BE543959A - - Google Patents

Description

       

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   The invention relates to novel useful antibiotic compounds and to methods of making them. More particularly, it relates to new antibiotics in various forms, and to the methods of making them by fermentation as well as of concentrating them, of purifying them and of isolating them, as well as of making their salts. In its free form, one of the two newer antibiotics that have been isolated is called Amphotericyn A, and the other antibiotic is called Amphotericyn B; and the unmodified term Amphotericyne is used to refer generically to these two antibiotics and their mixture.



   The antibiotics according to the invention are prepared by culturing, under controlled conditions, a hitherto undiscovered species of Streptomyces.



   The microorganism.-
The microorganism useful for the preparation of Am- photericyne is a recently discovered species of Streptomyces isolated from a sample obtained at Tembladora on the Orinoco River in South America. A culture of the living organism has been deposited and forms part of the stock culture collection at the Rutgers Institute of Microbiology (New Brunswick, New Jersey), where it is available; it received number 3694 in the Wakaman collection and is hereinafter referred to as Streptomyces sp. 3694.



   It is understood that the invention is not limited to the use of the particular organism described herein, but includes, among others, variants made from the organism described by agents. determining a change, such as x-rays, ultra-violet radiations

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 and nitrogen mustards.



   To isolate and characterize the microorganism, part of the sample taken from the soil is stirred in sterile distilled water and is applied to Henrici agar medium. This medium contains:
 EMI2.1
 
<tb> Caseinate <SEP> of <SEP> sodium <SEP> or <SEP> cas <SEP> N-Z <SEP> 5 <SEP> gm.
<tb>
 
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 f>: -3 ¯ -,: i "...
 EMI2.3
 
<tb> Glycerol <SEP> 5 <SEP> ml.
<tb>
<tb>



  K2HP0 <SEP> 2 <SEP> gm.
<tb>
 
 EMI2.4
 



  -MgSO .7H 2 gm FeSO 7Ii 0 Trace '
 EMI2.5
 
<tb> Agar-agar <SEP> 15 <SEP> gm.
<tb>
<tb> Distilled <SEP> water <SEP> 1 <SEP> liter
<tb>
 . This medium is adjusted to pH 7.0 and is sterilized in an autoclave at 121 ° C. for 20 minutes. After 7 to 10 days of incubation at 25 C, colonies of Streptomyces sp. 3694 are isolated from the ground on which they are applied. These isolated colonies are then cultivated on an agar-agar medium which contains:
 EMI2.6
 
<tb> Bacto-tryptone <SEP> 5 <SEP> gm.
<tb>
<tb>



  Extract <SEP> from <SEP> malt <SEP> 3 <SEP> gm.
<tb>
<tb>



  Glucose <SEP> 10 <SEP> gm.
<tb>
<tb>



  Yeast <SEP> <SEP> <SEP> 3 <SEP> gm extract.
<tb>
<tb> Distilled <SEP> water <SEP> up to <SEP> 1 <SEP> liter
<tb>
 The agar-agar is heated in an autoclave at 121 ° C. for 15 minutes.



   The microorganism, when tested by the method of banding onto brewer's yeast agar for antibiotic activity against both bacteria and fungi, does not destroy any of the bacteria. test bacteria, but destroys test fungi Saccha- romyces cerevisiae, Rhodotorula glutinis, Candida albicans

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 and Aspergillus niger.



     Below is a description of the colonies
 EMI3.1
 of. microorganism incubated for 13 1'6-different iodines and on different media: Potato plant:
After 15 days the culture spreads out, very heavy, damp, hardened, with appreciably the same color as the potatoes, white spores covering the part of the culture on the inclined portion.



  'Sunflower milk:
In 7 days, the nucleus of the culture on the surface is colorless. The milk turns blue-purple, gradually lightening from top to bottom and indicating proteolysis without coagulation.



  Czapek-Dox Agar:
 EMI3.2
 ZuaN03 '3 gas KH2PO, +, 1 gm .; KCl, 0.5 gm .; Mus04'7H0 0.5 gm .; FeSO. 7H0 '0 01 gm. - glucose, 40 gzn. ; acar- '' '4 f 2, 0.01 gm .; glacose, gm .; agar-agar, 15 gm .; distilled water up to 1,000 ml.



   No growth in 15 days despite two inoculations or inoculations.



  Purple Glucose Agar-Nutrient Medium-Bromine Cresol Plant:
Beef extract, 3 gm. ; protein peptone, 10 gm.
 EMI3.3
 glucose, 10 gm. ; NaCl, 5 gm .; agar-agar,, .20 gm. ; cresol bromine purple, 0.15 gm .; distilled water up to 1,000 ml. pH 7.0.



   Moderately heavy growth in 51 days, shiny wrinkles, asporogenic. The wrinkling on all the agar-agars is very characteristic. The wrinkles are parallel, with straight line serrations in the crop at right angles to each other, dosing a murky appearance.

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   m @ qonnerieë with rectangles of different dimensions.



   Sabouraud agar-agar flant:
Glucose, 40 gm .; neopeptone, 1C gm. ; agar-agar, 15 gm. ; distilled water 1,000 ml.



   After 4 days the culture is yellow, turning brown after 7 days and all brown after 15 days The culture is heavy, first shiny, then turning dull, wrinkled with a diffusible brown pigment visible in the after 3 to 4 days.



  Glucose-asparagine agar-agar plant:
Glucose, 10 gm. ; K2HPO4, 0.5 gm. ; asparagine 0.5 gm; agar-agar 15 gm .; distilled water, 1,000 ml.



   After 7 to 15 days, the culture is moderately heavy, non-wrinkled, colorless, with white spores turning gray, and a diffusible yellow pigment turning brown. In addition, a water insoluble, dark yellow material is deposited on the edges of the agar plant at the base of the plant. This material is soluble in methanol, propanol and dimethyl formamide.



   The microorganism is further characterized in that hydrogen sulfide is not produced, as shown by testing with lead acetate paper in medium containing proteose peptone, 20 gm .; glucose, 1 gm .; Na2HPO4, 2 gm .; and distilled water up to 1 liter. The culture develops as a white, porogenic film. In addition, Difco gelatin is liquefied. The Culture develops as a light brown nucleus and asporogenous on the surface. In addition, in N Rate Difco broth, the culture grows as a slightly brown, asporogenic film. The broth darkens with a diffusing brown pigment. Nitrate is strongly

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 reduced in nitrite.

   In addition, starch is hydrolyzed by the culture as it grows in a medium containing starch.



   To determine the carbon and hydrogen content of Streptomyces sp. 3694, tests were carried out as follows: four sets of agar tubes with mineral salts are prepared with 1 mg. per ml. of these four sources of nitrogen respectively: (NHSO, NaNO3, asparagine and casein hydrolyzate. In each series, each tube is reinforced with 10 mg per ml. of one of the sixteen possible sources of carbon and energy These are starch, inulin, dextrin, - raffinose, maltose, lactose, sucrose, sorbose, glucose, dulcitol, inositol, sorbitol, mannitol, xylose, arabinose and glycerol.



   The agar-agar in the tubes (10 ml.) Is enclosed and the plants are treated with a suspension of Streptomyces spores. 3694 in distilled water. After 9 days of incubation, the culture was examined and is described in Table I attached.



   The results indicate that the microorganism is capable of assimilating carbon from starch, dextrin, maltose, glucose and mannitol, regardless of the source of the hydrogen. With the sources organi. As nitrogen, lactose, sucrose, inositol and glycerol can also be used, although cultivation with these carbon sources may be lighter than with glucose and its polymers. Nitrates do not serve as a source of nitrogen along with any of the carbon and energy sources, while asparagine and casein hydrolyzate may serve as the sole sources of carbon, gene and energy.

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 EMI6.1
 



  Antibiotics8.



   Streptomyces sp. 3694 produces a mixture of antibiotics. The mixture itself, as well as the specific antibiotics isolated from the same mixture, exhibit a broad fungicidal spectrum, but no significant bactericidal properties.



   In order to manufacture Amphotericyne, Streptomyces sp. 3694 is developed at an appropriate temperature
 EMI6.2
 at 23 ° C, at 300C, preferably at about 25C, under aerobic conditions immersed in an aqueous nutrient medium containing a source of assimilable and fermentable carbohydrate and a source of assimilable nitrogen.



   Suitable sources of carbohydrate, as indicated above, include: starch, dextrin, sugars such as maltose, lactose and glucose, glycerol, etc. Suitable sources of nitrogen include: Asparagine, casein hydrolyzate, soybean meal, beef extract, yeast extract, etc. The fermentation is carried out for about 24 to 150 hours. .



  At the end of this period of time, a substantial amount of Amphotericyne has been prepared (as shown by bioassays), as will be described in more detail in the Examples.



   When the development of the culture has been completed, the Amphotericyne is separated from the culture by one of the following three alternative methods: (a) The mycelium is separated from all the broth by filtration or centrifugation, and the Amphotericyne is extracted from the mycelium after lowering the pH of the mycelium to about 2 to 3 by treatment with an acid. The extraction is carried out with a suitable solvent, such as a lower alkanol (eg isopropanol, n-propanol or n-butanol). Evaporation of the alkanol causes precipitation of Amphotericyne.

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 brute.



   (2) All the broth is alkalized at a pH of about 11, and preferably at a pH of about 12, using a base such as sodium hydroxide or potassium hydroxide. The broth is then stirred and filtered, and the filtrate is neutralized to a pH of about 7 with an acid such as a mineral acid (eg hydrochloric acid, sulfuric acid or. phosphoric acid) to precipitate crude amphotericine.



   (3) The pH of all the broth is adjusted either to a value of about 2 to 3 (by treatment with an acid) or to a value of 10 to II (by treatment with a base), since Amphotericyne is more soluble at these pH ranges. The broth is then extracted with a suitable extractant, such as the aforementioned alkanols, then is filtered and the phases are separated (if n-butanol is used). The crude Amphotericyne is then precipitated directly from the filtrate by neutralizing a pH of about 7 and removing part of the extractant by vacuum distillation.



   Subsequent purification of the crude Amphotericyne * isolated by one or the other of these processes results in its fractionation in its two constituents, Amphotéricyne A and Amphotéricyne B. This fractionation is carried out by one or the other of the following processes: (1) The precipitate of crude Amphotericyne is slurried in an alcohol, such as a lower alkanol (e.g. methanol, n-propanol, isopropanol and butanol) at pH low (obtained by treating the slurry with an acid such as a mineral acid), and is filtered.

   The insoluble matter consists mainly of crude Amphotericyne B; The filtrate is

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 neutralized with a base, such as sodium hydroxide, to cause the formation of a precipitate of purified crystalline material predominantly representing Amphoteryne A.



  (2) The precipitate of crude Amphotericyne is boiled up in a solvent, such as a di-lower alkanoic acid (lower alkyl) amide (for example dimethyl formamide, dimethyl acetamide or diethyl formaid), and is filtered. The insoluble matter consists mainly of crude Amphotericyne B. By treating the amide solution with a mixture of water and a polar organic solvent, such as an aqueous alcohol (e.g. a solution of methanol and water) or an aqueous ketone (e.g. a solution acetone and water), a crystalline precipitate is obtained comprising mainly Amohotericyne A.



   Amphotericines A and B are amphoteric substances which readily form salts with both bases and acids. Thus by treating Amphotericyne with an inorganic base, such as a base of an alkali metal (for example sodium hydroxide or potassium hydroxide) or a base of an alkaline earth metal, the salt of the corresponding metal is formed. By treating Amphoteryne with a salt of an alkaline earth metal (eg calcium chloride or magnesium chloride) in ;. an alcohol such as methanol, complexes are closed.



  By reacting Amphotericyne with an ammonium hydroxide or an organic nitrogen base, the corresponding ammonium or amine salt is formed.



   Each of the Amphotericynes further reacts with both mineral and organic acids to form the salt of the corresponding acid. Thus Amphotericyne can be treated with mineral acids, such as hydrochloric acid.

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   drric, sulfuric acid or phosphoric acid, to form the corresponding salt of hydrochloride, sulphate or phosphate; or else it can be treated with organic acids, such as acetic acid, citrus acid or tartaric acid, to form the salts of the corresponding acids.



   The following examples illustrate, without limiting them, suitable methods for preparing, purifying and fractionating Amphotericyne.



   Example 1.- Fermentation in a container of Streptomyces sp. 3694.-
A 3600 liter load of Streptomyces sp. 3694 is fermented with the sowing medium, for a period and under the conditions indicated below: Preparation of sowing A. - First phase
Seed source - culture of Streptomyces sp, 3694, grown on Gould agar plants.



   Staley 4 S Medium-3% Nutrient
2% glucose
 EMI9.1
 ÔJ0005% C CiZ.6HZ0
0.1% CaCO3 pH set to 7.0 -7.2 Sterilization for 30 minutes at 121 C Volume-100 ml. in a 500 ml flask.
 EMI9.2
 Temperature = 25 C
Inoculation 72 hours on a reciprocating shaker (170 cycles per minute).



  B. - Second phase
Source of incubation or seeding -10% of the first phase.



   Middle-the same as in the first phase.

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 Sterilization -40 minutes at 121 C.



    Volume-480 ml. in a 2,000 ml flask.
 EMI10.1
 Temperature -25 C
Inoculation-48 hours on reciprocating shaker (120 cycles per minute) Fermentation conditions Staley 4S medium-3% nutrient
2% glucose
0.25% CaC03
0.1% NaCl
0.0005% CoCl2
Sterilization-15 minutes at 121 C of full dilution.



   Temperature -25 C
Agitation -2 Hp / 454 1.



   Ventilation-60 cm / min. surface air speed
Fermentation cycle -144 hours
Premium Combustion Oil Defoamer (approx.0.5% of load)
Importance of inoculation -480 ml. of the second phase of the balloon (4%)
Volume -12,000 ml.



   The results of the fermentation are given in the table below (with regard to two charges):

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 EMI11.1
 
<tb> Load <SEP> 1
<tb>
<tb>
<tb>
<tb> Duration <SEP> of <SEP> Test <SEP> on <SEP> S. <SEP> Cerevisae <SEP> pH <SEP> Test <SEP> on <SEP> S.Cerevisae
<tb>
<tb>
<tb>
<tb> fermentation, <SEP> Units <SEP> of <SEP> dilution <SEP> Units <SEP> of <SEP> dilu-
<tb>
<tb>
<tb>
<tb> hours <SEP> Extract <SEP> 36 <SEP> Broth <SEP> (*) <SEP> tion
<tb>
<tb>
<tb>
<tb> hour <SEP> Extract <SEP> Broth <SEP> pH
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 0 <SEP> '<SEP> @ <SEP> 6.5 <SEP> - <SEP> - <SEP> 6.6 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 25 <SEP> 1600 <SEP> 120 <SEP> 7,2 <SEP> 640 <SEP> 120 <SEP> 7,2
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 49 <SEP> 1600 <SEP> 120 <SEP> 7,0 <SEP> 120 <SEP> 120 <SEP> 7,

  1
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 73 <SEP> 8000 <SEP> 180 <SEP> 7.1 <SEP> 12000 <SEP> 80 <SEP> 7.3
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 97 <SEP> 4000 <SEP> 80 <SEP> 7,0 <SEP> 3000 <SEP> 120 <SEP> 7.1
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 121 <SEP> 3000 <SEP> 40 <SEP> 6.9 <SEP> 4000 <SEP> 120 <SEP> 7.9
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 144 <SEP> 9706 <SEP> 2560 <SEP> 7,2 <SEP> 40 <SEP> 80 <SEP> 8,1
<tb>
   (@) Samples were centrifuged (the tests on the supernatant medium being reported as broth and the centrifugate extracted with a volume of butanol equal to the original sample (the tests on this extract being reported as an extract).



   Example 2. - Fermentation in a stirred flask, of Streptomyces sp. 3694
A suitable inoculation, prepaid according to the manner described in example 1, is introduced into a fermentation medium containing:
 EMI11.2
 
<tb> <SEP> soy flour <SEP> <SEP> 10 <SEP> gm
<tb>
<tb>
<tb>
<tb>
<tb> apples <SEP> from <SEP> earth <SEP> sprayed <SEP> 10 <SEP> gm
<tb>
<tb>
<tb>
<tb>
<tb> dextrose <SEP> 10 <SEP> gm
<tb>
<tb>
<tb>
<tb>
<tb> CoCl2.6H2O <SEP> 10 <SEP> ml <SEP> of a <SEP> solution
<tb>
<tb>
<tb> to <SEP> 0.05%
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> CaCO <SEP> 1 <SEP> gm <SEP>
<tb>
<tb>
<tb>
<tb> Distilled <SEP> water <SEP> 1 <SEP> liter <SEP>
<tb>
 
The medium is sterilized in an autoclave at 121 ° C. for 20 minutes prior to introduction into the seed,

   After four days, tests were carried out against Saccharomyces cerevisiae with a part

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 broth which has been lyophilized and reconstituted with water to about 2 1/3 times the original concentration (on both the clear broth that floats and on the alcohol extract butyl of the washed cells, reconstituted to the volume of the original sample) giving the following results:
S. cerevisiae trial
Dilution units / ml.



  Broth supernatant fluid 5000 Cell extracts 5000 As a result of these tests, the fermentation was carried out for 5 days and, at the end of this period, a test of the broth for its activity showed that it was visually active. - against Candida albicans in a test on a washer.



   Amphotericyne can be extracted from all the broth according to the manner illustrated by the examples below:
Example 3 Extraction of all the broth with isopropanol
The extraction of the Amphotéricyne prepared in Example 1 from all the broth is carried out by adding 80 to 170% of the volume of the broth in isopropanol and by adjusting to a pH of 2, 0 with sulfuric acid. After stirring for about 1/2 hour, the mixture is filtered, preferably using the aid of a filter. The pH of. The filtrate is readjusted to about 7 with sodium hydroxide and the isopropanol is removed by vacuum distillation at a temperature not higher than 35 C.

   The mixture is then left ..., in a cold room overnight, the precipitate which forms is removed by filtration, washed with acetone and dried in vacuum. A mixture of Amphotetyne A and Am- photerycyne B is obtained with a yield of about 40, .. The tests on the mixture were carried out at about 150U-2500.

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 du / mg (Saccharomyces cerevisiae).
 EMI13.1
 



  E1 # l.l.PJ..D 4. - Butanol extraction of all the broth.
A quarter of its volume of butanol is added to 9.4 liters of all the broth, test 3,000 du / ml. The pH is lowered to 2.0 with sulfuric acid and the mixture is stirred well for 1/2 hour. Hyflo (5% w / v) or other filter aid is then added and the mixture is filtered. The filtrate is placed in a separatory funnel and the butanol layer is separated and retained. The butanol solution is then distilled to 1/3 of its original volume under vacuum at a temperature not higher than 35 C. A precipitate forms which is removed by filtration, which is washed well with acetone. and which is dried in a vacuum.



  The product, which is obtained with a yield of approximately 22%,
 EMI13.2
 is a mixture of Amphotericyn A and Amphotericyn B and tests are carried out with about 1600 du / mg against Saccharomyces cervisiae and with about 960 du / mg against Candida albicans.



   EXAMPLE 5.-
 EMI13.3
 Extraction of Amphotericyne from mycelium -
1 liter of all the broth containing Amphotericyne is centrifuged to separate the filtrate and the mycelium.



  The wet mycelium cake is stirred with 200 ml of n-propanol, adjusted to pH 2.0 to 3.0 with sulfuric acid and left in a cold room overnight. The propanol is separated by centrifugation and the mycelial cake is extracted three more times with 100 ml portions of n-propanol. The combined propanol extracts are concentrated
 EMI13.4
 in vacuum at a low volume of about 130 mol. , volume, 'for which a precipitate forms. This precipitate is eliminated

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 by centrifugation and is dried in vacuum.

   About 1.114 gm were recovered, with a potency of about 1176 µl / mg against Candida. 820 mg of this solid product is finely pulverized and is dissolved by heating in a mixture of
80 ml of n-butanol and 16 ml of methanol, then 80 ml of water are added gradually. To this solution are added 48 ml of hexane, and the mixture is stirred, then is left to stand at room temperature overnight. A = fraction of pale yellow crystals of Amphotericyne is formed; it is filtered and dried in a desiccator. Other fractions of less pure material can be obtained by concentrating the mother liquors in vacuum.



   EXAMPLE 6. -
Extraction of all the basic broth.
To a 500 ml sample of all the broth containing Amphotericyne and giving a test of 11,000 du / ml, against Saccharomyces cerevisiae, is added an equal volume of isopropyl alcohol. The pH of this mixture is then raised to 10.5 using 20% sodium hydroxide.



   This mixture is then stirred for 1/2 hour, 2% Hyflo (w / v) is added, and the mixture is filtered. The pH of the filtrate is lowered to 7 using 20% sulfuric acid and the isorpopyl alcohol is removed in vacuo at a temperature not higher than 25 C. After allowing to stand overnight, the precipitate is removed. by filtration, is washed with water and then with acetone and is dried in vacuo. The yield is approximately 1.22 g (77) of a mixture of Amphoricyne A and Amphotericyne B, the test of which gives approximately
3400 du / mg (Saccharomyces cerevisiae).



   Mixtures of Amphotericyne can be fractionated into their constituents, Amphotericyne A and Amphotericyne 6

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 by the methods illustrated in the examples below: EXAMPLE 7. -
Crystallization using alcohol. The Amphotericyne obtained in Example 1) is placed in a slurry of 70% isopropyl alcohol and 30% water at a concentration of 32,000-35,000 u / ml. Candida albicans, while stirring; the pH is lowered to 2 with hydrochloric acid.

   Insoluble matter (Amnhotéricyne
5 crude) is removed by filtration and the pH of the filtrate is raised to about 7.5 'After leaving to stand overnight, the crystalline precipitate, consisting mainly of purified Amphotericyne A, is removed by filtration, is: washed with acetone and dried in vacuum.

   The yield of Ammonoteryne A is about 50% on a biological activity basis.
EXAMPLE 8 - Crystallization using dimethyl formamide.
The Amphotéricyne obtained in Example 5 is treated as a slurry in dimethyl formamide (lg / 20 ml). While stirring, the pH is lowered to 7, using concentrated hydrochloric acid. After stirring for 1/2 hour, the Hyflo product (1% w / v) is added and the mixture is filtered.



   To the filtrate is added one volume of methanol, followed by the slow addition of one volume of water. (Although a solution of methanol and water is preferred, ethanol, acetone or dioxane in water also exert an effective action). The mixture is then allowed to stand overnight with the mixture. resulting pH of 4.5. The precipitate thus formed is removed by filtration, is washed with acetone and is dried. It is composed of 85-90% Amphotericyn B, 5-10% Amphotericyn A and a small amount of other impurities.



   The pH of the mother liquor results from where the Amphoté

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   @ B was isolated is then raised to 8 by addition of sodium hydroxide, and a further 3 volumes of water are added. After allowing to stand overnight, the precipitate is removed by filtration, washed with acetone and dried. This precipitate is composed of 80-85% Amphotericyne A, 5-10% Amphotericyne B and a small amount of other impurities.



   The recovery of all activity is 90-95%.



   The fraction predominantly containing Amphoterichen B can be further purified by slurrying it in 70% aqueous isopropanol at a concentration of 1 g per 100 ml at pH 2 for 1/2 hour. The mixture is then filtered, the filtrate is heated to 45 ° C. and the pH is slowly raised to 5 with sodium hydroxide. The mixture is then allowed to cool slowly to room temperature and stand overnight. The crystallized product (yield of about 50%) is then removed by filtration, is washed with acetone and is dried. It essentially represents the pure Am- photeryne B.



   The fraction containing predominantly Amphotéri-. Cyne A can be further purified by slurrying it in dimethyl formamide at a concentration of 1 g per 20 ml, stirring for 1 hour, filtering off insolubles and adding the filtrate slowly to the mixture. an equal volume of 50% aqueous methanol. The crystallized precipitate is filtered off after standing overnight, washed with acetone and dried. Substantially pure Amphoterichen A is thus obtained in a yield of approximately 70-0%.



   The fractionation and crystallization of the products obtained in Examples 1 to 5 can also be carried out by the processes either of Example 7 or of Example

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 8, the latter being however preferred because of the higher yields of the pure fraction obtained.



   Salts and complexes of Amphotericyne can be prepared according to the methods given in the examples below.



   EXAMPLE 9. Preparation of Alkali Metal Salts of Amphotericyne.
Amphotericyne, either in a raw mixture, or in
 EMI17.1
 the purified crystal form of Artiphatéricyne A or B readily forms salts and the following method is also effective in forming either the sodium or potassium salt of the mixture or the purified components A or B.



   Crystallized Amphotericyne A is suspended in an amount of methanol such that the concentration of the antibiotic is about 250,000 u / ml (Saccharomyces cerevisiae). 2 equivalents of 1N methanolic sodium hydroxide are added and the mixture is stirred for 15 minutes to ensure complete solution of the antibiotic. The solution is filtered and 10 volumes of acetone are added to the wire.
 EMI17.2
 trat. A precipitate of sodium alone is thus formed which is removed by filtration, washed with acetone and dried in a desiccator. The yield of sodium salt is approximately 90% of the original Amphotericyne based on its biological activity. gic (in vitro).
 EMI17.3
 



  The salt q.! ¯'c? D2-.um'jO) IJ.el1te in water a solubili- t '..:.: R.zao JP..9 :: Z20t'E1,. ::: lfl ::: 9Y ':: k'pht;> 4ticyn, E: lez cr.istallized orio; i! 1IEi' .; f: tli "'pJ,"' r. $ t é4lc? m & .nt.iunë "Ibo'niie! Solubilibé danf. le: i'GlcrIlCl .Tu? 1: c ''; CY.r? Tl & I ?? 3 ..dQ. "(J '' .. 1., 'itCa: I'j ..: fIJ.2, 1-1 is rented From soluble Li (: 81113 eth ::" 1tlol pt l-' isoprooanol, but insoluble in? r: tYr acetone, bvtD1J01, chlorofo: r'J'18 ', benzene, hexf'lnc had dioxane.

 <Desc / Clms Page number 18>

 



   EXAMPLE 10. Preparation of the calcium chloride complex.
An Amphotericyne calcium chloride complex can be prepared by dissolving crystallized Amphotericyne either A or B (or a mixture of both) in 1% methanolic calcium chloride (1 gm / 20 ml.), Removing with filter. removing any insoluble material and adding 5 volumes of acetone to the filtrate. The precipitate is removed by filtration, is washed with acetone and is dried in vacuo. The solubility of the calcium chloride complex in organic solvents is similar to that of acid salts. However, in water the complex hydrolyses and its solubility is that of the original Am-photeryne.



   EXAMPLE 11. Preparation of an Acid Salt.
Crystallized Amphotericyne A or B (or a mixture of the two) is dissolved in dimethyl formamide (1g / 25 ml), and one equivalent of concentrated hydrochloric acid is added thereto. The mixture is filtered and 10 volumes of acetone are added to the neutral filtrate. The precipitate thus formed is removed by filtration, washed with acetone and dried in a desiccator in vacuo. The product, which is obtained with a yield of approximately 90%, contains an equivalent of acid and exhibits in vitro a biological activity equivalent to that of crystallized Amphotericyne. It is somewhat more solved. ble in water and much more soluble in methanol, ethanol, isopropanol and butanol than is crystallized Amphotericyne.

   However, the acidic salt is insoluble in acetone, chloroform, ether, benzene, ethyl acetate and hexane.



   The sulphate was prepared in the same manner by substituting one equivalent of sulfuric acid for the hydrochloric acid.

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 drric. It has properties similar to those of hydrochloride.
 EMI19.1
 



  Chemical and physical properties of i \ .mphotéri9l.!. 18.-
Crystallized Amphotericyne A has the following physical and chemical charac- teristics: Melting point:
 EMI19.2
 Pumice at 1 $ U-lt's5 C, browns and contracts at the $ -z0U C melts with decomposition at about 210 C.



  Elemental analysis: C - 59.28% H - 8.43% N - 1.81% 0 - 30.48% (by difference) No other element present.



  No methoxy or acetyl groups.



  Specific optical rotation:
 EMI19.3
 4 C + 1630 (pyridine); +931 (acetic acid); +136 (dimethyl formamide); +28 (0.1N HCl in methanol).



  Solubility: Good solubility in glacial acetic acid and dimethyl formamide. Soluble up to the proportion of about 1 mg / ml in methanol, aqueous isoppopyl alcohol and wet butanol, much more soluble in these solvents when acidified or strongly alkalinized (for example in a solution.
 EMI19.4
 50% isopropyl alcohol, the solubility of Amphotericynes A is, as indicated, approximately 1 mg / ml at a pH between 3 and 8; at pH 2.9 and 10, however, the solubility is 3 mg / ml, while at pH 11 the solubility is increased to 8-10 mg / ml).



   The antibiotic is insoluble in water at neutral or acidic pH under ordinary conditions, but is soluble at pH 10 and above. However, in dilute solutions,

 <Desc / Clms Page number 20>

 the antibiotic can be dissolved in a neutral aqueous solution by increasing the pH of a suspension of the material to 11, such as, for example, with sodium hydroxide. The solution thus formed can be neutralized with an acid without any precipitation occurring.
 EMI20.1
 



  Amphotericyne A is insoluble in ether, dioxane, ethyl acetate, amyl acetate, chloroform, benzene, acetone, hexane, absolute ethanol, l 'iso' propanol or butanol.



   Ultra-violet spectrum:
 EMI20.2
 The absorption maxima in ultraviolet and Amphoter1cyne A crystallized in methanol are:
 EMI20.3
 1, t Max. (m) F, 1% ff l jf 1
 EMI20.4
 
<tb> 228 <SEP> 280
<tb>
<tb>
<tb> 280 <SEP> 262
<tb>
<tb>
<tb> 291 <SEP> 520 <SEP>. <SEP>
<tb>
<tb>
<tb>



  304 <SEP> 780
<tb>
<tb>
<tb> 318 <SEP> 715
<tb>
<tb>
<tb> 343 <SEP> 24
<tb>
<tb>
<tb> 362 <SEP> 34
<tb>
<tb>
<tb> 381 <SEP> 55
<tb>
<tb> 405 <SEP>. <SEP> 66
<tb>
 Some absorption maxima in the 362- region
 EMI20.5
 405 can be said to the presence of a little Amphotericyne B Spe ctre dtinra-, i, ouge .1 t ', - The spectrum of 1 absorption in the infrared of Amphotericyne A in suspension' in the Nujol medium shows pointed geese (and steps indicated by "sh") for the following frequencies and wavelengths:

   

 <Desc / Clms Page number 21>

 
 EMI21.1
 
<tb> Frequency <SEP> Wave length <SEP> <SEP> Frequency <SEP> Wave <SEP> length
<tb>
 
 EMI21.2
 (am-1) '¯¯¯¯¯¯
 EMI21.3
 
<tb> 3333 <SEP> 3. <SEP> 00 <SEP>, <SEP> 1129 <SEP> 8.86
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1695 <SEP> 5.90 <SEP> 1105 <SEP> 9. <SEP> 05
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1656 <SEP> 6.04 <SEP> 1066 <SEP> 9.38
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1623 <SEP> 6. <SEP> 16 <SEP> sh <SEP> 1035 <SEP> 9.66
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1567 <SEP> 6.38 <SEP> sh <SEP> 1008 <SEP> 9.92
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1546 <SEP> 6.47 <SEP> 992 <SEP> 10.08
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1425 <SEP> 7.02 <SEP> sh <SEP> 977 <SEP> 10.24 <SEP> sh
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1403 <SEP> 7.

   <SEP> 13 <SEP> 940 <SEP> 10.54
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1374 <SEP> 7.28 <SEP> sh <SEP> 912 <SEP> 10. <SEP> 96
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1323 <SEP> 7.56 <SEP> 893 <SEP> 11.20
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1276 <SEP> 7.84 <SEP> sh <SEP> 877 <SEP> 11.40
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1232 <SEP> - <SEP> 8.12 <SEP> 850 <SEP> 11.76
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1201 <SEP> 8.32 <SEP> sh <SEP> 836 <SEP> 11. <SEP> 96
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1183 <SEP> 8.45 <SEP> 810 <SEP> 12.34
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1159 <SEP> 8.63 <SEP> 794 <SEP> 12.60
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 760 <SEP> 13.16
<tb>
 
Neutral equivalent of crystallized Ampbotéricyne A:

   
929 titling as a base
989 titrating as an acid
PH stability:. Stable for all pHs between 3 and 11 for at least a period of up to 24 hours. At a pH as low as 2, Amphotericyn A is stable for 3 hours with only a small amount of decomposition occurring.



   Thermal stability:
 EMI21.4
 Stable up to about 70 0 in a neutral solution of t 3.sopr6. 50% panol.



  Chemical Tests: Provides a yellow color with ferric chloride. Crystallized Amphotéricyne'B has the following physical and chemical characteristics:

 <Desc / Clms Page number 22>

 Melting point: No distinct melting point; darkens and chars.



   Elemental analysis:
VS . 56.70% H = 7.72%
N = 1.87% 0 - 33.71%
No other element present.



   Neither methoxy groups nor acetyl groups. Specific optical rotation: [- [alpha]] D24 C + 238 (dimethyl formamide); -52.2 (0.1 N HCl in methanol).



  Solubility: Good solubility in dimethyl formamide and in glacial acetic acid. Soluble up to the proportion of 0.5 mg / l in methanol, aqueous isopropyl alkoll and wet butanol, and much more soluble in these solvents when acidified- or strongly alkalized.



  Ultraviolet spectrum: maximum absorption lees in the ultraviolet of Amphotericyne B crystallized in methanol are:
 EMI22.1
 
<tb> # <SEP> Max. <SEP> (Mu) <SEP> E11% <SEP> cm
<tb>
<tb>
<tb>
<tb>
<tb> 225 <SEP> 230
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 263 <SEP> sh <SEP> 42.8
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 273 <SEP> 60.0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 283 <SEP> 76.0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 345 <SEP> 390
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 362- <SEP> 830
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 382 <SEP> 1380
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 405 <SEP> 1540
<tb>
 

 <Desc / Clms Page number 23>

 Infra-red spectrum:

   The infrared absorption spectrum of Amphotericyne B in suspension in the Nujol medium shows peaks (and stages indicated by "sh") for the following frequencies and wavelengths:
 EMI23.1
 
<tb> Frequency <SEP> Wave length <SEP> <SEP>, <SEP> Frequency <SEP> Wave <SEP> length
<tb>
<tb>
<tb>
<tb>
<tb> (cm-1) <SEP> () <SEP> (cm-1) <SEP> ()
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 3333 <SEP> 3.00 <SEP> 1042 <SEP> 9.

   <SEP> 60 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1709 <SEP> 5.85 <SEP> 1008 <SEP> 9.92
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1577 <SEP> 6.34 <SEP> 981 <SEP> 10.19
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1401 <SEP> 7.14- <SEP> 965 <SEP> 10.36 <SEP> sh
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1374 <SEP> 7.28 <SEP> '<SEP> 903 <SEP> 11.08
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1323 <SEP> 7.56 <SEP> sh <SEP> 886 <SEP> 11.29
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1266 <SEP> 7. <SEP> 90 <SEP> 852 <SEP> 11.74
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1232 <SEP> 8.12 <SEP> 812 <SEP> 12K32 <SEP> sh
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1192 <SEP> 8.39 <SEP> 794 <SEP> 12.60
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1174 <SEP> 8.52 <SEP> 758 <SEP> 13.

   <SEP> 20 <SEP>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1134 <SEP> 8.82
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1106 <SEP> 9.04
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1068 <SEP> 9. <SEP> 36
<tb>
 Neutral equivalent of crystallized Amphotericyne B: 760 / titrant as a base The pH stability and thermal stability of Amphotericyne B are analogous to the corresponding stabilities of Am- photericyne A.



  Biological properties of Amphotericynes.- Amphotericyne, considered as the mixture and known to me Amphotericyne A and Amphotericyne B purified, presents a broad fungicidal spectrum, as indicated by the following table:

 <Desc / Clms Page number 24>

 
 EMI24.1
 
<tb> Amphotericynes <SEP> Amphotericyne <SEP> A <SEP> Amphotericyne <SEP> B
<tb>
<tb>
<tb>
<tb> mixed
<tb>
<tb>
<tb>
<tb> MIC <SEP> in <SEP> / ml <SEP> MIC <SEP> in <SEP> / ml <SEP> MIC <SEP> in <SEP> # / ml
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Saccharomyces <SEP> 25
<tb>
<tb>
<tb>
<tb> cerevisiae <SEP> 3.1 <SEP> 3.1 <SEP> 25-
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Aspergillus
<tb>
<tb>
<tb>
<tb>
<tb> fumigatus <SEP> 3.1 <SEP> 3.1 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Aspergillus <SEP> niger <SEP> 3.

   <SEP> 1 <SEP> 3.1 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> - <SEP> Penicillium <SEP> notatum <SEP> 3.1 <SEP> 3. <SEP> 1 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Ceratostomella <SEP> Ulmi <SEP> 1.6 <SEP> 1.6 <SEP> 3.1
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Candida <SEP> albicans <SEP> 1.6 <SEP> 1.6 <SEP> 6. <SEP> 3
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Tiichophyton
<tb>
<tb>
<tb>
<tb> mentagrophytes <SEP> 9533 <SEP> 1.6 <SEP> 1.6 <SEP> 12.5
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Microsporum <SEP> audouii <SEP> 12.5 <SEP> 3. <SEP> 1 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Microsporum <SEP> canis <SEP> 3. <SEP> 1 <SEP> 3.1 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Botrytis <SEP> tulipae <SEP> 6.3 <SEP> 6.

   <SEP> 3 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Rhodotorula <SEP> glutinis <SEP> 3. <SEP> 1 <SEP> 3. <SEP> 1 <SEP> 100
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> -Fusarium <SEP> bulbigenium <SEP> 6. <SEP> 3 <SEP> 6. <SEP> 3 <SEP> 100
<tb>
 
The above table does not represent an exact indication of the activity of Amphotericyne B, since this antibiotic is extremely insoluble and, therefore, its efficiency in the agar-agar medium used to determine the fungicidal spectrum. above is greatly diminished.

   Thus, in the broth tube tests, when Amphora- ricyn B is compared to mixed Amphotericines for efficiency against Candida albicans and Sachha- romyces cerevisiae, it was determined that the Amphotericyne B was 8.10 and 6.17 times more effective, respectively, than mixed Amphotericines.



     Further tests were carried out on eggs and in mice to determine the efficiency of Amphotericyne as a mixture and in the form of pure Amphetericyne A and pure Amphotericyne B. screw of Candida albicans.



  The conditions and results of representative tests a-

 <Desc / Clms Page number 25>

 with eggs (see table II) and with back mice (see table III) are as follows:
In both trials, the antibiotics used were:

     (1) a mixture of Amphotericyn A and Amphotericyn B dissolved in dimethyl acetamide and then diluted in water so that the final ratio between the antibiotics and the solvent is from 1 to 10 (hereinafter referred to as name "base"), (2) a mixture of sodium salts of Amphotericyne A and Amphoteryne B dissolved in dimethyl acetamide and then diluted in water so that the final ratio between the antibiotics and the solvent is from 1 to 10 (hereinafter referred to as the “Na salt”), (3) A mixture of hydrochlorides of Amphotericyn A and Amphotericyn B dissolved in dimethyl acetamide and then diluted in water so that the final ratio of antibiotics to solvent is 1 to 10 (hereinafter referred to as the "HCl salt").

   



   The toxicity of Amphotericyne is shown in Table IV, in which Amphotericyne was used as a mixture for the purpose of testing.



     Amphotericyne, either as a mixture or individually, is useful in combating pathogenic fungi which cause dermatomycosis and generalized mycosis.



   Specifically, it is effective in the treatment of "athlete's foot", ooccidioidomycosis, moniliasis, blastomycosis, etc. Thus, for prophylaxis and therapy in monilial infection and other fungi. - gnons, Amphotéricyne can be administered to humans orally in the form of tablets containing 1/2 to 1 gm of the antibiotic;

     and thus can be used in combination with broad-based bactericidal antibiotics.

 <Desc / Clms Page number 26>

 spectrum, such as oxytetracycline, chlorotetracycline or tetracycline, in place of nystatin to overcome the development of the resulting fungi.



   Comparison of Amphotericyne with other antibiotics.
Since Amphotericyne, both as a mixture and individually, exhibits certain characteristics analogous to those of other antibiotics, comparative physical and biological tests have been carried out to establish that these are new, unprepared antibiotics. nor previously described.



     Amphotericyne exhibits a high range of activity compared to nystatin and Rimocidin. A comparison of a purified mixture of Amphotericynes A and B with a purified charge of nystatin vis-à-vis two control organisms gave the following result:
 EMI26.1
 
<tb> Witness organization <SEP> Activity report <SEP>
<tb> Amphotericyne / <SEP> nystatin <SEP> '
<tb>
<tb> Saccharomyces <SEP> cerevisiae <SEP> 2.86
<tb>
<tb> Candida <SEP> albicans <SEP> 5.62
<tb>
   Known, Rimocidin has a fungicidal spectrum analogous to that of nystatin and Amphotericyne, it has been tested in the form of its sulphate alongside Am- photeryne and nustatin against S. Cerevisiae.

   It is less active than nystatin or Amphotericyne on a weight basis, as shown in the following table .;

 <Desc / Clms Page number 27>

 
 EMI27.1
 
<tb> MIE <SEP> vis-à-vis <SEP> of <SEP> S. <SEP> cerevisia <SEP>
<tb> u./ml ..
<tb>
<tb>



  Nystatin <SEP> 0.512 <SEP> (8 <SEP> trials)
<tb>
<tb> Sulfate <SEP> of <SEP> Rimocidine <SEP> 0.915 <SEP> (9 <SEP> tests)
<tb>
<tb> Amphotericyne <SEP> 0.160 <SEP> (10 <SEP> tests)
<tb>
 
These results show the superior properties of Amphotericyne by comparison with nystatin and Rimocidin with respect to Saccharomyces cerevisiae and Candida albicans.



   Another comparison between Amphotericyne and nystatin is made by comparing their efficiency against Candida albicans in mice. A mixture of Amphotéricyne A and dtAmphotéricyne B is dissolved in pure dimethyl acetamine. The solution is then diluted with distilled water. For the test, male mice weighing 19-23 grams are infravenously inoculated with Candida albicans, each mouse receiving 2 x 106 cells as determined by readings in a colorimeter. These mice are then treated, either subcutaneously (s.c.) or by bone (p.o.) twice each day for two days with the drug indicated in Table V appended hereto.



  The mice are observed for 10 days (240 hours) and each of the animals which survived after the 240 hours is assigned a value of 240 hours for the purpose of calculating the average survival time *
The results are shown in Table V.



   Apart from the aforementioned biological differences ,. Amphotericyne differs physically from nystatin, Rimocidin and Asconsin as indicated by the following comparisons of specific optical rotation and absorption spectrum in the ultraviolet:

 <Desc / Clms Page number 28>

 
 EMI28.1
 Specific optical rotation of 1 * Amphotericyne A, 1 * Amphotericyne B, lanystatin, Rimocidin and 1 ASCOnSiTU C -7 1)
 EMI28.2
 
<tb> Solvent <SEP> 0.1 <SEP> N <SEP> HCl
<tb>
<tb>
<tb>
<tb>
<tb> Antibiotic <SEP> Pyridine <SEP> Acid <SEP> Formamide <SEP> in <SEP> on
<tb>
 
 EMI28.3
 ] ¯¯¯¯ ¯¯¯¯¯¯ Acetic dimethyl methanol Amphotericyne A + 163 "93 <> +136 28"
 EMI28.4
 
<tb> Amphotericyne <SEP> B <SEP> +238 <SEP> 52.2
<tb>
 
 EMI28.5
 Nystatin + 11 "7 +,

   5 - 3250
 EMI28.6
 
<tb> Sulfate <SEP>
<tb> Rimocidine <SEP> + <SEP> 102 <SEP> + <SEP> 67
<tb>
<tb> Ascosin
<tb>
 
 EMI28.7
 o; o Tr / w NaHCO3) +12.2 - 13
The data in this table are viewed in comparison with the known ultraviolet absorption data for the two Amphotericynes; it is evident that Amphotericyne A, although it has an ultraviolet spectrum which is analogous to that reported for nystatin and rimocidin, differs from it by the respective specific rotatory powers; and, likewise, although Amphotericyne B has an ultraviolet spectrum which is very similar to that of Asconsin, the two products are distinguished by the differences in their respective specific rotational powers.



   The conclusion to be drawn from these comparisons is therefore that Amphotericyne differs from nystatin, Rimocidin or Asconsin both in its biological effects and in its physical characteristics.



   The method according to the invention can be implemented with variants without departing from the scope of the invention.

 <Desc / Clms Page number 29>

 
 EMI29.1
 

 <Desc / Clms Page number 30>

 
 EMI30.1
 

 <Desc / Clms Page number 31>

 
 EMI31.1
 

 <Desc / Clms Page number 32>

 
 EMI32.1
 

 <Desc / Clms Page number 33>

 
 EMI33.1
 

 <Desc / Clms Page number 34>

 
 EMI34.1



    

Claims (1)

CLAIMS.- EMI35.1 :: 1 ========: === :::::::: 1. A process for preparing Amphotericyne, this process comprising cultivating a strain of Streptomyces sp. 3694 in an aqueous nutrient medium comprising a source of assimilable and fermentable carbon and energy and a source of assimilable nitrogen, under submerged aerobic conditions, until such time as substantial fungicidal activity is imparted to this medium. 2. - An antibiotic formed by the method of claim 1. 3.- A process for preparing Amphotericyne, this process comprising cultivating a strain of Streptomyces sp .. 3694 in an aqueous nutrient medium comprising a source of carbon and assimilable and fermentable energy and a source of assimilable nitrogen, under submerged aerobic conditions, until such time as substantial fungicidal activity is imparted to this medium, then to recover the Amphotericyne out of this medium by one. a process which comprises carrying out the extraction in an aqueous lower alkanol medium. 4. A process according to claim 3, wherein the pH of the extraction phase is adjusted to about 2-3. 5. - The method of claim 3, wherein the pH of the extraction phase is adjusted to about 10-11. 6. - Process according to claim 3, in which the Amphotericyne is recovered from the medium by extraction with butanol, by filtration and by concentration of the filtrate to precipitate the Amphotericyne. 7. - Process for preparing Amphotericyne, this process consisting of cultivating a strain of Streptomyces sp. 3694 in an aqueous nutrient medium comprising a source of carbon and assimilable energy and. fermentable and a source <Desc / Clms Page number 36> EMI36.1 of assimilable ilitrogen, under conditions:> o "! Jrob The immersed, until the moment when a forrt; icicl ub.'îbauti & l. activity is conferred on this medium, to filter this medium for one se- EMI36.2 parrying the mycelium and recovering from this mycelium the enrichment by a process which comprises the phase of extraction in a mixture of water and lower alkanol. EMI36.3 8. A process for preparing Amphotericyne, this process comprising cultivating a strain of Streptomyces sp. 3694 in an aqueous nutrient medium comprising a source of EMI36.4 assimilable nitrogen, under aerobic conditions im ',! f, rp / ef, until the moment when a substantial fungicidal activity * and conferred on this medium, to alkalize the medium, to filter the alkalinized medium and to neutralize the filtrate. 9. - Process for fractionating a mixture of Amphotericyne into its constituents, consisting in treating this mixture of Amphotericyne with an alcohol, with the result that one of the constituents is dissolved and the other constituent remains insoluble , then filter the alcoholic mixture. 10. A method according to claim 9, consisting in the fact that the alcoholic mixture of Amphotericyne is treated with: an acid prior to filtration, then is filtered, after which the filtrate is neutralized to precipitate the dissolved component. 11. A process for fractionating a mixture of Amphotericyne into its constituents, comprising treating this mixture of Amphotericyne with a lower di-alconic acid (lower alkyl) amide, with the result that one of the constituents; is dissolved and the other component remains insoluble, then filtering the mixture. 12. A process according to claim 11, wherein the filtrate is treated with an aqueous alcohol to precipitate the dissolved component. <Desc / Clms Page number 37> 13. - A substance capable of inhibiting growth Il /, of fungi chosen from the group comprising Amphotericyne and its salts, this Amphotericyne A being an amphoteric substance having substantially the following analytical composition: C 59.20%, H = 8.43%, N = 1.81% , O = 30.48%, which has a pure crystalline structure and has an absorption spectrum measured in methanolic hydrochloric acid with bands at the following wavelengths: 228, 291, 304 and 318 millimicrons . '14.- Amphotericyne A. 15. - Amphotericyne hydrochloride A. 16.- Salts of alkali metals of Amphotericyne A. 17.- Amphotericyne A Sulfate.- A substance capable of inhibiting the growth of fungus selected from the group consisting of Amphotericyne® and its salts, this Amphotericyne B being an amphoteric substance having substantially the composition following analytical: C = 56.70%, H = 7.72%, N = 1.87%, O = 33.71%, which has a struc. pure crystalline tide and has an absorption spectrum measured in methanolic hydrochloric acid with bands at the following wavelengths: 345, 362, 312 and; 405 millimicrons. ' 19.- Amphotericyne B. 20. - Alkali metal salts of Amphotericyne B.