The ultraviolet adsorption spectrum in methanol shows a maximum at 234, 425 and 443 ταμ, E} * _m values are 197, 85 and 85, respectively. The infrared spectrum of the antibiotic in potassium bromide shows maxima at the following points: 2.93, 305 (shoulder) , 3.42, 5.72, 6.04, 6.15, 6.31, 6.60, 6.73, 7.09, 7.35, 7.55, 7.65, 7.85. 8.15 (shoulder), 8.36, 8.50 (shoulder), 8.90 (shoulder), 9.15, 9.30, 9.45, 9.95, 10.15, 10.60, 11 , 00, 11.50 and 12.10 m «. Actinomycin is a neutral substance, slightly soluble in water and moderately soluble in lower alkanols, acetone, ethyl acetate, methylene chloride and benzene.

Example II

The raw product prepared in Example I is used in a dosage of 0.6 mg per kilogram Body weight administered parenterally to mice with a Sa-180 tumor. It makes you 65% decrease in the mean tumor weight of the treated mice compared to untreated comparison animals.

Claims (1)

PATENT CLAIM: The use of Streptomyces aureofaciens ATCC 13 336 or its mutants or variants for the production of actinomycin P ₂ by the usual fermentative route under submerged aerobic conditions and, if appropriate, separation of the antibiotic from the nutrient medium, if appropriate by solvent extraction. Considered publications:
German patents nos. 912 010, 925 064,
930579, 934715, 944395;
German interpretative document No. 1 021131. 1 sheet of drawings © 109 648/356 7.61