SE177855C1 - - Google Patents

Description

Inventors: M A Kaplan and I A Mydlinski Priority Beginned 9 Angsti 1955 (United States of America) The present invention relates to a method of producing a therapeutically beneficial substance, called Ayfactin, having antifungal activity and especially crystalline Ayfactin.

There are many infections in humans, animals and plants, which are caused by organisms, commonly referred to as yeast species and fungi, but Oven called Monilia, Saccharomycetes and fungi imperfecti. The result of such infections can vary from severe discomfort to death and entail great economic losses. In many cases, adequate means for controlling or having such infections are still not available. Cure cannot be obtained by using preparations belonging to the sulfanilamide class or by such antibiotics as. penicillin or tetracycline. Other Concerns are either ineffective or have excessive toxicity. There is a great need for an effective and therapeutically suitable antifungal agent.

According to the invention, a crystalline compound called AyfacLin has been discovered which has a therapeutically valuable and potent antifungal effect, especially against Candida albicana, but little antibacterial activity. The properties of hoc Ayfactin are described below.

The process according to the invention for the production of Ayfactin is characterized by the cultivation under submerged, aerobic conditions of a Streptomycet, which forms to produce terracycline and / or chlorotetracycline, the removal of the tetracycline and / or the chlorotetracycline. from the mycelium the new antibiotic, called Ayfactin, is extracted, preferably by extraction with an organic solvent, which forms a solution of at least 2.0 g / l thereof, and that, if desired, this antibiotic is further purified, which contains only the elements carbon, cotton, quay and oxygen In the following approximate amounts: C = 64.9%, H = 3.0%, N = 3.0% and 0 = 24.0% (difference), which in water at a pH of about 7 Or lbsligt in a amount less than 100 ug / ml, which is soluble in dimethylformamide, pyridine, morpholine and piperidine, and which is soluble in pyridine at a concentration of 0.01 mg / ml exhibits absorption maxima yid 410, 388, 366, 348 and 330 microns nima at 400, 376, 354 and 336 my.

Suspended in potassium bromide, Ayfactin, as shown in the accompanying drawing, exhibits characteristic absorption maxima in the infrared at the following frequencies expressed in microns: 3.1, 3.5, 5.9, 6.2 (ledge), 6.3, 7.2 7.3, 8.55, 9.4, 10.0-10.1, 11.85 and 13.0-13.2.

Ayfactin is obtained as nainnt by extraction of the mycelium of tetracycline and chlorotetracycline-producing species of Streptomyces, e.g. Streptomyces aureofaciens (eg strains NRRL2209, B-1286 and B-1288) and Streptomyces viridifaciens (eg strain ATCC-11989). This extraction from mycelium, which is generated by aerobic submerged fermentation, is facilitated by first removing the tetracycline and chlorotetracycline by acidifying the culture solution (eg to pH 2) in order to dissolve these antibiotics and then collect the mycelium (mat) on a filter. . Alternatively, the tetracycline and chlorotetracycline can be precipitated at substantially neutral or at alkaline pH on the mycelial mat and recovered. The tetracycline and chlorotetracycline are then separated from the carpet by dissolving them in acid (eg aqueous hydrochloric acid at pH 2), and the carpet is collected on a filter and treated to recover Ayfactin. The wet sum of the mycelium is installed at about pH 9.0-10.0 (eg with the aid of ammonium hydroxide) and each kilogram of water mycelium (including any filter aid, such as diatomaceous earth) is extracted with about 0, to 2.5 liters n-butanol. The butanol is separated by filtration in the Iran mycelium, installed at a neutral pH (eg pH 7 with sulfuric acid) and concentrated at least twenty times by azeotropic distillation (preferably vacuum distillation, removing water and keeping the temperature low). The ayfactin precipitates on cooling and is recovered by filtration.

In many cases, the Ayfactin is thus obtained in an oily mixture with the fuel oil or other antifoam medal used in the fermentation. By slurrying in a non-basic solvent, such as methyl isobutyl ketone, the fuel oil is dissolved but the Ayfactin is leached. After recovery by filtration, the solid Ayfactin is washed with water to remove the available neutral salts.

Alternatively, water and methyl isobutyl ketone are added to the mixture of fuel oil, neutral salts and Ayfactin, forming three layers: a) fuel oil in methyl isobutyl ketone, b) neutral salts in water and c) Ayfactin. A filter aid was added and the mixture was filtered. The filter cake is then extracted with pyridine. The pyridine-containing Ayfactin is concentrated and dried by distillation, preferably in vacuo, and solid Ayfactin is precipitated by the addition of a liquid precipitant for Ayfactin, e.g. toluene.

Further purification can, if desired, take place by dissolving this Ayfactin in dimethylformamide, pyridine, morpholine or piperidine and filtering to remove inactive, ancient impurities and filling in purified Ayfactin by adding to the filtrate other common organic solvents, e.g. toluene, aliphatic carbonates (Skelly solves) or isopropyl alcohol.

According to another purification method, the Ayfactin obtained above from n-butanol is mixed with aqueous acid (eg acetic acid or hydrochloric acid at pH 1.5) to remove contaminants which are soluble in acid water. Alternatively, the solid Ayfactin is slurried in formamide, methanol, acetone or mixtures daray, releasing additional impurities while the Ayfactin is released only to a small degree.

Crystalline Ayfactin has the above properties. The infrared absorption spectrum is shown in the accompanying drawing.

Ayfactin shows little or no activity against most gram-positive and gram-negative bacteria but is highly effective against certain fungi, such as the dermatophyte M. audouini, which occurs in clinical tinea capitis, the non-pathogenic plant fungus A. solani, the dermatophyte E. floccosum and especially against Candida albicans, the yeast organism that is filocased in the intestinal tract of man during antibiotic therapy, especially together with the widely active antibiotic substances chloramphenicol, tetracycline, chlorotetracycline. In the present description and in a large part of the medical literature, yeasts (Saccharomycetes) and fungi imperfecti, such as Manilla, and the infections caused by them are referred to as moniliasis or fungal infections. The remaining organisms of interest in this context are simply referred to as "other pathogenic fungi". Ayfactin has been shown to be effective in vivo against the pathogenic Candida albicans in moss, as it is injected intraperitoneally into neutral water suspension together with an intraperitoneal injection of chlorotetracycline (to protect against bacterial infection) and sufficiently by Candida albicans to kill all control moss Mom 48 hours. Thus, the minimum dose of Ayfactin required to ensure the survival of at least half of the bog for at least 12 days (CD 4) was 0.04 mg / kg. This shows the extremely fungicidal action of Ayfactin in the living animal body. In a similar experiment, in which Ayfactin was given orally, the CD 'amounted to Over 300 mg / kg, indicating that the Ayfactin joke was absorbed into the bloodstream through the lining of the intestinal tract. Ayfactin has a high therapeutic index (ratio between toxic and effective dose), which shows that its LD (minimum dose for killing half the number of experimental animals) in moss after intraperitoneal injection was set at about 0.8 mg / kg or twenty times the corresponding CD „.

Ayfactin according to the present application is suitable for controlling manga in humans, animals and plants caused by fungal infections. For such use, Ayfactin is combined with a substantial amount of a pharmaceutically acceptable carrier, which may be either a solid or a liquid. The compositions may take the form of tablets, effervescent tablets, powders, granules, capsules (both hard and soft capsules) or suspensions in releasable oils or other dosage forms, which may be administered separately for oral administration. Liquid diluents are added in a sterile state for parenteral use, i.e. by injection. Such a recgpt can be prepared by a sterile solution (eg 1-125 mg / ml dimethylacetamide) or by a suspension in water or an injectable oil, e.g. 0.5% in water containing 0.5% carboxymethylcellulose. The compositions may be in the form of active material, Ayfactin, mixed with solid diluents and / or tableting agents, such as corn starch, lactose, talc, stearic acid, magnesium stearate, gums or the like. An arbitrary Mom pharmaceutical practice custom encapsulation or tableting material can be used. If there is no incompatibility with Ayfactin and the material, Ayfactin can be tableted with or without aids. Alternatively, the Ayfactin may be applied in conventional capsule or resorbable material, e.g. in a gelatin capsule, and is given in this form, e.g. 1-500 and preferably 10-20 mg per capsule or tablet.

The ayfactin according to the invention is particularly valuable for topical application, e.g. in the form of lotions or ointments, yid fungal infections of the skin in humans and animals, and can be sprayed or mixed with fertilizers, as it applies to plants. Due to its low systemic absorption, low tmdcity--3 and strong activity against Candida albicans for Ayfactin, it is especially valuable for the treatment of infections by these organisms or other fungi, which often follow a treatment with antibiotics, as shown by J. Amer. With.

Assoc. 152: 206, 1953, or A. M. A. Arch. Int.

With. 95: 74-82 and 112-117, 1955, or The Practitioner 174: 29-38, 1955. Such fermentation of yeasts and especially of Candida albicans is common in the intestinal tract after antibiotic therapy and sometimes leads to fatal systemic infections. A particularly valuable form of Ayfactin for clinical use in combination with one or more of such Moms is a wide range of effective antibiotics which are as valuable for the treatment of bacterial infections but have the disadvantage of sometimes causing fungal infections, especially in the intestinal tract. usually given orally. For the breath, an average dose of tetracycline, chlorotetracycline, oxytetracycline or chloramphenicol (eg 50-250 mg / capsule) was added to a dose unit of Ayfactin as described above (eg 1-500 mg and preferably -50 mg per capsule). A special embodiment is a capsule containing 250 mg of tetracycline and 20 mg of Ayfactin. Such a product has an antibacterial effect, but a wide range and effective antifungal prophylaxis. If Ayfactin is not absorbed through the lining of the intestinal tract, the effectiveness of its antifungal effect is increased when the product is taken orally and the Ayfactin can exert its action primarily on the lumen of the intestinal tract.

The usual daily dose for adults of this product is 4-8 capsules. Except that such a combination in a capsule can be used to prevent moniliasis or pruritis ani, where the use in tablet form is valuable in the prevention of radiation fungus, the use of suppositories for the prevention of vaginitis or vulvitis and the use of ointments or lotions to prevent of corresponding symptoms in the growth of fungi.

Ayfactin is distinguished by the silt ultraviolet absorption spectrum and the lack of antibacterial activity has allowed all other antifungal agents produced by fermentation except from the substances of the trichomycin-ascosine-candicidine group (see Oroshnik et al, Science, 121, 147-149, 1955. , and Utahara et al., J. Antibiotics Japan, Ser. A 7 (4), 120-124, 1954, and Vining et al., Antibiotics Annual 1954-55, pp. 980-987). Ayfactin differs from candidine by the latter's solubility in water (Vining, ibid., P. 982) and by paper chromatography as well as by candidine's much greater activity against Sporotrichum schenkii (see Taber et al., Antibiotics and Chemotherapy, 4 (4), 455456, 1954). Ayfactin differs from Iran Candicidin by the higher nitrogen content of the latter (determined on a moisture-free and ash-free basis: C = 64.7, H = - 8.34, N = 4.24, 0 = 22.72 by difference; Ayfactin thus appears to contain and additional C7117_, 02-half). Ayfactin Or Above much less soluble in water On Candicidin and at least several times stronger than. Candicidin in the rudder dilution test against Candida albicans. Ayfactin differs from frail ascosine by the latter's much greater solubility in water (see Hickey et al., Antibiotics and Chemotherapy, 2 (9), 472-483, 1952). Ayfactin differs from frail trichomycin by the absence of nitrogen in the latter and by its great solubility in water (see Hosoya et al., J. Antibiotics Japan, 5, 564--566, 1955, and 7, 5-8, 1955). Ayfactin further differs from Iran trichomycin, ascosine, Candicidin and candidine by the differences in resp. infrared d-absorptionssp ektra.

The method for a suitable dilution test with Ayfactin Or as follows. Solid Ayfactin was dissolved in 1 mg / ml in methanol containing 10% hydrochloric acid, and aqueous pH 8 buffer was added to obtain a final pH of 7.3-7.4. The solution was diluted to a concentration of 200 ug / ml. Ten samples are each arranged containing 1 cm 3 of mycophilic wart (modified Sabouraud wart, eg from Baltimore Biological Laboratories) inoculated with a yeast culture (eg Saccharomyces 32) in 1: 10000 dilution. 1 ml of Ayfactin solution is then mixed into the first test tube. 1 cm3 of the first test tube is then transferred to the second tube and the same procedure is repeated throughout the journey. The tubes are then stored and observed for growth or inhibition of common salt. Ayfactin is found to have a minimum inhibitory concentration of less than 0.4 pg / m1 in delta cases and less than 1.0, ag / m1 against Candida albicans.

A typical commercial wart containing about 2500 pg / ml tetracycline contains per g of tetracycline about 1 g of Ayfactin with akt. 1.0 pg / m1 in the above rudder dilution test.

The invention is further damaged by the following examples, which are not to be construed as limiting.

Example 1. 1 kg of mycelium from a submerged aerobic culture of Streptanyces aureofaciens is obtained by acidifying the whole culture solution to pH2 and collecting on a filter. The mycelium was extracted with 3 liters of n-butanol at pH 9.5 and the butanol was separated and extracted with 1.5 liters of water at pH 1.5, discarding the aqueous wash liquor. The N-butanol was concentrated by distillation in vacuo to 200 ml, whereupon 6.2 g of solid Ayfactin precipitated. Delta was taken up in a filter, washed with an aliphatic carbonate (Skellysolve D) and found to inhibit yeast in the rO dilution sample at 1.6 pg / ml.

Example 2. 16 kg of mycelium from a submerged aerobic culture of S. aureofaciens erhollos by acidifying the whole culture solution to about pH 2 and recovering by filtration. The mycelium was extracted with 45 liters of acetone, warp. the mycelium (carpet) was removed by filtration and discarded. Hydrochloric acid was added to adjust the pH of the acetone to 6.0 whereupon 140 g of raft Ayfactin precipitated and collected on a filter, washed with acetone, dried in vacuo over 1330 and found to be active against yeast in the tube dilution sample at 6.3 pg / ml. The crude Ayfactin was slurried in 2.8 liters of water at pH 2.0 and the undissolved, purified Ayfactin was collected on a filter, washed with water and dried, yielding 9.0 g and vam. active against yeast in the rudder dilution sample at 1.6 pg / ml. By slurrying a portion of this Ayfactin in a mixture of equal volumes of hot formamide and methanol, impurities were removed, which were recovered by filling with ether and were found to contain very little Ayfactin when tested. About 7 g of purified Ayfactin remained undisturbed.

Example 3. 8 kg of mycelium from a submerged aerobic culture of S. aureofaciens recovered by acidifying the whole culture solution to about pH 2 and collecting on a filter. The mycelium was extracted with 20 L of n-butanol at pH 9.5 and filtered. The butanol was extracted with an equal volume of water at pH 9.5. The butanol was separated, concentrated by distillation and halided in four volumes of alkanes (Skellysolve C) to give 18.4 g, active at 0.5 pg / ml).

Example 4.

The ayfactin obtained in Example 2 (6.9 was slurried for 15 minutes in a mixture of 600 ml of water and 600 ml of n-butanol and adjusted to pH 10.0 with ammonium hydroxide. The butanol layer was separated and concentrated by vacuum distillation to a volume of 30 ml.) 1.8 g of crystalline Ayfactin precipitated and was recovered by filtration and was found to inhibit yeast in the rudder dilution sample at 0.2-0-0 pglml.

The crystalline Ayfactin has the following properties: Analysis: C = 63.7, H = 7.54, N residue 1.78, H 2 O = 8.26. Found for moisture-free, ash-free product: C = 64.85, H = 7.68, N = 3.01. This suggests a possible empirical formula: CH_ „NO7. Ayfactin is naranog insoluble in water but very soluble in dimethylformamide, pyridine, morpholine and piperidine.

Example 5. 200 g of mycelium obtained in submerged aerobic culture of S. aureofaciens by acidifying the whole culture solution to pH 2 and filtration were extracted with 1 liter of n-butanol. The butanol was separated and concentrated by vacuum distillation to 50 ml. To this solution was added 45 ml to 500 ml of alkanes (Skellysolve C), precipitating 500 mg of solid Ayfactin, which was taken up on a filter and found to inhibit the growth of Candida albicans yid flat samples.

Example 6.

Diatomaceous earth was added to 45 liters of culture fluid obtained in submerged aerobic culture of S. viridifaciens. After filtration, the mycelial cake was washed with water and extracted with 4 liters of acetone adjusted to pH 8.0 by adding ammonium hydroxide. The aqueous acetone solution thus obtained was concentrated by vacuum distillation with the addition of water to give 800 ml of aqueous residue, from which 5.4 g of solid Ayfactin with antifungal activity in the tube dilution sample of 1.8-3.7 pg / ml were precipitated.

The aqueous mother liquor was concentrated to 150 ml, adjusted to pH 1.5 with hydrochloric acid and extracted with 150 ml of n-butanol. The butanol was separated, concentrated by vacuum distillation to a volume of 50 ml and added to 500 ml of alkanes (Skellysolve C), whereupon 3.5 g of solid Ayfactin with antifungal activity precipitated in the quenching sample at 1.8-3.7 pg / ml.

The original mycelial cake was extracted after alkaline extraction with acetone with 4 liters of acetone adjusted to pH 2.0 by the addition of hydrochloric acid. The acidic acetone extract was neutralized to pH 7.0 with ammonium hydroxide, precipitating 27 g of solid product with negligible antifungal activity. The acetone-containing mother liquor was distilled with about 200 ml of water, whereby 2.0 g of oily Ayfactin with antifungal activity could be precipitated in the tube dilution sample at 0.39 pg / ml. Lyophilization of the remaining aqueous filtrate gay 8.0 solid product which did not show flagon antifungal activity at 100 pg / ml.

Example 7.

Ayfactin with antifungal activity at 1.5 pg / ml in the dilution sample was extracted with acetone to give a solid insoluble residue, active yid 0.39 pg / ml, and in addition a solid product by evaporation of the acetone and inactive yid 50 pg / ml.

Ayfactin (200 mg), active at 3.7 pg / ml, was extracted with 20 ml of methanol to give 60 mg of solid soluble Ayfactin, active at 3.1 pg / ml, and in addition 2 mg of solid product by adding the methanol extract to 200 ml. ml of ether and inactive at 10 .mu.g / ml.

Ayfactin (200 mg); active yid 3.7 pg / ml, extracted with 20 ml of n-butanol to give 70 mg of solid insoluble Ayfactin, active yid 6.3 pg / ml. Addition of the butanol extract to 10 volumes of alkanes (Skellysolve C) did not cause any fanning.

Ayfactin (500 mg), active at 3.7 pg / ml, was extracted with 10 ml of hydrochloric acid at pH 1.0 for 10 minutes, leaving 400 mg of solid, insoluble Ayfactin, active at 0.78 pg / ml. The acidic extract was lyophilized to give 80 mg of solid products, inactive at 50 pg / ml.

Example 8. 1 g of Ayfactin, active at 0.4 pg / ml, was suspended in a mixture of 100 ml of water and 100 ml of n-butanol. The mixture was adjusted to pH 1.5 with hydrochloric acid. After stirring for 10 minutes, 90 mg of brown solid products were recovered from the spruce layer, active at 4 pg / ml. After separation of the two phases, the aqueous layer was adjusted to pH 7.0, precipitating 80 mg of solid products, active at 60 ml. Lyophilization of the aqueous filtrate gay 170 mg, inactive at 1 g ml Butanol phase was extracted with an equal volume of water, and the pH was adjusted to 7.0 with ammonium hydroxide. The aqueous layer was separated and lyophilized to give 125 mg of solid Ayfactin, active at 1.2 .mu.g / ml. The butanol was concentrated and dried by distillation to a volume of 50 ml and saint was added to 500 ml of alkanes (Skellysolve C), precipitating 200 mg of solid Ayfactin, active at 4 pg / ml.

Example 9. 1 g of Ayfactin, active at 3.7, a g ml, was suspended in a mixture of 100 ml of water and 100 ml of isobutyl ketone. The mixture was adjusted to pH 2.0 with hydrochloric acid. After stirring, 150 mg of solid Ayfactin was actively recovered from the spruce layer at 0.45 .mu.g / ml. All solids recovered from the liquid phases (total 415 mg) were inactive in the tube dilution sample at concentrations as high as 50 pg / ml.

Example 10.

Ayfactin was not sublimed in a high vacuum at temperatures up to 200 ° C. Nor were any significant amounts of it soluble in large excesses of either chloroform or ethyl acetate. Ayfactin was completely soluble in pyridine and precipitated from ether, acetone or methanol. Ayfactin was completely soluble in piperidine and precipitated by the addition of ether.

Example 11. 6 g of Ayfactin, active at 3.2 μg / ml, were extracted with 100 ml of ammonium hydroxide at pH 10.5 for 15 minutes to give a black oily solution. The oil was removed by extraction with alkanes (Skellysolve C) and the aqueous phase was adjusted to pH 1.5 with hydrochloric acid to give 1 g of a black solid product, active at 1.6 pg / ml.

Example 12.

The study of 500 μg ml of commercial samples of tetracycline and chlorotetracycline by tube dilution showed no antifungal activity.

Claims (5)

Patent claim: 1. Introduced to produce a new antibiotic with fungicidal action, cthmetic drug, to cultivate under submerged, aerobic conditions a Streptomycet which produces tetracycline and / or chlorotracycline, to remove the tetracycline and / or the chlorotetracycline. , that the new antibiotic, called Ayfactin, is then recovered from the mycelium, preferably by extraction with an organic solvent, which forms a solution of at least 2.0 g / l of powder, and that this antibiotic, which contains only the elements carbon, , kydve and oxygen and these in the following approximate amounts: C = 64.9%, H = 3.0%, N = 3.0% and 0 = 24.0% (difference), which in water yid a pH of approx. Soluble in a quantity of less than 100 pg / ml, which is soluble in dimethylformamide, pyridine, morpholine and piperidine, and which dissolved in pyridine at a concentration of 0.01 mg / ml exhibits absorption maxima at 410, 388, 366, 348 and 330 m, uo ch absorption minima at 400, 376, 354 and 336 mo. 2. A claim according to claim 1, characterized in that the culture fluid is acidified, that the antibiotic-containing aqueous phase is separated from the mycelium, that the mycelium is extracted with the organic solvent, and that crystalline Ayfactin is recovered from the Iranian mycelium-separated liquid extract. Salt according to claim 1, characterized in claray, that the culture broth is treated with alkali, wherein the tetracycline and / or chlorotetracycline is carried out, that the aqueous phase is separated from the mycelium and the precipitated phase La amnena, that tetracycline and / or chlorotetracycline is treated with water. , that the mycelium is separated from the aqueous phase and in the aqueous state installed at a pH of about 9.0-10.0, and that the mycelium is finally extracted with the organic solvent 4. A seed according to claims 1-3, characterized in that the mycelium is extracted with n-butanol, dimethylformamide, pyridine, piperidine or morpholine. 5. Set according to claims 1, 2 and 4, Urinary drawing & ray, that the mycelium is extracted at a pH of about 9.0-10.0. Request publications: Stockholm 1962. Kungl. Coniferous. P. A. Norstedt & Sorter. 020089