JPS6241693B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to antifungal agents useful in the pharmaceutical field. More specifically, neoenactin-producing bacteria belonging to the Streptovertercilium family are cultured aerobically, and the antifungal substance neoenactin is separated from the bacterial cells from the culture solution, purified, and isolated by a pharmaceutically acceptable method. The present invention relates to an antifungal agent containing neoenactin as an active ingredient and its formulation. Diseases caused by fungi are generally considered incurable, and the number of fungal diseases has been rapidly increasing in recent years. Polyethylene antibiotics are currently commonly used as therapeutic agents for deep-seated mycoses. However, there is a narrow gap between the effective dose and the toxic dose, which poses a safety issue. Therefore, the present inventors developed a new screening method using antagonism between polyene antibiotics (e.g., trichomycin) and cholesterol [Chemical
and pharmaceutical Bulletin) Volume 21, 2057
Page, 1973] and searched for non-polyene antibiotics that have antifungal effects or substances that enhance the action of polyene antibiotics. Using this method, the present inventors were able to develop Streptomyces roseobiridis (Streptomyces roseobiridis).
roseoviridis) H646-SY3 is enactin [Journal of Antibiotics (Journal of Antibiotics)
of antibiotics) Vol. 30, p. 182, 1977]. Enactin itself does not have strong antibacterial activity against fungi, but it has been found to significantly enhance the antibacterial activity of trichomycin or amphotericin B. Furthermore, when we searched for microbial cultures using this screening method, we discovered that a strain belonging to Streptoverticillium produced neoenactin, a new substance with strong antifungal activity. The neoenactin of the present invention is a useful antifungal agent, exhibiting a minimum inhibitory concentration of 0.313 μg/ml against the fungus Candida albicans Yu-1200, and enhancing the antibacterial activity of the polyene antifungal agent tricomycin. Furthermore, it enhances the antifungal activity of trichomycin even in the presence of cholesterol, which is said to weaken the antibacterial activity of polyene antifungal agents. The present invention provides neoenactin or its functional derivatives and salts thereof. In addition, the present invention also relates to strains belonging to the genus Streptovertercilium, such as Streptovertercilium
Oribole Teikiyuri Subsp Neoenacteicus H829−MY10 (Streptoverticillium
An object of the present invention is to provide a method for culturing the antifungal substance neoenactin (subsp. Neoenactin is a white amorphous powder.
Shows a melting point of 60.5-64.5°C. Neoenactin is soluble in lower alcohols, ethyl acetate, ether, and chloroform, and insoluble in water, n-hexane, and petroleum ether. The percentage composition (average value) of enactin based on elemental analysis is as follows. C, 63.47% H, 10.04%, N, 7.44%, (O, 19.05%) Halogen and sulfur were not detected. Neoenactin methanol solution, 0.1N hydrochloric acid
The ultraviolet absorption in methanol solution and 0.1N caustic soda-methanol solution is shown in attached Figure 1. 208 nm in methanol solution (E ¹ % ₁ cm = 166) 0.1N hydrochloric acid methanol solution 208 nm (E ¹ % cm =
161) The infrared absorption spectrum of neoenactin potassium bromide tablets at 240 nm (E ¹ % cm = 138) in 0.1N caustic soda-methanol solution is shown in attached Figure 2. Specific optical rotation [α] ¹² _D = -14.2° (C = 3%, in methanol) Color reaction of neoenactin Ninhydrin reaction + Naphtresorcinol-phosphoric acid reaction + KMnO ₄ + Anthrone-phosphoric acid reaction - α-naphthol-phosphorus Acid reaction - Components of neoenactin Neoenactin was hydrolyzed with 1N hydrochloric acid at 110°C for 17 hours, and the hydrolyzate was extracted with ether and then ethyl acetate. The aqueous layer was evaporated to dryness, dissolved in water, passed through Amberlite IR-45, the passed liquid was collected and evaporated under reduced pressure to reduce the volume, and ethanol was added. The obtained white precipitate was identified as L-serine by paper chromatography and infrared absorption spectrum. Paper chromatography and silica gel G thin layer chromatography of neoenactin and enactin Candida albicans as the detected microorganism
The results of various paper chromatography using Yu-1200 and silica gel G thin layer chromatography are shown in Tables 1 and 2. For detection, a ninhydrin reaction and a method of spraying and heating 40% sulfuric acid were used as supplementary methods.

【table】

【table】

[Table] Specific examples of producing microorganisms include Streptovertecilium oliporeifolius subsp. neoenateicus H829-MY10, which belongs to the genus Streptovertercilium. This strain was isolated by the present inventors from soil in Nagasaki Prefecture, and its mycological properties are as follows. (1) Morphology: This strain forms well-branched basal hyphae under the microscope, but the growth of aerial hyphae is slow, and on most synthetic or organic media, aerial hyphae are not produced or even if they are produced. very poor. The sporophyte is mostly straight and produces primary whorls or rarely secondary whorls. The shape of the spore is cylindrical and monophalangeal, the size is about 1 to 1.2 microns, and the surface of the spore is smooth. Streptomyces Luteocolor
luteocolor) or Streptoverticillium
Ball-shaped structures found in vertioillus)
like body) is sometimes observed at the tip of the sporophyte. (2) Growth status on various media Table 3 shows the growth status on various media.

【table】

[Table] (3) Physiological properties Growth temperature range: Good growth on maltose-yeast extract agar medium in the temperature range of 20 to 37°C. Liquefaction of gelatin; moderate liquefaction ability. Hydrolysis of starch; Negative Coagulation of skim milk; Positive Peptonization of skim milk; Positive Production of melanin pigment; Negative (4) Utilization of carbon source (Pridham, Gotdlieb agar) Used; Meso-inositol, D-galactose , dextrin, maltose, glycerol Use is questionable; L-arabinose, D-
Fructose, raffinose, D-trehalose Not utilized; D-xylose, seuclose, D-mannitol, lactose To summarize the mycological properties of strain H829-MY10, it produces whorled branches, grows pale yellowish brown, has white aerial hyphae, and Non-production of soluble pigments. H829−
These characteristics of the MY10 strain are related to Streptoverticillium olivoreticuli, a member of the genus Streptoverticillium.
The properties match well with the properties of In other words, Streptovertercilium oriboleteikilily produces primary and secondary whorled branches, extends pale yellow-brown basal hyphae, and produces spherical structures with unknown physiological functions. However, Streptovertercilium oriboleteikilily is different from the H829-MY10 strain because it belongs to the chromodienic type and hardly uses inositol. Based on the above, the H829-MY10 strain is considered to be a subsp. of Streptovertecilium oriboleteikilily.
Streptoverticillium olivoreticuli subsp.
neoenacticus). The H829-MY10 strain is susceptible to changes in its properties, as seen in other strains of the genus Streptovertercilium or Streptomyces. For example, it is mutated by artificial means using ultraviolet rays, X-rays, radio frequency, radiation, chemicals, etc. Even such mutant strains can be used in the method of the present invention if they have the ability to produce neoenactin or neoenactin components. This strain has been deposited with the Institute of Microbial Technology, Agency of International Trade and Industry, and its FERM accession number is FERM-P 4376. In the method of the present invention, the above-mentioned bacterial strain is usually cultured in a medium containing nutrients that can be utilized by microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, starch, glycerin, dextrin, sucrose, starch syrup, molasses, soybean oil, etc. can be used as the carbon source. Also, as a nitrogen source, soya meal, dry dry mother, cornstarch liquor, wheat germ, cotton seed meal, ammonium sulfate, sodium nitrate, etc. can be used. In addition to adding other inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, phosphate, manganese chloride, copper sulfate, ferrous sulfate, and zinc sulfate as necessary, this also helps the growth of bacteria and promotes the production of neoenactin. Organic and inorganic substances can be added as appropriate. As a culture method, liquid culture is generally suitable, as is the case with antibiotic production methods. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 25 to 35°C, but in most cases it is cultured at around 28°C. Neoenactin production reaches its maximum in 1 to 3 days in both shaking culture and tank culture. The culture conditions described above can be applied by selecting the optimum conditions for each depending on the characteristics of the production strain used. Neoenactin is contained in both the bacterial cells and the culture solution, so it can be extracted from both. For example, the culture solution is extracted with ethyl acetate at pH 8, or the culture solution is adsorbed onto Amberlite XAD- and eluted with aqueous methanol. Furthermore, methanol extraction is performed from the bacterial cells, and after methanol is distilled off under reduced pressure, ethyl acetate extraction is performed at pH 8. Neoenactin contained in both ethyl acetate extracted fractions can be back-transferred to the aqueous layer at pH 2. Neoenactin transferred to the aqueous layer is extracted again into ethyl acetate fraction at pH 8. This ethyl acetate extracted fraction is concentrated under reduced pressure to obtain a highly viscous oily substance. oily substance 1/
20M phosphate buffer (PH8.0) was dissolved in saturated ethyl acetate, cellulose powder soaked in 1/20M phosphate buffer (PH8.0) and air-dried was developed in a column prepared using the same ethyl acetate solution. do. Collect the fraction containing neoenactin and wash with a small amount of water.
Evaporate to dryness under reduced pressure and dissolve 1/20M phosphate buffer in saturated ethyl acetate. This is again developed in a similar column of cellulose powder. The fractions containing neoenactin are collected, washed with a small amount of water, and evaporated to dryness under reduced pressure to obtain neoenactin powder. The antibacterial spectrum of neoenactin against various microorganisms is shown in Tables 4 and 5. The minimum inhibitory concentration was determined on plain agar medium containing glucose at 37°C.

【table】

【table】

[Table] As is clear from the above table, neoenactin exhibits almost no antibacterial activity against bacteria, but it has the property of exhibiting antibacterial activity against yeast and mold. It also enhances the antibacterial activity of known polyene antibiotics, trichomycin, and amphotericin B in the presence and absence of cholesterol. The above-mentioned physicochemical and biological properties are not found in any other known substances, and it is recognized as a new antibiotic with unique properties. Furthermore, neoenactin not only has an effect on yeast and mold, but also suppresses the uptake of 3 _H -thymidine or 3 _H -leucine against cancer cells (eg, Ehrlichi's ascites cancer cells), thereby exhibiting an antitumor effect. Next, we will discuss the formulation of this substance. When used as an antifungal agent, the substance may be used in any form convenient for obtaining its medicinal effect, depending on the type and symptoms of the disease, and may be used alone or in mixtures with pharmaceutically acceptable diluents and other agents. Can be used as The substances can be provided in dosage unit form.
Contains an effective amount of the active ingredient, and its forms include powders, granules, tablets, sugar-coated tablets, etc. for oral use.
Capsules, syrups, pills, suspensions, liquids,
Emulsions, etc. For parenteral use, it can be in the form of an ampoule or pin as an injection solution. Suppositories are also available. The diluent may be solid, liquid, or semi-solid, and examples include the following. That is, excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizing agents, solvents, and the like. Specific examples include lactose, sucrose, sorbitol, mannitrate, starch, precipitated calcium carbonate, heavy magnesium oxide, talc, calcium stearate, magnesium stearate, cellulose or its derivatives, amylopectin, polyvinyl alcohol, gelatin, surfactant, water,
These include physiological saline, ethanol, glycerin, propylene glycol, cacao butter, lauric fat, petrolatum, paraffin, and higher alcohols. The antifungal activity of the present invention can be produced by any known method. The active ingredient in the compositions used in the present invention generally comprises from 0.01% to 100% by weight, preferably from 0.05% to 80% by weight. The antifungal agent of the present invention can be administered orally or parenterally, but oral administration is preferred. Oral administration includes sublingual administration. Parenteral administration includes injection administration (eg, subcutaneous, intramuscular, intravenous injection, infusion), rectal administration, and the like. The dosage of the antifungal agent of the present invention depends on whether it is an animal or a human, and is influenced by age, individual differences, medical conditions, etc., so in some cases, doses outside the range shown below may be administered, but in general, humans are administered. In this case, the oral dosage of this substance is 0.1 to 500 per kg body weight per day.
mg, preferably 1 to 250 mg, the parenteral dosage is also 0.01 to 200 mg, preferably 0.1 to 100 mg per dose.
Administer in ~4 doses. Hereinafter, the present invention will be explained in more detail by showing examples and formulation examples of the substances of the present invention. Parts in the formulation examples below indicate weight. Example 1 Streptovertercilium oriboleteikilily subsp. Neoenacteicus H829−
Bacterium 1ase was inoculated from MY10 preservation slanted agar into 100 ml of a liquid medium (PH7.0) containing 1% maltose, 0.2% yeast extract, and 0.2% polypeptone and cultured with shaking at 27°C for 24 hours, which was used as a starter. 1.5% soluble starch, 1% in a 30 volume jar fermenter
Glucose, 2% Soya Meal, 0.5% Ebios, 0.25% NaCl, 0.3% _CaCO3 , 0.0008%
MnCl ₂ 4H ₂ O, 0.0007% CuSO ₄ 5H ₂ O, 0.0002
A medium containing 15% ZnSO ₄ .7H ₂ O and 0.0001% FeSO ₄ .7H ₂ O (PH 7.6, before sterilization) was prepared and the medium was sterilized by a conventional method. 300 ml of inoculum was transplanted and cultured with aeration at 27°C for 36 hours at an aeration rate of 10 per minute and a stirring rate of 250 rpm to obtain 1 kg of cultured bacteria. The bacterial cells were extracted three times with twice the volume of methanol, and the extracts were combined and dried under reduced pressure to form a syrup. The syrup was brought to pH 8 and extracted with an equal volume of ethyl acetate. Neoenactin extracted into the ethyl acetate layer was back-extracted with 200 ml of water at pH 2.0. This aqueous layer was adjusted to pH 8 again and extracted with an equal amount of ethyl acetate, and after washing the ethyl acetate layer with water,
It was evaporated to dryness under reduced pressure. The amount of crude enactin was 804 mg. Example 2 500 mg of crude neoenactin was soaked in 1/20M phosphate buffer (PH8.0), air-dried, and saturated with 1/20M phosphate buffer (PH8.0) in ethyl acetate (abbreviated below).
A column of cellulose powder prepared using
2 cm), and the active fraction was collected.
The activated fraction was washed with water and concentrated to dryness under reduced pressure.
93.6 mg of neoenactin was obtained. This neoenactin was added again to a similar column of cellulose powder (87x
1.5 cm), separate the active fraction,
After washing with water, it was concentrated to dryness under reduced pressure. The yield was 43.3 mg. Formulation example 1 10 (parts) of this substance (neoenactin), 15 parts of heavy magnesium oxide, and 75 parts of lactose are uniformly mixed and made into a powder or fine granules of 350μ
The following powder is used. Further, this powder was put into a capsule container to form a capsule. Formulation example 2 This substance (neoenactin) 45 (parts) Starch 15 Lactose 16 Crystalline cellulose 21 Polyvinyl alcohol 3 Water 30 After uniformly mixing and blending, crushing and granulating, drying, and then sieving to obtain granules with a size of 1410 to 177μ as a drug. Formulation Example 3 Granules are prepared in the same manner as in Formulation Example 2, and 4 parts of calcium stearate are added to 96 parts of the granules and compressed to form tablets with a diameter of 10 mm. Formulation Example 4 10 parts of crystalline cellulose and 3 parts of calcium stearate were added to 90 parts of the granules obtained by the method of Formulation Example 2, and compression molded to form tablets with a diameter of 8 mm, which were mixed with syrup gelatin and precipitated calcium carbonate. Add the suspension to make sugar-coated tablets. Formulation example 5 This substance (neoenactin) 0.6 (parts) Nonionic surfactant 2.4 Physiological saline 97 After heating and mixing, place in an ampoule and sterilize to make an injection.

[Brief explanation of the drawing]

Figure 1 shows the UV absorption of neoenactin. Figure 2 shows the IR absorption of neoenactin.

Claims (1)

[Scope of Claims] 1. An antifungal agent containing the antifungal substance neoenactin, its functional derivatives, and salts thereof as active ingredients, having the following physical and chemical properties. (a) Elemental analysis value The elemental analysis value (percentage composition (average value) % by elemental analysis is as follows. C: 63.47 H: 10.04 N: 7.44 (O: 19.05) However, halogen elements and sulfur are Not detected. (b) Melting point The melting point of neoenactin is 60.5 to 64.5°C. (c) Ultraviolet absorption spectrum The ultraviolet absorption spectrum of neoenactin is as shown in Figure 1. Also, the absorption and The values of E ¹ % ₁ cm are as follows: [Table] (d) Infrared absorption spectrum The infrared absorption spectrum of neoenactin measured by the potassium bromide tablet method is shown in Figure 2. (e ) Specific rotation The specific rotation of neoenactin in methanol is as follows: [α] ¹² _D = -14.2° (C = 3)% (f) Solubility in solvent The solubility of neoenactin is as follows: Soluble: lower alcohol, ethyl acetate, ether, chloroform Insoluble: water, N-hexane, petroleum ether (g) Color reaction The color reaction of neoenactin is as follows: Ninhydrin reaction + Naftresorcinol - Phosphoric acid reaction + KMnO ₄ + Anthrone-phosphoric acid reaction - α-naphthol-phosphoric acid reaction - (h) R _f value Single value obtained with various developing solvents in neoenactin paper chromatography and silica gel G thin layer chromatography Spoto R _f
The values are as follows. () Paper chromatography [Table] () Silica gel G thin layer chromatography [Table] (i) Appearance Neoenactin appears as a white amorphous powder. (j) Stability against acids and alkalis When Neoenactin is heated at 100℃ for 5 minutes at pH 9.0, 80% of Neoenactin is inactivated. However, approximately 90% of neoenactin's activity is retained even when neoenactin is heated at 100°C at PH2.6 for 5 minutes.