DE2021436A1 - Fungicidal antibiotic - Google Patents

Abstract

The antibiotic, known as BH 890, is prepd. by fermentation of a strain of Streptomyces misionensis in an aqs. nutrient medium in aerobic conditions. The crude fungicide is separated from the fermentation medium and exists in two forms alpha and beta. BH 890 is active in vivo against Candida albiscans and Cryptococcus neoformus and can be used to treat infections caused by these microorganisms.

Description

Antifungal agent BH890 and its preparation The invention refers to a new antifungal agent that is manufactured by Fermentation, on methods for its isolation and enrichment from raw solutions and procedures for its purification. The new products are against different ones Fungi effective and the effects of the new antifungal agent on certain Microorganisms in connection with their chemical and physical properties differ from known antifungal agents.

The new antifungal agent, which achieves the designation BH 890, arises during the breeding of a streptomycete from one from Pennsylvania forest soil sample was isolated. A viable culture of the new microorganism was obtained from the well-known culture collection facility Culture Collection Laboratory, Northern Utilization Research and Development Division, Inited States Department of Agriculture, On deposit in Peoria, Illinois and included in their permanent collection. The culture has been given the designation NRRL 3609 at this depository office and is freely available to everyone.

The description and identification of this new microorganism, which is also in the culture collection of Lederle Laboratories, Pearl River, New York is held, was held by the expert Dr. H. D. Tresner made these laboratories.

The culture was based on the cultural characteristics, physiological characteristics and morphological features examined according to the methods described by Shirling et al., ["Methods for Characterization of Streptomyces Species", Internat. journ.

of Syst. Bacterioi. 16: 313-340, (1966)]. The media used for the investigation were selected from among the media that of Pridham et al., £ A Selection of Media for Maintenance and Taxonomic Study of Streptomyces ", Antibiotics Annual (1956-1957), pp. 947-953] for the cultivation of Streptomycetes were recommended.

Details are given in Tables I through IV, and a general one Description of the culture is given below. The underlined color names are from Jacobson et al., ["Color Harmony Manula". 3rd edition Container Corp. of America, Chicago (1948)].

Growth Good on most media, weak on Czapek'a solution agar.

Aerial mycelium and / or spore color en masse aerial mycelium whitish to greyish or brownish; Spore masses in greyish tones, those of muted-brownish; Cover Tan (2 ge) to natural color; Natural (2 dc) to slate brownish; Slate Tan (2 ig) to opaque gray; Covert Gray (2 fe) to beige-brown; Beige-Brown (3 ig) are sufficient; Depending on the medium, sporulation weak to strong.

Soluble pigments There are no soluble pigments on the media used Pigments formed.

Revers color In yellowish to greyish to dark greenish tones.

Various physiological reactions Reduction of nitrates to nitrites in organic nitrate broth; complete gelatin liquefaction in 14 days; no Formation of melanoid pigments on peptone iron agar; tolerates up to 7% NaCl in growth medium. C-sources utilization spectrum according to Pridham et al., ["The Utilization of Carbon Compounds by Some Actinomycetales: an Aid for Species Determination ". J. Bacteriol. 56: 107-114 (1948U: Medium to good utilization of 1-arabinose, d-fructose, i-inositol, lactose, d-mannitol, d-melibiose, d-raffinose, 1-2hamnose, salicin, d-trehalose, d-xyloae and glucose; no utilization of adonite, d-melezitose and sucrose ..

Micromorphology Air mycelium strong, forms short, compact spiral chains from spherical to short cylindrical spores with the dimensions 0.5-0.6 / u x 0.9- 1.1 µ; Spore surfaces smooth (by electron microscopy at 8000x magnification certainly).

Because of the general characteristics observed, the culture is a representative of the genus Streptomyces. The grayish sporulation, the spiral chains of smooth spores and the absence of melanoid pigment connects the Culture with a relatively large group of Streptomyces species. Some features E.g. their compact spirals from spherical to occasionally short cylindrical ones However, spores are closely related to species of the Streptomyces hygroscopicus complex vicinity.

By comparison9, which were carried out with representatives of this group, it turned out that the new culture differs from it in that you the conspicuous hygroscopic character of the spore masses is absent, which is normally the case is associated with S. hygroecopicus. Perner are for this latter kind of spurs with short-cylindrical and finger-bone-like (phalangiform) appearance typical (cf. Trenner et al., E. "Morphological Spore Types in the Streptomyces hygroscopicuslike Complex ". Appl. Microbiol. 15: 637-639, (1967)], while the spores of NRRL 3609 often spherical aind. There was, however, a very striking resemblance to the species Streptomyces misionensis, Cercos et al., ["Misionina; antibiotico polienica producido por Streptomyces misionensis n @ sp. Revista de Investigaciones Agricolas 16: 5-28 (1962)] found.

Good agreement was found with regard to spore color, sporophore morphology, Spore shape, spore surface properties, melanin pigment formation and C-source utilization of fat found. Furthermore, such cultural features as growth habitus are parbe of the vegetative Mycelia and the speckled appearance of the culture undersides are noteworthy on some media similar, when comparing with the published descriptions and / or reference cultures of other species no species more similar to this culture than 5 was found. misionensis. Therefore, in the following, NRRL 3609 is considered to be a strain of this species.

Table I Culture characteristics of Streptomyces misionensis NRRL 3609 Incubation 14 days Temperature: 28 ° C Medium growth aerial mycelium and / or soluble reverse remarks Spore pigment color Czapek's solution weakly aerial mycelium whitish. none colorless Agar sporulation weak reverse Asparagine-dextrose aerial mycelium whitish, slate-speckled Agar good becomes covered, brownish-brown; lich; Covert Tan (2 none Slate Tan ge) in sporulation (2 ig) to un- zones. Named sporulation (18 pn) moderate [dark green] bamboo colors; Lapel Hickey and Tresner's good aerial mycelium is whitish Bamboo (2 gc) sprinkles; Agar to gray, will not be natural to unnamed moderate sector Colours; Natural (2 dc) (18 pn) education in sporulation zones. Weak sporulation Table I (cont.) Medium growth aerial mycelium and / or soluble reverse remarks Pigment color Spurs Yeast extract agar good aerial mycelium gray to none clay-brown; Lapel brownish, mottled with slate Adobe Brown; moderate carnation brownish; Slate Tan (2is) (3ig) to sector formation Brown in sporulation zones. Spo- Clowe Brown rulation strong (3 ni) to un- named (18 pn) Lapel Kuster's oatmeal good aerial mycelium gray to brown- light mustard-brown- speckles; measured lich, does not become slate-brown; Lt. Mustard sige sector lich, Slate Tan (2 ig) in Tan (2 ie) to un- education Sporulation Zones. Named Sporu (18 pn) lation moderate maple-yellow; Lapel Tomato paste and oat aerial mycelium gray to brown no yellow maple speckles Flour-agar good, turns slate-brown (3 ng) to un- lich; Slate Tan (2 ig) in named (18 pn) Sporulation Zones. Sporu- lation weak Potato dextrose aerial mycelium white to gray camel; Lapel speckled Agar good, becomes opaque gray; none Camel (3 ie) starch hydrolysis Convert Gray (2 fe) in to unnamed weak Sporulation Zones. Spoof (18 pn) rulation moderate Table I (cont.) Medium growth aerial mycelium and / or soluble reverse remark @ n Spore pigment color Benett's Agar good aerial mycelium gray to brown none covered-brown; Lapel Lich, is slate-tanned Covert Brown Speckles; lich; Slate Tan (2 ig) in (2 li) to excessive sec- Sporulation Zones. Tensioned (18 pn) gate formation rulation moderate Inorganic salts - aerial mycelium whitish, none becomes biscuit-colored; Lapel Starch agar good beige-brown; Beige Brown Biscuit (2 ec) speckles; (3 ig) in sporulation to slate-like sec- zones. Sporulation very brownish; Sla- tor formation te Tan (2 ig) until unnamed (18 pn) Table II Micromorphology of Streptomyces misionensis NRRL 3609 Medium aerial mycelium and / or spore shape Spore size Spore surface phore structures Yeast extract- aerial mycelium strong, forms short, spherical, jelly- 0.5 - 0.6 µ X smooth (electro- Agar compact, spiral spores - sometimes short - microscopic 0.9 - 1.1 µ chains cylindrical copy at 8000 times enlargement Right) Table III Various Physiological Responses of Streptomyces misionensis NRRL 3609 Temperature: 28 ° C Medium Incubation Growth Physiological Response duration Organic nitrate brew well weak reduction of nitrate for 7 days nitrite "Well for 14 days nitrate is reduced to nitrite Gelatin 7 days good weak liquefaction "14 days good complete liquefaction Peptone Iron Agar 24 hours good, no melanin pigment formation 4-13% NaCl in yeast extract agar 10 days up to 7% NaCl in growth medium are tolerated Table IV C source utilization spectrum of Streptomyces misionensis NRRL 3609 Incubation: 10 days Temperature: 28 ° C C-source recycling * Adonite O 1-arabinose 3 d-fructose 3 i-inositol 2 Lactose 3 d-mannitol 3 d-melezitosis Q d-melibiosis 3 d-raffinose 3 1-rhamnose 3 Salicin 3 Sucrose 0 d-trehalose 3 d-xylose 2 Dextrose 3 Negative control * 3-good recovery 1-poor recovery 2-medium recovery 0-no recovery It should be noted that, for the production of the new antifungal active ingredient, the invention is not limited to this specific microorganism or to microorganisms which have the growth characteristics given above for explanation and microscopic features fully correspond. Rather, the scope of the invention also includes the use of mutants which are obtained from the microorganism described by various known measures, for example

X-ray radiation, ultraviolet radiation, treatment with nitrogen mustard, Phage attack and the like are available.

The fermentation process The cultivation of the microorganism S. miaionensis can be performed in a variety of liquid culture media. Media that suitable for the production of the new antibiotic contain an assimilable Source of carbon, e.g. starch, sugar, molasses and glycerin, an assimilable one Source of nitrogen, e.g. protein, protein hydrolyzate, polypeptites, amino acids and corn steep liquor and inorganic anions and cations, e.g. potassium, sodium, Calcium, sulfate, phosphate, chloride and the like.

Trace elements like boron, molybdenum, copper and the like are considered Contamination of other components of the media supplied. For ventilation in Tanks and Plaschen is through. or passing sterile air through or on the surface of the permentation medium. Another mixture in tanks get a mechanical stirrer or impeller. If necessary, a defoamer can e.g. B. 1, octadecanol in lard oil can be added.

Inoculum Preparation A shake flask inoculum from S. misionensis is made by inoculating 100 ml of sterile liquid medium into 500 ml flasks with scraped off or washed off spores of an agar slant culture of the microorganism manufactured. Usually the following medium is used: Cerelose 20 g soy flour 10 g corn steep water 5 g calcium carbonate 3 g water ad 1000 ml The flasks are at a temperature of 25 to 299C, preferably 2600 and incubated on a Rotary shaker agitated intensively for 30 to 46 hours.

These 100 ml inocula are used to inoculate 1 l and 12 l batches of the same medium used in 21 and 20 l glass fermenters. The inoculum mash is ventilated with sterile air for a growing period of 30 to 48 hours. These inoculum approaches are used to inoculate tank fermenters.

Tank fermentation For the production of BH890 in tank fermenters usually the following fermentation medium used: starch 52.5 g corn flour 14.5 g corn steep liquor 15.0 g calcium carbonate 9.5 g ammonium sulfate 6.75 g Casein 3.0 g cottonseed flour 2.5 g ammonium chloride 2.0 g manganese sulfate 0.10 g water ad 1000 ml of lard oil is added to the medium in an amount of 0.8% (volume / volume). Each tank is filled with about 3 ffi of the inocululin prepared as described above inoculated.

Aeration takes place at a rate of 0.5 to 1.0 1 sterile air per liter of broth per minute and the fermentation mixture is through moved an impeller stirrer. which is operated at 300 to 800 rpm. The temperature is kept at 25-290C, usually 28 ° C.

In the cone, fermentation continues for 60 to 90 hours, whereupon the mash is harvested.

Isolation After the fermentation is complete, the fermented mash, containing the antifungal antibiotic according to the invention, filtered, preferably with the addition of dlatomaceous earth or another pilter aid. Usually the Nycel filter cake washed with a small amount of water. Filtrate and wash water are discarded. The antifungal product is made from the mycelial cake with methanol extracted, using about 1 l of methanol per 2.5 to 3.0 l of mash. One full extraction is usually done by a second extraction of the cake guaranteed with a smaller proportion of methanol.

The extracts are combined and formed into one under reduced pressure concentrated aqueous phase. The aqueous phase is left to stand at 4 ° C. overnight. When standing, a solid crude product is formed which contains components which are used as BH890a and BH89Oß. The solid is separated off and washed with acetone. The washed solid is first exposed to the air and then under reduced pressure dried over phosphorus pentoxide or another desiccant.

The a-component is made from part of the crude product thus obtained obtained by repeated recrystallization.

In practice, the ß-component is removed as an impurity, so that the purified α component remains in isolated form. To recrystallize a portion of the crude BH890 is dissolved in dimethylformamide and the solution is filtered Other solvents too, for example dimethylacetamide and dimethyl sulfoxide can be used. Ethyl acetate is used to separate the antifungal product or ethyl acetate and water are used, the mixture is usually placed in a "cold room" (Temperature around 0 to 5 5 ° C) set in order to achieve a complete separation.

The crystalline solid is filtered off with water and acetone washed and air dried. The recrystallization is usually done twice repeated to obtain a purified product BH890α free from is the ß-component. The ß component is made from part of the raw BH890 through Separation of the material into the α and β components by Lo by countercurrent distribution of the solvent won. A useful solvent system consists of ethyl acetate, sec-butanol, Methanol and buffer in a volume ratio of 900: 303: 160: 600. The buffer is used by mixing 6.3 ml of triethylamine with 2 l of water, adjusting with glacial acetic acid to pH 7.5 and diluting with water to 2,400 ml. Using a countercurrent distributor With 200 pipes, around 300 overpasses are sufficient for a good separation of the components aus0 The port stepping of the separation can be determined by measuring the optical density at 303 F and applying the measured values are tracked against the pipe numbers will. Appropriate fractions are combined, the liquid is evaporated and the α and β components are obtained by byophilization.

PHYSICAL PROPERTIES The two components BH890a and BH890ß can be distinguished from one another by certain physical properties will. The analytically pure samples of both components are practically ash-free. BH890a is under reduced pressure in an Abderhalden dryer over boiling Acetone 16 hours and BH890ß is under reduced pressure over P205 and boiling Acetone dried overnight. The components BHB90 «and BH890ß contain the elements Carbon, hydrogen, nitrogen and oxygen practically in the following percentages by weight: BH890α BH890β carbon 58.52 57.78 hydrogen 8.73 7.74 nitrogen 1.78 1.59 Oxygen (difference) 30.97 32.89 The component does not have a sharp melting point. A conventionally obtained infrared absorption spectrum of BH890α as KBr compact has characteristic absorptions in the infrared region of the spectrum at the following wavelengths, given in microns: 3.00, 3.42, 5.85, 6.13, 6.40, 6.48, 6.68, 6.87, 7.17, 7.57, 8.45, 8.87, 9.35, 9.65, 9.95, 10.02, 11.10, 11, 35, 11.82, 13.35 and 14.37, The infrared absorption curve is in Figure 1 of the accompanying drawings Drawings shown.

The a-component shows ultraviolet absorption maxima in Mothanol at: 230 mµ (E11% cm = 315) 279 mµ (E11% cm = 298) 290 mZ (E11% cm - 588) 302 mµ (E11% cm = 905) 318 mµ (E11% cm = 815) When titrating BH890a in glacial acetic acid with perchloric acid becomes a neutralization equivalent of 936 and when nitrated in pyridine with tetrabutylammonium hydroxide found a neutralization equivalent of 962.

The specific rotation value of BH89Oa. was in different solvents certainly. The results obtained are given in Table V below: Table V Specific rotation value of BH890a in various solvents Solvent Concentration [α] 25D dimethylformamide 0.988% + 19.2 ° dimethyl sulfoxide 1.031 * + 588 glacial acetic acid 0.863% + 10.4 ° pyridine 0.930% + 18.29 The ß-component has no defined melting point. An infrared absorption spectrum obtained in a conventional manner of BH890B as a KBr compact shows characteristic in the infrared region of the spectrum Absorptions at the following wavelengths, expressed in microns: 3.00, 3.45, 5.85, 6.36, 6.70, 6.90, 7.25, 8.50, 9.40, 10.00, 11.85, 13.33 and 14.40.

The infrared absorption curve is in figure? of the attached drawings shown.

The ß-component shows ultraviolet absorption maxima in methanol at: 230 mµ (E11% cm = 370) 278 my (E11% cm = 320) 289 mµ (E11% cm = 616) 302 mµ (E11% cm = 934) 318 mµ (E11% = 840) The specific rotation value of BH890B was determined in different Solvents determined. The values obtained are shown in Table VI below specified.

Table VI Specific rotation of BH890ß in various solutions average solvent concentration ga g 25 dimethylformamide 0.747% - 4.03 dimethyl sulfoxide 0.951 ffi - glacial acetic acid 0.993 * - 8.07 pyridine 1.038% - 9.7 The antifungal antibiotic BH890 differs from other antifungal agents by those listed above Labeling data and by s an antifungal activity. The activity of this new antifungal active ingredient in vitro is shown in Table VII below, in which the minimum inhibitory concentration indicated is representative of the inhibition of growth Microorganisms in a nutrient medium is required.

Table VII in vitro antifungal activity of BH890 * organism minimum inhibitory concentration mcg / ml Candida albicans 300 (E83) 2 Cryptococcus neoformans SP (E138) 2 Epidermophyton floccosum ATCC 10227 tE139) 1 Microsporum audouini ATCC 14057 (E140) 2 Microsporum canis ATCC 10214 (E55) 5 Microsporum gypseum ATCC 14683 (E130) 2.5 Phialophthora Jeanselmei NIH 8724 (E16) 5 Trichophyton rubrum (E97) 2.5 Trichophyton tonsurans NIH 662 (E10) 2 Trichophyton mentagrophytes (E11) 2.5 * standard agar dilution method Besides that BH890 shows high activity in vitro against phatogenic organisms, including strains of Blastomyces dermatitidis and Coccidioides immitis, the deep-seated mycoses cause.

Activity against fungi. the deep-seated mycoses cause organism and strain conc. mcg / ml minimal inhibitory fungicide conc. ** conc. * ~~~~~~~~~~~~~~~~~ Coccidioides immitis 658 0.78 1.56 660 0.78 1.56 691 0.20 0.20 Blastomyces dermatitidis 5.10 0.39 5.17 0.39 1.56 5.27 0.20 0.20 amphotericin B, which serves as a control, has an inhibitory effect on the three strains of Coccidioides immitis at 0.2 to 0.39 mcg / ml and at 0.39 to 0.78 mcg / ml fungicl.

Amphotericin B, which serves as a control, acts on the three strains inhibitory of Blastomyces dermatitidis at 0.1 to 0.2 mcg / ml and at 0.39 to 1.56 mcg / ml fungicidal.

* Necessary to inhibit the growth of the microorganism Concentration ** Concentration required to kill the microorganism.

The antifungal antibiotic BH890 is in vivo against Candida albicans and Cryptococcus neoformans are effective.

The new antifungal agent therefore comes as a therapeutic agent for the treatment of fungal infections in mammals by. these Microorganisms. Treating or combating such infections is beneficial by topical application or parenteral or oral administration of the new antifungal antibiotic possible.

The advantages of the new antifungal antibiotic can be seen in his Ability to contribute to lethal systemic infections by these or with these microorganisms Fight mice.

Tests with C. albicans were carried out on female mice, (Oarworth Farm), weighing about 20 g, performed intravenously at a lethal dose a 1:20 dilution of a 24 hour Tryptica, s, e soybean broth culture of the microorganism were infected. The infected mice were given various doses of BH890 by subcutaneous injection within one hour of infection, or by subcutaneous injection within one hour of infection and a second, Dose administered 4 hours later. The size of each group as well as the The number of mice that survived 6 days after infection was noted.

The following Table VIII shows the antifungal activity of BH890 against C. albicans.

Table VIII Activity of BH890. Amphotericin and nystatin mice infected with Candida albicans Subcutaneous dose of live / total mice (%) mg / kg 14 days after infection BH890 Amphotericin Nystatin 800 22/30 (73) 400 15/20 (75) 10/10 (100) 200 10/30 (33) 6/20 (30) 27/30 (90) 100 8/20 (40) 3/10 (30) 15/20 (75) 50 - 12/30 (40) 5/20 (25) 15/30 (50) 25 o / io (O) - 2/20 (10) 12 0/20 (0) 0/10 (0) 3/30 (10) Mean effective dose, mg / kg taae 200 (140-290) - 200 (estimated) 60 (40-70) infected, untreated control animals: 58/60 (97%) mice died within of 3 days after infection (mean survival time 1.3 days) (a) Limits for 95% probability in brackets.

The dose-response curves are not parallel.

Tests with 0. neoformans were performed on female mice, - (Carworth Farms CF 1), weighing about 20 g, performed intravenously with a lethal Dose of a 24 hour trypticase soybean culture of the organism. The infected mice were injected with BH890 at various doses by subcutaneous injection within one hour of infection or by subcutaneous injection within one hour after infection and a second 24 hours later Dose treated. 7 days after infection, the ratio was the Number of living mice to the total number of mice was found. The following table IX shows the antifungal activity of BH890 against 0. neoformans.

Table IX Activity of BH890, Amphotericin and Nystatin in with Cryptoccus Neoformans infected mice Subcutaneous dose, live / all mice (%) mg / kg 7 days after infection BH890 Amphotericin Nystatin 800 41/50 (82) 48/50 (96) Toxic 400 29/30 (97) 17/20 (85) 10/10 (100) 200 47/60 (78) 39/50 (78) 55/60 (92) 100 23/30 (77) 12/20 (60) 26/30 (87) 50 37/60 (62) 35/50 (70) 40/60 (67) 25 1 / io (io) - 13/30 (43) 12 - 13/30 (43) 17/50 (34) mean effective dose, mg / kg (a) 77 (24-250) 20 (12-32) 24 (18-32) infected, untreated control animals: 104/120 (87 *) Mice died between the 2nd and 7th day after infection.

(a) Limits for 95% probability in brackets. The dose-response curves are not parallel.

The invention is illustrated in more detail by the following examples.

B e i 8 p i e l Inoculllm production A typical medium for the The first inoculum is produced using the following recipe: Cerelose 20 g soya flour 10 g corn steep liquor calcium carbonate 3 g water ad 1000 ml of one Agar slant culture of S. misionensis washed or scraped off spores used to inoculate two 500 ml flasks, each 100 ml of the one described above sterile medium. Then the pistons are placed on a rotary shaker and agitated intensively for 48 hours at 28 ° C. The piston inoculum obtained is transferred to a 19 liter (5 gallon) glass fermenter containing 12 liters of sterile medium contains. The cultivation in the glass fermenter is about 48 hours with aeration sterile air. Then the contents of the fermenter are used to inoculate a 300 1 medium used in a tank fermenter.

Example 2 Fermentation A fermentation medium is made according to the following Recipe prepares: Starch 52.5 g corn flour 14.5 g corn steep liquor 15.0 g calcium carbonate 9.5 g ammonium sulfate 6.75 g casein 3.0 g cottonseed flour 2.15 g ammonium chloride 2.0 g manganese sulfate 0.10 g water ad 1000 ml Schma5öl used in the medium in an amount of 0.8% (volume / volume). The fermentation medium is at 12090 45 to 60 minutes with steam at a pressure of 1.4 kg / cm2 (20 pai) sterilized. After sterilization, the pH of the medium is around 6.6. 300 l sterile medium in a 400 l tank fermenter are mixed with 12 l of wie inoculum prepared in Example 1. The fermentation is at 28 ° C carried out using a defoamer (Hodag LG-8 oil). The ventilation takes place at a rate of 0.5 l of sterile air per liter of mash and Minute. The mash is moved by an impeller that rotates at around 300 revolutions operated per minute. After a fermentation period of about 66 hours harvested the mash.

Isolation 300 1 fermented mash with about 2% (weight / Volume) diatomaceous earth filtered as filter aid and the filter cake is with about Washed 30 l of water. The P entered and the wash water discarded. The washed one Cake is stirred in about 100 l of methanol for about 1/2 hour. The suspension will filtered and the kuchan becomes washed with a further 20 l of methanol. The methanol extract and the methanol washing solution are combined and form an aqueous, Phase concentrated, (volume about 6 l). This concentrate is left overnight at about 4 0C left to stand. A birefringent solid that forms when standing, is separated by centrifugation and washed with acetone. The remaining acetone is removed by evaporation in air and the solid is added under P205 dried under reduced pressure. The dried crystalline solid from raw BH890 weighs 118 g.

Example: 4 Extraction of BH890a A portion of the previous one Example obtained crude BH890 of 40 g is dissolved in 150 ml of dimethylformamide and the mixture is filtered. The filtrate is treated with ethyl acetate until the "cloud point" has been reached.

After adding about 150 ml of water, a further 150 ml ml of ethyl acetate were added. The resulting mixture is left to stand at 4UC overnight. The light yellow colored solid that forms and suspends in the two phase system is, is filtered off and washed first with water and then with acetone. The on the air-dried solid weighs 29 g. The crude product is recrystallized twice, by first dissolving it in dimethylformamide and then from that point on the above specified working method repeated. After three recrystallizations, about 4.4 g of purified BH890α was obtained which had the physical properties described above and chemical indicators.

Example 5 Obtaining BH890 Raw BH890, as obtained in Example 9 and used by solution mean countercurrent distribution separated into the a- and ß-component.

The solvent system consists of ethyl acetate, sec-butanol, methanol and buffer in the volume ratio 900: 303: 160: 600. The buffer is created by union of 6.3 ml of triethylamine and 2 1 of water with stirring, adjusting the pH of the Prepare the solution with glacial acetic acid to 7.5 and dilute with water to 2,400 ml. the Countercurrent distribution is performed in a 200 tube apparatus using 10 ml lower phase and 10 ml upper phase per tube and 310 transfers performed.

The sample loading is done in the first 15 tubes by manufacturing a saturated solution of BH890 in 150 ml lower phase and 150 ml upper phase given for a net load of 350 mg. After 310 experiences a portion of the lower phase of 1/2 ml is taken from some tubes. Every The sample is diluted with 10 Dl methanol and checked for its optical density at 303. By plotting the optical density against the pipe numbers, a curve is obtained, which first shows a smaller maximum and then a larger maximum. The pipe numbers 85 through 120 contain BH890ß and tube numbers 113 through 150 contain BH890α. The contents of the appropriate tubes are combined and the liquids become concentrated separately to an aqueous phase, filtered and lyophilized.

Each of the pesticides obtained by lyophilization is treated with acetone triturated, filtered, washed again with acetone and then air dried. Analytically pure samples of BH890β with a weight of 38 Ig and of BH890α weighing 140 mg.

B e i 5 p i e 1 6 Manufacture of tablets containing BH890 The antifungal active ingredient BH890 becomes -customary pharmaceutical according to the following recipe Tablets processed.

per tablet Ingredients mg for 10,000 tablets, g BH890 50 500 lactose 2? 5 2250 corn starch (: for mixing) 50 500 corn starch (for paste) 25 250 magnesium stearate 5 50 355: 3350 The active ingredient BH890, the lactose and corn starch to mix are mixed together. The cornstarch for paste is in water in a ratio suspended by 100 g of corn starch per 800 ml of water and heated by stirring converted into a paste. The paste obtained is then used to granulate the powder mixture related. If necessary, additional water is used.

The moist granulate is passed through a sieve with a clear mesh size 2.38 mm (No. 8 sieve) and dried at 49gC (120 ° F). The dry granules is through a sieve with a mesh size of 1.19. mm (sieve no.16) and made lubricious with the magnesium stearate.

The granules are then compressed into tablets in a tablet machine (Tablet weight 355 mg). Each tablet contains 50 mg of active ingredient.

Example 7 Preparation of hard shell capsules containing BH890 The active ingredient BH890 can be administered in the form of hard-shell capsules. For the production The following recipe is suitable for such capsules: Ingredients mg per capsule for 1000 Capsules, g BH890 50 50 Lactose 90 90 Magnesium stearate 1 1 141 141 The active ingredient, the lactose and magnesium stearate are mixed together. With the mix hard-shell capsules of corresponding size with a filling weight of 141 mg filled per capsule.

Example 8 Preparation of an Oral Sud tension Containing BH890 Ingredients Amount% (w / v) BH890 1.00 Magnesium Aluminum Silicate.Gel 0.50 Sucrose 60.00 methyl paraben 0.08 propyl paraben 0.02 flavoring distilled water t ad 100.00 The parabens and sucrose will be in about 2/3 of the final volume dissolved in distilled water at 809C and the gel (Veegum) is added with stirring. The suspension is cooled to 40UC and stirred with the active ingredient and the Flavoring added. The cooled suspension is made up with distilled water the final volume is set.

The suspension contains 50.mg of active ingredient per ml of suspension.

Example 9 Preparation of an Injection Supplement Containing BH890 The active ingredient BH890 can be made into an injectable suspension according to the following formulation are processed.

Ingredients. Amount% (w / v) BH890 5.0 Polyethylene Glycol 4000 U.S.P, 4.0 Sodium Chloride U.S.P., 0.90 Benzyl Alcohol, Reagent Grade 0.90 Water for Injections ad 100.0 The carrier is made by mixing all the above-mentioned ingredients, with the exception of Dz890. The carrier and the finely ground active ingredient are sterilized. A dispersion of the active ingredient in the carrier is prepared by the active ingredient is first slurried with part of the carrier and then the remainder of the carrier is added with stirring.

A dose of 1 ml of this suspension contains 50 mg of active ingredient.

Table X Oral activity of BH890-alpha and nystatin in mice with C. albicans intestinal infection Oral dose, number of mice with a decrease (2) in C. albicans by 90% mg / kg BH890-alpha nystatin test 1 test 2 test 1 test 2 25 7/10 9/10 10/10 9/10 12 - - 7/10 7/10 6 6/10 6/10 6/10 3 - - 7/10 6/10 1.5 2/5 6/10 4/10 0.8 - - 4/10 4/10 mean effective dose mg / kg (limit of 95% probability) 3.0 (1-9) 1.9 (0.5-8) (1) two doses in 0.5 ml of 0.85% NaCl, 3 hours apart. The first dose is given 1 hour after infection.

(2) Reduction of C. albicans in the faecal flora by at least 90% in comparison to the corresponding mean of the control mice treated with 0.85% NaCl; determined by colony counts on plates containing an aliquot of a One fecal pellet homogenate from each mouse was inoculated. The following average number C. albicans colonies in the fecal sample of the control mice were found: test I - 300 (standard deviation - 180); Test II- 60 (standard deviation - 45); Tabel XI Acuta toxicity of BH890-alpha, amphotericin and nystatin in mice dead / all Mice, 14 days after administration of a single dose, subcutaneously intraperitoneally orally mg / kg BH890 Ampho- Nysta- BH890 Ampho- Nysta- BH890 Ampho- Nystatericin tin tericin tin tericin tin 1600 0/20 0/10 - - 0/10 - 0/20 0/10 0/20 800 0/20 0/10 12/20 - 0/10 - - - -400 - - 5/20 9/10 - 10/10 - - -200 - - 3/20 * 5/10 - 9/10 - - -100 - - - 1/10 - 10/10 - - -50 - - - 0/10 - 4/10 - - -25 - - - - - 0/10 - - - * One dose of 200 mg / kg was not lethal in later tests with infected mice.

Claims (8)

Claims Antibiotic BH890a, characterized in that it (a) growth effectively inhibited by fungi, and in practically pure crystalline form (b) the following Elemental analysis values: C 58.52; H 8.73; N 1.78; 0 30.97; (c) ultraviolet maxima at 230 f (E11% cm = 315), 279 (E11% cm = 298), 290 mµ (E11% cm = 588), 302 mµ (E11% cm = 905), 318 mμ (E11% cm = 815) and (d) a characteristic as shown in FIG Has infrared absorption spectrum. 2. As a compound according to claim 1, antibiotic BH890α in practical pure form. 3. Antibiotic BH890a according to claim 1 and salts thereof. 4. Antibiotic BH890ß, characterized in that it (a) the wachatum effectively inhibited by fungi, and in practically pure crystalline form (b) the following Elemental Analysis Values: C, 57.78; H 7.74; N 1.59; 0 32.89; (c) ultraviolet maxima at 230 mµ (E11% cm = 370), 278 mµ (E11% cm = 320), 289 mµ (E11% cm = 616), 302 mµ (E11% cm # 934), 318 mµ (E11% cm = 840) and (d) one as shown in FIG has a characteristic infrared absorption spectrum. 5. As a compound according to claim 4, antibiotic BH890ß in practical pure form. 6. Antibiotic BH890ß according to claim 4 and salts thereof. 7. A method for the production of antibiotic BH890, characterized in that that an antifungal antibiotic BH890 producing strain of Streptomyces misionensis is grown in an aqueous nutrient medium under aerobic conditions until the nutrient medium as a result of the production of antifungal antibiotic BH890 has considerable antibacterial activity. 8. The method according to claim 7, characterized in that one antifungal antibiotic BH890 producing strain of Streptomyces misionensis cultured in an aqueous nutrient medium under aerobic conditions until the medium a significant antibacterial as a result of the production of the antifungal antibiotic BH890 Having activity, and thus produced antifungal antibiotic BH890 isolated.