DE1770204C - - Google Patents

Description

The present invention relates to adriamycin and its salts with pharmacologically acceptable inorganic and organic acids and therapeutic compositions containing them. They are new antibiotic substances, p which are particularly useful in therapy as anti-tumor products. Adriamycin is also called Antibiotic "B-106 F.I."

Adriamycin is made using a new microorganism that mutates from the known strain Streptomyces peucetius, which is described in British patent specification 1 003 383 and in Giorn. Microbiol., Vol. 11, 1963, pp. 109-118, biosynthetically produced. The new tribe is under the name F.I. 106 deposited in the microbiological collection of Farmitalia He is called Streptomyces peucetius var. caesius. He is also with the Commonwealth Mycological Institute, Ferry Lane, Kew, Surry (England) with index number I.M.I. 131 502 and at the institute of Microbiology of the Rutger University (U. S.A.) with index number I.M.R.U. 3920 deposited.

The new microorganism has the following microscopic, macroscopic and biochemical properties:

Microscopic properties

The vegetative mycelium on the usual culture media shows thin hyphae (0.5 to 0.9 μ thick), which are more or less long and branched. The branches form thicker hyphae (1.1 to 1.6 μ thick), the conidiophores are often collected in tuft-shaped forms that end in hooks. The conidia are spherical with a diameter between 1.8 and 3.3 μ, first arranged in small chains, then free.

Under the electron microscope, the conidia appear almost spherical, with irregular edges and a warty surface.

Macroscopic properties

In Table I the cultural properties are recorded which are determined on the indicated media in which the microorganism was grown at 28 ° C, the observations on the 3rd, 8th, 15th, 21st and 30th days after inoculation.

Biochemical properties Gelatin: slow and partial hydrolysis. Nitrates: no reduction to nitrites. Production of hydrogen sulfide: positive. Milk: no peptonization; no coagulation. Strength: very slow and weak hydrolysis.

Maltose, xylose, mannose, mannitol, glycerin, Glucose, sucrose, trchalose, raffinose, Fructose are used as a nutrient medium.

Lactose, adonitol, ramnose, sorbitol, arabinose, esculin and meso-inositol are considered to be Culture medium not used

Antibiotics: It creates substances with antibiotic and anti-tumor activity.

Classification of the mutant »F.I. 106 «

The mutant FI 106 has the following taxonomic position: In the classification system of Pridham et al. (Appl. Microbiol., Vol. 6, 1958, p. 52) the microorganism belongs to the group Retinaculum apertum, series RED. In the classification system of B a 1 dacci (Giorn. Microbiol., Vol. 6, 1958, p. 10), the microorganism belongs to the Albosporeus series and finally to the Waksman system (The Actinomycetes, Vol. II, 1961, p. 129) Row Ruber.

A comparison between the characteristics of the microorganism and those of the species belonging to the cited systematic groups (taxa) shows that none of them have the same properties as those of the correspond to the microorganism under test.

These comparative data are given in Table II. In the table, S. cinereoruber, S. cinereoruber var. Fructo-fermentans, S. caespitosus and S. antibioticus have been added, even if they are not part of the groups given above. The differences to the types of adriamycin that do not produce any substances are also listed.

The microorganism used according to the present invention differs from the species S. albosporeus (W aksman, The Actinomycetes, Vol. II, 1961, p. 171) because it does not produce soluble pigments, reduces nitrates and does not _{produce H 2} S; by S. cinnamomensis (Waksman, The Actinomycetes, Vol. II, 1961, p. 195) and by S. fradiae (Waksman, The Actinomycetes, Vol. II, 1961, p. 211) in the color of the vegetative mycelium and aerial mycelium; of the species S. ruber (Waksman,

The Actinomycetes, Vol. II, 1961, p. 271) because the latter coagulates the milk, produces _{no soluble pigments and no H 2 S.} It differs from S. rubescens (Waksman, The Actinomycetes,

"Vol. II, 1961, p. 271) in the color of the Luftmycels and because S. rubescens does not form soluble pigments and does not produce hydrogen sulfide; by S. oidiosporus (Waksman, The Actinomycetes, Vol. H, 1961, p. 251), because the latter does not reduce nitrates and milk does not peptonize. moreover generated

S. oidiosporus does not have any soluble pigments.

It follows that the mutant "FI 106" of S. peucetius differs from all species that produce similar substances, and more generally differs from all species belonging to the systematic subgeneric groups to which the strain itself belongs. In particular, the "FI 106" strain differs from the parent strain S. peucetius, which produces daunomycin (British patent specification 1 003 383), as it is a vegetative mycelium that is more intensely red in color and an aerial mycelium that sometimes takes on a blue-green-turquoise tint, and ultimately produces the antibiotic adriamycin, which has a different structure than daunomycin.

Table I. Cultural characteristics of the mutant »F. I. 106 "by S. peucetius

media growth Aerial mycelium Vegetative mycelium Soluble pigments Agar malt yeast small together very sparse, smooth, plentiful, yellowish, intense, first extract (according to flowing Kolo colored pale pink, then yellowish red Yellow-red, then Hesseltine never with a wrinkle no spirals Brownish red a.o. *) 1954) ligen wrinkles, and vortex hard, aloof plentiful Bennet agar sparse, single none sparse, first none yellowish little ones yellowish, then Colonies orange Emerson agar moderate, small none moderate, pale at first Reddish-clear brown together pink, then flowing reddish Colonies Kai toffel agar abundant in smooth plentiful, first plentiful, meat intense, first (according to more regular Pink, then Colours. Hardness yellowish red, then Hessel tine patina weak blue smooth patina of strong orange among others *) 1954) green turquoise. to bright red Hyphae with hook-shaped Ends and then spherical end up Agar peptone plentiful, in too none plentiful, colorless none + KNO ₃ flow together the small Colonies Agar Czapeck plentiful, in too sparse, first plentiful, weak none flow together Dirty white, colored pink the small then weak Colonies Blue green turquoise, weakly wadded, Hyphae with hook-shaped Ends or spherical end up Asparagine glucose sparse, in iso sparse, whitish sparse, colorless none Agar lated little ones pink. Very cool Colonies broken Mycelium, briefly without apical hooks Glycerine-glycine- plentiful, in none plentiful, of yellow none Agar smoother harder to orange -,> ^r ■ * '· patina Starch agar sparse, in none sparse, colorless none · individual then yellowish pink small ones Colonies gelatin moderate, at the none moderate, colorless to plentiful, brown up surface yellowish to deep black milk sparse none sparse, ring sparse, pink shaped upper flat, salmon pink colored

*) Hesseltine et al., Ann. N. Y. Acad, Sci. BtI. M), pp. 136 to 15! (W54).

SI

U; di

Ul A R

Oil

k < Zl

Ir rt S. df di A

IVI S. B ₁ m

Table II Comparison between the mutant »F.I. 106 «by S. peucetius and species, the substances similar to the Antibiotic producing adriamycin

mutant
»F. 1,106 " S. purpurescens S. bobiliae + S. cienereomber S. ceruleorubidus Sporophores straight or spiral-shaped spiral-shaped straight or double ge hook-shaped hook-shaped whirls,
spiral
shaped + oval, prickly, Spurs almost round, oval, prickly, - + oval, smooth, 0.6 to 0.9 warty 1.8 0.8 to 1 µ + 0.7 to 1 μ to 0.8 to to 3.3 μ to 0.4 to + to 0.9 to 1.2 0.5 µ + .2μ Yellow-red-brown Vegetative Yellow-red to Red Coral red + Yellow-red-brown Mycelium Intense red + Blue turquoise Aerial mycelium White pink, White pink White _ Ash gray sometimes - weak Cynerubin Blue green turquoise R'-uuction of / Nitrates _ / / Milk (pep. / Coag.) - - + + L-xylose + + + + L-arabinose .. - + ■ + + L-Ramnose ... - + -. + Fructose + + - + Sucrose ... + + - + Lactose - + + + Raffinose .... - + - + D-mannitol .... + + - + D-sorbitol - - - Rubidomycin Generated
Antibiotics Adriamycin Rhodomycin Rhodomycin

+ = positive reaction. - = negative reaction. / = no data.

(Continuation)

S.cinereoniber var.
fructofermentans S. caespitosus S. niveoruber S. galilaeus S. nogalater var.
nogalater Sporophores straight or
hook-shaped whorled spiral-shaped spiral-shaped straight or
hook-shaped Spurs oval, smooth,
0.7 to 1 μ
to 0.9 to 2 μ oval, ghtt,
0.5 to 1.5 µ
to 0.3 to
0.5 µ smooth . smooth more or
fewer
spherical,
smooth Vegetative
Mycelium Yellow-red-brown cream colored
to brown
to yellow
reddish Carmine Carmine Orange red Aerial mycelium Ash gray White
Yellowish gray Whitish White up
Ash gray Gray

continuation

S. cinereoruber var.
fructofermentans S. caespitosus S. niveoruber S. galilaeus S. nogalater var.
noga later Reduction of Nitrates j + j j milk (Pep. Koag.) + + - + L-xylose + - I. / + L-arabinose .. + - I. 7 ■ L-Ramnose ... · + - j -f Fructose + + I. / Sucrose ... + + j / . - Lactose + ■ / j / Raffinose .... - - I. / + D-mannitol .... + - j / + D-sorbitol . + + I. / + Generated Antibiotics ... Cynerubin Mithomycin Cynerubin Cynerubin Nogalamycin

+ = positive reaction. - = negative reaction. / = no data.

S. antibioticus / continuation S.A. 220 S. DOA 1205 / S.A. 1165 (Ashesh ο ν u. A ^ (Brockmann, just -t- (A s h e s h ο ν u. A., Antib. and Chemother., Chem. Ber., • 4- smooth, spherical / Antib. and Chemother., Vol. 4, 1954, p. 380) Vol. 92, 1959, p. 1880) / Yellow cream colored / Vol. 4, 1954, p. 38Ö) not described in a spiral shape
placed ■ / Sporophores White to mouse / not described not described not described / Spurs Gray I. not described not described Brick red, wine red / Vegetative mycelium I. not described not described Red-gray / Aerial mycelium I. not described / I. / Reduction of I. / · ' / Nitrates I. / / milk Cynertibine I ^l (Pep. Koag.) / I. Pyn «ni3rtii &. L-xylose / . I. L-arabinose / I. L-ramnose / I. Fructose ....... / I. Sucrose ..... / I ; Lactose .... ^; /: / Raffiöose ...... i /! / D-Mannitol ...... / / © 4kS * it ....... / RütiiantiH Generated Akfavin Antibiotics. · .. ν.

-t- = positive RöAtioii. - = iiegawe RiaktiOH. / = nagged filej.

I ^ Mutaöt "F.Ll06" kanfidiBrchLyop1iiIis} enihg a successor to you knftmerar ^ ä on a * fesft UHtef Verweödnng vor: Miki ader Milchsennn äb «s Mödimn, the ÖlacOse or emeii atttfere ^ Su ^ jendienniltei or düfcb Santröeln der Sporeö k ad ki ikfft

and storing them in a sterile substrate be stored. He also came through

Mödimn, that

Zacket and kcHäpitsfe stiekstdfftt-like SabstanzJ (iFiejgi extract, peptori, hydrated casein) geläjgi will. The medium can outside of SM

contain magnesium sulfate and potassium phosphate are particularly important.

The production of the antibiotic adriamycin is carried out in conventional manner and consists in the fact that the mutant "FI 106" in a liquid culture medium which has been sterilized beforehand (preferably at 28 ⁰ C) under aerobic conditions at a temperature between 25 and 37 ° C during for a period of 3 to 7 days, preferably 5 days, at a pH of 6.5 to 7.0 initially and 7.5 to 8.0 at the end of the fermentation process. The culture medium consists of a carbon and nitrogen source and mineral salts. The carbon source can consist of starch, dextrin, glucose, glycerine, mannitol, maltose, corn steep liquor, grains, soybean oil or soybean meal. The nitrogen source may consist of dry yeast, meat peptone and casein in addition to the nitrogen-containing complex substances mentioned above. Good results are also obtained when using ammonium salts such as ammonium nitrates, ammonium sulfates and diammonium phosphates.

The mineral salts useful for the manufacture of the antibiotic may vary depending on the one used Medium vary. In a medium that contains complex substances, such as various flours and fermentation residues, Contains the addition of potassium carbonate and sodium or potassium phosphates Proven useful. In media that contain glucose, yeast or ammonium salts are much higher Additions of mineral salts such as potassium, magnesium, iron, zinc, manganese and copper salts are required. The fermentation can take place in Erlenmeyer flasks or in laboratory or industrial fermentation apparatus of various capacities. The amount of adriamycin present in the broths is calculated by the following method: The culture solution is made with the help of 2% diatomaceous earth filtered. The filtered broth is washed with a 1N sodium hydroxide solution adjusted to pH 8.6 and then extracted twice with a mixture of chloroform and methanol (9: 1). the The extract is washed with water and then evaporated to dryness in vacuo. The residue is taken up with methyl alcohol and then via Whatman MM no. 3-paper that comes with a m / 15 phosphate buffer was buffered at pH 5.4, chromatographed, using as the eluent a mixture of propanol, ethyl acetate and water (7: 1: 2) is used. The red-colored one Part, which corresponds to the Rp value of Adriamycin, is with a mixture of methanol and water (9: 1) eluted, and the amount of in the filtered Adriamycins broth present is determined by spectrophotometric examination of a sample of the eluate of the wavelength of 495 πΐμ and calculated with a Sample of adriamycin, the titer of which is known, compared.

The amount of adriamycin present in the mycelium is calculated as follows: the mycelium is extracted with a mixture of acetone and 0.1 n-sulfuric acid (4: 1); the extract is neutralized and ^{concentrated under reduced pressure to 1} Z _{5 of} the original volume, the concentrate is with. 1 N sodium hydroxide solution adjusted to a pH value of 8.6 and then extracted twice with a mixture of chloroform and methanol (9: 1). The extract is washed with water and then evaporated to dryness in vacuo. The adriamycin content of a sample of the residue is determined by means of the method given above.

To isolate adriamycin, the antibiotic can be removed from either the Culture broth without filtering the mycelial mass or from the mycelium and the culture fluid that were previously used separated by filtration. If the extraction is performed separately, the following procedure is preferred. At the end of the fermentation, a silica adsorbent is used added to the culture broth; the mixture is filtered, and both the filter cake and that Filtrate are treated separately. Most of the antibiotic is found in the filter cake, the consists of the mycelium mixed with the silica adsorbent materials. This cake will pulped and in an alcohol such as methanol, ethanol, butanol; a ketone such as acetone, methyl ethyl ketone; a halogenated hydrocarbon such as chloroform, methylene chloride or in aqueous Solutions of organic or inorganic acids such as acetic acid, hydrochloric acid, sulfuric acid, stirred Mixtures of the organic solvents mentioned, such as alcohols and with Water-miscible ketones, and aqueous solutions of inorganic acids can be used.

In general, a mixture of acetone and 0,1 η-sulfuric acid in a ratio of 7: 1 to 3: 1, preferably 4: 1, is used.

The antibiotic can be obtained from the filtered broth which has previously been made alkaline to a pH of 8.5 to 9.0 with a water-immiscible alcohol, ketone or halogenated lower aliphatic Hydrocarbons, such as amyl alcohol, butyl alcohol, methyl isobutyl ketone, methylene chloride, Chloroform or mixtures thereof.

Another method of extracting the filtered broth is by making the broth itself The cationic exchange resin containing carboxy groups is passed through a chromatographic column (»Amberlite IRC 50«) in acidic form contains the product with an aqueous methanolic Solution of sodium chloride can be eluted.

The organic extracts of the broth and mycelium are collected, neutralized, water is added and the resulting solution is then concentrated under reduced pressure. The aqueous concentration is adjusted to a pH of 3 with 1 η-hydrochloric acid and then extracted with chloroform. Dei Extrakl containing various impurities is removed while the aqueous layer is on a The pH is adjusted from 8.5 to 9.0 and with a mixture of chloroform and methanol (9: 1 is extracted.

The extract is washed with water, over dried anhydrous sodium sulfate and then reduced to a small volume under reduced pressure. steamed. When ethyl ether is added, the concentrate becomes a raw product, which is used as the main component Contains nente adriamycin, obtained in the form of the free base. To get the adriamycin of various To purify water- and fat-soluble pigments came countercurrent distribution or column chromatography

be applied. In the first case, the mixture can be chloroform-methanol-m / 15-phosphate buffer used at pH 5.4 (2: 2: 1); better results are obtained, though chromatographed on a column of cellulose, which is buffered with phosphates to a pH of 5.4 and the mixture propanol-ethyl acetate-water as eluant (7: 1: 2) is used.

The fractions containing adriamycin are collected and, after adding water, evaporated.

The aqueous concentrate is adjusted to a pH of 8.6 with 1N sodium carbonate and then extracted with chloroform. The chloroform solution is dried over anhydrous sodium sulfate and evaporated to a small volume. By adding containing hydrochloric acid, anhydrous methanol adriamycin hydrochloride is obtained in the form of thin, orangerotgefärbten needles, the orange-red when recrystallized from anhydrous ethyl alcohol results in needles, mp. 204-205 ⁰ C (decomposition). It's optically active, O]? ⁰ = +248 ± 2 ° (c = 0.1 in methanol). The elemental analysis of a purified adriamycin hydrochloride sample gives the following values: C = 54.36%, H = 5.43%, N = 2.37%, Cl = 6.42%. The empirical formula corresponds to C ₂₇ H ₂ QNO _n ■ HCl, molecular weight = 579.98.

Adriamycin has the following structural formula

CH ₂ OH
O OH

The adriamycin hydrochloride is soluble but insoluble in water, methanol and aqueous alcohols in acetone, benzene, chloroform, ethyl ether and petroleum ether. Alcoholic solutions of antibiotic result in characteristic colorations with metal salts: scarlet red with magnesium salts, scarlet red with calcium salts, dark red with lead salts.

At an alkaline pH, the will turn to purple and precipitate pigmented substances observed. The aqueous solutions of adriamycin hydrochloride in acidic pH values are yellow-orange in color, red-orange at a neutral pH value and red-orange at a pH value of higher than 9 purple blue. The UV absorption spectrum and the spectrum in the visible ranges in methanol is characterized by the following maxima:

at 233 πΐμ (EU = 673)
at 252 πΐμ (EU = 450)
at 288 πΐμ (EU = 159)
at 479 ηΐμ (EJl = 219)
at 496 ηΐμ (EJl = 217)
at 529 m | i (EU = H8)

In the IR absorption spectrum, bands of the following wavelengths are found (in μ): 3.00, 3.44, 5.80, 6.17, 6.31, 6.55, 7.05, 7.78, 8.11, 8.24, 9.00, 9.35, 10.10, 10.98, 11.50, 12.68, 13.12, 14.60.

CH-CH ₂ -CH

O-

NH ₂ OH H

C-H

C-CH ₃

The antibiotic is a base that can form salts with inorganic and organic acids. The observed color change from red to blue-violet at a pH of ~ '9 occurs as a result of the formation of salts in the phenolic hydroxyl groups. Lstündiges by heating at 100 ⁰ C in 0.5 n-Adriamycin mineral acids results in a rotgefarbtes aglycone which is insoluble in water (adriamycinone), "and a water-soluble, basic, reducing · acting fraction (daunosamine). ·. · .-.

The water-soluble fraction consists of one known reducing amino sugar daunosamine of the following structural formula

-CHOH

CH ₂

HC-NH ₂
H-C-OH

1 ■■

-CH
CH ₃

60 Daunosamine hydrochloride melts at 168 ° C (decomposition); [a] ₀ = -54.5 "(in water); N-benzoyl derivative melts at 154" t) to 156 ° C. The structure of adriamycin is largely secured by these split pieces

Like the well-known Daunomycin (J. Am. Chem. Soa, Vol. 86, 1964, pp. 5334 to 5336) Adriamycin can be purified by chromatography. It shows however, a different behavior.

It became Whatman paper no. 1, buffered with m / 15 phosphate buffer (NaH ₂ PO * and K ₂ HPO ₄ ; pH 5.4), descending solution for development at room temperature.

Solvent A:

ButanoL saturated with "m / 15 phosphate buffer (pH 5.4);. ^ ,,

Solvent B:
Propanol — ethyl acetate — water (7: 1: 2).,

(HI)

Thin layer chromatography

Silica gel-G-layer (Merck), geouffeit with 1% oxalic acid in water. Oas töäto ^ Eäfife
would Ivan at room temperature

System C: methylene chloride-methanol

(100: 15);
System D: n-butanol-acetic acid-water

(4: 1: 5) upper phase;

System E: benzene - ethyl acetate - petroleum ether,
Kp 80 to 120 ⁰ C (80:50:20).

System F: benzene-ethyl formate-formic acid (50: 50: 1).

connection

Thin layer chromatography
system
CD EF

Adriamycin Rf

Daunomycin hydrochloride Rf
Daunomycinone Rf

0.17
0.35
0.95

0.33
0.40
0.85

0.00
0.00
0.15

0.00
0.00
0.40

The salts of adriamycin are prepared by conventionally reacting the base with pharmaceutically acceptable ones organic and inorganic acids such as hydrochloric acid, sulfuric acid, acetic acid, propionic acid, Valeric acid, palmitic acid, oleic acid, citric acid, Succinic acid, mandelic acid, glutamic acid or pantothenic acid. Be neutral salts by reacting the corresponding acid with the free base adriamycin, which is obtained by extraction of a aqueous solution of the hydrochloride at a pH of 8.6 with organic with water not miscible solvents such as butanol and chloroform ,, obtained. By evaporating the organic solvent, the antibiotic adriamycin is obtained in the form of the free base. the Salts can also be obtained by double conversion of the salts: for example, adriamycin sulfate becomes obtained with calcium pantothenate adriamycin pantothenate.

Although the antibiotic adriamycin opposed a remarkable bacteriostatic activity possesses various microorganisms (see Table III), it has proven to be particularly antitumoral Means proven effective.

Table III Antibiotic Activity of Adriamycin Hydrochloride

30th

35

40 The antibiotic shows a clear inhibitory effect on tumor growth in ascitic form, in which direct contact of the antibiotic with the neoplastic cells is achieved. A good inhibitory effect is also observed in solid tumors where the activity differs according to the mode of administration and the dose. The anti-tumor activity of adriamycin gives better results in terms of effectiveness and duration than does daunomycin. The antibiotic adriamycin and its pharmacologically acceptable salts, as well as their mixtures, can be used as medicinal agents. These contain the compounds in admixture with customary inert pharmaceutical carriers which are suitable for parenteral or local administration. For parenteral administration, lyophilized preparations are preferably used, which are to be dissolved at the time of use.

The study of the anti-tumor effect of the antibiotic using the strain Adriamycin, the Streptomyces »F.I. 106 «was produced in a few mouse and rat tumors made both in solid and ascitic form.

1. Ascitic tumors

Tribes . * Medium DIM μξ / τιύ Staph. aureus
cp. 209 P
Bacillus subtilis
Streptococcus faecalis
Sahnonella abortive
eqirina
Escherichia coli B ...!
Shigella flexneri
Candida albicans ■. Meatsoup
Meatsoup
Meatsoup
Meatsoup
Meatsoup
Meatsoup
Sabouraud '■■ 6.25
50
'50
3
> 50
> 50

Activity tests were carried out on mice which had Ehrlich's ascitic carcinoma and with solutions of the antibiotic in different concentrations for five consecutive days, starting with the day following tumor implantation, were treated intraperitoneally.

Table IV, in which the results obtained are summarized, shows that that to be tested

Antibiotikmn administered in equal doses from 1.75 ■ xtsd 2 ^ 0 mg / kg / Täg, eüien remarkable Hgseffekt atff the ascitic Tinsorwachstum exerts uad the average survival rate of the treated animals has increased considerably

Table IV

Ascitic Etnüch-Karzhionia

Groups of 10 animals

dose

mg / kg / day

(Days after implantation)

Medium Oifretztt (days)

Controls.

Adriamycin

Adriamycin

1J5 2 ^ 0

+7.5
-0.5
-1.8

+ 13.9 +3.8 +0.9

14th
34.6

This result is confirmed by a subsequent test in which the antibiotics are in doses of L25 and 2 ^ 0 mg / kgday (TabeDe V) was administered.

Table V Ehrlich's Ascitic Carcinomas

Groups of 10 animals

dose

mg / kg / day

Change in body weight in grams

(Days after implantation)

6 I 12

Medium
Survival time (days)

Controls.

Adriamycin

Adriamycin

1.25
2.50

+7.5
-0.6
-0.9

+ 13.2

+ 4.6

-4.3

17.8
31.8
51.2

A comparison of the results obtained under the same experimental conditions on mice, who exhibited Ehrlich's ascitic carcinoma, must Antibiotics daunomycin and adriamycin with respect to control mice allowed the determination that the latter is a more active product, as can be seen from Table VI, from which it can be seen that the ratio values which increase the survival time in the treated mice compared to control mice show higher levels of adriamycin at the same doses.

Table VI

Mean survival time of mice with ascitic Ehrlich carcinonia (as a ratio between the Survival time of the treated animals (mice) and the control animals; each value shows the mean of the obtained Results in groups of 10 animals).

dose
mg / kg / day

2.50

Daunomycin

1.8

Adriamycin

The antimycotic effect of the antibiotic to be tested was revealed in tests carried out on Mice were carried out, which ascitic tumors in the logarithmic growth stage (5th day) exhibited. These animals were treated intraperitoneally with adriamycin in the amount of 2 mg / kg was administered only once. Examination of the stains of the neoplastic exudate previously and at different times after the treatment (2, 4, S, 24, 32 and 48 hours), was able to show that the «antibiotic had a very rapid and complete termination of the multiple Effectiveness of the tumor causes that lasts up to the 32nd hour.

Forty-eight hours after the treatment, numerous cells were found in the myth, but their morphology was constantly changing.

2. Solid tumors

The tests for effectiveness against solid tumors were carried out with Sarcom 180 in mice and performed with Oberling-Guerin-Guerin myeloma in rats.

a) Sarcom 180

Mice implanted with a neoplastic tissue fragment were aged for 8 days treated subcutaneously starting the day following tumor implantation. The antibiotic was taken administered as a solution in various concentrations corresponding to the following doses in mg / kg / day: 7, 5, 3.5, 2.5 and U5.

On the 11th day of the experiment, all animals were sacrificed and their tumors removed and weighed.
The results are given in Tables VII and VIII below:

Table VII

Group and dose
(mg / kg / day)

Controls

Adriamycin

7th

3.50

1.75

Body weight:: change in weight gross I net

Tumor weight
G

inhibition
%

mortality

+ 4.78

-5.98
-2.31
+ 3.09

+ 0.86

-6.22 -3.01 + 1.10

Table VIII Sarcom 3,922

0.239
0.696
1,988

93.9
82.3
49.3

0/10

6/10
0/10
0/10

Group and dose
(mg / kgA day) Body weight
gross change
net Tumor weight
G inhibition
% mortality Controls + 5.65
-4.37
-1.60
+ 0.85
+ 1.52 + 3.19
+ 4.61
-2.26
-0.18
-0.23 2,461
0.239
0.656
1.029
1.745 90.3
73.4
58.2
29.1 0/10
0/10
0/10
'0/10
0/10 Adriamycin
■ 5 2.50 Daunomycin
5 2.50

From the two tables it can be seen that the antibiotic has a strong inhibition of tumor growth at all dosages used. A noticeable mortality in the treated animals was only used with higher doses (7 mg / kg / day) achieved.

Under the same experimental conditions, it was clear that adriamycin was compared to Daunomycin is superiorly effective for this type of tumor. This result is all the more vivid when the inhibition _{doses 50 (HD 50} ) are taken into account: daunomycin about 3.3 mg / kg, adriamycin about 1.5 mg / kg.

Toxicity experiments on healthy * Adriamycin was administered subcutaneously to mice for 8 days at doses of 10 to 1.25 mg / kg performed showed the following results:

20th

Toxicity of Table IX
Adriamycin at Mice ikeit on
15th day dose
(mg / kg / day) % Sterblic
10th day 100
100 10 ... 100
70 80
0
0
0 8.33 40
0
0
0 6.67
5 ... 2.50
1.25

30th

From the above data it can be seen, by means of a graphical method, that the lethal dose 10 (LD ₁₀ ) is 6.4 mg / kg. From the graph it can be seen that the inhibitory dose 90 (HD ₉₀ ) of adriamycin is equal to 5 mg / kg. With these data it is possible to calculate the therapeutic index of adriamycin according to Skipper (Cancer Chemotherapy Report, vol. 17, 1962, p. 1):

T. L =

LD,

Ίο

6.4

HD,

= 1.28.

'90

It should be noted that the therapeutic index of daunomycin under the same experimental conditions is 0.67 pointed out that according to the Skipper reference cited above, it appears that under the same experimental conditions the therapeutic index of other anti-tumor antibiotics that are already in use (actinomycin, Mitomycin, Actinobolin, Actidion) is lower than 1.

b) Oberling-Guerin-Guerin myeloma

Wistar rats which had been implanted with a fragment of tumor tissue were given intravenous injection 8 days starting with the day following tumor implantation. On the 12th day of the attempt the surviving animals were sacrificed and the tumors removed and weighed.

Table X Qberling-Guerin-Guerin myelomas

Group and dose
(mg / kg / day)

Body weight change (g) net gross + 3.3 + 15.7 - 1.0 + 9.2 + 14.6 + 25.2 - 5.6 - 1.3

mortality

Tumor weight

inhibition
%

Controls. Adriamycin

0.625

1.25

2.50

Table X shows that the antibiotic is also effective against this type of tumor. Under these experimental conditions, the HD ₅₀ of adriamycin is about 2 mg / kg.

The following examples are intended to explain the present invention in more detail.

0/10

3/10 0/10 1/10

12,447

10.253

10.649

4,295

17.7 14.5 65.5

example 1

Two 300 ml Erlenmeyer flasks, each containing 60 ml of the following culture medium for the vegetative phase, were prepared: peptone 0.6%, dry yeast 0.3%, hydrated calcium nitrate 0.05%, tap water. After the sterilization, the pH was 7.2. The sterilization was carried out by heating in the autoclave to 120 ^° C. for 20 minutes.

Each flask was injected with an amount of mycelium from the mutant »F. I. 106 "from Streptomyces peucetius inoculated corresponding to a suspension of the mycelium in sterile water; this mycelium comes from

55

60

65 of a 10 day old culture grown in a large test tube on the following medium: sucrose 2%, dry yeast 0.1%, dipotassium phosphate 0.2%, sodium nitrate 0.2%, magnesium sulfate 0.2%, agar 2 %, Tap water. The flasks were incubated for 48 hours at 28 ° C. on a rotary shaker with a stroke of 30 mm at 220 revolutions per minute.

2 ml of a vegetative medium drawn in this way were used to inoculate 300 ml ErIenmeyer flasks with 60 ml of the following medium for the production phase: glucose 6%, dry yeast 2.5%, sodium chloride 0.2%, dipotassium phosphate 0.1% , Calcium carbonate 0.2%, magnesium sulfate 0.01%, ferrous sulfate 0.001%, zinc sulfate 0.001%, copper sulfate 0.001%, tap water. The resulting pH is 7. The medium was sterilized for 20 minutes at 12O C ^0; the glucose was sterilized separately at 110 ° C. for 20 minutes. It was at 28 ° C under the same stirring conditions as for the

vegetative media described, incubates the maximum concentration of antibiotic was reached on day 6 of fermentation. The crowd after one Adriamycins produced at such a time corresponds to a concentration of 15 μg / ml.

Example 2

The experiment is carried out as described in Example 1, with the difference that the inoculum was grown on the following solid medium: 200 g of peeled potatoes were boiled in 500 ml of water for 20 minutes; the volume was returned to its original value and filtered through gases. 2% glucose, 0.1% Difco yeast extract and 2% agar were added. The volume was brought to 1000 ml; it was ^{sterilized at 120 °} C. for 20 minutes; pH 6.8 to 7.0. The maximum concentration of adriamycin of 12 μg / ml was reached in the 140th hour.

Example 3

The experiment was carried out as described in Example 2, with the difference that the vegetative and productive media had the following compositions:

Vegetative medium: starch 3%, calcium carbonate 0.4%, grains 0.3%, ammonium sulfate 0.1%, casein 0.5%, dipotassium phosphate 0.01%, in tap water. The pH value after the sterilization in the autoclave at 120 ^° C. for 20 minutes was 7.

Productive medium: starch 5%, calcium carbonate 0.8%, corn steep liquor 0.6%, casein 0.5%, Ammonium sulfate 0.1%, dipotassium phosphate 0.01%. The pH after the sterilization was 7. Die Sterilization was carried out as described for the vegetative phase. The maximum production was reached on the 7th day with 6.5 μg / ml.

Example 4

500 ml of the liquid medium of the vegetative phase, as described in Example 1, in a 2000 ml Pyrex glass flasks were included with a culture of the mutant »F. I. 106 «by Strept. peucetius on a solid medium, as in Example 2 described, inoculated. It was 48 hours at 28 ° C on a rotary shaker with a stroke of 3.5 mm incubated at 120 revolutions per minute;

100 ml of the culture broth thus obtained were then inoculated into 3000 ml of the same liquid medium. in a 5 1 neutral glass fermentation vessel, which is equipped with a propeller stirrer, an inlet pipe to the Injection of air ending under the propeller stirrer, a breakwater device, a pipe for inoculation, an air outlet pipe, a temperature test device and means for intermittent or continuous addition under sterile conditions was. The waxing took place at 28 ° C with a ventilation rate of 3 liters per minute and below Stir at a speed of 400 revolutions per minute (r.p.m.).

300 ml of the culture broth produced in this way were used to inoculate 61-des in Example 1 after 24 hours described productive medium, which in a

101 neutral glass fermentation device, which had the features described above, was included. During fermentation, carried out at a stirring speed of 350 revolutions per minute and at a ventilation rate of 51 per minute, the foaming was carried out by adding small amounts of silicone antifoam checked The maximum production achieved in 150 hours of fermentation corresponds to a concentration of 6 ^ g / ml adriamycin.

Example 5

With a culture as described in Example 1

ίο was obtained, 500ml of a medium of the The following composition was inoculated in a 2000 ml flask: Peptone 0.6%, granulated dry Yeast 0.5%, calcium nitrate in water 0.05%. The medium was on a rotary shaker for 48 hours stirred at 28 ° C by means of the culture thus obtained became an 801 fermenter with 501 of the above Inoculated with the medium. This medium was stirred at 230 revolutions per minute and with an air flow of 0.71 / 1 medium per minute at a temperature of 27 ° C. After 4 to The culture was brewed for 5 hours; used to put 500 ml of the culture medium in an approximately 8001 Occupy the fermentation vessel.

The fermentation medium had the following

2s composition: glucose 7%, chickpea flour 6.65%, calcium carbonate 0.2%, sodium chloride 0.2%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.02%, ferrous sulfate heptahydrate 0.00068%, manganese sulfate heptahydrate 0.001%, copper sulfate 0.002% ii tap water.

D '"medium was sterilized 30 minutes at 120 ⁰ C, cooled to 27 ° C and stirred by your inoculation at 250 revolutions per minute and aerated with a stream of air of 0.41 / 1 of medium per minute. After 145 hours the culture broth contained 6.5 μg / ml adriamycin.

Example 6

60 l of culture liquid, which was obtained from the fermentation according to Example 4, were from the mycelium filtered off by means of kieselguhr to give a cake and a filtrate, which were extracted separately became. The obtained cake was dissolved in acetone which was diluted with 0.1 N aqueous sulfuric acid (4: 1) was, suspended and stirred for 2 hours. The liquid was filtered and the cake more stirred twice. The extracts obtained were combined, neutralized and the acetone in vacuo evaporated. The concentrate, which contained about 0.25 g of adriamyein, was mixed with 1η hydrochloric acid to one pH 3 acidified and then extracted with chloroform, with some of the impurities was removed. The aqueous phase was with 1 II sodium hydroxide to a pH of 8.6 adjusted and then extracted with a mixture of chloroform and methanol (9: 1); the process has been taking so long repeated until the aqueous phase was colorless. The methanol-chloroform extracts were washed with water washed at pH 8.6, then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to a small volume, whereupon adriamycin by means of ethyl ether was precipitated in the form of the free base. 1.50 g of the crude product was obtained which was about 0.2 g Contained adriamycin.

The filtered broth was adjusted to pH 8.6 with 1N sodium hydroxide and thereafter with a mixture of chloroform and methanol (9: 1)

extracted. The process was repeated twice. The methanol-chloroform extracts were mixed with Washed water at a pH of 8.6 and then re-extracted with 0.01 η-hydrochloric acid until the aqueous phase turned a red color. The chloroform phase was removed. The aqueous phase was filtered and adjusted to a pH of 8.6 with 1N sodium hydroxide. After that she became with the mixture of chloroform-methanol (9: 1) extracted. The extract, which at this time, in addition to various impurities, 0.15 g of adriamycin was washed with water at pH 8.6, dried over anhydrous sodium sulfate, filtered and concentrated to a small volume under reduced pressure. By clogging of 10 parts by volume of ethyl ether was a precipitate of 1 g of the crude product, the 0.12 g of adriamycin contained, received.

A total of 0.320 g of adriamycin was obtained in the form of the crude base.

Example 7

0.5 g of crude product, containing about 15% adriamycin, was dissolved in 10 ml of an m / 15 phosphate buffer (pH value of 5.4) dissolved. The solution was adsorbed onto 10 g of cellulose powder (Whatman-CF-11), the mixture dried overnight in vacuo over anhydrous calcium chloride, then in a glass column for chromatography (100 cm high and 4 cm diameter), containing 225 g of cellulose powder (Whatman-CF-II), which had previously been buffered with m / 15 phosphate buffer (pH 5.4), introduced and dried in vacuo over anhydrous calcium chloride. It was made with a mixture of propanol- Ethyl acetate-water (7: 1: 2) eluted, and 25 ml fractions were collected with an automatic collector. The different factions were by chromatography over Whatman paper no. 1, buffered at a pH of 5.4, where the same mixture used to elute the column was used as the eluant was. Fractions 40 to 60 contained adriamycin. Fractions 40 to 60 were pooled and up 50 ml concentrated. The precipitated salts were filtered and 200 ml of water were added to the filtrate, and the pH of the solution was adjusted to 7 with 1N sodium hydroxide.

It was concentrated to 50 ml under reduced pressure, the concentrate was adjusted to pH of 8.6 and then extracted with chloroform. The process was repeated three times, then the chloroform extracts were combined and washed with water at pH 8.6 and then with washed with ordinary water, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to 5 ml under reduced pressure. Thereupon 0.15 ml of a 1 η solution of anhydrous Hydrochloric acid in methanol was added and the mixture was cooled. After a few minutes it formed A crystalline precipitate of adriamycin hydrochloride, which was filtered and washed with cold chloroform and anhydrous ethyl ether. 50 mg of the product were obtained from ethanol was recrystallized.

In this manner there were obtained 35 mg of the pure product having mp. 204-205 ⁰ C. A further 15 mg of an amorphous product of 90% purity were obtained from the mother liquors.

Patent claims:
1. Adriamycin of the formula

O OH

CO-CH ₂ OH

OH

H
O OH O NH ₂ OH

OCH ₃ CH-CH ₂ -CH-CH-CH-Ch ₃

-O-

and its salts with pharmacologically acceptable organic and inorganic acids.

2. Therapeutic compositions for tumor treatment, characterized by a content to adriamycin or its salts with pharmacologically acceptable organic or inorganic acids together with a usual inert carrier for parenteral or local Administration.